Revista de Saúde Pública
I SSN 0034- 8910 versão im pressa
Rev Saúde Pública 2004; 38(1)
Antischistosom al activity of artem ether in ex perim ental
Schistosomiasis mansoni
Susana Zevallos Lescanoa, Pedro Paulo Chieffia,b, Rosa Regina Canhassia, Marcos Boulosc an d Vicen t e
Am at o Net od
a
Laborat ório de I m unopat ologia da Esquist ossom ose ( LI M 06) doI nst it ut o de Medicina Tr opical de São Paulo. São Paulo, SP, Brasil. bFaculdade de Ciências Médicas da Sant a Casa de São Paulo. São Paulo, SP, Brasil. cDepar t am ent o de Doenças I nfecciosas e Par asit ár ias da Faculdade de Medicina da Universidade de São Paulo. São Paulo, SP, Brasil. dLabor at ór io de Par asit ologia ( LI M 46) do I nst it ut o de Medicina Tr opical de São Paulo. São Paulo, SP, Br asil
ABSTRACT
Objective
To ev alu at e t h e ef f ect of int r am uscular inj ect ion of ar t em et her in m ice ex per im ent ally infect ed w it h
Schist osom a m ansoni, at t he t im e of infect ion, dur ing schist osom ula m at ur at ion and aft er t he beginning of egg - laying.
Methods
w it h ar t em et her , accor ding t o t he follow ing schedule: t hr ee gr oups r eceiv ed doses of 100 m g/ k g on day s 0 , 2 0 or 6 0 aft er inoculat ion of t he cer car iae; t he ot her t hr ee r eceiv ed 5 0 m g/ k g of ar t em et her , also on day s 0, 20 or 60. At t he end of t he 9t h, 10t h and 11t h w eek s aft er infect ion all t he m ice infect ed w it h S. m ansoni w er e subm it t ed t o fecal ex am inat ion using t he Kat o- Kat z t echnique. On t he 80t h day of t he ex per im ent , t he sur v iv ing anim als w er e sacr ificed and subm it t ed t o per fusion of t he por t al sy st em in or der t o r ecov er t he w or m s. Body , liv er and spleen w eight s of each anim al w er e det er m ined at t h at t im e.
Results
A r educt ion in egg- lay ing and t he num ber of w or m s r ecov er ed w as obser v ed in m ice t r eat ed w it h ar t em et her ( 50 or 100 m g/ kg) on t he 20t h day aft er infect ion. The decr ease in t he num ber of w or m s w as m or e n ot able am on g S. m ansoni fem ales. A significant decr ease in liv er and spleen w eight s w as also seen on t he 20t h day am ong anim als t r eat ed w it h 50 or 100 m g/ kg of ar t em et her and also am ong t hose t hat r eceiv ed t he dr ug at a dose of 50 m g/ k g 60 day s aft er infect ion.
Conclusions
Ev idence of t he ant ischist osom al act iv it y of ar t em et her w as show n, ev en at a dose of 50 m g/ k g, w hen t he dr ug w as adm inist er ed dur ing t he schist osom ula m at ur at io n per iod in t he por t al sy st em of t he v er t ebr at e h ost .
Keywords
Schist osom a m ansoni. Art em isinins, t herapeut ic use. Schist osom iasis m ansoni, drug t herapy. Anim al experim ent at ion. Art em et her.
I N TROD U CTI ON
I t is believed t hat at least 2.5 m illion people in Brazil carry Schist osom a m ansoni and around 25 m illion individuals are exposed t o t he risk of cont ract ing it .9
Tw o dr ugs hav e been w idely used in t he t r eat m ent of t he disease, w it h good efficacy and low t ox icit y : oxam niquine and praziquant el. Over recent years, t he lat t er has been used preferent ially. I n view of t he possible dev elopm ent of t oler ance or r esist ance t o pr aziquant el, r esear ch int o and pr oduct ion of new dr ugs for t he pr ev ent ion and cur e of Schist osom a m ansoni has becom e j ust ified.4
I n 1982, Le et al7 confir m ed t he schist osom icide pr oper t y of ar t em et her . Mice or dogs infect ed w it h S.
j aponicum and t reat ed w it h t his drug at various doses and ut ilizing different adm inist rat ion rout es show ed a significant reduct ion in worm load. The larval phases ( schist osom ulae) of S. j aponicum w er e also show n t o be suscept ible t o ar t em et her . How ev er , no effect w as obser v ed on t r em at ode eggs.1 5 Subsequent st udies have confirm ed t hat ot her art em isinin derivat ives have schis t osom icide pr oper t ies: ar t esunat e,3,8 ar t eet h er1 4 and, recent ly, dihydroart em isinin.1 I n 1991 in Brazil, Araúj o et al2 st udied art em et her act ivit y in ham st ers and m ice experim ent ally infect ed wit h S. m ansoni, 45 day s aft er penet r at ion by t he cer car iae. Bet t er r esult s w er e also obser v ed w hen t he dr ug w as adm inist er ed via int ram uscular rout e.
The pr esent w or k has sought t o ev aluat e t he effect of t he int r am uscular adm inist r at ion of ar t em et her in m ice experim ent ally infect ed wit h S. m ansoni, at t h e t im e of in fect ion, during schist osom ula m at urat ion and aft er t he beginning of egg- laying.
METHODS
Eight y adult fem ale m ice of t he Balb/ c lineage were ut ilized, divided int o eight subgroups: six infect ed w it h S. m ansoni and t r eat ed using ar t em et her , one equally infected but not t r eat ed w it h ar t em et her and, finally , one ot her subgr oup t hat w as not infect ed and not t r eat ed, as show n in Table 1.
Table 1 – Artemether treatment schedule, via intramuscular route, for the mice infected with S. mansoni.
Subgroup No. of mice Dose Day of treatment
1 10 100 mg/kg of weight zero 2 10 100 mg/kg of weight 20 a.i. 3 10 100 mg/kg of weight 60 a.i.
4 10 50 mg/kg of weight zero
5 10 50 mg/kg of weight 20 a.i. 6 10 50 mg/kg of weight 60 a.i. 7 10 Infected control Not treated 8 10 Free of infection Not treated
a.i. = after infection
The m ice in subgr oups 1 t o 7 w er e each infect ed w it h 60 cer car iae of t he BH st r ain of S. m ansoni, v ia subcut aneous r out e. The BH st r ain of t his t r em at ode has been k ept for m or e t han 15 y ear s in t he s c hist osom iasis im m unopat hology laborat ory of I nst it ut o de Medicina Tropical de São Paulo ( São Paulo I nst it ut e of Tr opical Medicine) , by ut ilizing specim ens of Biom phalaria glabrat a and ham st ers. The inoculat ion of 60 cer car iae w as achiev ed by 1: 6 dilut ion of an aliquot t hat cont ained 1800 cer car iae per m l, wit h 0.2 m l inj ect ed int o each m ouse. The art em et her, at doses of 50 and 100 m g/ kg, was adm inist ered via int ram uscular rout e t o t he m ice in subgroups 1 t o 6.
The effect of t he ar t em et her w as ev aluat ed by m e ans of quant it at iv e ex am inat ion of feces, done accor ding t o t he Kat o- Kat z m et hod6 in t h e 9t h, 10t h and 11t h w eek s aft er t he infect ion. Eight y day s
Throughout t he experim ent t he m ice received feed and w at er ad libit um and t heir m anagem ent was in accordance wit h t he recom m endat ions of t he Colégio Brasileiro de Experim ent ação Anim al ( Brazilian College for Anim al Experim ent at ion) .
The r esult s w er e analy zed v ia non- par am et r ic st at ist ical t est s, ut ilizing a significance level of 95% ( p= 0.05) .
RESULT S
Table 2 show s t he average num ber of S. m ansoni eggs per gr am of feces obt ained fr om t he differ ent m ouse subgr oups in t he 9t h, 10t h and 11t h w eek s aft er infect ion. I n subgr oups 2 and 5, t r eat ed w it h ar t emet her on t he 20t h day aft er infect ion w it h doses of 100 m g/ kg and 50 m g/ kg, r espect ively, t her e was a m arked reduct ion in t he num ber of eggs found.
Table 2 - Results from the feces examinations (Kato Katz) between the 9th and 11th weeks after infection with S. mansoni, in the mice treated with artemether and untreated mice.
Subgroup No. of mice examined Eggs per gram of feces
(artemether dose) 9th week a.i. 10th week a.i. 11th week a.i.
1 (100 mg/kg, day 0) 10 239.0 168 158.4
2 (100 mg/kg, 20 days a.i.) 10 19.2 0 0
3 (100 mg/kg, 60 days a.i.) 6 75.4 85.7 0
4 (50 mg/kg, day 0) 9 163.0 125.3 112.0
5 (50 mg/kg, 20 days a.i.) 10 0 12.0 4.8
6 (50 mg/kg, 60 days a.i.) 10 103.2 138.0 7.2
7 (Untreated) 8 269.3 243.0 147.0
a.i. = after infection
Table 3 present s t he dat a r elat ing t o t he num ber of w or m s r ecover ed aft er per fusion of t he por t al syst em of t he surviving anim als.
Table 3 – Average number of worms recovered from the mice infected with S. mansoni and treated with different doses of artemether.
Subgroup No. of mice perfused Females Males Worms
N N N
1 10 3.0±3.85 9.0±4.07 12.0±6.97
2 10 0.1±0.31* 1.3±1.83* 1.4±1.9*
3 6 0.0 8.5±6.53 8.5±6.53
4 9 0.75±0.88 6.87±9.34 7.62±9.78
5 10 0.2±0.42* 2.8±6.83 3.0±6.81
6 10 0.125±0.35* 1.75±2.71* 1.85±2.95*
7 8 1.67±2.34 6.0±8.03 7.67±10.16
The r at ios bet w een t he liver and spleen w eight s and body w eight s of t he m ice in t he differ ent gr oups st udied ar e pr esent ed in Table 4. A significant differ ence w as obser v ed bet w een t he v alues found for anim als in subgr oups 2, 5 and 6 and t hose of t he m ouse subgr oup t hat w as infect ed but not t r eat ed w it h art em et her ( subgroup 7) .
Table 4 – Ratios of spleen weight X 100/ body weight and liver weight X 100/ body weight in mice infected with S. mansoni and treated with artemether.
Subgroup No. of mice examined Spleen weight x 100/ body weight Liver weight x 100/ body weight
1 10 1.137±0.44 6.817±1.68
2 10 0.675±0.09* 5.536±0.70*
3 6 1.078±0.24 6.732±0.56
4 9 1.262±0.80 7.426±2.31
5 10 0.704±0.22* 4.617±1.02*
6 10 0.705±0.18* 5.631±1.12*
7 8 1.46±0.60 8.021±1.85
8 10 0.402±0.09 4.443±0.80
* Significant difference
D ISCUSSION
Schist osom iasis cont inues t o occupy second posit ion in t he w or ld am ong par asit es, aft er m alar ia, in t er m s of t h e ex t ent of endem ic areas and num ber of people infect ed. Treat m ent using praziquant el is efficacious in t he r educt ion of m or bidit y , alt hough it fails t o pr ev ent r einfect ion. Thus, foci w it h high t ransm ission rat es st ill exist in endem ic areas, despit e t he regula r adm inist rat ion of t his drug.1 3 I n view of t he possible developm ent of t oler ance and/ or r esist ance t o t his dr ug, r esear ch int o new alt er nat iv es for t he pr ev ent ion and cur e of schist osom iasis hav e becom e j ust ified.4
Art em isinin ( qinghaosu) is t he m ain act iv e agent ex t r act ed fr om t he leav es of Art em isia annua, a plant t hat is widely dissem inat ed in China and which also grows nat urally in Cent ral Europe, t he Unit ed St at es and Argent ina. I t s ant im alarial act ivit y w as confirm ed in 1971 and m ore t han t w o million pat ient s w it h m alar ia have been t r eat ed w it h t his dr ug and it s der ivat ives ( ar t em et her , ar t esunat e and ar t eet her ) ov er t hat last 20 y ear s.1 1 The ant ischist osom al act iv it y of ar t em isinin w as
discovered in 1980, w hen it w as observed t hat it s adm inist rat ion t o anim als experim ent ally infect ed w it h S. j aponicum caused a m arked reduct ion in t he worm load, in com parison wit h cont rol anim als t h at w er e n ot t r eat ed .1 1 These r esult s w er e confir m ed by t he findings of Le et al7 ( 1982) . I n t heir t urn, Yu e et al1 5 ( 1984) v er ified t he suscept ibilit y of t he schist osom ulae of S. j aponicum t o ar t em et her .
They did not , how ev er , obser v e any effect of t his dr ug on t he eggs of t his t r em at ode.
r ecov er ed aft er t he per fusion of t he por t al sy st em . These r esult s w er e confir m ed by t he absence or low count s of eggs in t he feces of m ice in t hese subgr oups in t he 10t h and 11t h w eek s aft er infect ion ( Table 2) . These finding par t ially coincide w it h t hose of Xiao & Cat t o1 2 ( 1989) and Xiao et al1 3 ( 2000) , w ho w or k ed w it h m ice infect ed w it h S. m ansoni and obser v ed t hat t he par asit es aged 14 and 21 day s w er e t he ones m ost suscept ible t o ar t em et her . I t should be st r essed t hat low er m or t alit y w as obser v ed in t he subgr oups t r eat ed wit h ar t em et her ( w it h t he ex cept ion of subgr oup 3, w hich r eceiv ed t he drug when t he S. m ansoni specim ens were already fully developed) , t han in subgroup 7 ( m ice in fect ed w it h S. m ansoni and not t r eat ed w it h ar t em et her ) .
The anim als in subgroups 3 and 6 – t reat ed 60 days aft er infect ion, w it h 100 m g and 50 m g of art em et her per kg of weight – present ed lower num bers of fem ale worm s aft er t he perfusion ( Table 3) and t he egg count w as negat iv e in t he last w eek of t he ex per im ent ( Table 2) . Ev idence w as t hus show n of t he ant ipar asit ic act iv it y of ar t em et her , especially in r elat ion t o schist osom ulae and fem ales of S. m ansoni, even when lower doses of t his drug were ut ilized.
I n Brazil, t here is pract ically no overlap bet w een t he endem ic areas for m alaria and schist osom iasis. How ev er , in cer t ain r egions of t he Afr ican cont inent , t he t r ansm ission of t hese par asit oses t ak es place concom it ant ly, and t he result s from t he use of praziquant el have been less prom ising t han w hat w as ex pect ed5. I n t hese cir cum st ances, t he use of art em isinin derivat ives wit h t he aim of t reat ing m alaria cases m ay ex er t a significant influence on t he r educt ion of t he m or bidit y r at e due t o endem ic schist osom iasis.
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Address to correspondence
Pedr o Paulo Chieffi
I nst it ut o de Medicina Tr opical
Av. Dr. Enéas Carvalho de Aguiar, 500 2o andar Cerqueira César 05403 - 000 São Paulo, SP, Brasil
E- m ail: pchieffi@usp.br
Work not support ed by any sources ot her t han from t his laborat ory.
Pr esen t ed at t h e 3 6t h Brazilian Congress of Clinical Pat hology and Laborat ory Medicine, held in São Paulo, 2002.
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