Cellular uptake of a catechol iron chelator and chloroquine into Plasmodium falciparum infected erythrocytes

Article abstract of DOI:10.1016/S0006-2952(03)00042-X

Our study demonstrates the capacity of FR160, a catechol iron chelator, to reach and accumulate into infected Plasmodium falciparum erythrocytes and parasites (cellular accumulation ratio between 12 and 43). Steady-state FR160 accumulation is obtained aft

Full text of DOI:10.1016/S0006-2952(03)00042-X

Biochemical Pharmacology 65 (2003) 1351–1360
Cellular uptake of a catechol iron chelator and chloroquine into
Plasmodium falciparum infected erythrocytes
a
Akli Hammadi , Florence Ramiandrasoa , Veronique Sinou , Christophe Rogier ,
b
c,d
c,d
c,d
e
c,d
b
Thierry Fusai , Jacques Le Bras , Daniel Parzy , Gerhard Kunesch , Bruno Pradines
c,d,*
a
Unit e´ d’Enseignement Radioprotection, Biologie et M e´ decine, Institut National des Sciences et Techniques Nucl e´ aires,
Centre d’Energie Atomique de Saclay, Gif-sur-Yvette, France
b
Laboratoire de Chimie Bioorganique et Bioinorganique, FRE 2127 CNRS, Institut de Chimie Mol e´ culaire d’Orsay, Orsay, France
c
Unit e´ de Parasitologie, Institut de M e´ decine Tropicale du Service de Sant e´ des Arm e´ es, Marseille, France
d
Institut F e´ d e´ ratif de la Recherche n8 48, Marseille, France
Centre National de R e´ f e´ rence de Chimiosensibilit e´ du Paludisme, Laboratoire de Parasitologie,
H oˆ pital Bichat-Claude Bernard, Paris, France
e
Received 23 July 2002; accepted 8 January 2003
Abstract
Our study demonstrates the capacity of FR160, a catechol iron chelator, to reach and accumulate into infected Plasmodium falciparum
erythrocytes and parasites (cellular accumulation ratio between 12 and 43). Steady-state FR160 accumulation is obtained after 2 hr of
exposure. After 2 hr exposure, it reaches intracellular levels that are 4- to 10-fold higher in infected red blood cells than those attained in
normal erythrocytes. There is quite a good correlation between the accumulation of chloroquine and FR160 in the different strains
(
r ¼ 0:939) and in the IC values (r ¼ 0:719). In contrast, the accumulation of FR160 and its activity is poorly correlated (r ¼ 0:500),
5
0
suggesting that activity of FR160 may be independent of its penetration into infected erythrocytes. The mechanism of accumulation is yet
unknown but based on inhibitor studies, the uptake of FR160 seems to be not associated with the calcium pump or channel, the potassium
þ
þ
channel or the Na /H exchanger. Combinations of FR160 with verapamil, diltiazem, clotrimazole, amiloride, diazoxide, 4-aminopyr-
idine, and picrotoxin should be avoided (antagonistic effects). The potent in vitro activity of FR160 on chloroquine-resistant strains or
isolates, its lower toxicity against Vero cells, its mechanisms of action, its capacity to reach rapidly and accumulate into infected
erythrocytes suggest that FR160 holds much promise as a new structural lead and effective antimalarial agent or at least a promising
adjuvant in treatment of malaria.
#
2003 Elsevier Science Inc. All rights reserved.
Keywords: Malaria; Iron chelators; Siderophores; Chemotherapy; Accumulation
1
. Introduction
permeability properties of the erythrocyte membrane and,
in particular, how those properties differ in infected com-
pared to uninfected red blood cells.
The emergence and spreading of parasite resistance to
currently used antimalarial drugs indicate that novel com-
pounds need to be discovered and developed by identifica-
tion ofnovelchemotherapeutic targets [1]. Thedevelopment
of novel chemotherapies depends largely on the identifica-
tion of potential targets and elucidation of the mechanisms
by which the drugs gain access to intracellular parasites. The
strategy of targeting potential cytotoxic compounds to
infected cells first requires basic knowledge about the
Iron chelation therapy was considered a suitable treat-
ment for various infectious diseases, including malaria [2].
Iron is a prerequisite for all living organisms and is needed
for catalysis of DNA synthesis and for a variety of enzymes
concerned in electron transport and energy metabolism [3].
Recent experimental observations obtained in vitro [4,5] in
rodent [6] and in primate models [7], and in clinical studies
[8,9] showed antimalarial activity of desferrioxamine. The
antimalarial action of iron chelators is dictated by three
factors [10], i.e. iron(III)-binding capacity, chelator ingress
into parasitized erythrocytes, and chelator egress from
parasites after treatment. To reach the parasite cytosol, a
*
Corresponding author. Tel.: þ33-491-150-110; fax: þ33-491-150-164.
E-mail address: bruno.pradines@free.fr (B. Pradines).
Abbreviations: CQ, chloroquine; IC50, 50% inhibitory concentration.
0006-2952/03/$ – see front matter # 2003 Elsevier Science Inc. All rights reserved.
doi:10.1016/S0006-2952(03)00042-X

1352
A. Hammadi et al. / Biochemical Pharmacology 65 (2003) 1351–1360
drug would have to sequentially cross the concentric
compartments enclosed within the various membranes.
This process encompasses both membrane permeation
steps and diffusion across aqueous compartments.
2.1.2. Synthesis of isotopically labeled dicatechols
(Fig. 1)
2.1.2.1. Toluene-4-sulfonic acid non-3-ynyl ester (2). To a
cold solution (58) of toluene-4-sulfonyl chloride (6.0 g,
31 mmol) in 25 mL of dry CH Cl and 5 mL of NEt
2 2 3
Various iron chelators were assessed to improve the drug
lipophilicity leading to increased access of drug to intracel-
lular parasites and to faster speed of action [11,12]. We have
shown previously the in vitro activity on Plasmodium
falciparum of FR160, a catecholate siderophore derived
(36 mmol), 3.2 mL of non-3-yn-1-ol (1; 20 mmol) was
added dropwise, and then allowed to warmup to room
temperature. The mixture was stirred overnight. After
washing with water and brine, the organic layer was dried
4
1
8
from spermidine, the N -nonyl,N ,N -bis(2,3-dihydroxy-
benzoyl)spermidine hydrobromide [13]. FR160 was more
effective at the late trophozoite and young schizont stages,
although the drug affected rings and schizonts as well [14].
Combinations of FR160 and tetracyclines and norfloxacin
have synergistic or additive effects against P. falciparum
parasites[15]. We showed that theactivity ofFR160differed
significantly (P < 0:0001) between 75 isolates susceptible
to chloroquine (ic50 ¼ 1:13 mM) and 60 isolates resistant to
chloroquine (ic50 ¼ 2:07 mM) (Pradines, submitted).
The aim of this study was to assess the permeation of
FR160 into infected and uninfected erythrocytes. To explore
the cellular accumulation of FR160, we have prepared
(Na SO ) and filtered. The solvent was removed under
vacuum to give quantitatively 6.0 g of 1 as a viscous
2 4
yellow oil.
1
H NMR (CDCl ): 0.8 (t, 3 H) CH ; 1.3 (m, 6 H)
3
3
3
H) CH OTs; 7.3 (d, 2 H); 7.7 (d, 2 H).
ꢀ CH ; 2.0 (t, 2 H) CH -5; 2.4 (s, 3 H) CH ; 4.0 (t, 2
2
2
3
2
13
C NMR (CDCl ): 13.7 CH ; 18.3 CH ; 19.4 CH ; 21.3
2
3
3
2
CH ; 21.5 CH ; 21.9 CH ; 28.2 CH (Ts); 30.7 CH ; 68.1
2
2
2
3
2
CH OTs; 73.7 C-4; 82.6 C-3; 127.6 and 129.6 CH; 130.1
2
and 144.7 C.
Mass spectrum (CI), m/z 312 [M þ 18]þ.
3
radiolabeled FR160 ([ H]FR160). The accumulation of
2.1.2.2. 1-Bromo-non-3-yne (3). To a solution of 5.9 g
(20 mmol) of 1 in 50 mL of dry acetone was added 4.8 g
(35 mmol) of NaBr. The mixture was stirred at reflux for
72 hr. After usual workup, 6.6 g of crude product was
obtained and purified by distillation under vacuum
(15 mmHg) providing 3.34 g of 3 as a colorless oil
FR160 was compared with that of chloroquine. Chloroquine
is the most used antimalarial drug. It exerted a huge drug
pressure on P. falciparum populations and it is of major
importance to delineate the potential for cross-resistance.
Recent data suggest that iron chelation, by desferrioxamine,
in Plasmodium spp. also influences the efficiency of the
polymerization of hematin [16]. In addition, deprivation of
theiron supply by interferencewith hemoglobin metabolism
has also been proposed as a mode of action of chloroquine
(53%), b.p.: 130–1328.
1
H NMR (CDCl
): 0.9 (t, 3 H) CH
; 2.7 (m, 2 H) CH
2
; 1.2–1.6 (m, 6 H)
3
3
3 ꢀ CH2; 2.1 (m, 2 H) CH
; 3.4 (t, 2 H)
2
CH
Br.
2
13
[17]. Chloroquine may play a role in iron metabolism, in
particular, as an inhibitor of iron transport [17].
C NMR (CDCl
3
): 14.0 CH
; 31.0 CH
Mass spectrum (CI): m/z 202 (MH , Br), 204 (MH ,
81Br).
3
; 18.6 CH
; 22.2 CH ; 23.1
2 2
CH
; 28.5 CH
; 30.4 CH
Br; 76.8 and 82.7 CBC.
2
2
2
2
þ
79
þ
2
2
2
2
. Materials and methods
2.1.2.3. Preparation of N-alkylated spermidine (4). 1-
Bromo-non-3-yne (3; obtained in two steps from
3
.1. Synthesis of [ H]FR160
1
8
commercially available non-3-yn-1-ol (1) with N ,N -bis-
2,3-(bis(benzyloxybenzoyl)spermidine)] (1.5 g, 1.48 mmol)
was dissolved in dry acetone and to this Na CO (1.5 g) and
.1.1. Materials
1H and 13C NMR spectra were obtained on Bruker AC-
00 or 250 spectrometers. Chemical shifts are reported in
[
2
3
1-bromo-non-3-yne (3; 0.3 g, 1.48 mmol) in 10 mL of dry
acetone were added. The mixture was stirred at reflux
overnight. The solvent was removed under vacuum and
the residue was taken up in CH Cl . After usual workup
ppm relative to solvent CDCl or CD OD (d units). Mass
3
3
spectra were recorded in the chemical ionization (CI) mode
on a Riber-Mag R-10-10. Tritiated compounds were ana-
lyzed by chemical ionization-mass spectrometry (Nermag
R-10-10). HPLC analyses were performed on a Waters 600
chromatographic system supplied with a Waters 996 photo-
diode array detector and a radioactive flow monitoring
2
2
and further purification by MPLC (CH Cl , MeOH 3–10%)
2
2
product 4 was obtained in 40% yield.
1
H NMR (CDCl ): 0.9 (t, 3 H) CH ; 1.2–1.6 (m, 12 H)
3
3
CH2 ꢀ 6; 2.0–2.2 (m, 4 H) CH2 ꢀ 2, 2.4 (m, 4 H)
(LB507A analyzer; Berthold). Tritium determinations
2 ꢀ CH2N, 2.6 (t, 2 H) CH N; 3.2 (m, 4 H)
2
were made in an SL 3000 Intertechnique liquid scintilla-
tion counter. The automatic gas transfer unit used for
tritiation has been previously described [18]. Carrier-free
tritium gas was obtained from the Commissariat a` l’Ener-
gie Atomique (CEA).
2 ꢀ CH2NH; 5.0–5.2 (2 s, 8 H) 4 ꢀ CH2Br; 7.1–7.5 (m,
2 H) 2 ꢀ CH; 8.0 (2t, 2 H) 2 ꢀ NH.
13
C NMR(CDCl ):13.9CH ;16.6;18.6;21.5;22.1;24.1;
3
3
26.6; 27.0; 28.4; 28.6 CH ; 31.0 3 ꢀ CH2N; 37.9 and 39.3
2
CH NH; 46.2; 51.3; 52.7 and 53.1 4 ꢀ OCH3; 71.2 and 76.3
2

A. Hammadi et al. / Biochemical Pharmacology 65 (2003) 1351–1360
1353
Fig. 1. Synthesis of isotopically labeled dicatechols.
CBC; 116.8 CH; 123.1 CH; 124.3 CH; 136.3 C; 146.6 C;
1
suspended in EtOAc and deuterium gas was bubbled
through the mixture for 5 hr. The Pd–C catalyst was
filtered and the solvent was removed under vacuum to
give quantitatively product 5.
51.6 C; 164.9 and 165.1 C=O.
Mass spectrum (CI): m/z 899 (M ), 900 (MH ).
þ
þ
1H NMR (CDCl
): 0.8 (t, 3 H) CH ; 1.25 (m, 12 H)
3 3
2
.1.2.4. Preparation of deuterated product (5). Compound
(10 mg, 0.011 mmol) and 10% Pd–C (3 mg) were
4
2 ꢀ CD2 and 4 ꢀ CH2; 1.5–1.8 (m, 6 H) 3 ꢀ CH2; 1.9 (t, 2

1354
A. Hammadi et al. / Biochemical Pharmacology 65 (2003) 1351–1360
H) CH ; 2.6–2.9 (m, 6 H) 3 ꢀ CH N amine, 3.4 (t, 2 H)
2.4. In vitro assay
2
2
CN NH amide; 3.6 (t, 2 H) CH NH amide; 6.7 (dd, 2 H)
2
2
2
ꢀ CH; 6.9 (t, 2 H) 2 ꢀ CH; 7.1 (dd, 2 H) 2 ꢀ CH.
For in vitro isotopic microtests, 200 mL of the suspension
of erythrocytes parasitized with ring stages was distributed
in 96-well plates with antimalarial agents. Parasite growth
13
C NMR (CDCl ): 14.1 CH ; 21.6; 22.6; 23.5; 26.1;
3
3
2
9.1; 29.7 and 31.7 CH ; 37.9 CH NH; 52.6 CH N; 113.8
2
2
2
3
and 113.9 2 ꢀ CH; 116.9 and 117.2 2 ꢀ C; 118.1 and 118.7
was assessed by adding 1 mCi of [ H]hypoxanthine with a
2
1
ꢀ CH; 145.6 and 145.7 2 ꢀ C; 149.0 and 149.1 2 ꢀ C;
70.4 and 170.7 2 ꢀ C=O.
specific activity of 14.1 Ci/mmol (NEN Products) to each
well. Plates were incubated for 42 hr at 378 in an atmo-
sphere of 10% O , 6% CO , 84% N and a humidity of
þ
Mass spectrum (CI): m/z 908 (MH ).
2
2
2
95%. Immediately after incubation the plates were frozen
and then thawed to lyse erythrocytes. The contents of each
well were collected on standard filter microplates (Uni-
2
.1.2.5. Preparation of tritiated product (6). The same
batch of catalyst used for deuteration (10% Pd–C, 27 mg)
was added to a solution of 4 (6 mg, 0.007 mmol) in 1 mL of
EtOAc. The vial was connected to the tritiation apparatus [1].
TM
filter GF/B; Packard Instrument Company) and washed
TM
using a cell harvester (FilterMate Cell Harvester; Pack-
The solution was frozen in liquid N . Carrier-free tritium gas
2
ard). Filter microplates were dried and 25 mL of scintilla-
tion cocktail (Microscint O; Packard) was placed in each
TM
was introduced and compressed to 1.3 bar. After thawing, the
reaction mixture was kept at 208 and stirred for 4 hr. The 10%
Pd–C catalyst was filtered and labile tritium atoms were
exchanged by successive flash evaporation with 150 mL of
MeOH. The crude tritiated product 6 was dissolved in 1 mL
of MeOH (total radioactivity of 1.85 GBq) and purified by
reverse-phase chromatography (Vydac C18 column; eluent
A: 0.1% trifluoroacetic acid, eluent B: 90% acetonitrile in
well. Radioactivity incorporated by the parasites was
TM
measured using a scintillation counter (Top Count
Packard).
The 50% inhibitory concentration (IC ), i.e. the drug
;
5
0
concentration corresponding to 50% of the uptake of
[3H]hypoxanthine by the parasites in drug-free control
wells, was determined by non-linear regression analysis
of log-dose/response curves. Data were analyzed after
logarithmic transformation and expressed as the geometric
mean of IC50 and 95% confidence intervals (95% CI) were
calculated.
0
.1% trifluoroacetic acid; eluted at 1 mL/min with the
following gradient: isocratic 40% B for 5 min, linear
gradient from 40 to 60% B in 10 min and from 60 to
1
6
00% B in 15 min). Unambiguously, the tritiated product
was characterized by a retention time of 15 min and an
absorption band (lmax) at 215 nm. Compound 6 (111 MBq,
3
2.5. Measurement of the uptake of [ H]FR160 by
erythrocytes parasitized with ring stages
3
.33 TBq/mmol) was obtained and stored at ꢁ808 in
acetonitrile (33.3 MBq/mL) for several months without
degradation.
3
3
Accumulation of [ H]FR160 and [ H]CQ was carried
out essentially according to the protocol of Bray et al. [20]
with some modifications [21]. Infected erythrocytes were
suspended in RPMI 1640 medium buffered with 25 mM
þ
Mass spectrum (CI): m/z 553 (MH ).
2
.2. Other drugs
Radiolabeled chloroquine diphosphate ([ H]CQ) (26 Ci/
HEPES and 25 mM NaHCO , at a parasitemia of 2% and
3
3
hematocrit of 2%. Eppendorf microfuge tubes were loaded
with 400 mL of silicon oil 550, 1 mL of reaction buffer
mmol) was purchased from Amersham. Silicon oil 550 was
purchased from Dow Corning (BDH Laboratory Supplies).
Verapamil, clotrimazole, amiloride, diltiazem, diazoxide,
4-aminopyridine, and picrotoxin were obtained from
Sigma Chemical.
3
3
containing 10 nM [ H]FR160 or 3 nM [ H]CQ on top of
the oil, and then with 25 mL of appropriately concentrated
cell suspension. The cell suspension was mixed with the
reaction buffer and incubated for 2 hr at 378 in an atmo-
sphere of 10% O , 6% CO , 84% N and 95% relative
2
2
2
2
.3. Strains of Plasmodium falciparum
humidity. After 1 min of centrifugation at 13,000 g,
100 mL of the buffer was processed for scintillation count-
Two chloroquine-susceptible strains 3D7 (Africa) and
ing. Infected erythrocytes pellets were lysed by distilled
water and by 5:5:2 quaternary hydroxide:glacial acetic
acid:hydrogen peroxide and left in an oven at 508 for
2 hr. They were then processed for scintillation counting.
FR160 and chloroquine accumulation is expressed as the
cellular accumulation ratio, which is the ratio of the
amount of radiolabeled drug in parasitized erythrocytes
D6 (Sierra Leone) and two chloroquine-resistant strains
W2 (Indochina) and FCR3 (Gambia) were maintained in
culture. When required for drug assays, cultures were
synchronized by sorbitol lysis [19]. Susceptibility to the
iron chelator FR160 was determined after suspension in
RPMI 1640 medium (Life Technologies), supplemented
þ
3
3
with 10% human serum (pooled from different A or AB
sera from non-immune donors), and buffered with 25 mM
(amount of [ H]FR160 or [ H]CQ in parasitized
3
3
erythrocytes ꢁ amount of [ H]FR160 or [ H]CQ in unin-
3 3
HEPES and 25 mM NaHCO (hematocrit of 1.5%, para-
3
fected red cells) to the amount of [ H]FR160 or [ H]CQ in
a similar volume of buffer after incubation [22].
sitemia of 0.5%).

A. Hammadi et al. / Biochemical Pharmacology 65 (2003) 1351–1360
1355
2
.6. Measurement of the time course of the uptake of
H]FR160 by parasitized erythrocytes at ring stages
tested drugs alone and plotted on the horizontal axis.
The corresponding FR160 fractional IC50 was calculated
by dividing the IC50 of FR160 combined with fixed con-
centrations of blockers by the IC50 of FR160 alone and
plotted on the vertical axis. A curve was then drawn
through the resulting pairs of fractions from the ends of
both axes on the graph. Points lying above the straight
diagonal line (corresponding to the points where there is no
interaction between the drugs) are antagonistic, points
below the straight diagonal line are considered to be
synergistic [29].
3
[
The cellular accumulation ratio for FR160 was assessed
after several different times of exposure.
3
.7. Measurement of the uptake of [ H]FR160 by
2
parasitized erythrocytes at ring stages in presence of
pharmacological blockers
The cellular accumulation ratio of FR160 was assessed
in presence of several pharmacological blockers, such as
2þ
verapamil (L-type Ca -channel blocker) [23], clotrima-
þ
2þ
zole (inhibitor of the Ca -activated K channel) [24],
þ
3. Results and discussion
þ
amiloride (inhibitor of transmembrane Na entry and Na -
2þ
þ
K -ATPase) [25], diltiazem (L-type Ca -channel antago-
þ
The elucidation of the mode of action of antimalarial
agents involves the study of the routes and modes of entry
of drugs into infected red blood cells and drug interference
with parasite functions. It is of interest to assess whether
the permeation properties of normal vs. infected erythro-
cytes are sufficiently different to ensure preferential access
of drugs to infected cells. We show that the rapid inhibitory
potency of FR160 (less than 6 hr on ring stages and 3 hr on
trophozoite stages to reduce parasites growth by 50–70%
[14]) is dictated by its capacity for rapid penetration into
parasitized erythrocytes. Previous studies showed that
desferrioxamine enters the parasite through an aqueous
duct leading to the parasite outer membrane [30] and
permeates into parasite [31]. One of the salient features
of desferrioxamine and methylanthranilic-desferrioxamine
uptake into infected cells is that even after hours of
incubation with drug only 5–15% of the external concen-
tration is attained within the infected cell [31]. Results
demonstrated a 3-fold higher permeation into infected
compared with non-infected cells. The apparently poor
inhibitory effect of desferrioxamine on rings might merely
reflect its poor accessibility to the parasite at early stages of
growth.
nist) [23], diazoxide (ATP-sensitive K -channel opener)
þ
[
[
26], 4-aminopyridine (non-specific K -channel blocker)
ꢁ
27], and picrotoxin (antagonist of GABA–Cl –receptor–
ionophore complex) [28], at the concentration of 5 mM.
3
.8. Measurement of the uptake of [ H]FR160 by
2
parasites at ring stages
Infected erythrocytes were incubated in 10 nM
3
[3
After 1 min of centrifugation at 13,000 g, the supernatant
was discarded. Infected erythrocytes pellets were lysed by
1
parasites seem to be not altered in microscopy. However,
we are not sure that some drug had escaped out of the
parasite. The estimate of radioactivity associated with the
parasite may be underestimated. Eppendorf microfuge
tubes were loaded with 400 mL of silicon oil 550. One
hundred microliters of the supernatant with hemoglobin
was processed for scintillation counting. The pellet of
hemolysate and intact parasites was lysed by distilled
water and by 5:5:2 quaternary hydroxide:glacial acetic
acid:hydrogen peroxide and left in an oven at 508 for
H]FR160 or 3 nM [ H]CQ as described in Section 2.5.
mL 0.02% saponine to obtain intact parasites. The
3
We demonstrate here that [ H]FR160 is taken up by
2
Comparison was assessed between the amount ([ H]FR160
hr. They were then processed for scintillation counting.
3
erythrocytes infected with P. falciparum and that infected
red blood cells quickly exhibit accumulation of FR160
from medium (10- to 40-fold) (Table 1). The time course of
FR160 uptake by a chloroquine-susceptible and a chlor-
oquine-resistant P. falciparum at a fixed concentration of
10 nM is shown in Fig. 2. Steady-state FR160 accumula-
tion of the two strains is obtained after 2 hr. Steady state of
the resistant strain is one-fourth that of the susceptible
strain. Maximum uptake was attained after more than 8 hr
for desferrioxamine and after 10–15 min to 8 hr for
reversed siderophores [12]. The estimated amount of
[3H]FR160 in pellet which contained parasites (and ery-
throcytes membranes, hemozoin . . .) is 5-fold higher for
W2 to 18-fold higher for 3D7 than those measured in
pellets of uninfected erythrocytes (data not shown). These
results suggest that FR160 is taken up by parasites and not
only by infected erythrocytes.
3
in pellets of infected erythrocytes ꢁ amount of [ H]FR160
in pellets of uninfected red cells) and the amount
3
(
[ H]FR160 in hemoglobin supernatant of infected red
3
cells ꢁ amount of [ H]FR160 in hemoglobin supernatant
of normal red cells).
2
.9. Drug combinations
Combinations of FR160 with the several blockers were
tested three independent times against the P. falciparum
W2 clone. To evaluate drug interactions, isobolograms
were constructed by plotting a pair of fractional IC50 for
each combination of FR160 and blockers. The different
blocker fractional IC50 was calculated by dividing their
fixed concentrations (12 concentrations) by the IC50 of

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A. Hammadi et al. / Biochemical Pharmacology 65 (2003) 1351–1360
Table 1
Measurement of cellular accumulation ratio of FR160 at a fixed extracellular concentration of 10 nM and chloroquine at 3 nM in Plasmodium falciparum
infected erythrocytes
Strain
FR160
Chloroquine
Mean IC50 (nM)
Cellular accumulation ratio
Mean IC50 (nM)
Cellular accumulation ratio
(95% confidence interval)
(95% confidence interval)
(95% confidence interval)
(95% confidence interval)
3
D7
790 (760–820)
170 (130–210)
43 (27–60)
27 (19–37)
13 (7–18)
12 (9–16)
19 (9–29)
251 (232–271)
287 (247–333)
17 (13–22)
D6
W2
22 (18–27)
832 (665–999)
890 (762–1019)
1040 (970–1110)
1450 (1230–1670)
FCR3
11 (7–16)
Values are means of three to seven independent experiments.
After 2 hr of exposure, FR160 reaches intracellular levels
that are 4- to 10-fold higher in infected red blood cells than
those attained in normal erythrocytes. In a previous study,
between the accumulation values may suggest common
features in drug uptake and/or mode of action or resistance.
The mode of action of chloroquine and the mechanism
of resistance to it are not well understood. It has been
hypothesized that, as a weak base, chloroquine and its close
analogues follow the pH gradient and accumulate in the
food vacuole of the susceptible parasites [33]. The most
convincing explanation of its activity lies in its capacity to
interfere with hemoglobin degradation in food vacuole by
raising the vacuolar pH [34] and/or by inhibition of the
crystallization of the free heme by the formation of toxic
heme–chloroquine complex [35,36]. In previous study,
Vippagunta et al. [16] showed that desferrioxamine
increased the concentration of soluble forms of hematin,
enhanced the rate of hematin crystallization, and also could
initiate hematin crystallization. In contrast, chloroquine
decreased the concentration of soluble forms of hematin
and inhibited hematin crystallization. We have shown that
FR160 could act by generation of radical species and
enhancement of heme-catalyzed oxidation of lipid mem-
branes, but it neither affects the chemical heme polymer-
ization activity nor the production of hemozoin in P.
falciparum parasites (Pradines, submitted). Deprivation
of the iron supply by interference with hemoglobin meta-
bolism has also been proposed as a mode of action of
chloroquine [17]. Chloroquine may play a role in iron
1
3
Fritsch and Jung [32] showed that [ C]desferrioxaminewas
taken up by erythrocytes and after 5 hr exposure it reached
intracellular levels that were 3- to 4-fold higher than those
attained in normal red blood cells. The cellular FR160
accumulation ratio is 3- to 4-fold higher in parasites, which
are 2- to 5-fold more susceptible to FR160. Nevertheless, the
values of accumulation may be underestimated because
during the saponin lysis some drug could have escaped
out the parasite. The parasitized erythrocytes seemed to
be intact and not altered in microscopy. The parasites, which
present the highest accumulation ratio for FR160 and are the
more potent susceptible to FR160, are also the strains the
more susceptible to chloroquine (14-fold) and present a high
cellular accumulation ratio for chloroquine (20-fold higher
than accumulation in chloroquine-resistant parasites). There
is quite a good correlation between the accumulation of both
drugs in the different strains (r ¼ 0:939) and in the IC
5
0
values of both drugs (r ¼ 0:719). We have shown in pre-
vious study that the activity of FR160 differed significantly
(
P < 0:0001) between 75 isolates susceptible to chloro-
quine (ic50 ¼ 1:13 mM) and 60 isolates resistant to chlor-
oquine (ic50 ¼ 2:07 mM) (Pradines, submitted). A positive
correlation between the IC of two antimalarial drugs and
5
0
Fig. 2. Time course of FR160 accumulation by chloroquine-susceptible strain (3D7) and chloroquine-resistant strain (W2).

A. Hammadi et al. / Biochemical Pharmacology 65 (2003) 1351–1360
1357
Table 2
Measurement of cellular accumulation ratio of FR160 in combination with pharmacological channel blockers in Plasmodium falciparum parasites with
different susceptibilities to FR160 and chloroquine
Drug
3D7 strain
W2 strain
Cellular accumulation ratio
95% Confidence interval
Cellular accumulation ratio
95% Confidence interval
Without drug
Picrotoxin
Clotrimazole
Diazoxide
Amiloride
Diltiazem
43
50
40
30
58
42
38
41
27–60
35–65
24–55
24–36
46–70
30–54
27–51
29–53
13
16
8
7–18
8–24
5–11
6–14
6–16
10–17
5–14
10–18
10
11
13
9
Verapamil
4-Aminopyridine
14
Values are means of three independent experiments.
metabolism, in particular, as an inhibitor of iron transport
in Saccharomyces cerevisiae [17]. However, such mechan-
ism has not been shown in Plasmodium. The correlation
between IC50 of chloroquine and FR160 could be ascribed
to common features on iron homeostasis.
are responsible for the access of FR160 and chloroquine
into infected erythrocytes, we assessed the interaction of
FR160 with several channel blockers, which have been
known to reverse chloroquine resistance, such as verapamil
or diltiazem, or which inhibit chloroquine uptake as
amiloride and clotrimazole, or never used with chloroquine
as diazoxide, 4-aminopyridine, and picrotoxin. These
results show that accumulation of FR160 into infected
erythrocytes seems to be not associated with the calcium
The hypothesis that correlation between the accumula-
tion values of chloroquine and FR160 could be ascribed to
common mechanisms of uptake is significantly weakened
by the finding that the pharmacological blockers, which
modulate response to chloroquine, have no effect on the
accumulation of FR160 (Table 2) and antagonize its activ-
ity (Fig. 3). Chloroquine reaches the parasite food vacuole,
where it accumulates due to the local acid pH and the weak
base properties of the drug [33]. The chloroquine activity
depends on a high level accumulation within the parasite
and drug resistance stems from reduced drug accumula-
tion. Several compounds, like verapamil [37,38], desipra-
mine [39], and dihydroanthracene derivatives [40,41],
demonstrated in the past decade promising capability to
reverse the chloroquine resistance in parasite isolates in
vitro in animal models [42] and human malaria [43]. The
reversal of chloroquine resistance by verapamil was pro-
posed to occur by modulating the activity of the parasitic
þ
þ
pump or channel, the potassium channel or the Na /H
exchanger. The hypothesis that correlation between the
accumulation values of chloroquine and FR160 could be
ascribed to common mechanisms of uptake is significantly
weakened by the finding that the pharmacological block-
ers, which modulate response to chloroquine, have no
effect on the accumulation of FR160 and antagonize its
activity. Nevertheless, a larger number of strains should be
tested to provide a meaningful analysis. The weak differ-
ence of the accumulation values for FR160 between chlor-
oquine-susceptible and chloroquine-resistant strains (3- to
4-fold) associated to a weak difference of FR160 IC50 (2- to
5-fold) in contrast with the differences of the accumulation
values for chloroquine (20-fold) and IC50 (40-fold) also
suggest that cellular uptake of both drugs may involve
different transport systems. The identification of these
transport systems is now required.
It is poorly correlated between accumulation and IC50 for
FR160 (r ¼ 0:5), while it is highly correlated for chlor-
oquine (r ¼ 0:994). These data suggest that the activity of
FR160 seems to be independent of its accumulation in
infected erythrocytes. In contrast, desferrioxamine does
indeed penetrate the infected red cell and its antimalarial
activity is dependent on this [48].
þ
þ
Na /H exchanger via the calcium/calmodulin-dependent
pathway [44]. Nevertheless, Bray et al. [20] provided
definitive evidence that chloroquine uptake is determined
by the binding of chloroquine to ferriprotoporphyrin IX.
Recently, it has been shown that the digestive vacuolar pH
of a chloroquine-resistant strain is more acidic relative to
chloroquine-susceptible parasites [45]. Verapamil nor-
malizes the vacuolar pH of the chloroquine-resistant para-
sites to a value near that measured for chloroquine-
susceptible parasite (increase of the pH) and has no effect
on chloroquine-susceptible parasites [46]. In fact, it seems
that acidification of digestive vacuole contributes to drug
resistance via the effects that pH has on the solubility of
unpolymerized heme found in the vacuole [47]. Reversers
of chloroquine resistance increase the pH of acid vesicles
where ferriprotoporphyrin IX is generated and, therefore,
this increases the affinity of chloroquine-ferriprotopor-
phyrin IX binding. To evaluate if similar mechanisms
In conclusion, our study demonstrates the capacity of
FR160 to reach and quickly accumulate into infected
erythrocytes. The mechanism of accumulation is yet
unknown but it seems to be not associated with calcium
þ
þ
pump or channel, potassium channel or Na /H exchan-
ger. The potent in vitro activity of FR160 on chloroquine-
resistant strains or isolates, its lower toxicity, its mechan-
isms of action, its capacity to reach rapidly and accumulate

1358
A. Hammadi et al. / Biochemical Pharmacology 65 (2003) 1351–1360
Fig. 3. In vitro combination of FR160 with several pharmacological channels blockers against the Plasmodium falciparum W2 strain.
into infected erythrocytes suggest that FR160 holds much
promise as a new structural lead and effective antimalarial
agent or at least a promising adjuvant in treatment of
malaria and that it is suitable for in vivo studies.
Acknowledgments
We thank Professor Walliker (Cell Animal and Popula-
tion Biology, Edinburgh, UK), Professor Milhous (Walter

A. Hammadi et al. / Biochemical Pharmacology 65 (2003) 1351–1360
1359
antibiotic activities by a catecholate iron chelator against chloroquine-
Reed Army Institute of Research, Washington, DC, USA),
and Doctor Gysin (Laboratoire de Parasitologie Exp e´ ri-
mentale, Marseille, France) for clones and strains, and the
staff of the Blood Transfusion Centre (Military Hospital,
Toulon, France) for providing human erythrocytes. The
resistant Plasmodium falciparum. Antimicrob Agents Chemother
2
002;46:225–8.
[
16] Vippagunta SR, Dorn A, Bubendorf A, Ridley RG, Vennerstrom JL.
Deferoxamine: stimulation of hematin polymerisation and antagonism
of its inhibition by chloroquine. Biochem Pharmacol 1999;58:817–24.
17] Emerson LR, Nau ME, Martin RK, Kyle DE, Vahey M, Wirth DF.
Relationship between chloroquine toxicity and iron acquisition in
Saccharomyces cerevisiae. Antimicrob Agents Chemother 2002;46:
3
[
synthesis of radiolabeled FR160 ([ H]FR160) was
achieved in the Laboratoire de Marquage des Prot e´ ines,
D e´ partement d’Ing e´ nierie et d’Etude des Prot e´ ines, Centre
d’Energie Atomique. This work was supported by la
D e´ l e´ gation G e´ n e´ rale pour l’Armement (contrat d’objectif
n8 9810060), la Direction de la Recherche et de la Tech-
nologie/Services Techniques des Recherches et des D e´ vel-
oppements Technologiques (contrat n8 97/2509A), le
Groupe de Recherche en Parasitologie 1077, and la Direc-
tion Centrale du Service de Sant e´ des Arm e´ es.
7
86–7.
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de gaz (tritium, deut e´ rium, hydrog e` ne). J Labelled Compd 1975;11:
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