A
ItTfpsda(cl©
K
1
ianine2
0d
Journal of Ethnopharmacology 109 (2007) 258–263
Effects of canthin-6-one alkaloids from Zanthoxylum chiloperoneon Trypanosoma cruzi-infected mice
Maria Elena Ferreira a, Hector Nakayama a, Antonieta Rojas de Arias a, Alicia Schinini a,Ninfa Vera de Bilbao a, Elva Serna a, Delphine Lagoutte b, Flor Soriano-Agaton b,
Erwan Poupon b, Reynald Hocquemiller b, Alain Fournet c,∗a Department of Tropical Medicine, Casilla de Correo 2511, Instituto de Investigaciones en Ciencias de la Salud Asuncion,
Universidad Nacional de Asuncion, Paraguayb Laboratoire de Pharmacognosie, UMR 8076 CNRS, Faculte de Pharmacie, Universite Paris-Sud, rue J.B. Clement,
92296 Chatenay-Malabry Cedex, Francec IRD US 084, Laboratoire de Pharmacognosie, Faculte de Pharmacie, rue J.B. Clement, 92296 Chatenay-Malabry Cedex, France
Received 6 June 2006; received in revised form 18 July 2006; accepted 23 July 2006Available online 29 July 2006
bstract
Canthin-6-one (1), isolated from Zanthoxylum chiloperone (Rutaceae), possesses a broad sprectum of antifungal and leishmanicidal activities.n this study, we have examined the antiparasitic effects of canthin-6-one (1), 5-methoxycanthin-6-one (2), canthin-6-one N-oxide (3), as well ashat of the total alkaloids of Zanthoxylum chiloperone stem bark, in Balb/c mice infected either acutely or chronically with Trypanosoma cruzi.he compounds were administered orally or subcutaneously at 5 mg/kg/day for 2 weeks, whereas the alkaloidal extract was given at 50 mg/kg/day
or 2 weeks. The antiparasitic activity was compared with that of benznidazole given at 50 mg/kg/day for 2 weeks. In the case of acute infection,arasiteamia was significantly reduced following oral treatment with canthin-6-one (1). Moreover, the total alkaloids of Zanthoxylum chiloperonetem bark led to high levels of parasitological clearance. Seventy days post-infection, the serological response in the acute model was significantlyifferent between oral canthin-6-one (1) and benznidazole-treated mice. Chronic model of the disease showed that both canthin-6-one (1) and the
lkaloidal extract at the above dosage induced 80–100% animal survival compared to untreated controls. These results indicate that canthin-6-one1) exhibits trypanocidal activity in vivo in the mouse model of acute or chronic infection. This is the first demonstration of anti-Trypanosomaruzi activity for a member of this chemical group (canthinones). Considering the very low toxicity of canthin-6-one (1), our results suggest thatong-term oral treatment with this natural product could prove advantageous compared to the current chemotherapy of Chagas disease.2006 Elsevier Ireland Ltd. All rights reserved.
gas d
acay2ce
eywords: Zanthoxylum chiloperone; Rutaceae; Paraguay; Canthin-6-one; Cha
. Introduction
Eighteen to 20 million people in South and Central Amer-ca and 50,000–100,000 in the United State (WHO, 1997)re infected with Trypanosoma (Schizotrypanum) cruzi, whileearly 90 million persons live in zones where Chagas diseases endemic. Recent surveys indicate that there are ∼200,000
ew cases and 21,000 deaths associated with this conditionvery year (Morel, 2000; OPS/HCP/HCT/140/99, 1999; WHO,002). Chemotherapy of Chagas disease remains unsatisfactory,∗ Corresponding author. Tel.: +33 1 46 83 55 94; fax: +33 1 46 83 53 99.E-mail address: [email protected] (A. Fournet).
BtFgatc
378-8741/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.jep.2006.07.028
isease; Experimental treatment
nd is based exclusively on nitroimidazoles. Belonging to thislass of agents, benznidazole, developed by Roche three decadesgo, shows efficacy in the acute and short-term (up to a fewears) chronic phase of the disease (Vinhaes and Schofield,003). In recent years, great advances have been made in theontrol of the vectorial and transfusional transmission of the dis-ase, particularly through the Southern Cone Initiative involvingrazil, Argentina, Paraguay and Uruguay. However, the ques-
ion of the persons already infected remains of great concern.or a few years, Paraguayan and French groups have investi-
ated natural products from plants used in traditional medicine,s to find active compounds against Trypanosoma cruzi. Zan-hoxylum chiloperone var. angustifolium Engl. (syn. Fagarahiloperone Engl. Ex Chod. & Hassl.), Rutaceae) is a dioic
hnopharmacology 109 (2007) 258–263 259
t““daapibabcm1aaaccebobttdoocit
2
2
c6d2mtobRd
2
uAcocB
Tu
2
2
mFmit8eoiC6acai
2
ceoaeno
s
M.E. Ferreira et al. / Journal of Et
ree (up to 15 m) growing in South America, which is calledtembetary hu”, (“tembe = lip, ita = stone, y = abbreviation ofyvyra = tree, hu = black sa’yju) in Guarani (Spichiger and Stutze Ortega, 1987). A decoction of Zanthoxylum chiloperone var.ngustifolium root bark is used traditionally in Paraguay for itsntimalaric (Milliken, 1987), emmenagogue and antirheumaticroperties (ethnobotanical data collected in the field). Due tots also known antiparasitic properties, we evaluated herein theioactivity of various extracts of Zanthoxylum chiloperone var.ngustifolium against Trypanosoma cruzi, which is responsi-le for Chagas disease. We had previously reported that threeanthin-6-one-related alkaloids, isolated from the Paraguayanedicinal plant Zanthoxylum chiloperone, were active against
3 fungi (Thouvenel et al., 2003), and also effective in vivogainst Leishmania amazonensis (Ferreira et al., 2002). As fars their mechanism of action is concerned, the antifungal andntiprotozoal properties of canthin-6-one alkaloids might showertain similarities with those of the antifungal triazole ravu-onazole (Urbina et al., 2003b), albaconazole (Matta Guedest al., 2004), and posazaconazole (Urbina, 2002), which haveeen shown to induce parasitological cure in murine modelsf either acute or chronic Chagas disease. In a preliminaryiological screening, the alkaloidal extract of stem bark of Zan-hoxylum chiloperone showed activity against epimastigote andrypanomastigote forms of Trypanosoma cruzi (unpublishedata). In the present study, we compared the in vivo activityf canthin-6-one (1), 5-methoxycanthin-6-one (2), canthin-6-ne N-oxide (3), as well as the total alkaloids of Zanthoxylumhiloperone, with that of the reference compound benznidazole,n mouse models of acute and chronic Trypanosoma cruzi infec-ion.
. Materials and methods
.1. Compounds
The crude alkaloidal extract of Zanthoxylum chiloperone,anthin-6-one (1), 5-methoxycanthin-6-one (2) and canthin--one N-oxide (3) (see Fig. 1) were isolated as previouslyescribed (Schiplander and Mitscher, 1971; Thouvenel et al.,003). Physical and spectral data (1H and 13C NMR andass spectrometry) were used to determine the chemical struc-
ure of the compounds, and were in agreement with thosef reference samples and literature values. Benznidazole (N-enzyl-1,2-nitro-1-imidazole-acetamide) was purchased fromoche (Buenos Aires, Argentina), and used as a referencerug.
.2. Mice and parasites
Female and male Balb/c mice were purchased by the Fac-lty of Veterinary Sciences, National University (La Plata,rgentina), and bred at the Instituto de Investigaciones en Cien-
ias de la Salud (Asuncion, Paraguay). Animals were 6–8 weeksld when used. In all experiments, the CL clone of Trypanosomaruzi (Cano et al., 1995) was used, generously supplied by Dr.. Zingales (Sao Paulo, Brazil). Routine maintenance of the
ii5t
Fig. 1. Chemical structure of canthin-6-one alkaloids.
rypanosoma cruzi strain was carried out in BALB/c mice inoc-lated intraperitonally every 14 days.
.3. Drug treatment
.3.1. Acute infectionAll mice were infected intraperitonally with 5000 trypo-
astigotes from the CL strain following separation from blood.or the acute or chronic infection study, two separate experi-ents were conducted to evaluate the effectiveness of the drugs
n vivo. Treatments were started 12 days after inoculation ofhe parasites. The mice were randomly divided into groups of–12 mice. The reference drug benznidazole was used for allxperiments. All tested drugs were sampled in 50 �l aliquotesf phosphate buffered saline (PBS). Benznidazole was admin-stered to BALB/c mice orally at 50 mg/kg/day for 2 weeks.anthin-6-one (1), 5-methoxy-canthin-6-one (2) and canthin--one N-oxide (3) were administered orally or subcutaneouslyt 5 mg/kg/day for 2 weeks. The total alkaloids of Zanthoxylumhiloperone root bark was administered orally or subcutaneouslyt 50 mg/kg/day for 2 weeks. Each experiment was performedn duplicate.
.3.2. Chronic infectionRoutine maintenance of the Trypanosoma cruzi strain was
arried out in BALB/c mice following intraperitoneal infectionvery 11 days. All mice were infected with 1000 trypomastig-tes of CL strain from blood, allowing a slow-developing par-sitaemia peaking between 21 and 28 days. This infection wasffectively controlled since 70–80% animals survived showing aegative or subpatent parasitaemia, in spite of a slow degradationf their general physical conditions.
For the murine model of long-term infection, treatments weretarted 60 days post-infection when parasitaemia was sub-patent
n all mice. In this experiment, the mice were randomly dividednto groups of 5–8 animals. All tested drugs were sampled in0 �l aliquotes of phosphate buffered saline (PBS) and giveno BALB/c mice by the oral route in regimen of 50 mg/kg/day
2 hnopharmacology 109 (2007) 258–263
(5f
2
wt
EpmAPta
dtt
3
3e
cttiC2bpoc
F5i5f
neN
-oxi
de(3
),an
dcr
ude
Zan
thox
ylum
chil
oper
one
alka
loid
extr
acti
nm
ice
with
acut
eTr
ypan
osom
acr
uzii
nfec
tion
1(s
.c.)
(n=
20)
2(o
ral)
(n=
9)2
(s.c
.)(n
=8)
3(o
ral)
(n=
9)C
rude
Zan
thox
ylum
chil
oper
one
(ora
l)(n
=8)
Cru
deZ
anth
oxyl
umch
ilop
eron
e(s
.c.)
(n=
8)
15.3
±14
.80.
2±
0.3b
50.9
±0.
35.
5±
4.1b
20.5
±15
.85.
2±
2.3b
8.2
±8.
9b9.
7±
6.5b
49.0
±36
.065
.4±
26.0
28.3
±18
.8b
81.3
±41
.3c
38.5
±29
.535
.1±
28.8
15.5
±13
.9d
52.1
±20
.734
.3±
14.8
91.3
±50
.330
4.3
±21
5.2
130.
9±
72.5
34.2
±14
.630
.7±
11.7
8.6
±5.
3d43
.6±
15.8
8.3
±6.
724
1.3
±14
2.8
271.
3±
170.
02.
2±
1.5c
3.1
±14
.81.
7±
1.1d
5.7
±4.
6d20
6.7
±18
4.6
166.
6±
143.
40
3.3
±1.
3d1.
4±
0.8
5.7
±4.
3c13
.4±
10.4
d16
.6±
11.9
01.
2±
0.7a
5.6
±2.
4a
3.3
±2.
7c49
.3±
27.8
28.4
±23
.70
0.7
±0.
8a2.
7±
1.7
60 M.E. Ferreira et al. / Journal of Et
benznidazole, crude extract of Zanthoxylum chiloperone) or atmg/kg/day (canthin-6-one (1) by oral or subcutaneous route
or 20 days).
.4. Treatment outcome
Parasitaemia in control and treated mice was determined onceeekly for 145 days from samples of tail-vein blood. All mor-
ality rates were recorded.Sera from acute infected mice were analyzed in duplicate by
LISA (Enzyme Linked Immuno Sorbent Assay) 30 and 70 daysost-infection. In the chronic experiments, sera from infectedice were sampled prior to treatment, then 20 and 90 days after.locally produced ELISA kit (Chagas test, IICS, Asuncion,
araguay) was used following the procedure recommended byhe manufacturer. Values of optical density were obtained usingn ELISA plate reader (Titerek Unistan I).
The means and standard deviations were calculated, and theifferences between groups were determined using Student’s t-est, or the Kruskal–Wallis non-parametric analysis of varianceest. Significance level was established for a P value < 0.05.
. Results
.1. Effect of canthin-6-one and its derivatives in acutexperimental Chagas disease
Canthin-6-one (1), its derivatives 2 and 3, as well as therude extract of Zanthoxylum chiloperone, seemed to be wellolerated by the mice, and we could not observe any objec-ivable side-effect in the experiments. Following intraperitonealnfection of Balb/c mice with 5000 trypomastigotes of theL strain, the mean value of maximum parasitaemia obtained1–28 days post-infection was 323.3 ± 169.5 × 104 parasites/ml
lood in untreated mice (see Fig. 2 and Table 1). The meanarasitaemia at day 28 for benznidazole- and oral canthin-6-ne-treated mice was significantly reduced (P = 0.0001 versusontrols). Untreated mice infected with CL strain presented 45%ig. 2. Effects of benznidazole at 50 mg/kg/day and canthin-6-one (1) atmg/kg/day on parasitaemia in BALB/c mice with acute Trypanosoma cruzi
nfection (CL strain). BALB/c mice (20 animals per group,) were infected with× 103 trypomastigotes and drug treatment were initiated 12 days post-infection
or 2 weeks. Tabl
e1
Eff
ecto
fbe
nzni
dazo
le,c
anth
in-6
-one
(1),
5-m
etho
xy-c
anth
in-6
-one
(2),
cant
hin-
6-o
Day
spo
st-i
nfec
tion
Mea
npa
rasi
taem
ia(×
104
para
site
s/m
lblo
od±
S.D
.)
Con
trol
(n=
20)
Ben
znid
azol
e(n
=20
)1
(ora
l)(n
=20
)
1899
.2±
65.1
2.6
±3.
3a11
.0±
7.2b
2532
3.3
±16
9.5
13.9
±10
.7b
14.9
±11
.8b
3212
1.2
±91
.212
.7±
11.3
c8.
9±
7.5a
3961
.6±
52.1
9.0
±8.
25.
2±
5.0
4554
.8±
37.7
8.2
±9.
4c2.
7±
2.5c
5343
.4±
31.5
6.0
±5.
8d2.
1±
2.0c
6011
5.1
±85
.54.
0±
3.7c
1.1
±0.
8b
6871
.1±
57.5
0.6
±0.
5b0.
1±
0.2b
aP
=0.
002
vs.c
ontr
ol.
bP
=0.
0001
vs.c
ontr
ol;s
.c.:
subc
utan
eous
.c
P=
0.02
vs.c
ontr
ol.
dP
=0.
05vs
.con
trol
.
M.E. Ferreira et al. / Journal of Ethnopharmacology 109 (2007) 258–263 261
Table 2Cure rates in mice with Trypanosoma cruzi acute infection treated 2 weeks with benznidazole at 50 mg/kg/day, canthin-6-one (1), 5-methoxy-canthin-6-one (2) andcanthin-6-one N-oxide (3) at 5 mg/kg/day, crude Zanthoxylum chiloperone alkaloid extract at 50 mg/kg/day, as judged by absence of parasitaemia
Treatment n Absent parasitaemia/number of survivors (days post-infection)
18 32 45 56 68
Control 20 0/19 4/17 6/14 0/12 3/11Benznidazole 20 14/20 12/20 10/19 11/19 17/191 oral 20 5/20 11/20 15/20 17/20 19/201 s.c. 21 6/21 6/21 12/21 10/21 19/212 oral 9 8/9 2/7 1/7 4/7 3/72 s.c. 8 6/8 5/8 3/8 4/8 6/83 0C 2C 1
mbCbt
nirsts
a(pt
awgtTo2
optTa(c
3e
madcmte5n
TV(
E
N
N2(
a
by oral 10 0/1rude Zanthoxylum chiloperone alkaloid oral 12 1/1rude Zanthoxylum chiloperone alkaloid s.c. 12 1/1
ortality, whereas a survival rate of 100% was obtained withenznidazole or canthin-6-one (1) given orally (see Table 2).oncerning the drug-treated mice, the absence of parasites inlood was attested by control of parasitaemia, and a decrease ofhe antibody titer by ELISA analysis (see Table 3).
When the mice were treated with canthin-6-one subcuta-eously, the maximum parasitaemia appeared 39 days post-nfection, a mean value similar to that of the control group. Miceeceiving 5-methoxy-canthin-6-one (2) orally or subcutaneouslyhowed maximum parasitaemia 45 days post-infection, althoughhere were no significant differences in parasitaemia and ELISAerology when compared with the untreated group.
Oral treatment of mice with canthin-6-one N-oxide (3) ledsignificant reduction of parasitaemia at day 18 post-infection
P = 0.0001), and from day 45 to the end of experiment in com-arison with untreated mice. The mortality rate for the groupreated with compound 3 was 40%.
The total alkaloid extract of Zanthoxylum chiloperone waslso tested orally and subcutaneously at 50 mg/kg/day for 2eeks. Treatments were started 12 days post-infection. Theroup treated orally had 83% survival, and 75% of the subcu-
aneously treated group was alive at the end of experiment (seeable 2). Oral treatment with extract of Zanthoxylum chiloper-ne produced a significant reduction in mean peak parasitaemia5 and 39 days post-injection and during the three last weeksdzob
able 3alues of serological ELISA test in mice (n = 10) with acute Trypanosoma cruzi inf
2), and crude Zanthoxylum chiloperone alkaloid extract
xperiment Treatment
o. 1 (n = 10)
ControlBenznidazole 50 mg/kg/day × 14 days (oral route)(1) 5 mg/kg, 14 days, oral(1) 5 mg/kg, 14 days, s.c.Crude Zanthoxylum chiloperone alkaloid, 50 mg/kgCrude Zanthoxylum chiloperone alkaloid, 50 mg/kg
o.
n = 10)
ControlBenznidazole 50 mg/kg/d × 14 (oral route)(2) 5 mg/kg, 14 days, oral
P = 0.001 vs. control; bP < 0.05 vs. control.
0/7 0/6 6/6 6/60/10 5/9 5/9 8/91/11 10/11 10/11 5/10
f the experiment. Those results were confirmed by a completearasitological elimination in 88% of this group, and by a reduc-ion of ELISA serology 30 days post-injection (P < 0.05) (seeable 3). During all experiments, we could not observe anypparent side-effect resulting from treatment with canthin-6-one1), its derivatives 2 and 3, nor the total alkaloids of Zanthoxylumhiloperone.
.2. Effect of canthin-6-one and its derivatives in chronicxperimental Chagas disease
In the chronic model, we carried out two distinct experi-ents. Animals were infected with 1000 trypomastigotes/mice
nd surviving animals (60–70%) were treated with drugs 60ays post-infection for 2 weeks. Following this treatment, noirculating parasites could generally be observed. After 20 days,ice treated with benznidazole at 50 mg/kg, oral or subcu-
aneous canthin-6-one (1) at 5 mg/kg, or with the alkaloidalxtract of Zanthoxylum chiloperone orally or subcutaneously at0 mg/kg, showed high (90–100%) survival level, without a sig-ificant difference between those groups (see Table 4). Ninety
ays post-treatment, 100% mice treated orally with benznida-ole or canthin-6-one (1) was parasite-free. Consistent levelsf antibodies, as determined using ELISA, were obtained withenznidazole (P = 0.02, 20 days post-treatment and P = 0.001,ection treated with benznidazole, canthin-6-one (1), 5-methoxy-canthin-6-one
ELISA (optical density mean ± standard deviation)days post-treatment
30 70
0.399 ± 0.092 1.598 ± 0.3820.169 ± 0.118a 0.793 ± 0.861b
0.111 ± 0.039a 0.395 ± 0.753b
0.215 ± 0.145b 1.347 ± 0.633b
, 14 days, oral 0.142 ± 0.089a 1.187 ± 0.641, 14 days, s.c. 0.283 ± 0.152 1.360 ± 0.449
1.339 ± 0.216 1.779 ± 0.1860.919 ± 0.188b 1.741 ± 0.1481.228 ± 0.187 1.683 ± 0.654
262 M.E. Ferreira et al. / Journal of Ethnopharmacology 109 (2007) 258–263
Table 4Absent parasitemia in mice with Trypanosoma cruzi chronic infection treated with benznidazole, canthin-6-one (1) and crude Zanthoxylum chiloperone alkaloidextract, as judged by absence of parasitaemia and serological response (ELISA testing)
Treatment Negative parasitological/no of survivals(days post-treatment)
ELISA (optical density mean ± standard deviation)days post-treatment
20 40 60 90 0 20 90
Experiment 1 (n = 8)Control 2/5 2/4 1/1 1/1 1.805 ± 0.075 1.913 ± 0.115 1.793a
Benznidazole 50 mg/kg, 20 days, oral 5/5 2/5 5/5 5/5 2.072 ± 0.220 1.712 ± 0.473 1.979 ± 0.350(1) 5 mg/kg, 20 days, oral 7/8 8/8 8/8 8/8 1.878 ± 0.348 1.621 ± 0.547 1.799 ± 0.333(1) 5 mg/kg, 20 days, s.c. 7/8 6/8 6/8 6/8 1.916 ± 0.368 1.850 ± 0.405 1.870 ± 0.268Crude Zanthoxylum chiloperone alkaloid
extract, 50 mg/kg, 20 days, oral7/7 7/7 7/7 6/7 1.932 ± 0.227 1.890 ± 0.288 1.961 ± 0.172
Crude Zanthoxylum chiloperone alkaloidextract 50 mg/kg, 20 days, s.c.
6/6 3/5 5/5 3/5 1.718 ± 0.285 1.703 ± 0.469 1.815 ± 0.374
Experiment 2 (n = 12)Control 8/11 7/11 6/11 11/11 ND 1.482 ± 0.211 1.779 ± 0.186Benznidazole 50 mg/kg, 20 days, oral 11/12 7/12 11/12 10/12 ND 1.128 ± 0.214a 1.741 ± 0.148a
(1) 5 mg/kg, 20 days, oral 7/11 10/11 9/11 10/11 ND 1.484 ± 0.074 1.683 ± 0.654a
(1) 5 mg/kg, 20 days, s.c. 9/11 11/11 5/10 10/10 ND 1.295 ± 0.243 1.833 ± 0.110a
Crude Zanthoxylum chiloperone alkaloidextract, 50 mg/kg, 20 days
5/11 11/11 11/11 11/11 ND 1.408 ± 0.165 1.683 ± 0.654a
Crude Zanthoxylum chiloperone alkaloidextract 50 mg/kg, 20 days, s.c.
8/10 8/10 8/10 8/10 ND 1.085 ± 0.179b 1.833 ± 0.110b
N
9(ot9wwc
4
qpwmaPiawcsawBrt
ost
mortatoractwdodcmec
uidatDwt
D: not determinated; aP = 0.02 vs. control; bP = 0.001 vs. control.a One survival.
0 days post-treatment), oral or subcutaneous canthin-6-one1) (P = 0.02, 90 days post-treatment), and with total alkaloidsf Zanthoxylum chiloperone given subcutaneously (P = 0.001,wice at 20 and 90 days post-treatment) or orally (P = 0.02, at0 days post-treatment). Here again, during all experiments,e could not observe any side-effect resulting from treatmentith canthin-6-one (1) nor the total alkaloids of Zanthoxylum
hiloperone.
. Discussion
This study has compared benznidazole, the drug most fre-uently used for the treatment of acute and congenital Try-anosoma cruzi infection (Cerecetto and Gonzalez, 2002),ith canthin-6-one (1), two canthin-6-one derivatives (5-ethoxycanthin-6-one (2) and canthin-6-one N-oxide (3)),
nd the alkaloidal extract of Zanthoxylum chiloperone, thearaguayan plant from which canthin-6-one (1) was previously
solated (Thouvenel et al., 2003). For the first time, an activitygainst Trypanosoma cruzi in a mouse model of Chagas diseaseas found. The antiprotozoal activity (Ferreira et al., 2002) of
anthin-6-one (1), together with its antifungal properties demon-trated in vitro against 13 strains of fungi, suggested that 1 couldlso be active against Trypanosoma cruzi in vivo. For this study,e chose a non-lethal Trypanosoma cruzi strain (i.e. clone CLrener), along with a non-lethal inoculum of parasites, which
esemble in many respects the chronic features of the humanrypanosomal disease.
The present study showed that oral canthin-6-one treatmentf mice acutely infected with Trypanosoma cruzi induced aignificant decrease of parasitaemia compared to benznidazolereatment. Although the immune response in this model was
Tf
e
ore evident in animals treated with benznidazole, canthin-6-ne (1), or the alkaloidal extract Zanthoxylum chiloperone (oraloute) than in untreated mice, it has to be pointed out that the cri-erion of cure is the negativization of serological tests or at least,progressive, persistent regression of antibody titer. In contrast,
he effectiveness of 5-methoxycanthin-6-one (2) or canthin-6-ne N-oxide (3) against acute Trypanosoma cruzi infection wasather limited, although we observed that compound 2 inducedrapid reduction of parasitemia and circulating parasites. Con-
erning the chronic Trypanosoma cruzi infection, we observedhat the results obtained with benznidazole or canthin-6-one (1)ere very similar. In a perspective of chemotherapy of Chagasisease, our results, together with the low toxicity of canthin-6-ne (1), suggest possible treatments of long duration, e.g. 2–3aily administrations for 30–60 days. In comparison, the effi-acy of the total alkaloids of Zanthoxylum chiloperone in ourodels of acute or chronic infection was limited. However, this
xtract constitutes an interesting alternative as to elaborate aheap treatment in regions where Chagas disease is endemic.
The mechanism of action of canthin-6-one is entirelynknown, but its antifungal and trypanocidal activities couldndicate, as first hypothesis, the inhibition of sterol C-14aemethylase in intracellular Trypanosoma cruzi amastigotes,mechanism of action similar to that proposed for antifungal
riazoles (Molina et al., 2000; Lira et al., 2001; Urbina andocampo, 2003; Urbina et al., 2003a,b). In the future studiesith canthin-6-one as anti-Trypanosoma cruzi drug, we must
ake into account other criteria such as the clonal evolution of
rypanosoma cruzi in different hosts, as well as specificitiesound in different geographical areas.In conclusion, this study indicated that canthin-6-one (1)xhibits an important trypanocidal activity in vivo in a mouse
hnoph
msclcOo
ut
A
Pr
R
C
C
F
L
M
M
M
M
O
S
S
T
U
U
U
U
V
M.E. Ferreira et al. / Journal of Et
odel of acute or chronic infection. This is the first demon-tration of anti-Trypanosoma cruzi activity for this class ofompound. Furthermore, this molecule has a potential for use inong-term oral treatment, due to its low toxicity, and is a goodandidate against drug-resistant strains of Trypanosoma cruzi.ur results are stimulating enough to continue the developmentf canthin-6-one (1) as a possible treatment for Chagas disease.
Also, the present study exemplifies how trypanocidal prod-cts can be discovered with the help of ethnomedical informa-ion, gained from populations using traditional remedies.
cknowledgements
We are grateful to Drs. Marisel Maldonado (IICS, Asuncion,araguay) and Romain Duval (Paris XI, France) for a criticaleading of this manuscript.
eferences
ano, M.I., Gruber, A., Vazquez, A.M., Cortes, A., Levin, M.J., Gonzalez,A., Degrave, W., Rondinelli, E., Zingales, B., Ramirez, J.L., Alonso, C.,Requena, J.M., Silveira, J.F., 1995. Molecular karyotype of clone CL Brenerchosen for the Trypanosoma cruzi genome project. Molecular and Biochem-ical Parasitology 71, 273–278.
erecetto, H., Gonzalez, M., 2002. Chemotherapy of Chagas’ disease: status andnew developments. Current Topics in Medicinal Chemistry 2, 1187–1213.
erreira, M.E., Rojas de Arias, A., Torres de Ortiz, S., Inchausti, A., Nakayama,H., Thouvenel, C., Hocquemiller, R., Fournet, A., 2002. Leishmanicidalactivity of canthin-6-one alkalloids isolated from Zanthoxylum chiloperonevar. angustifolium. Journal of Ethnopharmacology 80, 199–202.
ira, R., Contreras, L.M., Rita, R.M., Urbina, J.A., 2001. Mechanism of action ofanti-proliferative lysophospholipid analogues against the protozoan parasiteTrypanosoma cruzi: potentiation of in vitro activity by the sterol biosyn-
thesis inhibitor ketoconazole. Journal of Antimicrobial Chemotherapy 47,537–546.atta Guedes, P.M., Urbina, J.A., Lana, M., Afonso, L.C.C., Veloso, V.M.,Tafuri, W.L., Machado-Coelho, G.L.L., Chiari, E., Bahia, M.T., 2004.Activity of the new triazole derivative albaconazole against Trypanosoma
W
W
armacology 109 (2007) 258–263 263
(Schizotrypanum) cruzi in dog hosts. Antimicrobial Agents and Chemother-apy 48, 4286–4292.
illiken, W., 1987. Plants for malaria for fever, medicinal species in LatinAmerica. The Royal Botanical Garden, Kew, London.
olina, J., Brener, Z., Romanha, A.J., Urbina, J.A., 2000. In vivo activity of thebis-triazole D0870 against drug-susceptible and drug-resistant strains of theprotozoan parasite Trypanosoma cruzi. Journal of Antimicrobial Chemother-apy 46, 137–140.
orel, C.M., 2000. Reaching maturity—25 years of the TDR. ParasitologyToday 16, 522–525.
PS/HCP/HCT/140/99, 1999. Tratamiento etiologico de la enfermedad de Cha-gas. Conclusiones de una consulta tenica. Revista de Patologia Tropical 28,247–279.
chiplander, M.T., Mitscher, L.A., 1971. A facile one-step synthesis ofbenz[4,5]canthin-6-one. Journal of Heterocyclic Chemistry 8, 695–696.
pichiger, R., Stutz de Ortega, L., 1987. Flora del Paraguay: Rutaceae. Ed.Conservatoire et Jardin botaniques de la Ville de Geneve. Missouri BotanicalGarden, 19–36.
houvenel, C., Gantier, J.C., Duret, P., Fourneau, C., Hocquemiller, R., Fer-reira, M.E., Rojas de Arias, A., Fournet, A., 2003. Antifungal compoundsfrom Zanthoxylum chiloperone var. angustifolium. Phytotherapy Research17, 678–680.
rbina, J.A., 2002. Chemotherapy of Chagas disease. Current PharmaceuticalDesign 8, 287–295.
rbina, J.A., Docampo, R., 2003. Specific chemotherapy of Chagas disease:controversies and advances. Trends of Parasitology 11, 495–501.
rbina, J.A., Payares, G., Sanoja, C., Lira, R., Romanha, A.J., 2003a. In vitroand in vivo activities of ravuconazole on Trypanosoma cruzi, the causativeagent of Chagas disease. International Journal of Antimicrobial Agents 21,27–38.
rbina, J.A., Payares, G., Sanoja, C., Molina, J., Lira, R., Brener, Z., Romanha,A.J., 2003b. Parasitological cure of acute and chronic experimental Cha-gas disease using the long-acting experimental triazole TAK-187. Activityagainst drug-resistant Trypanosoma cruzi strains. International Journal ofAntimicrobial Agents 21, 39–48.
inhaes, M.C., Schofield, C.J., 2003. Trypanosomiasis control: surmountingdiminishing returns. Trends of Parasitology 19, 112–113.
orld Health Organization, 1997. Thirteenth Programme Report, UNDP/WorldBank/World Health Organization for Research and Training in Tropical Dis-eases. World Health Organization, Geneva.
orld Health Organization, 2002. Control of Chagas Disease, TechnicalReports. Ser. 2905, pp. 1–109.
Recommended
