toxicity of the antimalarial artemisinin and its...
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Critical Reviews in Toxicology
ISSN: 1040-8444 (Print) 1547-6898 (Online) Journal homepage: https://www.tandfonline.com/loi/itxc20
Toxicity of the antimalarial artemisinin and itsdervatives
Thomas Efferth & Bernd Kaina
To cite this article: Thomas Efferth & Bernd Kaina (2010) Toxicity of the antimalarialartemisinin and its dervatives, Critical Reviews in Toxicology, 40:5, 405-421, DOI:10.3109/10408441003610571
To link to this article: https://doi.org/10.3109/10408441003610571
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(Received 14 August 2009; revised 11 January 2010; accepted 11 January 2010)
ISSN 1040-8444 print/ISSN 1547-6898 online © 2010 Informa UK LtdDOI: 10.3109/10408441003610571 http://www.informahealthcare.com/txc
R E V I E W A R T I C L E
Toxicity of the antimalarial artemisinin and its dervatives
Thomas Efferth1, and Bernd Kaina2
1Department of Pharmaceutical Biology, Institute of Pharmacy and Biochemistry, University of Mainz, Mainz, Germany, and 2Institute of Toxicology, University of Mainz, Mainz, Germany
AbstractAs long as no effective malaria vaccine is available, chemotherapy belongs to the most important weapons fighting malaria. One of the most promising new drug developments is the sesquiterpene artemisinin (ARS) and its deriva-tives, e.g., artemether, arteether, and sodium artesunate. Large clinical studies and meta-analyses did not show serious side effects, although proper monitoring of adverse effects in developing countries might not be a trivial task. There is a paucity of large-scale clinical trials suitable to detect rare but significant toxicity. Therefore, a final and definitive statement on the safety of artemisinins still cannot be made. In contrast, animal experiments show considerable toxicity upon application of artemisinins. In the present review, the authors give a comprehensive overview on toxicity studies in cell culture and in animals (mice, rats, rabbits, dogs, monkeys) as well as on toxicity reported in human clinical trials. The authors emphasize the current knowledge on neurotoxicity, embryotoxic-ity, genotoxicity, hemato- and immunotoxicity, cardiotoxicity, nephrotoxicity, and allergic reactions. The lesson learned from animal and human studies is that long-term availability rather than short-term peak concentrations of artemisinins cause toxicity. Rapid elimination of artemisinins after oral intake represents a relatively safe route of administration compared to delayed drug release after intramuscular (i.m.) injection. This explains why consider-able toxicities were found in the majority of animal experiments, but not in human studies. In addition, there are drug-related differences, i.e., intramuscular application of artemether or arteether, but not to artesunate, which is safe and gives good profiles after i.m. administration in severe malaria. Although there is no need to increase doses of artemisinins for uncomplicated malaria, this has to be taken into account for cerebellar involvement in severe malaria. It might also be important in determining dose limitations for treatment of other diseases such as cancer.
Keywords: Allergic reactions; Artemisia annua; cardiotoxicity; embryotoxicity; hematotoxicity; malaria; nephrotoxicity; neurotoxicity
Abbreviations: ACT, artemisinin-containing combination therapy; ARS, artemisinin; ARE, arteether; ARL, artelinate; ARM, artemether; ART, artesunate; DHA, dihydroartemisinin; ECG, electrocardiogram; i.m., intramuscular; i.v., intravenous; p.o., per os; ROS, reactive oxygen species.
Contents
Abstract ............................................................................................................................................................................................. 405 1. Introduction............................................................................................................................................................................. 406 2. Neurotoxicity ........................................................................................................................................................................... 407 2.1. In vitro studies .................................................................................................................................................................. 407 2.2. In vivo studies ................................................................................................................................................................... 410 2.3. Human studies ................................................................................................................................................................. 410 3. Embryotoxicity ........................................................................................................................................................................ 411 3.1. In vitro studies .................................................................................................................................................................. 411 3.2. In vivo studies ................................................................................................................................................................... 411 3.3. Human studies ................................................................................................................................................................. 411 4. Genotoxicity ............................................................................................................................................................................ 413 5. Hemato- and immunotoxicity ............................................................................................................................................... 413
Critical Reviews in Toxicology, 2010; 40(5): 405–421Critical Reviews in Toxicology
2010
405
421
14 August 2009
11 January 2010
11 January 2010
1040-8444
1547-6898
© 2010 Informa UK Ltd
10.3109/10408441003610571
Address for Correspondence: Prof. Dr. Thomas Efferth, Chair, Department of Pharmaceutical Biology, Institute of Pharmacy and Biochemistry, University of Mainz, Staudinger Weg 5, 55128 Mainz, Germany. E-mail: [email protected]
TXC
461566
406 T. Efferth and B. Kaina
5.1. In vitro studies .................................................................................................................................................................. 413 5.2. In vivo studies ................................................................................................................................................................... 415 5.3. Human studies ................................................................................................................................................................. 415 6. Cardiotoxicity .......................................................................................................................................................................... 416 6.1. In vitro and in vivo studies .............................................................................................................................................. 416 6.2. Human studies ................................................................................................................................................................. 416 7. Other toxicities ........................................................................................................................................................................ 416 8. Conclusions and perspectives ............................................................................................................................................... 416Declaration of interest ..................................................................................................................................................................... 417References ......................................................................................................................................................................................... 417
1. Introduction
Malaria represents a major disease burden in developing countries, accounting for annually 1.1–2.7 million deaths (WHO Expert Committee on Malaria, 2000). Over 40% of the world’s population are at risk of malaria infection, and 350–500 million people are infected every year (Korenromp et al., 2005). The economic impact of malaria in the Third World is tremendous. The gross national product per capita has been estimated to be reduced by more than 50% in countries suf-fering from malaria compared to countries without malaria (Sachs and Malaney, 2002). Despite decades of immunologi-cal research, there is currently no effective malaria vaccine available, although there is reason for hope in the foresee-able future (Matuschewski and Mueller, 2007). Until then, chemotherapy and chemoprophylaxis will belong to the most important weapons to fight malaria.
One of the most promising new developments in the field of malaria chemotherapy is the sesquiterpene artemisinin (ARS), a natural product from the herb Artemisia annua L. After resolution of the chemical structure, semisynthetic modifications were introduced to improve pharmacologi-cal features of the natural lead compound (Krishna et al., 2004). ARS derivatives, such as artemether (ARM), arteether (ARE), and sodium artesunate (ART), are metabolized to dihydroartemisinin (DHA) (Krishna et al., 2004), which is highly effective and can rapidly reduce parasite burden. The chemical structures of these compounds are shown in Figure 1.
The highly reactive endoperoxide moiety in artemisinins is thought to be crucial for their mode of action, although the exact mechanism remains elusive. In a Fenton-type reaction, artemisinins generate reactive oxygen species and carbon-centered radical molecules that modify proteins of the Plasmodium parasites (Eckstein-Ludwig et al., 2003; Krishna et al., 2006). Some studies suggest that artemisinins inhibit the Ca2+-dependent SERCA-like ATPase PfATP6 upon activation by Fe2+ from hemoglobin (Eckstein-Ludwig et al., 2003). Another mechanism is the disruption of the mitochon-drial membrane potential, as suggested from data of a yeast model (Li et al., 2005).
A drawback of this class of drugs is the short plasma half-life in the body and, hence, possible recrudescence if used as monotherapy (Menard et al., 2005). As a strategy to
minimize this problem, artemisinins are generally applied in combination therapy together with mefloquine, lumefan-trine, piperaquine, amodiaquine, or sulfadoxine-pyrimeth-amine (artemisinin combination therapies [ACTs]) (Olliaro and Taylor, 2003). However, there is some skepticism about certain ACTs, where the partner drug already has been compromised by development of drug resistance (Duffy and Mutabingwa, 2006). Even more serious, there is some recent evidence that resistance to one of the major ACTs, ARM-lumefantrine, has developed in parts of East Africa (Sisowath et al., 2005; Dokomajilar et al., 2006). Recently, alarming results have been published demonstrating that field isolates with reduced susceptibility to ARM carried a specific S769N mutation in PfATP6. These isolates were from French Guiana, where artemisinins are used with-out control (Jambou et al., 2005). These results imply that PfATP6 plays a prominent role in the artemisinins mode of action, although it may possibly not be the only target. Interestingly, resistance to ARS and ART has been induced in the rodent parasite Plasmodium chabaudi, although without mutations or copy number changes in potential resistance gene homologues, including pfatp6 (Afonso et al., 2006). This might be due to differences between diverse Plasmodium species and their host system. On the other hand, these results could be taken as a forewarning for development of resistance and other resistance mecha-nisms yet undiscovered.
ARS and its semisynthetic derivatives, not only exert antimalarial activity but also profound cytotoxicity against tumor cells (Moore et al., 1995; Efferth et al., 1996, 2001; Efferth, 2005; 2006, 2007). The inhibitory activity of ARS and its derivatives towards cancer cells is in the nano- to micromolar range (Efferth et al., 2004a; Kelter et al., 2007). The addition of ferrous iron enhances the cytotoxicity of ART. Candidate genes that may contribute to the sensi-tivity and resistance of tumor cells to artemisinins were identified by pharmacogenomic and molecular pharma-cological approaches (Efferth et al., 2002a, 2003a). Target validation was performed using cell lines transfected with candidate genes or corresponding knockout cells. These genes are from classes with different biological functions, for example, regulation of proliferation (BUB3, cyclins, CDC25A), angiogenesis (vascular endothelial growth factor
Toxicity of artemisinins 407
and its receptor, matrix metalloproteinase-9, angiostatin, thrombospondin-1), or apoptosis (BCL-2, BAX) (Efferth et al., 2002a, 2003a). ART triggers apoptosis both through p53-dependent and -independent pathways (Efferth et al., 2003b). Antioxidant stress genes (thioredoxin, catalase, γ-glutamylcysteine synthetase, glutathione S-transferases) as well as the epidermal growth factor receptor (EGFR) and its downstream kinases confer resistance to ART (Efferth and Oesch, 2004; Efferth et al., 2003a, 2004b; Konkimalla et al., 2009). ART also induces DNA damage in mammalian cells, notably base modifications and DNA single-strand breaks (Li et al., 2008a). Furthermore, ART inhibits the Wnt/β-catenin pathway (Li et al., 2007a; Konkimalla et al., 2008). Cell lines overexpressing genes that confer resist-ance to established antitumor drugs (MDR1, MRP1, BCRP, dihydrofolate reductase, ribonucleotide reductase) were not cross-resistant to ART, indicating that ART is not involved in multidrug resistance of tumors (Efferth et al., 2001, 2002b, 2003a). The anticancer activity of ART has been shown in human xenograft tumors in mice (Dell’Eva et al., 2004). First encouraging therapeutic effects have also been achieved in patients with uveal melanoma (Berger et al., 2005) and non-small cell lung cancer (Zhang et al., 2008).
In addition to combination therapies, strategies to over-come drug resistance include the application of increased drug doses or the use of alternative application routes. With improved therapeutic efficacy, concomitant toxicities on healthy nonaffected tissues and organs in the body may also increase. Over the past years, several million patients received artemisinins without evidence of considerable toxicity (Taylor and White, 2004). Large studies and meta-analyses of thousands of patients did not show serious side
effects (Meshnick et al., 1996; Ribeiro and Ollario, 1998; Adjuik et al., 2004; Staedke et al., 2008), although proper monitoring of adverse side effects in developing countries might not be a trivial task (Staedke et al., 2008). Common side effects were nausea, vomiting, and diarrhea, which are also symptoms of malaria itself. Therefore, it is frequently not possible to distinguish between disease-specific symp-toms and treatment-related adverse events. Tenesmus has been found in 6% of patients administered ART sup-positories (Hien and White, 1993). Serum transaminases rose, whereas reticulocyte count and neutrophil counts decreased (Hien and White, 1993). One quarter of healthy volunteers developed fever under treatment with artem-isinins and a single case of rush has been reported (Hien and White, 1993; White, 1994; Barradell and Fitton, 1995). This indicates that side effects are mild and severe cases are rare as yet. In many cases, side effects are not distinguish-able from the symptoms of malaria. In contrast, animal experiments show considerable toxicity upon application of artemisinins.
The aim of the present review is to give a comprehensive overview on toxicity studies in cell culture and in animals (mice, rats, rabbits, dogs, monkeys) as well as on toxicity reported in human clinical trials.
2. Neurotoxicity
2.1. In vitro studiesNeurotoxicity has been most frequently investigated as a possible adverse side effect of artemisinin-type drugs. Cell culture studies were performed to unravel cellular and molecular mechanisms of neurotoxic effects (Table 1).
Artemether (ARM)MW: 298.38
Arteether (ARE)MW: 312.41
Dihydroartemisinin (DHA)MW: 284.35
CH3
CH3
H3C
H
H
O
O
OO
O
CH3
CH3
H3C
H
O
OHO
H
H
O
O
O
OO
CH3
CH3
OCH3
H3C
H
H H
O
O
O
O
CH3
O-CH2-CH3
CH3
H3C
H
HH
O
O
O
O
CH3
CH3
H3C
H
H H
OH
OO
O
O
Artemisinin (ARS)MW: 282.33
Artesunate (ART)MW: 384.42
Figure 1. Chemical structures of artemisinin-type compounds.
408 T. Efferth and B. KainaTa
ble
1. N
euro
toxi
city
of a
rtem
isin
in a
nd
its
der
ivat
ives
.
Spec
ies
Dru
gD
ose
Ap
plic
atio
n m
ode
Eff
ect
Ref
eren
ce
Cel
l cu
ltu
res
AR
M
in v
itro
Feta
l rat
pri
mar
y n
euro
nal
cu
ltu
res;
feta
l rat
sec
ond
ary
astr
ocyt
e
cult
ure
s; N
G10
8-15
rat n
euro
blas
tom
a ce
lls; N
B2a
mou
se
neu
robl
asto
ma
cells
: Neu
ron
al c
ell t
ype-
spec
ific
toxi
city
; no
toxi
city
to
glio
ma
cells
Wes
che
et a
l., 1
994
AR
E
DH
S
Der
ivat
ives
NB
2a a
nd
C6
cells
AR
S, D
HA
in v
itro
Inh
ibit
ion
of n
euri
te o
utg
row
th fr
om d
iffer
enti
atin
g N
B2a
n
euro
bla
stom
a ce
llsFi
shw
ick
et a
l., 1
995
AR
M, A
RE
NB
2aD
HA
± h
emin
in v
itro
Hem
in p
oten
tiat
ed in
hib
itio
n o
f NB
2a n
euri
te o
utg
row
thSm
ith
et a
l., 1
997
AR
M
AR
E
NB
2a a
nd
C6
cells
DH
A ±
hem
inin
vit
roIn
hib
itio
n o
f diff
eren
tiat
ing
cells
; hem
in in
crea
sed
D
HA
bin
din
g to
cor
tex
and
cel
l pro
tein
sFi
shw
ick
et a
l., 1
998a
Rat
cer
ebra
l cor
tex
NB
2a c
ells
DH
A
in v
itro
Dam
age
of m
itoc
hon
dri
al c
rist
ae a
nd
en
dop
lasm
ic
reti
culu
m; d
eple
tion
of fi
lop
odia
-lik
e p
roce
sses
Fish
wic
k et
al.,
199
8b
NB
2a c
ells
AR
T ±
hem
inin
vit
roIn
hib
itio
n o
f neu
rite
ou
tgro
wth
from
diff
eren
tiat
ing
NB
2a c
ells
Smit
h e
t al.,
199
8
NB
2a c
ells
DH
A
in v
itro
Inh
ibit
ion
of n
euri
te o
utg
row
th fr
om d
iffer
enti
atin
g N
B2a
cel
ls in
the
ord
er D
HA
> A
RM
/AR
E; d
iffer
enti
ated
neu
ron
al c
ells
are
mor
e
vuln
erab
le th
an d
iffer
enti
ated
glia
l cel
ls; d
amag
e of
mit
och
ond
rial
m
emb
ran
es a
nd
en
dop
lasm
ic r
etic
ulu
m
McL
ean
an
d W
ard
, 199
8
AR
M
AR
E
NB
2a c
ells
DH
A
in v
itro
Inh
ibit
ion
of n
euri
te o
utg
row
th fr
om d
iffer
enti
atin
g N
B2a
cel
ls
glu
tath
ion
e d
eple
tion
by
AR
M +
hem
in, b
ut n
ot D
HA
Smit
h e
t al.,
200
1
AR
M ±
hem
in
Bra
in c
ell c
ult
ure
sA
RS
in
vit
roD
iffer
enti
al e
ffec
ts o
n A
RS-
sen
siti
ve n
euro
nal
bra
in s
tem
pri
mar
y
cell
cult
ure
s an
d A
RS-
resi
stan
t cor
tica
l neu
ron
an
d a
stro
cyte
p
rim
ary
cell
cult
ure
s; c
orti
cal n
euro
ns
and
ast
rocy
tes:
oxi
dat
ive
st
ress
, exc
itot
oxic
eve
nts
, red
uce
d A
TP
leve
ls, a
nd
mit
och
ond
rial
m
emb
ran
e p
oten
tial
; all
cell
typ
es: e
ffec
ts o
n c
ytos
kele
ton
, m
itoc
hon
dri
al a
nd
met
abol
ic d
efec
ts
Sch
mu
ck e
t al.
2002
Dog
sA
RE
10 a
nd
20
mg/
kg/d
ayi.m
. for
8 d
ays
Gai
t dis
turb
ance
, los
s of
sp
inal
an
d p
ain
res
pon
se
refl
exes
, pro
min
ent l
oss
of b
rain
ste
m a
nd
eye
refl
exes
; n
euro
pat
hic
lesi
ons
limit
ed to
pon
s an
d m
edu
lla
Bre
wer
et a
l., 1
994a
Dog
sA
RE
20 m
g/kg
/day
i.m. f
or 8
day
sG
ait d
istu
rban
ce, l
oss
of s
pin
al a
nd
pai
n r
esp
onse
;B
rew
er e
t al.,
199
4b
Rat
sA
RE
12.5
mg/
kg/d
ayi.m
. for
7 d
ays
No
clin
ical
ch
ange
s, b
ut s
ign
s of
his
tolo
gica
l dam
age
Gen
oves
e et
al.,
199
5
Mon
keys
AR
E16
an
d 2
4 m
g/kg
/day
i.m. f
or 1
4 d
ays
Ext
ensi
ve b
rain
ste
m n
ucl
ei o
f (1)
ret
icu
lar
form
atio
n
(cra
nia
l an
d c
aud
al p
onti
ne
nu
clei
, med
ulla
ry g
igan
to
cellu
lar
and
par
agig
anto
cellu
lar
nu
clei
); (
2) v
esti
bu
lar
sy
stem
(m
edia
l, d
esce
nd
ing,
su
per
ior,
and
late
ral n
ucl
ei);
le
ss in
jury
in (
3) a
ud
itor
y sy
stem
(su
per
ior
oliv
ary
nu
clea
r
com
ple
x an
d tr
apez
oid
nu
clea
r co
mp
lex)
Pet
ras
et a
l., 1
997
Rat
sA
RE
50 m
g/kg
/day
i.m. f
or 5
–6 d
ays
Neu
ron
al n
ecro
sis,
vac
uol
ariz
atio
n, a
nd
foca
l axo
nal
sw
ellin
g in
the
neu
trop
hil
in v
esti
bu
lar
nu
clei
an
d r
ed
nu
clei
; sca
tter
ed s
wol
len
neu
ron
s in
cer
ebel
lar
nu
clei
an
d r
etic
ula
r fo
rmat
ion
Kam
chon
won
gpai
san
et a
l., 1
997
Rat
sA
RE
12.5
mg/
kg/d
ayi.m
. for
7 d
ays
Ch
ange
s of
beh
avio
ral p
erfo
rman
ce in
au
dit
ory
d
iscr
imin
atio
n ta
sk te
sts;
his
top
ath
olog
ical
dam
age
in
nu
clei
trap
ezoi
des
, su
per
ior
oliv
e, a
nd
ru
ber
Gen
oves
e et
al.,
199
8a
Tab
le 1
. con
tin
ued
on
nex
t pag
e
Toxicity of artemisinins 409
Spec
ies
Dru
gD
ose
Ap
plic
atio
n m
ode
Eff
ect
Ref
eren
ce
Rat
sA
RE
12.5
mg/
kg/d
ayi.m
. for
7 d
ays
Ch
rom
atol
ysis
in n
ucl
eus
trap
ezoi
des
an
d n
ucl
eus
su
per
ior
oliv
e; n
euro
pat
hy
in n
ucl
eus
rub
erG
enov
ese
et a
l., 1
998b
Mou
seA
RM
50–1
00 m
g/kg
/day
i.m. f
or 2
8 d
ays
Sele
ctiv
e d
amag
e of
bra
in s
tem
cen
ters
pre
dom
inat
ely
in
volv
ed in
au
dit
ory
pro
cess
ing
and
ves
tib
ula
r re
flex
esN
ontp
rase
rt e
t al.,
199
8
Dog
sA
RM
20 m
g/kg
/day
i.m. f
or 3
0 d
ays
Axo
nal
dam
age
in c
ereb
ella
r ro
of, p
onti
ne,
an
d v
esti
bu
lar
n
ucl
ei a
nd
in r
aph
e/p
aral
emn
isca
l reg
ion
Cla
ssen
et a
l., 1
999
40/8
0 m
g/kg
/day
i.m. f
or 8
day
s
50–6
00 m
g/kg
/day
p.o.
for
8 d
ays
No
nu
clea
r d
amag
e af
ter
p.o.
AR
M
Rat
sA
RE
75 a
nd
125
mg/
kg/d
ayi.m
. for
11
day
sC
hro
mat
olys
is in
nu
cleu
s tr
apez
oid
es a
nd
nu
cleu
s su
per
ior
oliv
eG
enov
ese
et a
l., 1
999
Mon
keys
AR
E8
or 2
4 m
g/kg
/day
i.m. f
or 7
or
14 d
ays
Neu
rolo
gica
l les
ion
s in
pre
cere
bel
lar
nu
clei
of m
edu
lla
oblo
nga
ta, l
ater
al r
etic
ula
r n
ucl
ei, p
aram
edia
n r
etic
ula
r n
ucl
ei,
and
per
ihyp
oglo
ssal
nu
clei
; beh
avio
ral c
han
ges:
imp
aire
d p
ostu
re,
gait
an
d a
uto
nom
ic r
egu
lati
on a
nd
eye
mov
emen
t
Pet
ras
et a
l., 2
000
Mou
seA
RM
300
mg/
kg/d
ayp.
o. fo
r 28
day
sB
alan
ce/g
ait d
istu
rban
ces
and
mor
talit
yN
ontp
rase
rt e
t al.,
200
0
AR
T30
0 m
g/kg
/day
p.o.
for
28 d
ays
Bal
ance
/gai
t dis
turb
ance
s an
d m
orta
lity
AR
M50
mg/
kg/d
ayi.m
. for
28
day
sB
alan
ce/g
ait d
istu
rban
ces
and
mor
talit
y
Rat
sA
RE
25 m
g/kg
/day
i.m. f
or 7
day
sP
rogr
essi
ve a
nd
sev
ere
dec
line
of b
ehav
iora
l per
form
ance
in
au
dit
ory
dis
crim
inat
ion
task
test
s an
d b
ehav
iora
l to
xici
ty (
trem
or, g
ait d
istu
rban
ce, a
nd
leth
argy
) by
AR
E,
bu
t not
AR
T a
nd
AR
L
Gen
oves
e et
al.,
200
0
AR
T31
mg/
kg/d
ay
AR
L36
mg/
kg/d
ay
Mar
ked
his
tolo
gica
l dam
age
in th
e b
rain
ste
m n
ucl
ei
rub
er, s
up
erio
r ol
ive,
trap
ezoi
deu
s, a
nd
infe
rior
ves
tib
ula
r
(ch
rom
atol
ysis
, nec
rosi
s, g
liosi
s)
Rat
sA
RE
25 m
g/kg
/day
i.m. f
or 7
day
sB
ehav
iora
l ch
ange
s: d
efici
t in
maz
e te
st,
dam
age
in n
ucl
eus
trap
ezoi
des
Gen
oves
e et
al.,
200
1
Rat
sA
RM
400
mg/
kg/d
ayp.
o. 4
-nig
htl
y fo
r 5
mon
ths
No
neu
roto
xici
tyX
iao
et a
l., 2
002a
Mou
seA
RM
50-1
00 m
g/kg
/day
i.m. f
or 2
8 d
ays
Dam
age
of tr
apez
oid
nu
cleu
s, g
igan
toce
llula
r re
ticu
lar
n
ucl
eus
and
infe
rior
cer
ebel
lar
ped
un
cle;
b
ehav
iora
l ch
ange
s: g
ait d
istu
rban
ce
Non
tpra
sert
et a
l., 2
002a
AR
M30
0 m
g/kg
/day
p.o.
No
dam
age
AR
T30
0 m
g/kg
/day
p.o.
or
i.m.
No
dam
age
Mou
seD
HA
50-3
00 m
g/kg
/day
p.o.
for
28 d
ays
Neu
roto
xic
effec
ts >
200
mg/
kg/d
ayN
ontp
rase
rt e
t al.,
200
2b
AR
M
AR
T
Rat
sA
RE
25 m
g/kg
/day
i.m. f
or 7
day
sC
once
ntr
atio
n-
and
exp
osu
re ti
me-
dep
end
ent n
euro
toxi
c ch
ange
sLi
et a
l., 2
002
Rat
sA
RL
160
mg/
kg/d
ayp.
o. fo
r 9
day
sB
ody
wei
ght l
oss,
neu
ron
al in
jury
Si e
t al.,
200
7
288
mg/
kg/e
very
ot
her
day
, 5 ti
mes
Rat
sA
RM
25 m
g/kg
/day
i.m. f
or 7
day
sD
amag
e of
trap
ezoi
d n
ucl
ei,
abn
orm
alit
ies
in b
alan
ce a
nd
coo
rdin
atio
nA
kin
lolu
an
d S
hok
un
bi,
2008
Not
e. A
RS,
art
emis
inin
; AR
E, a
rtee
ther
; AR
L, a
rtel
inat
e; A
RM
, art
emet
her
; AR
T, a
rtes
un
ate;
DH
A, d
ihyd
roar
tem
isin
in.
Tabl
e 1.
Con
tin
ued
.
410 T. Efferth and B. Kaina
Wesche et al. (1994) found that neuronal cell types, but not glioma cells, were vulnerable to artemisinins. The water-soluble ART and DHA were more cytotoxic than oil-soluble derivatives. Subsequent studies confirmed cytotoxic effects towards neuronal cells. ARS derivatives inhibited the neu-rite outgrowth of differentiating NB2A neuroblastoma cells (Fishwick et al., 1995, 1998a, 1998b, McLean and Ward, 1998; Smith et al., 1998, 2001; Schmuck et al., 2002). Oxidative stress may explain this effect. Hemin as a source for ferrous iron ions enhanced the neurotoxic effects of artemisinins (Smith et al., 1997; Fishwick et al., 1998). Ferrous iron is thought to facilitate the generation of reactive oxygen species (ROS) and carbon-centered radical molecules by breaking the endoper-oxide moiety of artemisinins in a Fenton-type reaction (van Agtmael et al., 1999).
2.2. In vivo studiesWhereas the more water-soluble derivatives were more cytotoxic in vitro than water-insoluble artemisinins, the contrary seems to be true in laboratory animals. Neurotoxic effects in mice, rats, dogs, and monkeys include behavioral changes (tremor, restlessness, lethargy), abnormalities in balance and coordination (gait disturbance, jerking limb movements), changes in auditory discrimination task tests, loss of spinal and pain response reflexes, and loss of brain-stem and eye reflexes (Table 1). Histological examinations of treated animals revealed extensive damage (chroma-tolysis, necrosis, swollen cell bodies, nuclear shrinkage, vacuolization of cytoplasm, axonal degeneration, etc.) in brainstem nuclei of the reticular formation (medullary nucleus gigantocellularis, lateral reticular, and reticulote-gmental nuclei), the vestibular system (inferior and lateral nuclei), and the auditory system (trapezoid and superior olivary nuclei). Forebrain regions (cerebral cortex, basal ganglia, thalamus, and hypothalamus) were not or rarely affected.
A repeatedly observed result is that the water-soluble ART shows less neurotoxicity in laboratory animals than the oil-soluble ARE and ARM. Several explanations have been discussed (Gordi and Lepist, 2004):
Intravenous injections of water-soluble drugs cause 1. a rapid distribution in the general compartment (i.e., plasma), whereas oil-drug suspensions are distributed and released in a slower manner (Rowland and Tower, 1994). The elimination half-life of the water-soluble DHA is less than 1 h after intravenous (i.v.) administration in rats (Li et al., 1998). Half-life times of 7 h for ARE solu-bilized in cremophor and of 17 h for ARE solubilized in sesame oil have been observed after i.v. injection (Li et al., 2002). These results emphasize that the choice of the oil vehicle impacts the biodistribution and half life of ARE in the body.
Oil-ARE-formulations that have been intramuscularly 2. (i.m.) injected showed that 90% of the drug dose was still present at the site of injection 24 h after the 7th (last) day of injection (Li et al., 1999). This indicates that oil may
act as a depot, resulting to retarded release of the drug and prolonged half-life of the compound.
The oral administration route also considerably influ-3. ences effective drug doses. Orally applied ART was rap-idly metabolized in the liver by the cytochrome P450 monooxygenases CYP2B6 and CYP3A4 (Svensson et al., 1999). Liver metabolism may, therefore, significantly contributes to the elimination of ARE from the body, reducing the bioavailability of the drug.
In conclusion, neurotoxicity of artemisinins seems to depend at least in part on kind of administration, the serum half-life, and the duration of exposure, but also on the chemical properties of the compounds, which are able to form reactive oxygen species and carbon-centered radical molecules upon breakage of the endoperoxide moiety. The delayed release observed after i.m. administration mainly related to ARM and ARE, but not to ART, which was safe and gave good profiles after i.m. administration in severe malaria (Nealon et al., 2002). The toxicities were probably not related to increased area-under-the-curve (AUC)-time profiles, when the i.m. route was used, but were more likely to result from the chemi-cal properties of the oil-soluble derivates that can only be used by the i.m. route (particularly as the peak levels of ARM were blunted compared with ART, and conversion to DHA was less for ARM than ART) (Kissinger et al., 2000).
2.3. Human studiesNeurotoxicity was not a common side effect of artemisinins in malaria treatment. The most common application route for artemisinins to treat uncomplicated malaria is the oral administration. Comparable to laboratory animals, i.m. injec-tion of ARM or DHA to human subjects resulted in a storage with longer elimination periods than per os (p.o.) applica-tion (Titulaer et al., 1990; Teja-Isavadharm et al., 1996). This indicates that different toxicities can occur if different appli-cation routes are chosen. Furthermore, the doses applied in human studies are much less than the one used in laboratory animals. Whereas patients normally receive 2–8 mg/kg body weight for 3–5 days, concentrations in laboratory animals ranged from 12.5 to 600 mg/kg/day. Hundred-fold higher dosing may explain neurotoxicity in animals, which is not apparent at malaria-effective doses.
Clinical trials designed with special emphasis on neuro-logical symptoms such as movement, hearing, vestibular, or cerebellar abnormalities did not show any differences between controls and test groups (Park et al., 1998; Kissinger et al., 2000). Van Vugt et al. (2000) described one neurological abnormality, which was, however, without clinical relevance. Ataxia and slurred speech was found after ART treatment (Miller and Panosian, 1997). This event was, however, assigned by the authors to disease-related symptoms rather than to drug-related effects. The same is true for a case report on tremor and unsteadiness after ARM treatment (Elias et al., 1999; White, 2000).
Prolonged length of time to recovery from coma in ARM-treated patients affected with severe malaria after ARM
Toxicity of artemisinins 411
treatment compared to quinine therapy has been observed in three clinical trials (Hien et al., 1996; Tran et al., 1996; van Hensbroek et al., 1996). A meta-analysis of seven studies with a total of 1919 patients did not report significant differences of coma recovery times between both drugs (Stepniewska et al., 2001). An audiometry study reported ototoxicity of artemisi-nins in healthy volunteers by comparing results before and after ARE treatment (Kager et al., 1994). A case of brainstem encephalopathy after ARS-containing herbal treatment for breast cancer has been described (Panossian et al., 2005; White et al., 2006a). The patient exhibited reversible ataxia, nystagmus, and slurred speech.
A very large placebo-controlled community-based trial of rectal artesunate in children with malaria gave quality evidence regarding the lack of neurotoxicity of artesunate (Gomes et al., 2009). Other studies of rectal artesunate con-firmed that the exposure of children is higher to parent and a metabolite, DHA, without obvious evidence of neurotoxicity (Krishna et al., 2001; Simpson et al., 2006)
An association of ARM-lumefantrine combination therapy for uncomplicated falciparum malaria and reduced hear-ing abilities in audiometric tests has been reported in 50 patients (Toovey and Jamieson, 2004a, 2004b). Subsequently, a case report on the ototoxicity after ARM-lumefantrine appeared (Toovey, 2006). Another study did not find such an association (Hutagalung et al., 2006) with the same drug combination in a case-controlled study with 128 matched pairs of ARM-lumefantrine–treated patients versus controls. Although this trial was controversially discussed (Toovey, 2007), a meta-analysis of 16 clinical trials did also find that decreased hearing (16/2318 cases = 0.7%) was not more after ARM-lumefantrine treatment as compared to other antima-larial treatments (Reinhart et al., 2005). Hearing decrease was mild to moderate and reversible.
3. Embryotoxicity
3.1. In vitro studiesThere is an urgent need for safety control of artemisinins for pregnant women treated with ARS-containing combinations therapies (ACTs). Available information on the safety of ACTs in pregnant women, especially in their first trimester, was limited in the past (Nosten et al., 2006; White et al., 2006b). To address this issue, several toxicity investigations have recently been performed. A synopsis of these experiments indeed pro-vided perturbing hints for embryotoxicity by artemisinins.
As shown in Table 2, ARS increased ROS levels and inhib-ited angiogenesis in mouse embryonic stem cell–derived embryonic bodies (Wartenberg et al., 2003). In addition, ARS caused impairment of laminin organization and of expression of matrix metalloproteinase (MMP)-1, -2, and -9. The hypoxia-inducible factor 1α (HIF-1α) and the vascular endothelial growth factor (VEGF) were down-regulated in response to ARS (Wartenberg et al., 2003). In a Xenopus frog embryo teratogenesis assay, a reduction of primitive red blood cells was observed (Longo et al., 2008).
3.2. In vivo studiesEarly investigations of Chinese scientists on embryotoxicity and teratogenicity date back to the mid 1980s (Chen et al., 1984). Subsequently many groups worldwide observed embryotoxic effects of artemisinins in mice, rats, rabbits, and monkeys (Table 2). Typical observations were
deletion of primitive erythroblasts, reduction of •maternal reticulocyte count, anemia;
embryonic death and fetal resorption during •organogenesis;
retardation of fetal growth among surviving fetuses; •and
cardiovascular malfunction, skeletal defects, and •delays in limb and tail development.
These embryotoxic effects may be due to oxidative stress as indicated by increased ROS levels, decreased glutathione levels, decreased embryonal and placental glutathione per-oxidase, and increased malondialdehyde.
3.3. Human studiesAgain Chinese scientists were the first to address embryotox-icity in human studies. In two investigations, no evidence for toxicity was found on human fetuses in 6 and 17 pregnan-cies, respectively (Li et al., 1990; Wang et al., 1990). Later on, ART or ARM were used to treat pregnant women suffering from infection with multidrug-resistant falciparum malaria. Both drugs were well tolerated without affecting the rate of live births or of congenital abnormalities among newborn children (McGready et al., 1998). A subsequent study of the same group confirmed these results. A total of 539 episodes of acute falciparum malaria in 461 pregnant women, including 44 first trimester episodes, were treated with artemisinins. Birth outcomes of this group of treated women did not sig-nificantly differ from general community rates for abortion, stillbirths, congential abnormality, and mean gestation at delivery (McGready et al., 2001a). Franco-Paredes et al. (2005) reported a case of neurotoxicity due to antimalarial therapy associated with misdiagnosis of malaria. Tremor, restlessness, hyperreflexia, and spasticity were observed after ART and chloroquine treatment for each 10 days. Symptoms disappeared after cessation of medication.
Interestingly, pregnancy was observed to be associ-ated with reduced blood concentrations both of ARM and lumefantrine compared to levels of previously reported non-pregnant adults, indicating preferential drug elimination in pregnant women (Mc Gready et al., 2006).
A meta-analysis of 14 studies investigated a total number of 945 women exposed to artemisinins during pregnancy, of whom 123 were in the first and 822 in the second or third trimester (Dellicour et al., 2001). In none of these studies was a risk for adverse pregnancy outcomes found. However, none of these studies was large enough to detect small differ-ences in event rates important for public health. Despite the unlikelihood of fetal loss or malformations among newborn
412 T. Efferth and B. Kaina
Tabl
e 2.
Em
bry
otox
icit
y of
art
emis
inin
an
d it
s d
eriv
ativ
es.
Spec
ies
Dru
gD
ose
Ap
plic
atio
n m
ode
Eff
ect
Ref
eren
ce
Rat
sA
RT
4–60
mg/
kg/d
ays.
c.E
mb
ryo
abso
rpti
on, d
ecre
ased
em
bry
onal
an
d p
lace
nta
l gl
uta
thio
ne
per
oxid
ase,
an
d in
crea
sed
mal
ond
iald
ehyd
eL
ou e
t al.,
200
3
Mou
se e
mb
ryoi
dB
odie
sA
RS
in
vit
roIn
crea
sed
leve
ls o
f rea
ctiv
e ox
ygen
sp
ecie
s an
d in
hib
itio
n o
f an
giog
enes
is in
mou
se e
mb
ryon
ic s
tem
-cel
l der
ived
em
bry
oid
b
odie
s; im
pai
red
lam
inin
org
aniz
atio
n a
nd
exp
ress
ion
of M
MP-
1, -
2,
an
d -
9; d
own
-reg
ula
tion
of H
IF-1
α a
nd
VE
GF
War
ten
ber
g et
al.,
200
3
Rat
s an
d R
abb
its
AR
T5–
7 m
g/kg
/day
p.o.
on
ges
tati
onal
day
s 6–
17E
mb
ryon
ic lo
ss, c
ard
iova
scu
lar
mal
form
atio
ns,
sk
elet
al d
efec
tsC
lark
et a
l., 2
004
Rat
sA
RM
1.5,
7.5
, or
15 m
g/kg
/day
i.p. f
or 7
day
sN
o eff
ect o
n r
ate
of c
once
pti
on, p
artu
riti
on, p
rete
rm d
eliv
ery,
or
litte
r
size
; no
effec
t on
bir
th w
eigh
t or
grow
th r
ate
of p
up
s; r
edu
ced
ox
ytoc
in-i
nd
uce
d c
ontr
acti
on in
ute
rin
e ti
ssu
es
Ejio
for
et a
l., 2
006
Rat
sD
HA
7.5
or 1
5 m
g/kg
/day
p.o.
on
ges
tati
onal
day
s
9–10
or
11–-
20R
edu
ctio
n o
f pri
mit
ive
red
blo
od c
ells
from
yol
k sa
c, a
cute
an
emia
, m
alfo
rmat
ion
, em
bry
onic
dea
th, o
xid
ativ
e st
ress
, an
d d
ecre
ased
gl
uta
thio
ne
leve
ls in
red
blo
od c
ells
Lon
go e
t al.,
200
6
Rat
sA
RT
17 m
g/kg
/day
day
s 10
an
d 1
1 p
ostc
oitu
mPa
ling
and
em
bry
onic
ery
thro
bla
st d
eple
tion
, im
pai
red
hem
e sy
nth
esis
m
alfo
rmat
ion
, hyp
oxia
, nec
rosi
s, e
mb
ryon
ic d
eath
, hea
rt
abn
orm
alit
ies,
car
dio
myo
pat
hy,
del
ays
in li
mb
an
d ta
il d
evel
opm
ent
Wh
ite
et a
l., 2
006
Rat
sA
RS
35 a
nd
70
mg/
kg/d
ayp.
o. o
n g
esta
tion
al d
ays
7–
13 o
r 14
–20
Pos
tim
pla
nta
tion
loss
of e
mb
ryos
; re
du
ctio
n o
f mat
ern
al p
roge
ster
ons
and
test
oste
ron
eB
oare
to e
t al.,
200
8
Xen
opu
sD
HA
in v
itro
24 h
aft
er fe
rtiz
iliza
tion
for
48h
Red
uct
ion
of p
rim
itiv
e re
d b
lood
cel
ls in
the
frog
em
bry
o te
rato
gen
esis
as
say;
frog
larv
ae w
ith
hea
rt d
efec
tsL
ongo
et a
l., 2
008
Rat
sA
RT
30 m
g/kg
/day
i.m. f
or 3
day
sH
igh
er A
RT
/DH
A a
ccu
mu
lati
on in
pre
gnan
t th
an in
non
pre
gnan
t rat
sLi
et a
l., 2
008
Rat
sA
RS
1.5–
3 m
g/kg
Sin
gle
i.v. a
dm
inst
rati
on o
r
p.o.
day
11
pos
tcoi
tum
Pos
tim
pla
nta
tion
loss
of e
mb
ryos
Cla
rk e
t al.,
200
8a
DH
AA
RS:
red
uct
ion
of m
ater
nal
ret
icu
locy
te c
oun
t
AR
T
AR
M
AR
E
Mon
keys
AR
T30
mg/
kg/d
ayp.
o. fo
r 37
day
sR
edu
ctio
n o
f em
bry
onic
ery
thro
bla
sts,
car
dio
myp
ath
y, e
mb
ryon
al
dea
th, n
o m
alfo
rmat
ion
s, r
edu
ctio
n o
f ret
icu
locy
te c
oun
tC
lark
et a
l., 2
008b
Rat
sA
RT
17 m
g/kg
Sin
gle
dos
e p.
o.E
mb
ryo
leth
alit
y, c
ard
iova
scu
lar
mal
form
atio
ns,
ske
leta
l def
ects
; m
ost s
ever
e eff
ects
at d
ay 1
1 p
ostc
oitu
mC
lark
et a
l., 2
008c
Rat
sA
RM
7 m
g/kg
p.o.
on
ges
taga
tion
al d
ays
0–
6, 7
–14,
or
14–2
0Fe
tal r
esor
pti
on d
uri
ng
orga
nog
enes
is; r
etar
dat
ion
of f
etal
gro
wth
am
ong
surv
ivin
g fe
tuse
s; n
o m
alfo
rmat
ion
sE
l-D
akd
oky
et a
l., 2
009
Rat
s an
d m
onke
ysA
RT
12 m
g/kg
/day
, >12
day
sp.
o.D
elet
ion
of p
rim
itiv
e er
yth
rob
last
s, e
mb
ryon
ic d
eath
Cla
rk e
t al.,
200
9
Not
e. A
RS,
art
emis
inin
; AR
E, a
rtee
ther
; AR
M, a
rtem
eth
er; A
RT,
art
esu
nat
e; D
HA
, dih
ydro
arte
mis
inin
.
Toxicity of artemisinins 413
under ACT, complete safety still cannot be assured. This points to the need of larger clinical trials and postmarketing pharmacovigilance.
One mechanism of embryotoxicity seems to be the inhibi-tion of erythropoiesis. Because reduced reticulocyte counts were found both in animal experiments and human studies (see below), an embryotoxic risk for human malaria patients cannot be ruled out, even if no cases are reported thus far in the literature. Embryos may get lost at early pregnancy stages, where mothers still are unaware of their pregnancy. For this reason, it is possible that embryotoxic effects were not recorded in previous studies. Malformations may occur in damaged but surviving embryos.
The danger of embryotoxicity may be higher in the first 3 months of pregnancy. Based on animal experimentation data, the World Health Organization (WHO) does not recommend the use of artemisinins in the first trimester (WHO, 2007). A problem is, however, that many women are unaware of their pregnancy in the first trimester and take artemisinins, if they are infected with malaria.
4. Genotoxicity
Another potentially toxic mechanism of ART is its genotoxic-ity. The cleavage of ART’s endoperoxide bridge leads to the formation of ROS and carbon-centered radical molecules. These highly reactive molecules target several proteins in Plasmodia, resulting in death of the microorganism.
Artesunate has been shown to be cytoxic for mammalian cells, including DNA repair–defective Chinese hamster cell lines (Li et al., 2008), and a large panel of cell lines of different tumor origin (Efferth et al., 1996; 2001; Kelter et al., 2007). The main route of cell kill was shown to be apoptosis (Efferth et al., 1996, 2007; Li et al. 2008a). Therefore, it is conceivable that during malaria therapy with artesunate side effects would be appearing due to apoptosis induction in normal cells. Cell kill in vitro was observed in DNA repair–competent cells in a dose range of 1–10 µg/ml for the most sensitive endpoint colony formation, and 5–50 µg/ml for the endpoint apoptosis. DNA single-strand breaks were observed in Chinese hamster ovarian (CHO) cells with doses >30 µg/ml, and γ-H2AX foci formation was evident at dose levels >5 µg/ml. Thus, cyto-toxic and genotoxic effects were observed in cultivated cells with doses >5 µg/ml (Li et al., 2008a). Interestingly, in DNA repair–defective cells such as DNA polymerase β and Ku80 mutants, cytotoxic effects were observed at much lower dose levels (>0.1 µg/ml) (Li et al., 2008a). We have also data at hand to show that ART induces the mutagenic oxidative DNA dam-age 8-oxo-guanine (Kaina et al., unpublished data). Overall, the data suggest that DNA damage induced by ART may not only contribute to its therapeutic effect against Plasmodia and cancer cells. It has also to be considered that ART is potentially genotoxic and mutagenic.
It is important to note that in vitro cyto- and genotoxicity (e.g., DNA strand breaks) were only observed upon chronic treatment of cells with the drug, whereas 1-h pulse treatment was without any effect. Although it is difficult to translate
these findings to the in vivo situation, it would be interest-ing to compare the doses with the serum levels of ART in malaria patients. The IC
50 for Plasmodium falciparum for
ART is in the range of 3–30 ng/ml, which is much lower than the cytotoxic dose in repair-competent mammalian cells (>1 µg/ml). The plasma level for ARS after oral administration of patients of 500 mg was determined at 200 µg/ml (de Vries and Dien, 1996). For ART administered i.m. at 2 mg/kg, it was 510 ng/ml (C
max) and after i.v. administration, 2640 ng/ ml
(deVries and Dien, 1996). The latter dose approaches the concentration that caused cytotoxic effects in cell culture experiments (in which colony formation was measured as the most sensitive endpoint). However, it is important to note that the serum half-life in the body was determined with T
1/2 = 0.49 h for ART, and with several hours for the still-active
degradation product DHA, whereas such short exposures remained without cytotoxic effect in mammalian cells in vitro (Kaina et al., unpublished data). Overall, comparison of dose-response data with plasma levels during therapy indicates that inactivation of P. falciparum is achieved with much lower dose levels and shorter treatment times of ART than those causing cytotoxic and genotoxic effects in mammalian cells. This is presumably due to high uptake of ART and DHA in the parasite, since more than 150-fold higher levels were found in parasited erythrocytes than in noninfected erythrocytes (Navarathnam et al., 2000). Therefore, a low plasma level of ART (∼500 ng/ml) is already effective in malaria therapy, whereas normal cells do not respond yet to this dose.
Toxicological studies in animals revealed that an acute lethal dose caused neurological syndroms and signs of cardiotoxicity (deVries and Dien, 1996). After chronic treatment also hema-totoxicity was observed, with decrease in reticulocyte counts and adverse effects on erythropoiesis. Therefore, it cannot be excluded that some cell types in the human body (e.g., some blood cell populations and neurons) respond in a highly sen-sitive way causing side effects upon ART administration. We should also note that comparison of plasma dose levels with in vitro toxicity has not been done with the other ARS derivatives, DHA, ARM, and ARE, which exhibit a longer half-life in the body. Thus, it will be important in future work to elucidate the cytotoxicity of ARS and its derivatives on the different normal cell populations in the human body.
5. Hemato- and immunotoxicity
5.1. In vitro studiesIn the Chinese literature, hematopoietic and immunotoxic effects have already been described in the 1980s (Shen et al., 1984; Gu et al., 1989). Later on, the activity of artemisinins towards hematopoietic cells has widely been addressed by many other groups. Hematopoietic effects can be categorized as erythropoietic or leukopoietic toxicities according to the differentiation lineage. Whereas in vitro effects of artemisinins on erythropoiesis have not been analyzed as yet, artemisinins are shown to both enhance and inhibit leukocyte function (Table 3). ART inhibited phythemagglutinin-stimulated proliferation of lymphocytes in vitro (Chen et al., 1994).
414 T. Efferth and B. Kaina
Tabl
e 3.
Hem
ato-
an
d im
mu
not
oxic
ity
of a
rtem
isin
in a
nd
its
der
ivat
ives
.
Spec
ies
Dru
gD
ose
Ap
plic
atio
n m
ode
Eff
ect
Ref
eren
ce
Hu
man
ly
mp
hoy
tes,
neu
trop
hils
AR
T
in v
itro
Inh
ibit
ed p
hyt
ohem
aggl
uti
nin
-sti
mu
late
d ly
mp
hoc
yte
pro
lifer
atio
nC
hen
et a
l., 1
994
Hu
man
neu
trop
hils
DH
A0.
1–50
mg/
Lin
vit
roD
ecre
ased
ph
agoc
ytic
act
ivit
y of
neu
trop
hils
; in
crea
sed
gen
erat
ion
of r
eact
ive
oxyg
en s
pec
ies
Wen
isch
et a
l., 1
997
AR
S
AR
T
Hu
man
bon
emar
row
Div
erse
AR
S
der
ivat
ives
in v
itro
Hig
her
toxi
city
to p
roge
nit
or c
ells
of t
he
gran
ulo
cyte
-mon
ocyt
e
linea
ge (
CFU
-GM
) th
an to
can
cer
cells
Bea
kman
et a
l., 1
998
Mon
ocyt
esA
RS
in
vit
roD
own
-reg
ula
tion
of m
onoc
yte
rece
pto
rsG
old
rin
g an
dN
emoa
ran
i, 19
99
Mon
keys
AR
S24
mg/
kg/d
ayi.m
.D
ecre
ase
in r
etic
ulo
cyte
an
d e
ryth
rocy
te c
oun
tC
hin
ese
Coo
p. R
es. G
rou
p o
n
Qin
hao
su, 1
982
Dog
sA
RM
6, 1
9, a
nd
32
mg/
kg/d
ayi.m
. for
15
day
sD
ecre
ase
of T
, Tµ,
Tγ,
an
d B
lym
ph
ocyt
esG
u e
t al.,
198
9
Mic
eA
RT
10 m
g/kg
i.p. f
or 7
–10
day
sE
nh
ance
d T
lym
ph
ocyt
e–m
edia
ted
imm
un
e re
spon
ses:
en
han
ced
DN
A
syn
thes
is o
f sp
leen
cel
ls, i
ncr
ease
of I
L-2
pro
du
ctio
n, e
nh
ance
d D
TH
re
spon
se a
nd
an
tib
ody
resp
onse
up
on c
hal
len
ge; a
ccel
erat
ed im
mu
ne
re
con
stit
uti
on a
fter
syn
gen
eic
bon
e m
arro
w tr
ansp
lan
tati
on
Yan
g et
al.,
199
3
Mic
eA
RT
75 m
g/kg
/bid
aily
i.m. f
or 7
day
sSu
pp
ress
ed im
mu
ne
resp
onse
: dec
reas
ed h
um
olys
in-f
orm
ing
cap
acit
y
and
ser
um
IgG
up
on im
mu
nog
enic
ch
alle
nge
; en
han
cem
ent o
f cel
l-
med
iate
d im
mu
nit
y: e
nh
ance
d P
HA
-in
du
ced
lym
ph
ocyt
e tr
ansf
orm
atio
n
rate
, in
crea
sed
sp
leen
wei
ght,
bu
t red
uce
d th
ymu
s w
eigh
t; e
leva
ted
D
NFB
-in
du
ced
del
ayed
-typ
e h
yper
sen
siti
vity
; red
uce
d p
hag
ocyt
osis
of
per
iton
eal m
acro
ph
ages
Lin
et a
l., 1
995
Dog
sA
RM
20 m
g/kg
/day
i.m. f
or 3
0 d
ays
Hyp
och
rom
ic, m
icro
cyti
c an
emia
Cla
ssen
et a
l., 1
999
40/8
0 m
g/kg
/day
i.m. f
or 8
day
s
50–6
00 m
g/kg
/day
p.o.
for
8 d
ays
Rat
sA
RT
20 m
g/kg
/day
p.o.
for
14 d
ays
No
effec
t on
ret
icu
locy
te a
nd
ery
thro
cyte
cou
nt
Kn
igh
ts, 2
002
Rat
sA
RM
80 m
g/kg
p.o.
on
ce e
very
2w
eeks
for
5 m
onth
s71
% d
ecre
ase
in r
etic
ulo
cyte
cou
nt,
re
vers
ible
incr
ease
in b
lood
hem
oglo
bin
Xia
o et
al.,
200
2b
Rat
sA
RT
240
mg/
kg/d
ayi.v
. for
3 d
ays
Rev
ersi
ble
red
uct
ion
in re
ticu
locy
te a
nd
ery
thro
cyte
cou
nt,
hem
atoc
rit,
an
d h
emog
lob
in in
per
iph
eral
blo
od;
red
uct
ion
of m
yelo
id/e
ryth
roid
rat
io in
bon
e m
arro
w b
y A
RT
Xie
et a
l., 2
005
AR
L80
mg/
kg/d
ayi.v
. for
3 d
ays
Rat
sA
RT
17 m
g/kg
p.o.
sin
gle
dos
e on
ge
stat
ion
al d
ay 1
1T
ran
sien
t dec
reas
e in
ret
icu
locy
te c
oun
tC
lark
et a
l., 2
008a
Mon
keys
AR
T40
mg/
kg/d
ayp.
o. fo
r 14
day
sR
edu
ced
ret
icu
locy
te c
oun
tC
lark
et a
l., 2
008b
Not
e. A
RS,
art
emis
inin
; AR
M, a
rtem
eth
er; A
RT,
art
esu
nat
e; D
HA
, dih
ydro
arte
mis
inin
.
Toxicity of artemisinins 415
Decreased phagocytic activity of neutrophils and accompa-nied increased ROS generation were found in neutrophils upon exposure to DHA, ARD, or ART (Wenisch et al., 1997). Remarkably, diverse derivatives exhibited higher cytotoxicity to hematopoietic progenitor cells of the granulocyte-mono-cyte lineage (CFU-GM) than to cancer cells (Beakman et al., 1998), indicating that myelosuppression might be an issue of artemisinins in cancer therapy.
5.2. In vivo studiesIn animal experiments, damage of erythropoiesis frequently occurs (Table 3). A reduction of maternal and embryonic erythroblasts was described, the latter contributing to embryotoxicity (see above). It seems that erythropoi-esis represents a sensitive target for artemisinins (Chinese Cooperative Research Group on Qinhaosu and Derivatives as Antimalarials, 1982; Classen et al., 1999; Xiao et al., 2002b; Xie et al., 2005; Clark et al., 2008a, 2008b, 2008c, 2009). A sensitive measure for inhibition of erythropoiesis is the number of reticulocytes. Reticulocytes are bone marrow-derived erythrocyte precursors in peripheral blood. The reticulocyte number represents a reliable indicator for effec-tive erythropoiesis. Reports of reduced reticulocyte counts after treatment with artemisinins (Table 3) are supplemented by binding studies using radioactively labeled artemisinins. 14C-artelinic acid was preferentially accumulated in bone marrow and spleen of rats (Noker and Simpson-Herren, 1998; cited in Navaratnam et al., 2000). Bone marrow is the site of erythropoiesis, and spleen is the major organ of degradation of erythrocytes and hemoglobin by macrophages. In another study, embryonic erythroblasts were specifically labeled by 3H-ART, as determined by high-resolution microautoradiog-raphy (White et al., 2007).
Whereas the inhibitory action of artemisinins towards erythropoiesis is clearly documented, reports on their toxicity towards leukocytes are contradictory (Table 3). An early report by Gu et al. (1989) recorded decreased lymphocyte counts in dogs after ARM treatment. Yang et al. (1993) found enhanced T lymphocyte–mediated immune responses, enhanced DNA synthesis of spleen cells, increase of interleukin (IL)-2 pro-duction, as well as enhanced delayed-type hypersensitivity (DTH) response and antibody response upon immunogenic challenge. Furthermore, the authors observed accelerated immune reconstitution in mice after syngeneic bone marrow transplantation.
Mixed reaction patterns in mice were also described by Lin et al. (1995). On the one hand, the authors described suppressed immune response, whereas on the other hand an enhancement of cell-mediated immunity was found. Increased spleen weight contrasted with reduced thy-mus weight. Furthermore, elevated dinitrofluoro benzene (DNFB)-induced delayed-type hypersensitivity and reduced phagocytosis of peritoneal macrophages were observed.
Other contrasting results have been described in the literature. ARS and derivatives have been shown to enhance immune responses. ARS can increase phagocytosis of perito-neal macrophages and interferon production. The drug can
also enhance the delayed-type hypersensitivity response and acid phosphatase activity in macrophages (Qian et al., 1981, 1987; Ye et al., 1982). Furthermore, ART stimulated sheep erythrocyte–induced antibody formation (Chen et al. 2002).
Although possible suppressive effects of ARS-like compounds on T cells may be favorable to the development treatment strategies for autoimmune and chronic inflamma-tory diseases, e.g., lupus erythematosus (Gladman et al., 1983; Tam et al., 2000; Li et al., 2006), autoimmune encephalomy-elitis (Wang et al., 2007), rheumatoid arthritis (Xu et al., 2007), acute pancreatitis (Zhao et al., 2007), and contact dermatitis (Chen et al., 1994), immunosuppression may counteract the cytotoxic activity of artemisinins towards malaria and tumors.
In order to clarify the effect of artemisinins on immune functions in the context of cancer therapy, we used a trans-genic mouse spontaneous melanoma model, in which the ret transgene was expressed in melanocytes under the control of metallothionein-I promoter. Ret transgenic mice are known to accumulate melanoma-specific effector memory T cells and natural killer (NK) cells in the primary tumors and meta-static lymph nodes. We monitored ART’s effects on CD4+ and CD8+ T cells as well as Treg and NK cells from ret transgenic tumor-bearing C57BL/6 mice and nontransgenic littermates in vivo and did not find considerable effects of ART on the immune function, as measured by major cell populations of the immune system, i.e., CD4+ and CD8+ T cells as well as Treg and NK cells, both from mice treated for 2 weeks with a daily dose of 1 mg ART (Ramacher et al., 2009). These results indicate that the cytostatic and apoptotic effects of ART were not diminished by concomitant immunosuppression.
5.3. Human studiesReduced reticulocyte counts have also been observed in humans. Guo et al. (1990) found reduced reticulocyte numbers after application of 3 mg/kg/day ART for 4 days in 2/4 subjects. This effect was reversible. Similar observations were made in 284 malaria patients, who received 4 mg/kg ARM i.m. followed by 2 mg/kg every 8 h for at least 72 h (Hien et al., 1996). Reduced reticulocyte counts were found both in male healthy volunteers (Phase I trial) and adult patients affected with uncomplicated falciparum malaria and treated with either ART (4 mg/kg) alone or ART + chlorproguanil + dapsone (Phase II trial) (Wootton et al., 2008). Reduced reticulocyte numbers were reversible and returned to nor-mal or even higher counts a few days after therapy. Reviews on a total of 4062 patients from published and unpublished clinical trials of artemisinins revealed a reduced reticulocyte count in 25 patients (Ribeiro and Olliaro, 1998; Taylor and White, 2004). Furthermore, hemoglobin urea was observed in patients receiving artesunate for malaria (Nealon et al., 2002; Ezzedine et al., 2007).
In conclusion, toxic effects of artemisinins on erythropoiesis were detectable both in animal experiments and human studies. The reduction of reticulocyte numbers by artemisinins was mild to moderate and returned to normal levels after therapy.
416 T. Efferth and B. Kaina
6. Cardiotoxicity
6.1. In vitro and in vivo studiesThe generation of ROS and carbon-centered radical molecules by artemisinins raises the questions about cardiotoxicity, as it is a well-known unwanted side effect for established cancer drugs such as anthracyclines (Mordente et al., 2009). Using guinea pig ventricular myocytes, increase in Ca2+ levels has been observed after exposure of cell cultures with ARS (Ai et al., 2001) (Table 4). Although no cytotoxicity of ARS has been measured in this investigation, Ca2+ homeostasis was affected in myocytes.
These in vitro data represent a supplement for an in vivo study on dogs, where cardiotoxicity appeared (Brewer et al., 1994). ARE caused progressive cardiorespiratory collapse and death in 5/6 dogs treated with 20 mg/kg/day. Furthermore, a prolongation of QTc intervals on electrocardiograms (ECGs) with bizarre ST-T segment changes has been measured. The QTc interval is a measure for heart function: the faster the heart rate, the shorter the QT interval.
However, it cannot be concluded that the dose alone may determine risk of cardiotoxicity. Some subjects may be more likely to manifest toxicity and dosing may be related to allo-metric scaling.
6.2. Human studiesA toxicity study on ARM-lumifantrine combination treatment versus halofantrine was conducted in 13 healthy male volunteers in a randomized double-blind crossover study (Bindschedler et al., 2002). ECGs were recorded before, during, and after treatment. Halofantrine, known to cause QTc prolongations, was used as positive control. In contrast to halofanrine, ARM-lumefantrine (80/480 mg) did not affect QTc intervals.
Electrocardiographic monitoring over 24 h was performed in 53 patients suffering from with severe falciparum malaria (Bethell et al., 1996). Nine out of 53 patients (17%) died during the monitoring period, but none due to cardiac arrhythmia. No cardiac abnormalities were detected. This indicates that cardiac arrhythmias are rare in severe malaria and that artemisinins did not affect cardiac function in malaria
patients. A similar conclusion can be drawn from an ECG analysis in 31 severe falciparum malaria patients treated with an i.m. loading dose of 160 mg ARM followed by 80 mg daily for another 6 days or with quinine in the control group (19 patients). No significant ARM-related ECG changes appeared nor were the ECGs from the patients who died different from those of survivors (Karbwang et al., 1997). ACT with ARM and lumefantrine (n = 150) or ART and mefloquine (n = 50) did not result in clinically significant changes in ECG intervals (van Vugt et al., 1999).
In summary, although cardiotoxicity has been detected in dogs, human trials did not show signs of impairment of heart function by artemisinins. This is presumably due to the low dose of ART applied in malarial therapy, and may be different in cancer therapeutic trials.
7. Other toxicities
Nephrotoxicity. Renal failure and tubular necrosis have been found in healthy rats treated with artelinate (ARL) or ART (Li et al., 2007b). Interestingly, less pathological lesions induced by artemisinins were found in malaria-infected rats (Table 4).
Allergic reactions. Six patients treated p.o. with ART dis-played allergic reactions (Leonardi et al., 2001). These six cases were the only ones among 17,000 patients treated with artemisinins over a decade. Each of them developed urticarial rush, two of whom had a severe clinical picture requiring appropriate treatment (adrenaline, high-dose antihistamines, steroids).
8. Conclusions and perspectives
In conclusion, the lesson learned from animal and human studies is that long-term rather than short-term peak con-centrations of artemisinins cause toxicity. Furthermore, the chemical nature of the drug, in addition to its pharmakokinetic properties, also represents a major factor of toxicity. Oral intake represents a relatively safe route of administration compared to delayed drug release after i.m. injection. This explains why considerable toxicities were
Table 4. Other toxicities of artemisinin and its derivatives.
Species Drug Dose Application mode Effect Reference
Cardiotoxicity
Guinea pig ARS in vitro Increase of intracellular Ca2+ levels in Guinea pig ventricular myocytes
Ai et al., 2001
Dogs ARE 10 or 20 mg/kg/day i.m. for 8 days Neurological defect with progressive cardiorespiratory collapse and death in 5 of 6 high dose–treated dogs; prolongation of QTc intervals on electrocardiograms with bizarre ST-T segment changes
Brewer et al., 1994
Dogs ARM 20 mg/kg/day i.m. for 30 days Prolongation of QTc interval Classen et al., 1999
40/80 mg/kg/day i.m. for 8 days
50–600 mg/kg/day p.o. for 8 days
Nephrotoxicity
Rats ARL 40 mg/kg/day ARL i.v. for 3 days Renal failure and tubular necrosis in noninfected rats; less pathological lesions in malaria-infected animals
Li et al., 2007
ART 240 mg/kg/day ART
Note. ARS, artemisinin; ARE, arteether; ARL, artelinate; ART, artesunate.
Toxicity of artemisinins 417
found in the majority of animal experiments, but not in human studies. Brainstem toxicity of artemisinins is not considered to be a high risk in malaria, This may have to be revisited for patients with cancer. Also, for cancer (as indeed for malaria) it is not clear what the in vitro versus in vivo determinants of efficacy might be. It is likely that they cannot be easily related to drug levels. Concentrations of artemisinins in the nano- to micromolar range were necessary to kill cancer cells in vitro and in vivo, whereas Plasmodia are killed at nanomolar concentrations (Efferth, 2005, 2006, 2007).
A general theme throughout the literature is that clinical use of artemisinins is safe. There is, however, a paucity of large-scale clinical trials suitable to detect rare but significant toxicity. Integration of pharmacovigilance in public health programs will greatly facilitate monitoring of safety of artemisinins (WHO, 2006; Pirmohamed et al., 2007; Brabin et al., 2008). This points to a general problem of many coun-tries with high malaria infection rates. Infrastructure and resources for pharmacovigilance and efficacy programs may be limited (WHO, 2006; Talisuna et al., 2006; Pirmohamed et al., 2007).There are some concerns whether cumulative toxicity might occur upon several drug treatment courses for separate episodes of malaria. Furthermore, the vulner-ability of children and pregnant women to toxic effects of artemisinins in comparison to adults has to be elaborated in more detail.
Furthermore, it is still not sufficiently analyzed whether ACTs exert synergistic toxicity as compared to artemisinins alone. This is not only true for combinations with estab-lished antimalarials, but also for novel, investigational compounds. Looareesuwan et al. (1996) tested the toxicity of a combination of ART and desferroxamine B. The latter compound is an iron chelator that also reveals antimalar-ial activity (Bunnag et al., 1992). Therefore, the question is whether desferroxamine B would decrease neurotoxicity, because of its iron-chelating effect, which diminishes ART’s activity. The authors did not find specific toxicity assign-able to the combination treatment in a total of 31 malaria patients. It could be suspected that this drug combination also reduces activity towards Plasmodia, because of a pos-sible antagonism of both compounds. Looareesuwan et al. (1996) clearly showed that desferrixaomine B did not alter the parasite clearance kinetics of artesunate and there was no in vivo antagonism.
Declaration of interest
There is no conflict of interest. There was no financial support for preparation of the review. One of the authors (T.E.) holds a patent on the “Combined treatment with artesunate and an epidermal growth factor receptor inhibitor” (US Patent, US60/619,829). The authors prepared this paper during the normal course of their academic employment. The authors have sole responsibility for the writing and content of the paper.
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