OF IR-(COD)-PENTAMIDINE TETRAPHENYLBORATE

(1)

IN VIVO ANTILEISHMANIAL ACTION

OF IR-(COD)-PENTAMIDINE TETRAPHENYLBORATE

O N LEISHMANIA DONOVANI A N D LEISHMANIA MAJOR MOUSE MODELS

LOISEAU P.M.*, MBONGO N.*, BORIES C.*, BOULARD Y.** & CRACIUNESCU D.G.***

Summary:

lr-(COD)-pentamidine tetraphenylborate which has previously been studied on promastigote forms of Leishmania, was investigated for its antileishmanial properties compared with pentamidine used as reference compound. In vitro, the iridium complex had the same I C5 0 value on intracellular forms of Leishmania as pentamidine (15 µM). In vivo, the compound could not be injected intravenously due to the DMSO excipient so that the treatments were performed intraperitoneally or subcutaneously. On the L. donovani LV9 /Balb/C mouse model, the iridium complex was not toxic after intraperitoneal treatment at 232 mg/kg/day x 5 or 147 µmoles/ kg/day x 5, whereas all the mice died within five days when treated at the same dose with pentamidine isethionate.

However, only 23 % of parasite suppression was observed with the iridium complex. On a L. major MON 74/Balb/C mouse model, susceptible to intravenously administered pentamidine at 6.7 umoles/ kg/day x 5 (54 % of parasite suppression), the iridium complex exhibited 32 % of parasite suppression after a treatment at 76 umoles/ kg/day x 5 administered subcutaneously.

This slight activity is of interest since pentamidine isethionate is not active under these conditions. Transmission electron microscopy of amastigotes from infected and treated mice show aggregation of ribosomal material, distension of the nuclear membrane and kDNA depolymerization. The mechanism of action therefore involves several targets: membranes, ribosomes and kDNA. According to our results, the Iridium complex is a suitable candidate to be encapsulated in drug carriers such as liposomes or nanoparticles.

KEYWORDS :

Iridium pentamidine complex, pentamidine, Leishmania, electron microscopy, mechanism of action.

MOTS CLÉS :

complexe iridié de pentamidine, pentamidine, Leishmania, microscopie électronique, mécanisme d'action.

Résumé :

ACTION ANTILEISHMANIENNE IN VIVO DU TÉTRAPHÉNYLBORATE D'IR-(COD)-PENTAMIDINE SUR DES MODÈLES MURINS À LEISHMANIA DONOVANI ET LEISHMANIA MAJOR

L'action antileishmanienne du tétraphénylborate d'Ir-(COD)- pentamidine qui a précédemment été étudiée sur les (ormes promastigotes de Leishmania est ici évaluée in vivo en comparaison de l'iséthionate de pentamidine, produit de référence. In vitro, le complexe iridié présente une valeur d'IC50

sur la forme intracellulaire de Leishmania équivalente à celle de la pentamidine (15 µM). In vivo, puisque le composé ne pouvait pas être injecté par voie intraveineuse du fait de l'utilisation du DMSO comme excipient, les traitements ont été réalisés par voie intrapéritonéale ou sous-cutanée. Sur le modèle à L. donovani LV9 chez la souris Balb/C, le complexe iridié ne s'est pas montré toxique après un traitement par voie intrapéritonéale à la dose de 232 mg/kg/jour x 5 soit 147 µmoles/kg/jour x 5, tandis que toutes les souris traitées avec l'iséthionate de pentamidine aux mêmes doses sont mortes dans les cinq jours. Cependant, on observe une suppression de la charge parasitaire de 23 % seulement avec le composé iridié. Sur le modèle à L. major MON 74 chez la souris Balb/C, qui est sensible à l'iséthionate de pentamidine administré par voie intraveineuse à la dose de 6,7 µmoles/kg/jour x 5 (54 % de suppression de la charge parasitaire), le complexe iridié a montré 32 % de suppression de la charge parasitaire après un traitement par voie sous-cutanée à la dose de 76 µmoles/kg/jour x 5. Cette activité, bien que légère est intéressante puisque l'iséthionate de pentamidine n'est pas actif dans ces conditions. La microscopie électronique d'amastigotes prélevés chez les souris traitées montre une aggrégation du matériel ribosomal, une distension de l'enveloppe nucléaire et une dépolymérisation de l'ADN kinétoplastique. Par conséquent, le mécanisme d'action de ce composé met en jeu plusieurs cibles : les membranes, les ribosomes et l'ADN kinétoplastique. A la suite de cette étude, le complexe iridié mériterait d'être encapsulé dans des vecteurs de médicaments tels que les liposomes ou les nanoparticules.

INTRODUCTION

V

isceral a n d c u t a n e o u s leishmaniases are c a u s e d by hemoflagellate p r o t o z o a w h i c h are obligate parasites o f the m o n o n u c l e a r p h a g o c y t e system

* Biologie et Contrôle des Organismes Parasites, Faculté de Phar- macie, 92296-Châtenay-Malabry, France.

** Biologie parasitaire, Protistologie, Helminthologie, Muséum National d'Histoire Naturelle, 75231 Paris, France.

*** Quimica Inorganica y Bioinorganica, Universidad Complutense, Madrid, Spain.

Correspondence: P.M. Loiseau.

(MPS). Leishmaniases c a u s e c o n s i d e r a b l e morbidity and mortality worldwide. T h e first line treatments are pentavalent antimonials a n d amphotericin B , both o f w h i c h s h o w high potential toxicity. Moreover, failure a n d relapse u n d e r antimonials and amphotericin B have also b e e n reported ( M o d a b b e r , 1992). T h e treat- m e n t with pentamidine is not fully effective and has also t o x i c side effects ( S o t o et al, 1994). Thus, the search for n e w antileishmanial drugs remains a prio- rity s i n c e n o currently available drug is both safe a n d active. Several antitumor agents directed towards DNA as their m o l e c u l a r target have b e e n s c r e e n e d for their

Parasite, 2000, 7, 103-108

Mémoire 103

Article available athttp://www.parasite-journal.orgorhttp://dx.doi.org/10.1051/parasite/2000072103

(2)

trypanocidal activity, since the metabolic pathways o f Kinetoplastidae and tumor cells present some similarities.

B y this means, organometallic c o m p l e x e s w e r e found to have antitrypanosomal activity (Kinnamon et al, 1979).

T h e association o f a metallic structure with an organic c o m p o u n d decreases the toxicity and usually increases the biological activity (Fanell et al, 1984). S o m e metallic drug c o m p l e x e s have b e e n found to b e active against Leishmania donovani amastigotes in m o u s e peritoneal macrophages in vitro (Croft et al, 1992). Moreover, some organometallic compounds derived from pentamidine are active against African trypanosomes (Loiseau et al, 1992).

In addition, c o m p o u n d s combining platinum and pen

tamidine appeared promising in the T. b. bruceil'sheep model (Dreyfuss etal, 1988; 1993). An antitrypanosomal s c r e e n i n g using Trypanosoma b. brucei CMP strain allowed a very active metallic pentamidine derivative, called pentamidine di-(iridium cyclo-octadiene) tetraphenylborate or Ir-COD-pentamidine tetraphenylborate, to b e selected (Loiseau et al, 1992). This c o m p o u n d was found to b e 16-fold m o r e active than pentamidine isethionate and was well tolerated. Organometallic com

plexes have interesting antimicrobial effects which could b e exploited, particularly against drug resistant Kineto- plastida strains. Previous pharmacokinetic studies in sheep plasma have shown that the metal part increases the half-life o f the drug compared to pentamidine isethionate, explaining the higher activity at low doses (Dreyfuss et al, 1993; 1995; Loiseau et al, 1997). Such c o m p o u n d s could b e o f potential interest as antileishmanial drugs, to replace pentamidine which is sometimes used in cases o f antimonial-resistance.

MATERIALS AND METHODS

PARASITES

T

w o strains o f Leishmania w e r e used throughout this study:

• L. donovani ( M H O M / E T / 6 7 / L 8 2 ) or LV9 was routinely maintained in male golden hamsters (Wright's strain). After six-eight w e e k s amastigotes w e r e isolated from the spleen o f infected animals for in vitro and in vivo studies. This strain was provided by Dr S.L. Croft (London School o f Hygiene and Tropical Medicine, UK).

• L. major ( M H O M / P T / 9 2 / C R E 2 6 ) , zymodeme MON 74, w a s a c u t a n e o u s strain w h i c h regularly d e v e l o p e d a visceral infection in the hamster w h e n infected by the intraperitoneal route. This strain, kept in liquid nitro

gen, was provided by Prof. M. Deniau (CHU Henri Mon- dor, Creteil, F r a n c e ) .

ANIMALS

F e m a l e B A L B / c m i c e ( 1 8 ± 2 g ) w e r e p u r c h a s e d from Iffa Credo ( L A r b r e s l e , F r a n c e ) .

Fig. 1. - Chemical structure of Ir-(COD)-pentamidine tetraphenylborate. COD = cyclo-octadiene.

DRUGS

T h e structure o f Ir-(COD)-pentamidine tetraphenylbo

rate is s h o w n in Figure 1. Pentacarinat® (Pentamidine isethionate) was kindly supplied b y Roger Bellon Labo

ratory ( F r a n c e ) . Glucantime®, a pentavalent antimonial u s e d as the reference antileishmanial drug, w a s sup

plied b y R h ô n e - P o u l e n c Rorer, France.

ACTIVITY ON INFECTED MACROPHAGES In vitro evaluation

Leishmanicidal activity against intramacrophagic amas

tigotes w a s performed o n infected m o u s e peritoneal m a c r o p h a g e s according to the m e t h o d o f Neal & Croft ( 1 9 8 4 ) .

In vivo evaluation

F e m a l e B A L B / c m i c e w e r e infected with 1 07 amasti

gotes o b t a i n e d from the s p l e e n o f an infected hamster and injected into m i c e b y the retro-orbital sinus. O n e w e e k after infection, the m i c e w e r e randomly divided into groups o f ten. Ir-(COD)-pentamidine tetraphenyl

b o r a t e in D M S O and pentamidine isethionate in ste

rile water w e r e given b y the s u b c u t a n e o u s or intra

peritoneal routes at d o s e s u p to 2 3 2 m g / k g ( 5 0 mg pentamidine equivalent/kg) and 8 7 m g / k g respectively.

Mice w e r e treated o n c e a day for five consecutive days and sacrificed three days after the e n d o f treatment.

Drug activity was estimated b y counting the n u m b e r o f amastigotes/500 liver cells in Giemsa stained impres

s i o n s m e a r s p r e p a r e d from t h e w e i g h e d livers o f treated and untreated m i c e (Neal & Croft, 1 9 8 4 ) .

TRANSMISSION ELECTRON MICROSCOPY

Liver samples w e r e p r e p a r e d for electron m i c r o s c o p y after intraperitoneal administration o f Ir-(COD)-pentamidine tetraphenylborate to infected B A L B / c m i c e at 2 3 2 m g / k g ( 5 0 mg pentamidine e q u i v a l e n t / k g ) . Sam

ples w e r e also prepared from m i c e treated with e x c i - pient a l o n e .

Small portions o f liver w e r e cut into 1 m m³ p i e c e s and fixed at r o o m temperature for 6 0 min. in a 2.8 % glu- taraldehyde solution in a 0.1 M S o r e n s e n p h o s p h a t e buffer (pH 7 . 4 ) , w a s h e d twice in this buffer and post-

104

Parasite, 2000, 7, 103-108

LOISEAU P.M., MBONGO N.? BORIES C., BOULARD Y. & CRACIUNESCU D. G.

Mémoire

(3)

fixed for o n e h o u r in a 2 % o s m i u m tetroxide solution in the s a m e buffer. Potassium ferricyanide w a s a d d e d to b o t h fixatives. T h e s a m p l e s w e r e then stained for 12 hours in 0.5 % uranyl acetate in distilled water. Fol

lowing dehydration, the material w a s e m b e d d e d in a 1:1 mixture o f Araldite-Epon®. 5 0 n m sections w e r e cut with a d i a m o n d knife a n d stained with uranyl acetate and lead citrate. T h e preparations w e r e e x a m i n e d with a Phillips EM 201 electron m i c r o s c o p e ( 1 0 0 Kv) at the Centre Interuniversitaire de M i c r o s c o p i e Électronique, (Université d e J u s s i e u Paris V I ) .

RESULTS

IN VITRO ACTIVITY ON AMASTIGOTES FORMS IN MACROPHAGES

T iridium c o m p l e x h a d the s a m e IC50 value o n intracellular forms o f Leishmania as pentamidine ( 1 5 µM ) .

IN VIVO EFFECT

In vivo, the c o m p o u n d c o u l d not b e injected intrave

nously due to the D M S O e x c i p i e n t so that the treat

ments w e r e performed intraperitoneally or subcutaneously. T h e iridium c o m p l e x w a s not toxic towards the L. donovani LV9 / B a l b / C m o u s e model after a treat

m e n t at 2 3 2 m g / k g / d a y x 5 o r 147 u m o l e s / k g / d a y x 5 administered intraperitonealy w h e r e a s all the m i c e died within five days w h e n treated with the same d o s e with pentamidine isethionate. However, only 23 % o f parasite inhibition w a s o b s e r v e d with the iridium c o m p l e x ( T a b l e I ) .

In the L. major CRE 2 6 / B a l b / C m o u s e model, s u s c e p  tible to intravenously administered p e n t a m i d i n e at 6.7 u m o l e s / k g / d a y x 5 ( 5 4 % o f parasite inhibition), the iridium c o m p l e x e x h i b i t e d 32 % o f parasite inhi

bition after a treatment at 7 6 p m o l e s / k g / d a y x 5 subcutaneously administered ( T a b l e II). This slight activity is o f interest since pentamidine isethionate is not active under t h e s e conditions.

: n = 10 mice per group.

b : toxic dose: death of 4 mice at day 2 3 mice at day 3 2 mice at day 4 1 mouse at day 5.

c : Treatment with 0.2 ml excipient per day for five days.

Table I. - Effect of Ir-(COD)-pentamidine tetraphenylborate on the L. donovani LV9/BALB/C mouse model compared with reference drugs.

232 mg/kg of Ir-(COD)-pentamidine tetraphenylborate corresponds to 50 mg equivalent pentamidine/kg. 200 mg/kg of Glucantime® cor

responds to 56 mg SbVkg.

a : n = 10 mice per group.

b : Treatment with 0.2 ml excipient per day for five days by the subcutaneous route.

Table II. - Effect of Ir-(COD)-pentamidine tetraphenylborate on the L. major CRY. 26/BALB/c mouse model compared with reference drugs.

Parasite, 2000, 7, 103-108

Memoire ¹⁰⁵

PENTAMIDINE METALLIC COMPLEX AS ANTILEISHMANIAL

Drug mg/kg/day

T r e a t m e n t

aDose (i.p.)

umol/kg/day

Stauber count x 1 0^s (± SEM)

Percentage of parasites

Inhibition (mean)

Ir-(COD)-pentamidine t e t r a p h e n y l b o r a t e 232 x 5 147 x 5 5.13 ± 2.31 23

bPentacarinat® 87 x 1 147 x 1 / /

Glucantime® 200 x 5 546 x 5 0.02 ± 0.02 97

54 147 x 5 6.01 ± 1.04 10

c C o n t r o l / / 6.67 ± 1.33 0

Drug

T r e a t m e n t

aDose

mg/kg/day umol/kg/day

Stauber count x IO⁸ (± SEM)

Percentage of parasites

inhibition (mean)

Ir-(COD)-pentamidine t e t r a p h e n y l b o r a t e 120 x 5 (s.c.) 76 x 5 (s.c.) 1.50 ± 0.71 32

60 x 5 (s.c.) 38 x 5 (s.c.) 1.73 ± 0.94 22

Pentacarinat® 4 x 5 (i.v.) 6.7 x 5 (i.v.) 1.02 ± 0.72 54

bC o n t r o l / / 2.21 ± 1.33 0

(4)

LOISEAU P.M., MBONGO N.. BORIES C , BOULARD Y. & CRACIUNESCU D.G.

Fig. 2. - Electron micrograph of sections of L. donovani LV9-infected liver of BALB/c mice three days after treatment with Ir-(COD)-pen- tamidine tetraphenylborate administered intraperitoneally at 50 mg pentamidine equivalent /kg/day x 5. Within these treated parasites, the most stricking features are:

a) a clear cytoplasm due to a depleted ribosomal material, a nuclear membrane convoluted and a kinetoplast presenting major alterations:

swollen mitochondrion, low electron density of probably depolymerized kDNA (x 15,000).

b) fragmentation of the kDNA in a deflated mitochondrion. In addition, the alterations already observed in Figure 2a can also be noted here (x 20.000).

ELECTRON MICROSCOPY

T h e in vivo treatment with the Iridium c o m p l e x at 50 mg equivalent p e n t a m i d i n e / k g / d a y x 5 induced significant d a m a g e in a m a s t i g o t e s c o m p a r e d with controls treated with D M S O only. In the cytoplasm o f the parasite, many electron-lucent areas surrounded by aggregated ribosomes are observed. T h e peri-nuclear space (between the two nuclear membranes) is swollen.

T h e mitochondrion, deflated or swollen, appears amor

phous and n o longer organized. T h e kinetoplast has b e c o m e less electron-dense and is sometimes disrupted into several fragments (Fig. 2a and 2b). Croft & Brazil ( 1 9 8 2 ) h a v e o b s e r v e d similar structural d a m a g e in L. m. amazonensis p r o m a s t i g o t e s and a m a s t i g o t e s treated with 10 pM o f pentamidine in vitro.

DISCUSSION

A

previous study has s h o w n that Iridium-(COD)- pentamidine tetraphenylborate enters and accu

mulates within promastigotes ( M b o n g o et al, 1 9 9 8 ) . T h e apparent Km value w a s l o w e r than that o f p e n t a m i d i n e for the s a m e parasite strain (Basselin et al., 1 9 9 6 ) . T h u s , the apparent Km values o f Iridium- C O D - p e n t a m i d i n e tetraphenylborate and pentamidine

106

isethionate w e r e 17.4 pM and 7 3 pM, respectively and the apparent Vm w e r e 1.3 and 2 n m o l e s / m g protein per t w o hours, respectively ( M b o n g o et al, 1 9 9 8 ) . D a i g - m e t a l c o m p l e x e s are sensitive to pH and their structure can b e modified in the p r e s e n c e o f water (Van der V e e r & Reedijk, 1 9 8 8 ) . Several h y p o t h e s e s could explain the behavior o f Iridium-COD-pentamidine tetraphenylborate under acidic conditions o f extraction:

a) partial dissociation o f iridium c o m p l e x cation and tetraphenylborate anion; b) release o f ligands (i.e pen

tamidine); c) formation o f a n e w c o m p l e x with hyper- chlorate anion (Fig. 3 ) . Considering the results obtained from HPLC experiments, w e c o n c l u d e d that the iridium

H+

(a) [Ir2(COD)2(L)](BPh4)2 > [Ir2(COD)2(L)]2+ + 2(BPh4)-

<

H+

(b) [Ir2(COD)2(L)](BPh4)2 > L + 2Ir + + 2 COD + 2(BPh4)-

2 ClOY

(c) [Ir2(COD)2(L)](BPh4)2 > [Ir2(COD)2(L)](C104)2 + 2H+ + 2(BPh4)- 2 H+

Fig. 3- - Hypothetical dissociation of Ir-COD-pentamidine tetraphe

nylborate in acidic medium (HC104).

COD = 1,5 cyclooctadiene; L = pentamidine; Ph = C⁶H⁵.

Mémoire Parasite, 2000, 7, 103-108

(5)

PENTAMIDINE METALLIC COMPLEX AS ANTLLEISHMANIAL

c o m p l e x is taken u p b y the cells in its undissociated neutral form. This is consistent with the lipophilic pro

perties o f tetraphenylborate w h i c h e n a b l e s the iridium c o m p l e x to cross the parasite m e m b r a n e s .

T h e s e previously obtained results ( M b o n g o etal., 1 9 9 8 ) suggest that the iridium c o m p l e x is relatively stable within the cells, p r o b a b l y b e c a u s e o f strong binding to intracellular c o m p o n e n t s such a s m a c r o m o l e c u l e s . Therefore, it could act as a carrier for pentamidine. T h e electron m i c r o s c o p y o f amastigotes reported in the pre

sent study indicates an action o n several targets within the parasite: m e m b r a n e s , ribosomes and kDNA.

T h e undissociated neutral form o f the c o m p l e x c o u l d interact easily with m e m b r a n e s a n d t h e alterations o b s e r v e d b y e l e c t r o n m i c r o s c o p y confirm s u c h a n interaction.

W e h a v e previously c o m p a r e d t h e interaction o f pen

tamidine a n d the iridium-complex with isolated ribo

s o m e s . T h e o r g a n o m e t a l l i c c o m p l e x stabilised ribo

s o m e s , w h e r e a s pentamidine h a d n o effect u n d e r t h e s a m e conditions. This difference b e t w e e n t h e b e h a viour o f pentamidine and o f the iridium c o m p l e x is due to t h e distribution o f positives c h a r g e s a r o u n d t h e cation structure and their accessibility to anions groups o f n u c l e o p r o t e i n s . T h e iridium c o m p l e x is positively charged, thus it m a y interact m o r e strongly with o p p o site c h a r g e s o f r i b o s o m e s than d o e s p e n t a m i d i n e . C h a n g e s in the cytoplasmic r i b o s o m e content o f drug treated- amastigotes in vivo a n d the effect o f iridium c o m p l e x o n stability in vitro w e r e not associated with a n y inhibition o f m a c r o m o l e c u l a r biosynthesis, indi

cating that r i b o s o m e s are not t h e main target o f either the iridium c o m p l e x o r pentamidine.

As far as interactions with kDNA are c o n c e r n e d , they w e r e similar to t h o s e o b s e r v e d with p e n t a m i d i n e , w h i c h interacts with the AT b a s e s o f kDNA in Kine- toplastida ( B e l l et al., 1 9 9 0 ) .

D e s p i t e its toxicity, pentamidine remains a m o l e c u l e o f great interest for curing early stages o f African try

p a n o s o m i a s i s , antimonial-refractory leishmaniasis a n d p n e u m o c y s t o s i s ( S a n d s et al, 1 9 8 5 ) . T h u s , n e w , less toxic, p e n t a m i d i n e derivatives w o u l d b e valuable in antiparasitic c h e m o t h e r a p y . Since organometallic c o m p l e x e s h a v e previously s h o w n activity against L. dono- vanipromastigotes (Mesa-Valle et al, 1 9 8 9 ) a n d amas

tigotes (Croft et al, 1992), the combination o f a metallic- structure with t h e p e n t a m i d i n e m o l e c u l e a p p e a r e d promising. In vitro, o n the both Leishmania strains eva

luated, the advantage o f the iridium c o m p l e x c o m p a r e d with p e n t a m i d i n e c o u l d not b e e s t a b l i s h e d as clearly as in the case o f the trypanosomes ( M b o n g o etal, 1997,

1998; Loiseau et al, 1 9 9 2 ) .

Although its activity in vivo in m i c e is not strong e n o u g h , t h e iridium c o m p l e x is less toxic than penta

midine isethionate. T h e relative selectivity o f these

c o m p o u n d s towards parasite cells remains to b e inves

tigated. P h a r m a c o k i n e t i c data carried out in s h e e p h a v e s h o w n that after a single d o s e , the c o m p o u n d w a s eliminated slowly from the b l o o d (Loiseau et al, 1 9 9 7 ) . Further studies will b e undertaken, for e x a m p l e encapsulation in nanoparticles a b l e to c o n c e n t r a t e the active principle in the reticulo-endothelial system since previous encapsulation strategies w e r e successful for pentamidine ( D u r a n d et al, 1997; Fusai et al, 1 9 9 7 ) .

REFERENCES

BASSELIN M., LAWRENCE F. & ROBERT-GERO M. Pentamidine uptake in leishmania donovani and Leishmania amazo- nensis promastigotes and axenic amastigotes. Biochemical Journal, 1996, 315, 6 3 1 - 6 3 4 .

BELL CA., HALL J.E., KYLE D.E., GROGL M., OHEMENG K.A., ALLEN M.A. & TIDWELL R.R. Staicture-activity relationships of ana

logs of pentamidine against Plasmodium falciparum and Leishmania mexicana amazonensis. Antimicrobial Agents and Chemotherapy, 1990, 34, 1 3 8 1 - 1 3 8 6 .

CROFT S.L. & BRAZIL R.P. Effect of pentamidine isethionate on the ultrastructure and morphology of Leishmania mexi

cana amazonensis in vitro. Annals of Tropical Medicine and Parasitology, 1982, 76, 37-43.

CROFT S.L., NEAL R.A., CRACIUNESCU D.G. & CERTAD-FOMBONA G.

The activity of platinum, iridium and rhodium drug com

plexes against Leishmania donovani. Tropical Medicine and Parasitology, 1992, 43, 24-28.

DREYFUSS G., GAYRAL P., DUBOST G., NICOLAS J.A. & CRACIU- NFSCI D.G. Activité antiparasitaire du cis-PtdD-pentamidine dans la trypanosomose experiméntale du mouton ä Try

panosoma brucei brucei. Bulletin de la Société de Patho

logie Exotique et de ses Filiales, 1988, 81, 5 6 1 - 5 7 0 . DREYFUSS G., PÉMCALT B., NICOLAS J.A., CRACIUNESCI; D . G . &

LOISEAU P.M. Trypanocidal activity and platinum plasma kinetics of cis-Pt pentamidine iodide in Trypanosoma brucei sheep model. Tropical Medicine and Parasitology, 1993, 44, 9 5 - 9 8 .

DREYFUSS G., LOISEAU P.M., LACHATRE G., PÉNICAUT B., NICOLAS J.A. & CRACIUNESCU D.G. Trypanosoma brucei brucei: anti

trypanosomal evaluation of stilbamidinium hexachloroiri- diate on the murine CNS model and iridium serum kine

tics in infected sheep. Tropical Medicine and Parasitology, 1995, 46, 4 1 - 4 4 .

DURAND R., PAUL M., RIVOLLET D., FESSI H., HOUIN R., ASTIER A.

& DENIAU M. Activity of pentamidine-loaded poly(D,L-lac- tide) nanoparticles against Leishmania infantum in a murine model. Parasite, 1997, 4, 3 3 1 - 3 3 6 .

FARRELL N.P., WILLIAMSON J . & MCLAREN D.J.M. Trypanocidal and antitumor activity of platinum metal and platinum- metal-dmg dual function complexes. Biochemical Phar

macology, 1984, 33, 9 6 1 - 9 7 1 .

FUSAI T., BOULARD Y., DURAND R., PAUL M., BORIES C , RIVOLLET D., ASTIER A., HOUIN R. & DENIAU M. Ultrastructural changes in parasites induced by nanoparticie-bound pentamidine in a Leishmania major/mouse model. Parasite, 1997, 2, 133-139.

Parasite, 2000, 7, 103-108

M é m o i r e ^{1 0 7}

(6)

L O I S E A U P . M . , M B O N G O N.. B O R I E S C , B O U L A R D Y . & C R A C I U N E S C U D . G .

KINNAMON K.E., STECK E.A. & RANE D.S. Activity of antitumor

drugs against African trypanosomes. Antimicrobial Agents and Chemotherapy, 1979, 15, 157-160.

LOISEAU P.M., CRACIUNESCU D.G., DOADRIO-VILLAREJO J.C., CERTAD- 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. Tropical Medicine and Parasitology, 1992, 43, 110- 114.

LOISEAU P.M., DREYFUSS G., DAULOUEDE S., LACHÂTRE G., VIN- CENDEAU P. & CRACIUNESCU D.G. Trypanocidal effect of Ir-

(COD)-pentamidine tetraphenylborate on T. b. brucei and T. b. gambiense rodent models and serum kinetics in the sheep. Tropical Medicine and International Health, 1997, 2, 19-27.

MBONGO N., LOISEAU P.M., LAWRENCE F., BORIES C , CRACIUNESCU D.G. & ROBERT-GERO M.

In vitro sensitivity of Leishmania donovani to organometallic derivatives of pentamidine.

Parasitology Research, 1997, 83, 515-517.

MBONGO N., LOISEAU P.M., LAWRENCE F., CRACIUNESCU D.G. &

ROBERT-GERO M.

Uptake into Leishmania donovani pro- mastigotes and antileishmanial action of an organometallic complex derived from pentamidine. Arzneimittel For

schung Drug Research, 1998, 48, 850-855.

MESA-VALLE C M . , CRACIUNESCU D.G., PARRONDO-IGLESIAS E. &

OSUNA

A. In vitro action of Platinum (II) and Platinum (IV) complexes on Trypanosoma cruzi and Leishmania dono

vani. Arzneimittel Forschung/Drug Research, 1989, 39, 838-842.

MESA-VALLE C M . , MORALEDA-LlNDEZ V. & CRACIUNESCU D. In

vitro action of new organometallic compounds against Try- panosomatidae protozoa. Arzneimittel Forschung/Drug Research, 1993, 43, 1010-1013.

MODABBER F. Leishmaniasis, in: WHO Tropical Disease Research, Progress 1991-1992, Eleventh Program Report., 1992, pp. 77-81.

MOSSMAN T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays.

Journal of Immunological Methods, 1983, 65, 55-63.

NEAL

R.A. &

CROFT

S.L. An in vitro system for determining the activity of compounds against the intracellular amas- tigote form of Leishmania donovani. Journal of Antimi

crobial Chemotherapy, 1984, 14, 463-475.

SANDS M., KRÖN M. & BROWN R.B. Pentamidine: a review.

Review of Infectious Diseases, 1985, 7, 625-634.

SOTO J . , BUFFET P., GROGL M. & BERMAN J . Successful treat

ment of Colombian cutaneous leishmaniasis with four injections of pentamidine. American Journal of Tropical Medicine and Hygiene, 1994, 50, 107-111.

Reçu le 25 novembre 1999 Accepté le 9 mars 2000

108 Parasite, 2000, 7, 103-108

Mémoire

OF IR-(COD)-PENTAMIDINE TETRAPHENYLBORATE

IN VIVO ANTILEISHMANIAL ACTION