Article

Design of novel artemisinin-like derivatives with cytotoxic and anti-angiogenic properties. J Cell Mol Med

Dafra Pharma Research & Development, Slachthuisstraat, Turnhout, Belgium.
Journal of Cellular and Molecular Medicine (Impact Factor: 4.01). 05/2011; 15(5):1122-35. DOI: 10.1111/j.1582-4934.2010.01120.x
Source: PubMed

ABSTRACT

Artemisinins are plant products with a wide range of medicinal applications. Most prominently, artesunate is a well tolerated and effective drug for treating malaria, but is also active against several protozoal and schistosomal infections, and additionally exhibits anti-angiogenic, anti-tumorigenic and anti-viral properties. The array of activities of the artemisinins, and the recent emergence of malaria resistance to artesunate, prompted us to synthesize and evaluate several novel artemisinin-like derivatives. Sixteen distinct derivatives were therefore synthesized and the in vitro cytotoxic effects of each were tested with different cell lines. The in vivo anti-angiogenic properties were evaluated using a zebrafish embryo model. We herein report the identification of several novel artemisinin-like compounds that are easily synthesized, stable at room temperature, may overcome drug-resistance pathways and are more active in vitro and in vivo than the commonly used artesunate. These promising findings raise the hopes of identifying safer and more effective strategies to treat a range of infections and cancer.

Full-text

Available from: Gert Fricker, Jul 29, 2014
Design of novel artemisinin-like derivatives with cytotoxic
and anti-angiogenic properties
Shahid Soomro
a
, Tobias Langenberg
b
, Anne Mahringer
c
, V. Badireenath Konkimalla
d
,
Cindy Horwedel
d
, Pavlo Holenya
d
, Almut Brand
d
, Canan Cetin
d
, Gert Fricker
c
,
Mieke Dewerchin
b
, Peter Carmeliet
b
, Edward M. Conway
e
, Herwig Jansen
a
, Thomas Efferth
f,
*
a
Dafra Pharma Research & Development, Slachthuisstraat, Turnhout, Belgium
b
KU Leuven, VIB Vesalius Research Center, Herestraat, Leuven, Belgium
c
Institute for Pharmacy and Molecular Biotechnology, University of Heidelberg, Im Neuenheimer Feld, Heidelberg, Germany
d
German Cancer Research Center, Pharmaceutical Biology, Im Neuenheimer Feld, Heidelberg, Germany
e
Centre for Blood Research, Department of Medicine, University of British Columbia, Vancouver, BC, Canada
f
Department of Pharmaceutical Biology, Institute of Pharmacy and Biochemistry, University of Mainz, Mainz, Germany
Received: October 10, 2009; Accepted: April 28, 2010
Abstract
Artemisinins are plant products with a wide range of medicinal applications. Most prominently, artesunate is a well tolerated and effec-
tive drug for treating malaria, but is also active against several protozoal and schistosomal infections, and additionally exhibits anti-
angiogenic, anti-tumorigenic and anti-viral properties. The array of activities of the artemisinins, and the recent emergence of malaria
resistance to artesunate, prompted us to synthesize and evaluate several novel artemisinin-like derivatives. Sixteen distinct derivatives
were therefore synthesized and the
in vitro
cytotoxic effects of each were tested with different cell lines. The
in vivo
anti-angiogenic prop-
erties were evaluated using a zebrafish embryo model. We herein report the identification of several novel artemisinin-like compounds
that are easily synthesized, stable at room temperature, may overcome drug-resistance pathways and are more active
in vitro
and
in
vivo
than the commonly used artesunate. These promising findings raise the hopes of identifying safer and more effective strategies to
treat a range of infections and cancer.
Keywords: malaria
cancer
drug resistance
P-glycoprotein
chemotherapy
J. Cell. Mol. Med. Vol 15, No 5, 2011 pp. 1122-1135
© 2011 The Authors
Journal of Cellular and Molecular Medicine © 2011 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd
doi:10.1111/j.1582-4934.2010.01120.x
Introduction
Artemisinin is a natural product of the plant
Artemisia annua L
.
Reduction of artemisinin yields the more active dihydroartemisinin
(DHA), a compound which is thermally less stable [1]. DHA
can be further converted to different derivatives, including, for
example, artesunate and artemether, which are generally referred
to as artemisinins. Artemisinins are widely known for their
potent anti-malarial activity [2], but also have efficacy in the
treatment of several protozoal and schistosomal infections
[3, 4]. Indeed, artemisinin-like compounds exhibit a wide
spectrum of biological activities, including, for example, anti-
angiogenic, anti-tumorigenic and even anti-viral, all of which are
medically relevant [5–11].
Insights into the mechanisms of action of artemisinins are
increasingly being gained. The anti-tumorigenic activity of the
drug is believed to be partly due to iron-dependent generation of
reactive oxygen species (ROS) [8], as well as alkylation of proteins
and DNA [12, 13]. The underlying molecular mechanisms by
which artemisinins suppress angiogenesis, which in turn, likely
contributes to the anti-tumour activities, are less clear.
Nonetheless, direct effects on angiogenesis and lymphangiogenesis
have been described. Artemisinins inhibit endothelial cell
proliferation, cell migration and endothelial tube formation, at least
partly by inducing apoptosis. They also interfere with synthesis of
*Correspondence to: Thomas EFFERTH,
Department of Pharmaceutical Biology,
Institute of Pharmacy and Biochemistry,
University of Mainz, Staudinger Weg 5,
55128 Mainz, Germany.
Tel.: 49-6131-39-25751
Fax: 49-6131-39-23752
E-mail: efferth@uni-mainz.de
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1123
© 2011 The Authors
Journal of Cellular and Molecular Medicine © 2011 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd
vascular endothelial growth factors [14–22], possibly
via
suppres-
sion of hypoxia inducible factor activation [23].
In spite of its therapeutic utility in treating malaria, resistant
strains of the malaria parasites are emerging, mostly in western
Cambodia, where treatment failure rates after combination therapy
have exceeded 10% [24]. The mechanisms of resistance are largely
unknown, but may replicate some of those that become active in
cancer cells as they develop chemoresistance. These include,
among others, mutations in target proteins, resistance to apopto-
sis and increased drug efflux
via
transporters [25]. Interestingly,
the latter mechanism is known to be used by parasites to enhance
the clearance of drugs, and the multidrug resistance-conferring
ATP-binding cassette (ABC) transporter, P-glycoprotein (P-gp) [26]
has been implicated. Increased expression of ABC transporters
such as P-gp may also enable tumour endothelial cells to escape
from anti-angiogenic treatment. Because artemisinin-like com-
pounds are generally well tolerated, with potentially wide clinical
applications beyond malaria, it is important to identify alternative
forms that do not induce host resistance.
In this report, we synthesized several novel artemisinin-like
compounds, and tested their
in vitro
cytotoxic effects
,
their capac-
ity to alter P-gp function and finally their
in vivo
anti-angiogenic
properties. Our strategy was based on the generally accepted con-
cept that DHA, a breakdown product of artesunate (see structures,
Fig. 1), provides the biological activity of all the artemisinin-related
compounds. Our biochemical approach was feasible, because the
lactol of DHA can be converted to different derivatives, such as
ethers and esters, allowing us to synthesize a range of different
DHA derivatives. The findings provide new insights that will hope-
fully lead to the development of more effective treatment options
for a variety of diseases.
Material and methods
Chemistry
Materials and reagents were purchased from Acros Organics (Beerse,
Belgium) or Aldrich (Taufkirchen, Germany). Tris-(2-aminoethyl)-amine
polystyrene resin was obtained from Nova biochem (Merck, Darmstadt,
Germany). Nuclear magnetic resonance (NMR) spectra were recorded on a
Bruker Avance DRX-400 spectrometer (400 MHz) (Bruker Daltonik, Bremen,
Germany). Coupling constants (
J
) are reported in Hz. Column chromatog-
raphy was performed on a Flashmaster II (Jones Chromatography) with
Isolute columns pre-packed with silica gel (30e90 mM) for normal phase
chromatography. Melting points were determined with a capillary melting
point apparatus (Büchi 510, BUCHI, Flawil, Switzerland) and are uncor-
rected. Electrospray ionization mass spectra were acquired on an ion trap
mass spectrometer (Bruker Daltonics esquire 3000 plus, Bruker Daltonik).
LC-MS spectra were recorded on an Agilent 1100 Series HPLC system
(Agilent Technologies, Böblingen, Germany) equipped with a HILIC Silica
column (2.1 100 mm, 5 mm, Atlantis HILIC) (Waters, Eschborn, Germany)
coupled with a Bruker Daltonics esquire 3000 plus mass spectrometer
(solvent A: H
2
O with 0.1% formic acid, solvent B: acetonitrile (ACN) with
0.1% formic acid, gradient 2: 90% B to 40% B, 12 min., 0.2 ml/min.).
Analytical TLC was done on pre-coated silica gel plates (60 F254, 0.2 mm
thick, VWR International, Darmstadt, Germany), visualization of the plates
was accomplished using UV light and/or iodine staining. The dried solvents
were purchased from Acros Organics. Artemisinin, DHA and artesunate
were provided by Dafra Pharma R&D (Turnhout, Belgium), anhydrodihy-
droartemisinin (4) [27], deoxoartemisinin (3) [28], 10-dihydroartemisinyl
acetate (7) [29], compound 5a synthesized by a modified procedure,
NaOH/H
2
O
2
were used as oxidizing agents [30], 10-dihydroartemisinyl
benzoate [13, (29) with small modification, instead of benzoylchloride, the
benzoic anhydride was used with catalytic amount of 4-(dimethylamino)-
pyridine (DMAP)] were prepared as previously described.
Fig. 1 Structure of artemisinin, DHA and artesunate.
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1124 © 2011 The Authors
Journal of Cellular and Molecular Medicine © 2011 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd
Synthesis of 10-dihydroartemisinyl 2,
2-dichloroacetate (8)
DMAP (0.6 g, 4.9 mmol) and dichloroacetic anhydride (6.0 g, 25 mmol)
were added to a stirred solution of DHA (5 g, 17.6 mmol) in
dichloromethane (300 ml) at 0C and the reaction mixture was slowly
brought to room temperature and stirred for 6 hrs, during which time, all
DHA was consumed. The solvent was removed under reduced pressure
and the residue was purified by flash chromatography with ethyl acetate/
hexane (10:90 to 50:50) to provide the product dense liquid (3.82 g, 55%).
1
H-nuclear magnet resonance (HNMR) (400, CDCl
3
) d 0.86 (d,
J
7.0 Hz,
3 H, 9-Me), 0.97 (d,
J
5.95 Hz, 20 3 H, 6-Me), 1.45 (s, 3 H, 3-Me),
1.23–1.94 (m, 9 H), 2.04 (ddd,
J
14.5, 5.0, 3.0 Hz, 1 H), 2.39 (ddd,
J
14.5,
5.0, 3.0 Hz, 1H), 2.55 (m, 1 H, H-9), 5.40 (s, 1 H, H-12), 5.90 (d,
J
10.0 Hz, 1H, H-10), 6.25 (s, 1H, COCHCl
2
); electron ionization mass
spectrometry (EIMS) (m/z) 396.3 (MH)
.
Synthesis of 10-dihydroartemisinyl
butyrate (9)
DMAP (0.6 g, 4.9 mmol) and butyric anhydride (4.0 g, 25 mmol) were added
to a stirred solution of DHA (5 g, 17.6 mmol) in dichloromethane (300 ml)
at 0C and the reaction mixture was slowly brought to room temperature and
stirred for 8 hrs, during which time, all DHA was consumed. The solvent was
removed under reduced pressure and the residue was purified by flash chro-
matography with ethyl acetate/hexane (10:90 to 50:50). Re-crystallization
from ethyl acetate/hexane provided white big crystals (5.9 g, 95%), m. p.
81–85C.
1
HNMR (400, CDCl
3
) d 0.86 (d,
J
7.0 Hz, 3 H, 9-Me), 0.97 (d,
J
5.95 Hz, 3 H, 6-Me), 1.17–1.24 (m, 6 H), 1.45 (s, 3 H, 3-Me), 1.23–1.94
(m, 9 H), 2.04 (ddd,
J
14.5, 5.0, 3.0 Hz, 1 H), 2.39 (ddd,
J
14.5, 5.0,
15 3.0 Hz, 1H), 2.55 (m, 1 H, H-9), 2.68 (m, 1 H, COCH), 5.45 (s, 1 H,
H-12), 5.850 (d,
J
10.0 Hz, 1 H, H-10); EIMS (m/z) 355.4 (MH)
.
10-dihydroartemisinyl propionate and butyrate are similar.
1
HNMR of
10-dihydroartemisinyl propionate
1
HNMR (400, CDCl
3
) d 0.91 (d,
J
7.0 Hz, 3 H, 9-Me), 1.03 (d,
J
5.95 Hz, 3 H, 6-Me), 1.17–1.24 (m, 6 H),
1.50 (s, 3 H, 3-Me), 1.23–1.94 (m, 7 H), 2.04 (ddd,
J
14.5, 5.0, 3.0 Hz,
1 H), 2.39 (ddd,
J
14.5, 5.0, 15 3.0 Hz, 1H), 2.55 (m, 1 H, H-9), 2.68 (m,
1 H), 5.51 (s, 1 H, H-12), 5.87 (d,
J
10.0 Hz, 1 H, H-10).; EIMS (m/z)
350.0 (MH)
.
Synthesis of 10-dihydroartemisinyl
i
butyrate (10)
DMAP (0.6 g, 4.9 mmol) and isobutyric anhydride (4.0 g, 25 mmol) were
added to a stirred solution of DHA (5 g, 17.6 mmol) in dichloromethane
(200 ml) at 0C and the reaction mixture was slowly brought to room tem-
perature and stirred for 8 hrs, during which time, all DHA was consumed.
The solvent was removed under reduced pressure and the residue was
purified by flash chromatography with ethyl acetate/hexane (10:90 to
50:50) to provide the product dense liquid (5.2 g, 84%).
1
HNMR (400,
CDCl
3
) d 0.86 (d,
J
7.0 Hz, 3 H, 9-Me), 0.97 (d,
J
5.95 Hz, 3 H, 6-Me),
1.17–1.24 (m, 6 H) 1.45 (s, 3 H, 3-Me), 1.23–1.94 (m, 9 H), 2.04 (ddd,
J
14.5, 5.0, 3.0 Hz, 1 H), 2.39 (ddd,
J
14.5, 5.0, 15 3.0 Hz, 1H), 2.55
(m, 1 H, H-9), 2.68 (m, 1 H, COCH), 5.45 (s, 1 H, H-12), 5.850 (d,
J
10.0 Hz, 1 H, H-10); EIMS (m/z) 355.4 (MH)
.
Synthesis of 10-dihydroartemisinyl
2-propylpentanoate (11)
DMAP (0.5 g, 4.1 mmol) and triethylamine (3.03 g, 30 mmol) were added to
a stirred solution of DHA (7.1 g, 25 mmol) in dichloromethane (400 ml).
2-Proplypentanlychloride (4.87 g, 30 mmol) at 30C was added, and the
reaction mixture was continuously stirred for 2 hrs and slowly brought to
room temperature and stirred overnight. The solvent was removed under
reduced pressure and the residue was purified by flash chromatography with
ethyl acetate/hexane (10:90 to 50:50) to provide the product as a white solid.
Re-crystallization from ethyl acetate/hexane resulted in a colourless liquid
(8.19 g, 80%).
1
HNMR (400, CDCl
3
) d 0.86 (d,
J
7.0 Hz, 3 H, 9-Me), 0.90
(t, 6H), 0.97 (d,
J
5.95 Hz, 3 H, 6-Me), 1.33 (m, 4H), 1.45 (s, 3 H, 3-Me),
1.64 (m, 4H), 1.23–1.94 (m, 9 H), 2.04 (ddd,
J
14.5, 5.0, 3.0 Hz, 1 H), 2.29
(t, 1 H), 2.39 (ddd,
J
14.5, 5.0, 15 3.0 Hz, 1H), 2.55 (m, 1 H, H-9), 5.45
(s, 1 H, H-12), 5.850 (d,
J
10.0 Hz, 1H, H-10); EIMS (m/z) 411.5 (M H)
.
Synthesis of 10-dihydroartemisinyl 2,
2-dimethylpropianate (12)
DMAP (0.5 g, 4.1 mmol) and trimethylacetic anhydride (5.59 g, 30 mmol)
were added to a stirred solution of DHA (7.1 g, 25 mmol) in
dichloromethane (400 ml) at 0C. The reaction mixture was slowly brought
to room temperature and stirred overnight, during which time all DHA was
consumed. The crude material was washed with water (2 100 ml), and
the solvent was removed under reduced pressure. The product was then
re-crystallized from ethyl acetate/hexane, yielding white crystals (5.17 g,
75%), m. p. 101–104C.
1
HNMR (400, CDCl
3
) d 0.86 (d,
J
7.0 Hz, 3 H,
9-Me), 0.97 (d,
J
5.95 Hz, 3 H, 6-Me), 1.25 (s, 9H C(CH)
3
), 1.45 (s, 3 H,
3-Me), 1.23–1.94 (m, 9 H), 2.04 (ddd,
J
14.5, 5.0, 3.0 Hz, 1 H), 2.39
(ddd,
J
14.5, 5.0, 15 3.0 Hz, 1H), 2.55 (m, 1 H, H-9), 5.45 (s, 1 H, H-
12), 5.850 (d,
J
10.0 Hz, 1H, H-10); EIMS (m/z) 369.5 (MH)
.
Synthesis of 10-dihydroartemisinyl N,
N-dimethylacetamide (14)
DMAP (0.5 g, 4.1 mmol) and dimethylcarbomoyl chloride (3.23 g,
30 mmol) were added to a stirred solution of DHA (7.1 g, 25 mmol) in
dichloromethane (400 ml) at 0C. The reaction mixture was slowly brought to
room temperature and stirred for 8 hrs, during which time all DHA was con-
sumed. The crude material was washed with water (2 100 ml) and the sol-
vent was removed under reduced pressure. The residue was purified by flash
chromatography with ethyl acetate/hexane (10:90 to 90:10), yielding a white
dense liquid (5.8 g, 65%).
1
HNMR (400, CDCl
3
) d 0.86 (d,
J
7.0 Hz, 3 H,
9-Me), 0.97 (d,
J
5.95 Hz, 3 H, 6-Me), 1.45 (s, 3 H, 3-Me), 1.23–1.94 (m,
9 H), 2.04 (ddd,
J
14.5, 5.0, 3.0 Hz, 1 H), 2.39 (ddd,
J
14.5, 5.0, 15 3.0
Hz, 1H), 2.55 (m, 1 H, H-9), 2.92 (s, 3H, N(CH
3
)
2
, 2.98 (s, 3H, N(CH
3
)
2
, 5.45
(s, 1 H, H-12), 5.68 (d,
J
10.0 Hz, 1H, H-10); EIMS (m/z) 356.4 (MH)
.
Synthesis of 10-(2-butyloxy) dihydroartemisinin (15)
Boron trifluoride-diethyl ether (3 ml) was added to a stirred solution of
DHA (1, 2.56 g, 9.0 mmol) and
i
butanol (2.2 g, 30 mmol) in diethyl ether
(100 ml). After 6 hrs, the reaction mixture was quenched with saturated
aqueous NaHCO
3
and dried with MgSO
4
. Filtration and concentration of the
Page 3
J. Cell. Mol. Med. Vol 15, No 5, 2011
1125
© 2011 The Authors
Journal of Cellular and Molecular Medicine © 2011 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd
filtrate gave a residue which on flash chromatography with ethyl
acetate/hexane (5:95 to 10:90), yielded a white microcrystalline powder
(2.05 g, 67%), m.p. 100–101C.
1
HNMR (400, CDCl
3
) d 0.86 (d,
J
7.0 Hz,
3 H, 9-Me), 0.97 (d,
J
5.95 Hz, 3 H, 6-Me), 1.08 (d,
J
6.1Hz, 3H),
1.20 (d,
J
6.2 Hz, 3H), 1.45 (s, 3 H, 3-Me), 1.23–1.94 (m, 9 H), 2.04
(ddd,
J
14.5, 5.0, 3.0 Hz, 1 H), 2.39 (ddd,
J
14.5, 5.0, 15 3.0 Hz, 1H),
2.55 (m, 1 H, H-9), 4.0 (m, 1 H, OCH(CH
3
)
2
), 4.87 (d,
J
3.5 Hz, 1 H,
H-10), 5.44 (s, 1 H, H-12); EIMS (m/z) 341.5 (MH)
.
Synthesis of 10-dihydroartemisinyl
thioethylamine (16)
DHA (7.1 g, 25 mmol) and cysteamine (2.7 g, 35 mmol) were dissolved in
300 ml dichloromethane and boron trifluoride-diethyl ether (10 ml) was
added slowly at 0C. The reaction mixture was stirred for 3 hrs at 0C and
an additional 1 hr at room temperature. The reaction was quenched with
5% NaHCO
3
and extracted with dichloromethane. The solvent was
removed under reduced pressure and the residue was purified by flash
chromatography with ethyl acetate/hexane (10:90) to yield a brown wax
product (4.7 g, 55%).
1
HNMR (400, CDCl
3
) d 0.86 (d,
J
7.0 Hz, 3 H, 9-
Me), 0.97 (d,
J
5.95 Hz, 3 H, 6-Me), 1.25, 1.45 (s, 3 H, 3-Me), 1.23–1.94
(m, 9 H), 2.04 (ddd,
J
14.5, 5.0, 3.0 Hz, 1 H), 2.39 (ddd,
J
14.5, 5.0,
15 3.0 Hz, 1 H), 2.55 (m, 1 H, H-9), 2.9 (t, 2H), 3.1 (t, 2H), 4.56 (d,
J
10.0 Hz, 1 H, H-10), 5.31 (s, 1 H, H-12); EIMS (m/z) 344.5 (MH)
.
XTT cytotoxicity assay
Multidrug-resistant, P-gp-overexpressing CEM/ADR5000 cells and their
parental, drug-sensitive counterpart, CCRF-CEM cells were used. The cell
lines were provided by Dr. Daniel Steinbach (University of Ulm, Ulm,
Germany). Doxorubicin resistance of CEM/ADR5000 was maintained as
described [31]. CEM/ADR5000 cells have previously been shown to selec-
tively express multidrug resistance (MDR)1 (ABCB1), but none of the other
ABC transporters [32]. The cell lines were maintained in Roswell Park
Memorial Institute (RPMI) medium (Life Technologies, Carlsbad, CA, USA)
supplemented with 10% foetal calf serum in a humidified 7% CO
2
atmos-
phere at 37C. Cells were passaged twice weekly. All experiments were
done with cells in the logarithmic growth.
Cytotoxicity was assessed using the 2,3-bis[2-methoxy-4-nitro-5-
sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt (XTT) assay kit
(Roche, Indianapolis, IN, USA), which measures the metabolic activity of
viable cells [33, 34]. Toxicity of compounds was determined with the Cell
Proliferation Kit II (Roche Diagnostics, Mannheim, Germany), according to
the manufacturer’s instructions. Fresh stock solutions of each compound
were prepared in dimethyl sulfoxide (DMSO) at a concentration of 100 mM,
and a dilution series was prepared in Dulbecco's minimal essential
medium (DMEM). Cells were suspended at a final concentration of 1
10
5
cells/ml, and 100 ml were aliquoted per well into a 96-well culture plate
(Costar Corning, Lowell, MA, USA). Marginal wells were filled with 100 l
of media to minimize evaporation. A row of wells with cells was left
untreated and another row of wells with cells was treated with 1 l DMSO,
the latter serving as a solvent control. All studies were performed in dupli-
cate, in a range of concentrations and in two independent experiments with
different batches of cells. Quantification of cytotoxicity was achieved with
an ELISA plate reader (Bio-Rad, München, Germany) at 490 nm with a ref-
erence wavelength of 655 nm, and reported as a percentage of viability
compared to untreated cells. The ligand binding module of Sigma plot soft-
ware (version 10.0) was used for analysis.
HUVEC proliferation/viability assay
Single donor human umbilical vein endothelial cells (HUVECs) cells were
purchased from Lonza (Breda, Netherlands). Cells were seeded at 5000 cells
per well in 96-well microtitre plates in endothelial cell growth medium
(EGM)-2EV medium (Invitrogen, Darmstadt, Germany). Upon adherence the
cells were gently washed twice with phosphate-buffered solution and starved
overnight in EGM-2EV medium with reduced foetal bovin serum (FBS) con-
tent (0.1%; starvation medium). The medium was then aspirated and
replaced with starvation medium with or without 30 ng/ml recombinant
human vascular endothelial growth factor (VEGF)165 (R&D System,
Wiesbaden, Germanys) and with or without increasing concentrations of
compound 1 (artemisinin), 7 and 10 (0.5–100 M). Due to its precipitation
from the cell culture medium, artesunate could not be used as a reference
compound. After 96 hrs, cell growth was quantified using the water-soluble
tetrazolinum (WST)1 Rapid cell proliferation kit (Calbiochem, Merck,
Darmstadt, Germany), and was expressed in percentage of the control value
(VEGF alone). Experiments were carried out in triplets.
Isolation of porcine brain capillary
endothelial cells (PBCECs)
PBCECs were isolated from porcine brains as reported [35]. Briefly, freshly
isolated porcine brains were collected from the local slaughterhouse, cleaned
of meninges, choroid plexus and superficial blood vessels. After removal of
grey matter, the tissue was minced into cubes 2 mm
3
and incubated in
Medium 199, supplemented with 0.8 mM L-glutamine, penicillin/strepto-
mycin (100 U/ml), 100 g/ml gentamicin and 10 mM 2-(4-(2-hydroxyethyl)-
1-piperazinyl)-ethane sulfonic acid (HEPES), pH 7.4 (Biochrom, Berlin,
Germany) with dispase II (0.5%) (Roche Diagnostics) for 2 hrs at 37C. After
centrifugation at 1000
g
for 10 min. at 4C, the supernatant was discarded
and the pellet was re-suspended in media containing 15% dextran (Sigma-
Aldrich, Taufkirchen, Germany). Micro-vessels were separated by centrifuga-
tion at 5800
g
for 15 min. at 4C and incubated in 20 ml medium contain-
ing collagenase–dispase II (1 mg/ml) (Roche Diagnostics) for 1.5–2 hrs at
37C. The resulting cell suspension was filtered through a 150 m
Polymon
®
mesh (NeoLab Migge, Heidelberg, Germany) and centrifuged for
10 min. at 130
g
at 4C. The cell pellet was re-suspended in media con-
taining 9% horse serum (Biochrom) and separated on a discontinuous
Percoll (Sigma-Aldrich) gradient consisting of Percoll
®
1.03 g/ml (20 ml)
and 1.07 g/ml (15 ml) by centrifugation at 1000
g
for 10 min. at 4C.
Endothelial cells were enriched at the interface between the two Percoll solu-
tions. Cells were collected, washed in media with 9% horse serum at 4C,
and stored with 10% DMSO in liquid nitrogen until use.
Calcein–AM assay
Freshly isolated or recently thawed PBCECs were incubated in
DMEM/HAM’s F12 1:1 (Biochrom) for 1 hr at 37C at a cell density of
2.5 10
6
cells/10 ml. Test compounds were dissolved in DMSO as stock
solutions and further dilutions were made with DMEM/Ham’s F12 1:1
(Biochrom). DMSO concentration in the cell suspension did not exceed
1%, a concentration that was determined not to affect the assay. A range
of concentrations of test compound in a volume of 300–600 l cell sus-
pension were added, followed by a 15 min. incubation at 37C.
Calcein–acetoxymethyl ester (AM) (300 l) (MoBiTec, Göttingen,
Germany) in DMEM/HAM’s F12 1:1 was added to a final concentration of
Page 4
1126 © 2011 The Authors
Journal of Cellular and Molecular Medicine © 2011 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd
1 M and incubated for 30 min. at 37C. Suspensions were then centrifuged
at 200
g
for 5 min. cells were washed with 4C DMEM/HAM’s F12 1:1,
and centrifuged again at 200
g
for 5 min. at 4C. The supernatant was
discarded and cells were lysed with 600 l 1% Triton X100 for 10 min. on
ice. 100 l of clarified cell lysate was added to 1 well of a 96-well
microplate. Fluorescence was detected with a Fluoroskan Ascent plate
reader (Labsystems, Helsinki, Finland) (l [excitation] 485 nm and l [emis-
sion] 520 nm). All concentrations and controls were measured 10–12
times, at least three experiments were performed per test compound.
Flow cytometry
For the calcein–AM assay using flow cytometry, the cell density of suspen-
sions in DMEM/Ham’s F12 1:1 was 2.5 10
7
cells/ml. Intracellular fluo-
rescence was measured using a fluorescence-activated cell sorting system
(FACS: Calibur flow cytometer, Becton-Dickinson, Franklin Lakes, NJ, USA)
with l (excitation) 488 nm and a 530/30 band-pass filter to collect
emitted fluorescence. Gating on forward and side scatter in concert with
propidium iodide staining allowed us to distinguish live endothelial cells.
Twenty thousand cells were sorted in each run, and data were processed
and analysed with CellQuest (Franklin Lakes, NJ, USA). All fluorescence
signals were corrected for background fluorescence. Calcein–AM auto-
hydrolysis was measured in control samples (
n
6) without cells. The
increase in intracellular fluorescence induced by a test compound was
compared to control fluorescence levels (100%), and results are reported
as percentage of control.
In vivo
experiments
Tg(fli1:EGFP) zebrafish, which express enhanced green fluorescent protein
(GFP) in their endothelial cells, were used as an
in vivo
model for angio-
genesis [36]. At 20 hrs after fertilization (hpf), zebrafish embryos (10 per
well/condition) were bathed in fish media, containing a concentration
range of each of the compounds or control. Compounds had been
dissolved as stock solutions in DMSO, stored at room temperature and
serially diluted in fish media prior to use. The anti-angiogenic tyrosinase
kinase inhibitor SU5416 (Pfizer, Berlin, Germany) [37], and a vehicle-alone
control containing the maximum concentration of DMSO were used as
controls in all experiments. In the first sets of experiments, a broad range
of concentrations were used to identify the maximum tolerable dose, based
on toxicity to the embryos, visualized directly by light microscopy.
Subsequent experiments were performed a minimum of two times. Live
analyses of the embryos were performed under light and fluorescence
microscopy at 28 and 48 hpf to monitor viability, overall morphology and
pattern of swimming. Angiogenesis was evaluated visually by fluorescence
microscopy. The developmental growth and patterning of the dorsal aorta,
posterior cardinal vein, intersomitic vessels (ISV) and vascular plexus were
monitored, as was the heart rate and blood flow.
Results
Synthesis of compounds
To identify novel artemisinin-like compounds for evaluation of
efficacy in different models, we synthesized several acetal and
non-acetal derivatives of DHA. Esters (Fig. 2) were made by react-
ing DHA with corresponding anhydrides or acid chloride in basic
medium in the presence of triethylamine, as reported [29]. The
ether and amine (Fig. 3) were made by reacting DHA with a Levis
acid forming an oxonium ion [38], that reacts with nucleophiles,
such as alcohol or amine, and converts to ether (or amine) deriv-
atives. In the absence of nucleophiles, it forms an anhydro prod-
uct 4, or it can be further reduced in the presence of Et
3
SiH to
obtain the product 3. Compound 4 was further converted to alco-
hol 5a-b (5a major product) by addition of borane followed by
hydrogen peroxide and aqueous NaOH (Fig. 4). A similar reaction
was previously reported [39].
Cytotoxicity (XTT assay)
All compounds were tested both towards drug-sensitive CCRF-CEM
leukaemia cells and their multidrug-resistant subline, CEM/
ADR5000. The IC
50
values are summarized in Table 1. Acetal type C-
10 derivatives were more active than non-acetal derivatives 3 and 4.
The degree of cross-resistance of CEM/ADR5000 cells towards the
various compounds ranged from 0.06 (compound 4) to 22.46
(compound 8). Substitution played an important role in C-10 deriv-
atives. In general, alkyl side chains showed high efficacy in terms
of activity and cross-resistance when compared to aromatic side
chain 13 and dichloroacetate side chain 8. Branched side chain
substances possessed more activity than their straight-chain coun-
terparts as in the case of compounds 9 and 10. When C-10 ether
15 is compared with ester 10, the activity remains the same in both
cases, but ether shows slightly less drug resistance than ester.
Calcein assays
As a next step, we analysed whether the transport of calcein was
affected by artemisinin and its derivatives to answer the question,
whether artemisinin-like compounds act as P-gp inhibitors. As can
be seen in Figure 5, the calcein fluorescence in CCRF-CEM and
CEM/ADR5000 cell is low and not different in both cell lines after
exposure to artemisinin or artesunate. This indicates that these
two drugs do not act as P-gp inhibitors. In contrast, all other com-
pounds tested led to an intracellular accumulation of calcein in
multidrug-resistant CEM/ADR5000 cells, indicating an inhibition
of the efflux activity of P-gp. Their EC50 values were in a range
from 17.35 1.3 M (11) to 61.8 9.62 M (15). Intracellular
calcein fluorescence increased from 916% (7) up to 3343% (14)
compared to untreated controls, suggesting high affinities of these
compounds to P-gp (Table 2). Well-known P-gp inhibitors were
chosen as controls,
e.g.
verapamil and PSC-833 [38, 39].
Inhibition of blood brain barrier function
The inhibitory potential of artemisinin derivatives towards P-gp
expressed in porcine capillaries was analysed by confocal
microscopy. Figure 6 shows control capillaries, where luminal
Page 5
J. Cell. Mol. Med. Vol 15, No 5, 2011
1127
© 2011 The Authors
Journal of Cellular and Molecular Medicine © 2011 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd
localized P-gp effluxes a fluorescent P-gp substrate [40],
N--(4-nitrobenzofurazan-7-yl)-d-Lys8 (NBD)-cyclosporin A
(NBD-CSA) back into the capillary lumen (green). Exposure to
both 8 and 15 resulted in an almost empty lumen, indicating that
the P-gp substrate NBD-CSA accumulated in the endothelial cells,
indicating inhibition of P-gp (Fig. 6). Luminal P-gp was inhibited
by a well-known selective P-gylcoprotein inhibitor, PSC-833 (data
not shown) [41].
The inhibition of luminal P-gp in porcine brain capillaries by
seven artemisinin derivatives was quantified by fluorospectrome-
try (Fig. 6, bottom row).
Inhibition of angiogenesis
in vivo
Eight compounds (4, 7, 8, 9, 10, 11, 12 and 15) were compared
to artesunate for their anti-angiogenic potential using an
in vivo
zebrafish embryo model system (Fig. 7 and Table 3). DMSO at
concentrations of 0.5%, 1% and 2% were used as vehicle control.
No effects were observed on overall morphology, heart rate, blood
flow or angiogenesis in control embryos. The anti-angiogenic
agent SU5416 was used as a positive control [37]. At a concentra-
tion of 10 g/ml, SU5416 completely blocked formation of ISVs at
28 hpf. At 48 hpf ISVs sprouted only minimally as compared to
Fig. 2 Conversion of DHA into esters.
Fig. 3 Conversion of DHA into ether and amine.
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1128 © 2011 The Authors
Journal of Cellular and Molecular Medicine © 2011 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd
control embryos. The heart rate was not affected by SU5416, and
oedema was rarely observed.
Of the compounds tested, 7, 8, 9, 10, 11 and artesunate exhib-
ited dose-dependent anti-angiogenic effects. Although there was
some inter-experimental variability in the dose–response, com-
pounds 7 and 8 consistently had distinct anti-angiogenic properties.
Similarly, compounds 9, 10 and 11 also suppressed angiogenesis,
but there was more toxicity than with compounds 7 and 8 at higher
doses. Compound 12 was the most toxic at comparable doses, and
a specific anti-angiogenic effect was not observed. All of the com-
pounds that did suppress angiogenesis were more effective, on a
dose basis, than artesunate. Of note, all compounds induced brady-
cardia in a dose-dependent manner, and this occurred irrespective
of effects on angiogenesis. Oedema, a typical consequence of heart
insufficiency, coincided with the bradycardia. Preliminary experi-
ments on rabbit hearts indicate that the bradycardia is unique to the
zebrafish and not observed in mammalian models (data not shown).
Inhibition of VEGF-induced HUVEC proliferation
To further test the anti-angiogenic potential of our novel compounds,
we assayed proliferation and survival of HUVECs treated with VEGF
and two compounds that were very active in the zebrafish model.
Artemisinin and VEGF alone served as control and reference com-
pound (Fig. 8). In this assay, despite the presence of the proliferation-
inducing VEGF, compounds 7 and 10 inhibited the proliferation and
survival of HUVECs significantly stronger than artemisinin. Notably, the
survival rate of HUVECs was very poor after more than 48 hrs expo-
sure to the compounds when VEGF was omitted (data not shown).
Discussion
By synthesizing several artemisinin-like derivatives, we have iden-
tified a range of unique compounds that may ultimately be of clin-
ical value. It is well known that C-10 derivatives of DHA can act as
pro-drugs, and that the introduction of bulky substitutes at this
position decreases the rate of hydrolysis beginning with the pro-
pionate and isopropionate and different substitutes. Thus the
resultant compound derivatives may be released more slowly,
potentially increasing the circulating half-life and possibly the ther-
apeutic efficacy. Indeed, compound 10 is branch substituted, likely
reducing the rate of hydrolysis at C-10, which may contribute to
its greater cytotoxicity as compared with compound 9.
Fig. 4 Synthetic scheme of compounds 3, 4 and 5. Reagents and conditions: (A) NaBH
4
, THF; (B) BF
3
.OEt
2
/Et
3
SiH, CH
2
Cl
2
; (C) BF
3
.OEt
2
, CH
2
Cl
2
; (D) i.
BH
3
, THF; ii. 3 M NaOHaq, H
2
O
2
30%, THF.
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1129
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Journal of Cellular and Molecular Medicine © 2011 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd
Table 1 Cytotoxicity of artemisinin derivatives towards drug-sensitive
CCRF-CEM and multidrug-resistant CEM/ADR5000 leukaemia cell lines
Compound
CCRF-CEM
(M)
CEM/ADR5000
(M)
Degree of
resistance
1
148.05 16.64 94.92 30.46
0.64
2
0.87 0.13 1.84 0.31
2.11
3
240.73 50.68 117.38 8.20
0.49
4
83.36 7.51 33.64 0.68
0.4
5a
156.15 52.05 90.03 4.57
0.58
6
0.55 0.03 0.46 0.03
0.84
7
0.18 0.43 2.36 0.64
12.83
8
1340.96 1268.63 87.46 96.84
0.06
9
106.00 25.41 54.99 16.00
0.51
10
12.30 3.86 276.30 213.41
22.46
11
2.68 0.10 3.31 0.24
1.23
12
6.65 1.17 17.64 4.56
2.65
13
171.00 97.58 1333.54 507.76
7.79
14
1.05 0.14 20.75 8.05
19.76
15
16.68 7.34 7.96 3.76
0.47
16
0.86 0.19 3.34 0.64
3.88
Fig. 5 Inhibition of calcein ametoxymethylester efflux from human leukaemia CCRF/CEM and CEM/Adr5000 cells by different concentrations of the test-
ing substances – derivatives of artesunate. The intracellular accumulation of calcein inside the cells is measured by using FACS analysis. The points indi-
cate mean values of fluorescent effect, vertical lines show standard error calculated on the base of two independent experiment replicates. The effect cor-
responds to a control of cells which were treated only with calcein.
Table 2 EC50 and EC max values of artemisinin derivatives in the
calcein-AM assay using multidrug-resistant CEM/ADR5000 cells and
flow cytometry
Compound
EC50 (M)
EC max (%)
1 n.d. 114.1 ± 2.85
2 n.d.
114.3 10.19
7
50.23 48.5 916 829.9
8
19.47 5.25 1380 199.3
9
36.37 13.86 1387 393.8
10
26.45 3.12 1240 78.2
11
17.35 1.3 2224 94.7
12
35.0 8.65 1776 292.5
13
17.97 5.51 2645 423.6
14
27.51 2.59 3343 197
15
61.8 9.62 1180 178.7
16
27.17 4.69 1011 106.3
n.d.: not detectable.
Page 8
1130 © 2011 The Authors
Journal of Cellular and Molecular Medicine © 2011 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd
Fig. 6 (A) High-magnification micrographs of porcine brain capillaries incubated with substance 8. (first row) negative control (capillaries stained only with
NBD-CSA in the same time course); (second row) (1) transmitted light image of several capillaries; (2) confocal fluorescent micrograph of several capillar-
ies; in addition to the capillary endothelium, these vessels may contain pericytes within the wall of the capillary and blood cells within the lumen; (3) over-
lay of 1 and 2 (B)Transport of the P-gp substrate NBD-CSA into porcine brain capillary lumens in the absence of control and presence of testing substances.
Page 9
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© 2011 The Authors
Journal of Cellular and Molecular Medicine © 2011 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd
Additional factors that likely impact on the activity of these
compounds are solubility and conversion to DHA. In contrast to
artemisinin, artesunate is water soluble and metabolized to DHA.
These distinct properties may at least in part explain the greater
cytotoxicity of artesunate as compared to that of artemisinin. This
is exemplified by our findings that C10 derivatives, which are
metabolized to DHA, were more cytotoxic towards cancer cells
than C9 derivatives, which cannot be metabolized to DHA. Overall,
most of the new derivatives presented in this report are not only
generally more active than artemisinin, but were easily synthe-
sized and are stable at room temperature.
In the treatment of cancer, drug resistance remains a major
impediment to success. One well-characterized pathway that pro-
motes drug resistance is the P-gp transfer system [42, 43].
Its relevance in clinical oncology is well known. For example,
P-gp is expressed at the blood brain barrier, thereby hindering the
delivery of functionally active anti-tumour drugs to the central
nervous system [44–46].
Unfortunately, overcoming drug resistance by using compounds,
such as verapamil or PSC-833 that interfere with P-gp function, has
not successfully entered the clinic due to excess toxicity [47].
Notably, artemisinin and artesunate are well tolerated in clinical
malaria studies [48], and we have determined that the artemisinin-
like compounds that we synthesized also modulate P-gp function,
as measured with the calcein assay. Thus, in combination with clas-
sical chemotherapeutic, P-glycoproptein substrates such as vin-
blastine, paclitaxel and other anti-tumour drugs, these novel
artemisinin-like derivatives may enhance tumour cell killing, with
lower toxicity, less drug resistance and improved response rates.
As the ABC transporter, P-gp, is not the only drug resistance
mechanism, the question arises about the cross-resistance of
artemisinin-type compounds to anticancer drugs and about the rel-
evance of other members of the ABC transporter family. In addition
to the doxorubicin-resistant P-gp overexpressing CEM/ADR5000
cell line, artemisinin and derivatives were not cross-resistant to
MRP-1-overexpressing HL60 leukaemia cells and breast cancer
resistance protein (BCRP)-overexpressing MDA-MB-231 breast
cancer cells [10]. They do not exhibit cross-resistance in cell lines
selected for vincristine or epirubicin resistance [49], nor to cell lines
selected for methotrexate or hydroxyurea [9]. Furthermore, we
found that cisplatin resistant ovarian carcinoma cells were also not
cross-resistant to artemisinins (Sertel
et al
., submitted for publica-
tion). There was no relationship between expression of P-gp, MRP1
and BCRP and the sensitivity or resistance to artemisinin and eight
different artemisinin derivatives in 55 cell lines of different tumour
types (leukaemia, colon Ca, breast Ca, lung Ca, prostate Ca, renal
ca, brain cancer, ovarian Ca) [5, 9, 50, 51]. This result has been con-
firmed in another cell line panel with 39 cell lines of different tumour
origin [52] and investigation using cell lines derived from Kaposi
sarcoma [19], medularry thyroid carcinoma [53] and non-Hodgkin
lymphoma [54]. All these data indicate that artemisinin-type
compounds may be active in otherwise drug-resistant cancer cells.
In the present study, we found that some artemisinin deriva-
tives exert collateral sensitivity,
i.e
. doxorubicin-resistant P-gp
overexpressing CEM/ADR5000 cells were more sensitive to these
compounds than the parental wild-type CCRF-CEM cells. Collateral
sensitivity is a well-known phenomenon in multidrug-resistance
cancer cells for more than three decades [54] and led to the devel-
opment of treatment strategies with compounds that selectively
kill multidrug resistant cancer cells [56, 57], although the mecha-
nisms are still poorly understood. It has been proposed that
compounds extruded by P-gp consume ATP and repletion of ATP
Fig. 7 Artemisinin derivatives inhibit angiogene-
sis
in vivo
. Lateral views of the trunk region of
zebrafish embryos are shown at 48 hrs after fer-
tilization (hpf) (head to the left) that were treated
with 1% DMSO (A, control) or artemisinin deriv-
atives (BD) from 19–48 hpf. (A) At 48 hpf, sev-
eral blood vessels can clearly be distinguished in
the trunk of the embryo: dorsal aorta (DA), pos-
terior cardinal vein (PCV), ISVs, dorsal longitudi-
nal anastomosing vessel (DLAV). (B) In an
embryo treated with 25 g/ml of compound 9,
the ISVs are stunted (arrowheads) and the DLAV
has not formed. (C, D) Compound 11 has a
dose-dependent effect on blood vessel forma-
tion. At 1 g/ml, ISVs are thin and the DLAV is
incomplete (asterisks). At the higher dose of
5 g/ml, several ISVs are severely reduced and
the DLAV has many gaps (asterisks).
Page 10
1132 © 2011 The Authors
Journal of Cellular and Molecular Medicine © 2011 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd
from ADP by oxidative phosphorylation generates ROS [58]. ROS
production may lead to increased cell killing. This view is conceiv-
able with the fact that cell with high P-gp expression exhibit higher
collateral sensitivity than cells with low P-gp levels. It is well
known that Artemisinin derivatives produce ROS leading to
apoptosis [59, 60]. Hence, it can be speculated that some of our
derivatives produced more ROS than others leading to higher
degrees of collateral sensitivity.
In addition to collateral sensitivity, the question about possible
synergistic effects in combination treatments can be asked.
Indeed, we previously found that the combination of artesunate
increased the apoptosis-inducing effects of doxorubicin in
CEM/ADR5000 leukaemia cells [59]. A comparable result has also
been reported for
Plasmodium falciparum
, doxorubicin and
artemisinin showed synergistic interaction as compared to each
drug alone [61].
Furthermore, it should be pointed out that the CEM/ADR5000
cell line represents a suitable model for MDR analyses. We have
previously shown that that this cell line selectively expresses
ABCB1, but not other ABC transporters. This has been shown in a
microarray-based study with a biochip carrying the genes of the
ABC transporter family and validated by real-time RT-PCR [32].
Hence, the CEM/ADR5000 cell line provides a comparable genetic
background as transfected cell lines do.
Although
in vitro
evidence supports the notion that several of
the artemisinin-like compounds that we synthesized have potential
benefits, it was important to examine their role in an
in vivo
model.
Because artemisinins have been implicated in suppressing angio-
genesis
via
several mechanisms, we utilized the zebrafish embryo
model to directly visualize effects on vascular development. We
chose the zebrafish due to the availability of a transgenic line that
expresses GFP in all endothelial cells [34], which allows direct
observation of blood vessel formation. Despite the seemingly
distant relationship of human beings and zebrafish, there is a
remarkable homology at the genetic level: most human genes
have zebrafish orthologues and the zebrafish is more and more
recognized as a valuable model for human diseases [62, 63].
Our findings support the hypothesis that several of the
artemisin-like compounds have anti-angiogenic properties (Figs 7
and 8). For example, compounds 9 and 11 suppressed ISV forma-
tion at concentrations as low as 1 g/ml, above which toxicity
became evident. Similarly, compounds 7 and 8 also exhibited anti-
angiogenic effects, with somewhat lesser toxicity. When tested in
a HUVEC proliferation/survival assay, compounds 7 and 10 were
more effective at inhibiting cellular proliferation than artemisinin,
despite the presence of the strong proliferation inducing growth
factor VEGF.
The results of the present panel of novel artemisinine deriva-
tives are in accord with previous reports that artemisinin, DHA and
artesunate act in an anti-angiogenic manner by interfering with
angiogenesis-regulating genes such as vascular endothelial growth
factor receptor (VEGFR), thrombopplastin, thrombospondin 1,
plasminogen activator, matrix metalloproteinase 9, etc. [18–22].
Overall, our findings demonstrate that these synthesized
artemisinin-like compounds are not only endowed with different
Table 3 Anti-angiogenic effects in the zebrafish
in vivo
assay.
N
total
number of embryos tested (in multiples of 10); Dead number of
dead embryos up to 48 hpf; Vasc. defects number of surviving
embryos with vascular defects; Other other defects observed:
bradycardia (B) or oedema (E).
Compound
Conc.
(g/ml)
N Dead
Vasc.
defects
Other
Artesunate 25 20 1 0 B
50 20 1 2 B, E
100 20 1 6 B, E
200 10 1 9 B, E
4 50 10 2 0 B
75 10 5 0 B, E
7 0.1 10 1 1 -
1.0 20 2 3 B, E
10 20 3 4 B, E
25 10 0 1 B, E
50 20 4 8 B, E
75 10 3 7 B, E
100 20 4 16 B, E
8 1.0 10 0 2 -
10 10 0 1 B
25 10 0 10 B, E
50 10 0 10 B, E
9 1.0 20 2 4 B
5.0 10 2 2 B, E
10 20 3 6 B, E
15 10 1 9 B, E
25 30 17 13 B, E
50 10 5 5 B, E
10 0.5 10 2 3 B, E
1.0 20 5 7 B, E
10 20 5 10 B, E
11 1.0 30 3 9 B, E
5.0 20 9 11 B, E
10 30 22 8 B, E
12 1.0 10 0 0 B, E
10 10 1 0 B, E
25 10 9 0 B, E
15 1.0 10 0 0 -
10 10 1 0 B
25 10 0 0 B, E
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properties in terms of stability and P-gp modulating activity, but
that they retain potent
in vivo
biologic anti-angiogenic
properties, that provide strong rationale for further examination
of their effects in treating a range of diseases in larger
animal models.
Acknowledgements
P.C. is supported by grants from ‘long-term structural funding:
Methusalem funding by the Flemish Government’. The authors thank Joris
Souffreau for technical assistance.
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  • Source
    • "As a consequence of this awareness, increased research efforts are focused on new derivatives with innovative applications and improved properties. Major pharmaceutical companies are beginning to take an interest in developing new trioxane compounds [18] [19] [20] [21]. Current analytical techniques describe derivatisation-based methods [22], gas chromatography (GC) [23], thin layer chromatography (TLC) [24], supercritical fluid chromatography (SCFC) [25], spectroscopic [26] and immunological techniques [27] [28], but it is clear that the main-stream methods are mainly based on high performance liquid chromatography (HPLC), coupled to ultra violet (UV), evaporative light scattering detector (ELSD), electron capture detection (ECD) or electrospray ionisation (ESI)–mass spectrometry (MS) detection [29] [30] [31] [32]. "
    [Show abstract] [Hide abstract] ABSTRACT: A highly selective and stability-indicating HPLC-method, combined with appropriate sample preparation steps, is developed for β-artemether assay and profiling of related impurities, including possible degradants, in a complex powder for oral suspension. Following HPLC conditions allowed the required selectivity: a Prevail organic acid (OA) column (250 mm×4.6 mm, 5 μm), flow rate set at 1.5 mL/min combined with a linear gradient (where A=25 mM phosphate buffer (pH 2.5), and B=acetonitrile) from 30% to 75% B in a runtime of 60 min. Quantitative UV-detection was performed at 210 nm. Acetonitrile was applied as extraction solvent for sample preparation. Using acetonitrile–water mixtures as extraction solvent, a compartmental behaviour by a non-solving excipient-bound fraction and an artemether-solubilising free fraction of solvent was demonstrated, making a mobile phase based extraction not a good choice. Method validation showed that the developed HPLC-method is considered to be suitable for its intended regulatory stability-quality characterisation of β-artemether paediatric formulations. Furthermore, LC–MS on references as well as on stability samples was performed allowing identity confirmation of the β-artemether related impurities. MS-fragmentation scheme of β-artemether and its related substances is proposed, explaining the m/z values of the in-source fragments obtained.
    Full-text · Article · Feb 2014 · Journal of Pharmaceutical Analysis
  • Source
    [Show abstract] [Hide abstract] ABSTRACT: P-Glycoprotein/MDR1 represents an important component of the blood brain barrier and contributes to multidrug resistance. We investigated two derivatives of the anti-malarial artemisinin, SM616 and GHP-AJM-3/23, concerning their ability to interact with P-glycoprotein. The ability of the two compounds to inhibit P-glycoprotein (P-gp) activity was examined in sensitive CCRF-CEM and P-gp over-expressing and multidrug-resistant CEM/ADR5000 cells as well as in porcine brain capillary endothelial cells (PBCEC) by means of calcein-AM assays. Verapamil as well-known P-gp inhibitor was used as control drug. CEM/ADR5000 cells exhibited cross-resistance to GHP-AJM-3/23, but slight collateral sensitivity to SM616. Furthermore, SM616 inhibited calcein efflux both in CEM/ADR5000 and PBCEC, whereas GHP-AJM-3/23 did only increase calcein fluorescence in PBCEC, but not CEM/ADR5000. This may be explained by the fact that CEM/ADR5000 only express P-gp but not other ATP-binding cassette transporters, whereas PBCEC are known to express several ABC transporters and calcein is transported by more than one ABC transporter. Hence, SM616 may be the more specific P-gp inhibitor. In conclusion, the collateral sensitivity of SM616 as well as the inhibition of calcein efflux in both CEM/ADR5000 cells and PBCEC indicate that this compound may be a promising P-gp inhibitor to treat cancer therapy and to overcome the blood brain barrier.
    Full-text · Article · Apr 2012
  • [Show abstract] [Hide abstract] ABSTRACT: Nowadays, artemisinins are the mainstay of malaria treatment, but initial indications of resistance against clinically used derivatives are present. In this study, ten new artemisinin derivatives were tested in vitro against Plasmodium falciparum laboratory strains as well as clinical isolates from Gabon. All derivatives were highly active, with 50% inhibitory concentrations (IC(50) values) <13 nM in the clinical isolates. The activity of one fluoro-containing derivative did not correlate with that of the parent compound, suggesting a different activity profile. New artemisinin derivatives with different activity profiles are of special interest as they represent an important class of candidates for pre-clinical testing in clinical parasite isolates adapted to currently used artemisinins, since derivatisation is one possible strategy to prolong the clinical usefulness of this important class of antimalarials.
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