Research note In vitro antifilarial activity of organometallic complexes against infective larvae of Molinema dessetae and adult females of Brugia pahangi

International Journal for Parasitology 17 "0887# 0168Ð0171

Research note

In vitro anti_larial activity of organometallic complexes against infective larvae of Molinema dessetae and adult females of Brugia pahangi P[M[ Loiseaua\\ J[J[ Ja}eb\ D[G[ Craciunescuc a

Biologie et Contro¼le des Organismes Parasites\ Faculte de Pharmacie\ Universite de Paris!Sud\ Cha¼tenay!Malabry\ France b Department of Pharmacology\ College of Medicine\ University of Vermont\ Burlington\ U[S[A c Departamento de Quimica Inorganica y Bioinorganica\ Facultad de Farmacia\ Universidad Complutense\ Madrid\ Spain Received 5 November 0886^ received in revised form 00 March 0887^ accepted 00 March 0887

Abstract New organometallic complexes having protozoocidal properties were evaluated for their in vitro anti_larial activity using two models] infective larvae of Molinema dessetae and adult females of Brugia pahangi[ The compound most active on the M[ dessetae model was Ir"I#!COD!pentamidine tetraphenylborate with an EC49  520 mM after 6!day!incubation[ In the 1!aminobenzothiazole series\ Ruthenium was more potent than Iridium for anti_larial activity[ A dithiocarbamate function signi_cantly enhanced the anti_larial activity[ The compounds derived from benzimidazole were inactive whatever the metal "Iridium or Rhodium#[ The other compounds exhibited EC49 ranging from 09 to 20 mM[ On adult female Brugia pahangi in vitro\ Pt!DDH!N!acetylleucine\ Pt!diminazene and Pd!Cl3!piperazine at 19 mM began to kill both micro_lariae and the developing embryos within the mothers on day 1[ The compounds\ except for Pd!Cl3! piperazine\ killed the adults after 4 days[ Rh!Cl!1!chloropyridine caused obvious slowing of the adults from day 2 onward but did not a}ect the viability of adults\ micro_lariae or developing embryos[ In vivo anti_larial investigations are necessary to appreciate the real advantage of heavy metal complexes in the experimental treatment of _lariasis[ Þ 0887 Australian Society for Parasitology[ Published by Elsevier Science Ltd[ Keywords] Molinema dessetae^ Brugia pahangi^ Organometallic complexes^ Anti_larial activity

Organometallic complexes are known as anti! tumor agents ð0Ł[ Moreover\ some of them have interesting properties against parasitic protozoa such as Leishmania spp[ ð1Ł and african trypano! somes ð2Ł[ Their mode of action remains unknown but the low toxicity of these compounds compared with that of their parent organic moieties led us

Corresponding author[

to investigate their potential e}ect against other parasites such as helminths[ We report here for the _rst time an in vitro anti_larial study of organ! ometallic complexes using two models] infective lar! vae of M[ dessetae and adult female of B[ pahangi[ These models are complementary since the sen! sitivity of M[ dessetae infective larvae to chem! otherapeutic agents is similar to those of Onchocerca spp[ adult worms ð3Ł and the sensitivity of B[ pahangi is similar to those of lymphatic _lariae such as Brugia malayi ð4Ł[

S9919!6408:87 ,08[99¦9[99 Þ 0887 Australian Society for Parasitology[ Published by Elsevier Science Ltd[ Printed in Great Britain PII] S9919!6408"87#99961!0

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P[M[ Loiseau et al[ : International Journal for Parasitolo`y 17 "0887# 0168Ð0171

Eleven organometallic compounds were synthe! sised to be evaluated in the M[ dessetae model and compared with 00 compounds used as references[ The reference compounds\ 1!aminobenzo! thiazole\ piperazine hydrochloride and albendazole were purchased from Sigma[ Pentamidine ise! thionate was kindly supplied by Roger Bellon Lab! oratories "Paris#\ ivermectin by MSD laboratories "Rahway# and potassium melarsonyl from Rho¼ne Poulenc Rorer "Vitry sur Seine#[ Other reference compounds were synthesised by us[ Moreover\ we present here the activity of other organometallic complexes on the B[ pahangi model[ Several of the compounds tested possess organic moieties having either anthelmintic properties either protozoocidal activity[ The metallic structures were derived from Iridium\ Rhodium\ Ruthenium\ Platinum and Pal! adium[ The contribution of the metallic complexes

could be evaluated with regard to the anti_larial activity of the organic moiety alone "Table 0#[ The _laria M[ dessetae was maintained in the laboratory in its natural host\ Proechimys oris\ a small Brazilian rodent[ The intermediate host was the mosquito Aedes aegypti GKEP strain[ This model has been shown to be remarkably sensitive to the major known _laricides\ and has been used to screen for anti_larial activity ð3Ł[ The in vitro test using infective larvae has similar sensitivity to those of adult worms ð5Ł and is used as primary screening ð6\ 7Ł[ Brie~y\ infective larvae were isolated by dis! section from heads of mosquitoes A[ aegypti which were infected 19 days previously[ The in vitro test was performed in plastic ~at!bottomed 13!well trays[ Each well contained 0[4 ml of a culture medium consisting of Hepes "14 mM# bu}ered

Table 0 In vitro anti_larial activity of organometallic complexes on Molinema dessetae infective larvae

Compounds

E[C[ 49)2S[D[ "mM# on the Molinema dessetae infective larvae Day 1 Day 3 Day 5 Day 7

Ir "III#!benzimidazole dithiocarbamate Ir "III#!1!aminobenzothiazole Ir "III#!1!aminobenzothiazole dithiocarbamate Ir "III#!pyrolidine dithiocarbamate Ir "I#!COD!pentamidine tetraphenylborate Rh "III#!1!amino 5!ethoxybenzothiazole Rh "III#!Cl!1!aminonaphtothiazole Rh "III#!1!chloropyridine Rh "II#!benzimidazole dithiocarbamate Ru "II#!n!dihexyl dithiocarbamate Ru "III#!1!aminobenzothiazole Reference Compounds

×199 ×199 036201 065204 5324 021201 ×199 086219 ×199 082207 ×199

×199 ×199 4423 4424 2123 7627 6925 7425 ×199 7325 019202

×199 ×199 1121 3123 0220 4523 2422 3123 ×199 0221 1121

×199 000201 0221 2022 520 1722 1622 1422 ×199 0920 0320

Albendazole 1!aminobenzothiazole 1!aminobenzothiazole dithiocarbamate 1!amino 5!ethoxybenzothiazole 1!amino 5!ethoxybenzothiazole dithiocarbamate 4!carboxypyrolidine dithiocarbamate Pentamidine isethionate Piperazine Ivermectin Levamisole hydrochloride Potassium melarsonyl

×199 ×199 ×199 ×199 ×199 ×199 058204 ×199 1[029[1 0[229[0 1321

×199 012209 8927 025209 7625 8327 0621 ×199 9[729[0 9[8629[97 0621

×199 7026 1921 6225 0721 0821 229[1 ×199 9[3429[94 9[6129[96 0120

×199 2422 0621 4423 0321 0421 0[129[0 ×199 9[2029[93 9[3829[92 729[6

P[M[ Loiseau et al[ : International Journal for Parasitolo`y 17 "0887# 0168Ð0171

RPMI 0539 supplemented with 09) foetal calf serum and 0999 IU ml−0 penicillin and 099 mg ml−0 streptomycin[ The ~asks were maintained at 26>C and in an atmosphere air 84)ÐCO1 4)[ Twenty larvae were put into each well after decon! tamination in sterile medium[ Compounds dis! solved in DMSO or water were added to the well in a volume of 09 ml[ Each experiment was run in triplicate[ Controls were treated with DMSO only and observed under identical conditions[ The samples were tested in a randomised order[ Larvae viability was estimated at day 1\ 3\ 5 and 7 by direct observation of the motility of infective larvae using a microscope "×39#[ The percentage of surviving larvae corrected by comparison with the control was determined[ In these conditions\ more than 84) of worms were alive[ In order to exclude poten! tial reversible paralysis induced by the drugs\ infec! tive larvae were washed at the end of the assay and put into fresh medium for 13 h[ Immobilised worms were then considered as dead[ EC492SD "e.cient concentration which killed 49) of worms# are expressed in mM[ The most active compound was Ir"I#!COD!pen! tamidine tetraphenylborate with an EC49  520 mM after a 6!day!incubation time[ Never! theless\ this compound remained 4!fold less active than pentamidine isethionate[ Moreover\ this com! pound was the most promising against African trypanosomes ð2Ł and a polyvalent activity could be of interest[ Its chemical structure is presented in Fig[ 0[ 1!aminobenzothiazole had an EC49  2422 mM whereas Ir"III#!1!aminobenzothiazole had an EC49  000201 mM and Ru"III#!1!aminobenzo! thiazole had an EC49  0320 mM[ Ruthenium appears therefore as more potent than Iridium for anti_larial activity[

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A dithiocarbamate function signi_cantly enhanced the anti_larial activity[ Thus\ 1!amino! benzothiazole and 1!amino!5!ethoxybenzothiazole had EC49  2422 and 4423 mM\ respectively[ When a dithiocarbamate group was added\ they became more active "1!aminobenzothiazole dithi! ocarbamate and 1!amino!5!ethoxybenzothiazole dithiocarbamate with EC49  0621 and 0321 mM\ respectively#[ This e}ect was also observed with organometallic derivatives^ thus\ Ir"III#!1!ami! nobenzothiazole dithiocarbamate with a dithi! ocarbamate group was more active than Ir"III#!1! aminobenzothiazole "EC49  0221 and 0002 01 mM\ respectively#[ The compounds derived from benzimidazole "Ir"III#!benzimidazole dithiocarba! mate and Rh"III#!benzimidazole dithiocarba! mate were inactive whatever the metal "Iridium or Rhodium#[ The other compounds exhibited EC49 ranging from 09 to 20 mM[ Twelve other organometallic compounds were tested for activity against adult female B[ pahangi in vitro[ These compounds were chosen from a large series of derivatives for the following reasons] 0[ broadly representative complexes involving four heavy metals could be compared[ 1[ the diminazene ligand would represent the other aryldiamidines[ 2[ the piperazinyl ligand is known to be active against intestinal nematodes and the substituted piperazine diethylcarbamazine is a standard anti_larial drug[ 3[ it was of interest to compare the activity of pip! erazinyl and pyridinyl moieties[ 4[ _lariae take up exogenous leucine e.ciently[ Jirds "Meriones unguiculatus# infected i[p[ with B[

Fig[ 0[ Chemical structure of Ir!"COD#!pentamidine tetraphenylborate\ the most active compound on the M[ dessetae infective larvae in vitro model[

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P[M[ Loiseau et al[ : International Journal for Parasitolo`y 17 "0887# 0168Ð0171

pahangi were supplied by Dr John W[ McCall "University of Georgia\ USA#[ In vitro evaluation on B[ pahangi adult worms was performed as follows] each of the compounds was dissolved in 099) DMSO at a concentration of 1 mM\ and 19 ml was added to each of two sterile tubes containing 1 ml MEM Eagle "with balanced salts¦2) bovine serum albumin# and two sexually mature adult female B[ pahangi freshly recovered from the peritoneal cavity of infected jirds[ Thus\ each drug was tested in triplicate at an initial con! centration of 19 mM\ which is realistic in the context of achievable blood levels[ Identical volumes of DMSO were added to the tubes housing the controls[ The ~uid phase was removed and replaced with fresh drug!containing medium every 13 h for 4 consecutive days[ Each day the motility and viability of the adults were evaluated[ In this system\ the adult worms remained highly motile throughout the 4!day!period of observation "more than 84) viable#[ Each female usually releases approximately 0999 micro_lariae "mf#:day for the _rst 1 days^ thereafter the daily output of mf progressively falls[ Under the conditions described above\ Pt!DDH! N!acetylleucine began to kill both mf and the developing embryos within the mothers on day 1[ The adults slowed dramatically on day 1\ two out of four died on day 2 and all were dead by day 3[ Pt!diminazene began to kill both mf and developing embryos on day 1 but the cumulative e}ect on these forms was not as great as with the former compound[ The adults slowed dramatically on day 2 and two of four died by day 4[ Pd!Cl3!piperazine began to kill both mf and developing embryos on day 1\ with a cumulative e}ect as great as Pt!DDH! N!acetylleucine[ The adults slowed dramatically from day 1 onward but none died by day 4[ Rh! Cl!1!chloropyridine caused obvious slowing of the adults from day 2 onward but did not a}ect the viability of adults\ mf or developing embryos[ Apparently\ the response curve is steep since none

of these compounds a}ected the worms in any detectable way at 1 mM[ This is the _rst report on the use of organo! metallic complexes as anti_larials[ It seems clear that at least some heavy metal complexes have appreciable anti_larial activity[ Nevertheless\ the complexes did not exhibit higher activity than organic moieties\ except for Ru"III#! 1!aminobenzothiazole[ Useful antiparasitic drugs must have fairly wide margins of safety[ In vivo anti_larial investigations should now be performed to determine the real advantage of heavy metal complexes in experimental treatment of _lariasis[ References ð0Ł Farrell NP\ Williamson J\ McLaren DJM[ Trypanocidal and antitumor activity of platinum metal drug dual function complexes[ Biochem Pharmacol 0873^22]850Ð860[ ð1Ł Croft SL\ Neal RA\ Craciunescu DG\ Certad!Fombona G[ The activity of platinum\ iridium and rhodium drug com! plexes against Leishmania donovani[ Trop Med Parasitol 0881^32]13Ð17[ ð2Ł Loiseau PM\ Craciunescu DG\ Doadrio!Villarejo JC\ Cer! tad!Fombona G\ Gayral P[ Pharmacomodulations on new organometallic complexes of Ir\ Pt\ Rh\ Pd\ Os] in vitro and in vivo trypanocidal study against Trypanosoma brucei brucei[ Trop Med Parasitol 0881^32]009Ð003[ ð3Ł Gayral P\ Bories C\ Loiseau PM\ Gueyouche C[ Molinema "ex Dipetalonema# dessetae for in vitro and in vivo evalu! ations of _laricidal activities[ Trop Med Parasitol 0876^27]54[ ð4Ł Striebel H[ Filariasis[ In] Zak O\ Sande MA\ editors[ Exper! imental models in antimicrobial chemotherapy[ Academic Press\ 0875]2]170Ð221[ ð5Ł Bories C\ Loiseau PM\ Gueyouche C\ Gayral P[ Utilisation des larves infectantes de Dipetalonema dessetae en survie pour l|etude et la recherche de substances _laricides[ J Phar! macol "Paris# 0875^06]290Ð296[ ð6Ł Loiseau PM\ Mettey Y\ Vierfond JM[ Anti_larial and trypanocidal properties of phenothiazines and related poly! cyclics as new lead structures[ Int J Parasitol 0885^15]0004Ð 0006[ ð7Ł Loiseau PM\ Bourass J\ Letourneux Y[ Lymphotropic anti! _larial agents derived from closantel and chlorambucil[ Int J Parasitol 0886^16]332Ð336[