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ACTIVITY OF PENTAMIDINE-LOADED POLY (D,L-LACTIDE) NANOPARTICLES AGAINST

LEISHMANIA INFANTUM

IN A MURINE MODEL

D U R A N D R.*, PAUL M.**, RIVOLLE'T D.*, FESSI H.***, HOUIN R.*, ASTIER A.** & DENIAU M.*

S u m m a r y :

The activity of pentamidine-loaded poly(D, L-lactide) nanoparticles was compared, by determination of median effective doses (ED5 0), to that of free pentamidine in a murine model of visceral leishmaniasis induced by Leishmania infantum. BALB/с mice were infected intravenously on day 0 with promastigotes and then treated on days 14, 16, and 18. Groups of 5 mice received either 0 . 5 7 , 1.14 and 2.28 mg/kg of free pentamidine (expressed in pentamidine base) or 0 . 0 5 5 , 0 . 1 1 , 0 . 2 2 and 0 . 4 4 mg/kg of pentamidine-loaded nanoparticles. In the control group, 1 2 mice received normal saline. The liver parasite burden was evaluated using the Stauber method 7 2 h after the last injection and drug levels in livers and spleens were determined.

Bound pentamidine was 3.3 times more active than free drug ( E D5 0 value = 0 . 3 2 mg/kg versus 1.05 mg/kg for free drug].

Drug levels showed a weak accumulation in hepatic and splenic tissues following bound pentamidine administration. A lack of acute toxicity was noted in all groups treated by bound

pentamidine. Results obtained with this biodegradable carrier may be of particular interest as no new major antileishmanial compound is today available.

KEY W O R D S : pentamidine, poly (D,L-lactide) nanoparticles, mouse model, effective doses, Leishmania infantum, visceral leishmaniasis.

Résumé : EFFICACITÉ DE NANOPARTICULES D'ACIDE POLYLACTIQUE CHARGÉES EN PENTAMIDINE SUR UN MODELE MURIN INFECTÉ PAR L. INFANTUM

L'efficacité de nanoparticules d'acide polylactique chargées en pentamidine a été comparée, au moyen de la détermination des doses efficaces 50 % ( D E5 0) , à celle de la pentamidine libre dans un modèle murin de leishmaniose viscérale induite par Leishmania infantum. Des souris BALB/c ont été infectées par voie

intraveineuse J0 avec des formes promastigotes et ont ensuite été traitées J14, J16 et J18. Des lots de 5 souris ont reçu soit 0,57,

1, 14 et 2,28 mg/kg de pentamidine libre (exprimée en pentamidine base) soit 0,055, 0,11, 0,22 et 0,44 mg/kg de pentamidine vectorisée. Douze souris ont reçu du chlorure de sodium isotonique dans le groupe témoin. La charge parasitaire a été évaluée dans le foie par la méthode de Stauber 72 heures après la dernière injection et les concentrations en pentamidine ont été dosées dans les tissus hépatiques et spléniques. La pentamidine vectorisée a été 3,3 fois plus active que la pentamidine libre avec une DE50 égale 0,32 mg/kg versus

1,05 mg/kg pour la pentamidine libre. Les dosages dans le foie et la rate ont montré une faible accumulation du principe actif dans ces organes après administration de la pentamidine vectorisée comparativement à la forme libre. Une absence de toxicité aiguë a été notée pour tous les groupes traités par la pentamidine vectorisée.

MOTS CLÉS : pentamidine, nanoparticules, acide polylactique, modèle murin, doses efficaces, Leishmania infantum, leishmaniose viscérale.

INTRODUCTION

T

h e leishmaniases are parasitic diseases c a u s e d by haemoflagellate protozoa w h i c h are obligate parasites o f the m o n o n u c l e a r phagocyte system o f mammalian hosts. Clinical manifestations are multi- faceted, ranging from self-healing cutaneous ulceration to progressive and lethal visceral infection. T h e leish- maniases cause important morbidity and mortality in the Old and New Worlds (Desjeux, 1 9 9 6 ) . Leishmania/HW co-infections are regarded as emerging diseases, e s p e -

* Laboratoire de Parasitologic Faculté de Médecine de Créteil, 94010 Créteil.

** Laboratoire de Pharmacotechnie, Service Pharmacie, CHU Henri- Mondor, 94010 Créteil.

*** Faculté de Pharmacie Claude-Bernard, Lyon 1, 8, avenue Rocke- feller, 69373 Lyon, France.

Correspondence: Rémy Durand.

Tel: (33) 1 49 81 36 31 - Fax: (33) 1 49 81 36 01.

e-mail: rjdurand@worldnet.fr.

cially in southern Europe, w h e r e 25 to 7 0 % o f adult visceral leishmaniasis (VL) c a s e s are related to HIV infection, and 1.5 to 9 % o f AIDS c a s e s suffer from newly acquired or reactivated VL (Gradoni et ai, 1995).

In the M e d i t e r r a n e a n basin, Leishmania infantum usually causes the visceral type o f infection, which is fatal if not treated (Rosenthal et ai, 1 9 9 5 ) . Pentavalent antimonials, available as sodium stibogluconate (Pen- tostam®) or N-methylglucamine antimoniate (Glucan- time®) have b e e n the standard first line treatment o f VL for over 4 0 years (Neal, 1 9 8 7 ) . Pentamidine, a diamidine c o m p o u n d , and amphotericin B , an anti- fungal agent also active against Leishmania, are the s e c o n d line treatments after antimony failure or into- lerance. T o d a y n o other n e w major antileishmanial drug is available. However, clinical relapses occur regu- larly in HIV patients and resistances to antimonials are increasingly reported for s o m e species as L. donovani (Modabber et al., 1992). Therefore, alternative therapies using n e w formulations o f existing drugs are o f poten- Article available athttp://www.parasite-journal.orgorhttp://dx.doi.org/10.1051/parasite/1997044331

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tial interest. Drug delivery systems, already used in c a n c e r treatment (Astier et al, 1988), may reduce the toxicity and improve the activity o f drugs as antileish- manial c o m p o u n d s . This approach is also applicable to diseases involving intracellular pathogenic agents as bacteria, viruses or parasites (Fattal et al, 1989).

T h e different classes o f carriers that have b e e n used to target antileishmanial c o m p o u n d s are red cell ghosts, l i p o s o m e s and nanoparticles. Pentamidine containing h u m a n red cell ghosts gave promising results in vitro but their biological origin and their delicate m o d e o f p r e p a r a t i o n p r e v e n t e d t h e i r further d e v e l o p m e n t ( B e r m a n et al, 1 9 8 6 ) . L i p o s o m e s loaded with antimo­

nial drugs led to an improved anti-leishmanial activity but the c o m m e r c i a l production o f liposomal S bv was a b a n d o n e d b e c a u s e o f toxicity in m o n k e y s ( N e w et ai,

1 9 8 0 ) . L i p o s o m e s loaded with amphotericin В (AmBi­

some®) p r o v e d to b e a progress in the fight against VL (Torre-Cisneros et al., 1 9 9 3 ; Davidson et al., 1 9 9 6 ) . Results o f clinical trials with AmBisome® in Europe sug­

gest that short courses and low doses may reliably cure i m m u n o c o m p e t e n t VL patients, w h e r e a s patients c o - infected with HIV may require m a i n t e n a n c e therapy.

H o w e v e r the prohibitive cost o f the currently available formulation o f liposomal amphotericin В is a major i m p e d i m e n t to its u s e . O t h e r lipid f o r m u l a t i o n s (Amphocil", Abelcet®) less e x p e n s i v e than AmBisome®

are currently being evaluated ( H i e m e n z et al., 1996).

Nanoparticles have b e e n used to target antileishmanial drugs in experimental studies. Amphotericin В w a s incorporated into poly(D,L-lactide-coglycolide) n a n o ­ particles and s h o w e d a g o o d activity in vitro (Venier- J u l i e n n e et al., 1 9 9 5 ) . Primaquine-loaded poly(d,/-lac- tide) nanoparticles (PLA) mice w e r e m o r e active than free drug in a m o d e l associating Leishmania donovani and B A L B / c m i c e (Rodrigues et al, 1 9 9 4 ) . In our pre­

vious studies, pentamidine loaded o n polymethacrylate nanoparticles (PM) was 6 fold m o r e active than free drug in a model associating L. infantum and B A L B / c m i c e (Durand et al, 1 9 9 7 ) . T h e main problem o f these nanoparticles was their low biodegradability. Conver­

sely, lactide polymers and c o - p o l y m e r s are k n o w n to b e b i o d e g r a d a b l e ( M a k i n o et al, 1985; Lewis et al., 1 9 9 0 ) . T h e s e polymers are slowly hydrolysed in D and L lactic acid. T h e latter is a metabolite o f the Krebs cycle (Bazile et al., 1992). T h e aim o f the present study was to evaluate the activity o f pentamidine-bound PLA nanoparticles by determination o f median effective doses against Leishmania infantum in a murine model.

MATERIAL AND METHOD

P

e n t a m i d i n e i s e t h i o n a t e w a s p u r c h a s e d from Roger Bellon ( F r a n c e ) . T h e phospholipid mix­

ture (Lipoid S 7 5 ) was supplied by Lipoid G m b H

(Ludwigshafen, G e r m a n y ) . P o l o x a m e r ( S y m p e r o n i c PE/F-68®) was purchased from ICI (Clamart, F r a n c e ) . Polymer poly(D,L-lactide) (PLA, mol. wt. (GPC) 200,000) w a s supplied by B o e h r i n g e r Ingelheim ( I n g e l h e i m , G e r m a n y ) . Other reagents w e r e analytical grade.

PENTAMIDINE-LOADED ON POLY (D,L-LACTIDE) NANOPARTICLES

Pentamidine b a s e was previously obtained by precipi­

tation o f pentamidine isethionate solution in alkaline medium ( 2 5 % a m m o n i u m hydroxide) at 4 °C. T h e pre­

cipitate was filtered and dried under vacuum. PLA n a n o p a r t i c l e s w e r e p r e p a r e d by adaptation o f t h e m e t h o d reported by Fessi (Fessi et al., 1 9 8 7 ) . O n e mil­

ligram o f pentamidine b a s e , 125 mg o f phospholipids and 125 mg o f PLA w e r e dissolved in a c e t o n e ( 2 5 ml).

This solution was m i x e d to the alkaline a q u e o u s solu­

tion (1 ml o f 25 % a m m o n i u m hydroxide) containing 125 mg o f Poloxamer. After 15 minutes o f stirring, the a c e t o n e and part o f the water w e r e evaporated under vacuum. T h e final volume was 10 ml. T h e colloidal sus­

pension was monodispersed. T h e size o f nanoparticles was 150 ± 2 0 nm. This formulation was stable for at least o n e month at 4 °C. Before use, nanoparticles w e r e diluted in normal saline to obtain a final concentration o f 8 pg o f pentamidine b a s e per mg o f polymer.

PENTAMIDINE ISETHIONATE

Concentrations w e r e e x p r e s s e d in b a s e w h i c h repre­

sented 57 % o f the salt m o l e c u l a r weight. T h e diffe­

rent dilutions were carried out in 5 % dextrose b e c a u s e o f the low stability o f pentamidine in normal saline.

LEISHMANIA STRAIN

The strain o f Leishmania was identified by the Reference Center o f OMS (Montpellier) as Leishmania infantum MON 1 (reference M H O M / P T / 9 3 / C R E 4 1 ) . T h e zymo- d e m e o f this strain is usually responsible for visceral leishmaniasis. Leishmania w e r e injected in the hamster by intraperitoneal route to increase its virulence and maintained in NNN-medium at 27 °C for 8 days. Bulk culture o f the infectious promastigote forms was initiated and propagated in RPMI 1640 medium (Eurobio, France) supplemented with 0.15 % sodium bicarbonate ( B i o - Mérieux, France), 20 % heat-inactivated fetal calf serum (DAP, France), 20 % Schneider-medium ( G i b c o Ltd, United Kingdom), 1 % L-glutamine (BioMérieux, France) and 0.066 % gentamicine (Pharmacie Centrale des Hôpi­

taux, Paris, France). T h e promastigotes reached their infectious metacyclic phase after 8 days, at 27 °C.

ANIMALS

Male adult B A L B / c mice ( 5 - w e e k old, 20 g ± 2 g ) w e r e purchased from IFFA CREDO, (L'Arbresle, France). T h e infection model used was modified from Neal et al, 1985. T h e main difference was the use o f stationary pro-

332- Mémoire Parasite, 1997, 4, 331-336

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PENTAMIDINE NANOPARTICLES IN LEISHMANIA INFECTED MICE

mastigotes instead o f amastigotes obtained from ham­

ster spleen. Promastigotes w e r e obtained by centrifu- gation and resuspended in normal saline solution. O n day 0, mice w e r e infected by intravenous injection (tail vein) with 1 07 infective Leishmania infantum promas­

tigotes in a 0.1 ml volume. This procedure induced a heavy Leishmania liver burden after 12 days. T h e mice w e r e randomly assigned into three groups: the control group (normal saline), the group treated with free pen­

tamidine (pentamidine isethionate) and the group treated with pentamidine-loaded poly(D.L-lactide) nanoparticles.

GROUPS

In the control group, 12 m i c e w e r e tested. T h e diffe­

rent d o s e s w e r e c h o s e n according to our previous stu­

dies to obtain the median effective dose ( E D5 0) for each regimen (Fusai et al., 1 9 9 4 ) . In the treated groups, 5 mice r e c e i v e d 0.57, 1.14 or 2 . 2 8 m g / k g o f free pen­

tamidine ( e x p r e s s e d in pentamidine b a s e ) o n DM, D1 ( ) and D1 K and 5 m i c e r e c e i v e d 0 . 0 5 5 , 0 . 1 1 , 0.22 or 0 . 4 4 m g / k g o f p e n t a m i d i n e - l o a d e d n a n o p a r t i c l e s ( e x p r e s s e d in pentamidine b a s e ) in 0.1 ml by tail vein.

T w e n t y - o n e days after the initial infection, the animals w e r e killed b y cervical dislocation. T h e Guiding Prin­

ciples for Biomedical Research involving animals w e r e followed during all procedures (CIOMS, 1 9 8 5 ) . T h e following parameters w e r e u s e d to assess treat­

ment efficacy:

• Parasite burden: the liver parasite burden was eva­

luated after Giemsa staining o f the smears. T h e number o f amastigotes per 5 0 0 h e p a t o c y t e s was calculated and related to the liver weight ( m g ) , following the Stauber formula (Stauber et al, 1 9 5 8 ) . T h e p e r c e n t a g e o f parasite suppression was calculated as follows:

- P e r c e n t a g e o f parasite suppression = [ l - ( m e a n o f Stauber count o f the treated g r o u p / m e a n o f Stauber count o f control group)] χ 1 0 0 .

• Median effective doses: the E D2 5 and E D5 0 ( d o s a g e s o f drug calculated to eliminate 25 % and 5 0 % o f orga­

nisms c o m p a r e d to c o n t r o l s ) w e r e determinated using a Michaelis-Menten model.

• Levels o f pentamidine in tissues: w e i g h e d aliquote o f liver and spleen were immersed into liquid nitrogen.

Pentamidine levels in liver and spleen o f mice treated with pentamidine isethionate and pentamidine-loaded (D,L-lactide) nanoparticles were detenriined by high-per­

formance liquid chromatography. Briefly, after thawing, samples were homogenized in 0.5 ml o f phosphate buffer saline and pentamidine was extracted by aceto- nitrile (4 ml) containing hexamidine as an internal stan­

dard. T h e extract was introduced on a solid-phase column (Bond-Elut C„, 1 ml) under pressure. Pentami­

dine was eluted with a 1 ml o f a solution c o m p o s e d o f 0.5 % heptane-sulfonic acid, 0.02 % tetramethylammo- nium chloride and 0.1 % phosphoric acid in methanol.

The eluate was injected onto a reverse-phase C1 K column (Shandon, Hypersil, 5 pm, 2 5 0 mm x 4 . 6 mm, ID, France). T h e mobile phase was c o m p o s e d o f 7 0 0 ml o f methanol and 3 0 0 ml o f a 0.05 M o f heptane-sulfonic acid and 0.014 M o f diethylamine aqueous solution. T h e pH o f the aqueous solution was adjusted to 3 using phosphoric acid. T h e flow rate was 0.9 ml/min. Detec­

tion o f pentamidine was perfonned by ultraviolet absorp­

tion at 2 8 0 nm. T h e limit o f detection was 25 ng/g.

STATISTICAL ANALYSIS

Results w e r e e x p r e s s e d as m e a n ± standard error. An o n e - w a y analysis o f variance or a IJ-test was per­

formed to c o m p a r e the influence o f the various para­

meters. A p value l o w e r than 0.05 was considered as statistically significant.

RESULTS

CONTROL EXPERIMENTS

I

n m i c e treated with normal saline solution, there was a mean o f 3-5 (range = 3-3 - 3-7, N = 12) Leishmania infantum amastigotes per liver cell nucleus at the e n d o f the 21-day period o f experi­

mentation. T h e average liver parasite burden reached w a s 13 x 10* a m a s t i g o t e s ( S t a u b e r c o u n t ) for the control group for 1 07 promastigotes inoculated 21 days before as described in table I.

Amastigotes Percentage

c o u n t e d p e r Stauber o f sup­

5 0 0 hepato­ c o u n t p r e s s i o n

Group cytes 1 0s %

Control 1,745 ±90 12.75 ±0.65

Pentamidine-loaded nanoparticle dose (mg/kg)

0.055 1,496 ±42 9.63 ±0.68 24.4±5.4a b c 0.11 1,302 ± 3 8 8.34 ±0.33 3 ί . 6 ± 2 . 6 ac 0.22 1,210 + 49 7.62 + 0.42 40.2 ±3.3b

0.44 887 ±64 5.31 ±0.44 58.2±3.5a Pentamidine

isethionate dose (mg/kg)

0.57 1,053 ±24 7.39 ±0.43 42.1 ±3.4''

1.14 864 ±87 6.22 ±0.67 51.2 ±5.3

2.28 758 + 21 5.39 ±0.26 57.7±2.1

a 0.055 vs 0.22 and 0.57; 0.11 vs 1.14; 0.44 us 0.57 = ρ < 0.05.

b 0.055 VS 1.14; 0.22 vs 0.44 and 2.28; 0.57 VS 2.28 = p < 0.01.

c 0.055 vs 0.44 and 2.28; 0.11 VS 0.44 and 2.28 = p < 0.001.

Table I. — Suppression of experimental leishmaniasis in mice treated with pentamidine loaded polvi D-L-lactic acid) nanoparticles and free pentamidine. The suppression percentage was calculated using Stauber count VS untreated mice. Data are expressed as mean ± standard error for 5 mice per group except control group (n = 12).

U-test was used to compare the different groups.

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Fig. 1. — Semilogarithmic plot of suppression of experimental leish- maniasis in mite treated with pentamidine loaded (¿/./-lactide) nano- particles (·: doses ranging from 0.055 to 0.44 mg/kg) or with pen- tamidine isethionate (o : doses ranging from 0.57 to 2.28 mg/kg). The suppression percentage was calculated using the Stauber count vs untreated mice as described in the experimental section. Suppres- sion was expressed as the percentage of the maximal infestation. Data were fitted to a Michaelis-Menten model (r= 0.963) for pentamidine loaded (D.I.-lactide) nanoparticles and (r = 0.999) for pentamidine isethionate. Each point represents the mean ± SE for 5 mice per group.

Treatment ED

25

ED,

Free pentamidine 0.20 ±0.06 1.05 ±0.102*

(F)

Pentamidine-loaded nanoparticles 0.070 ±0.009 0.32 ±0.07 (B)

Ratio ED (F/B) 2.9 3.3

*ED,

0

(F) us ED,,, (B) = p < 0.001.

Table II. — Activity of pentamidine (bound or free) in a leishmania infantum infected mice. The effective doses (ED) were calculated using a Michaelis ¡vienten model and were expressed in mg/kg.

Pentamidine-loaded poly(lactide)

Free pentamidine nanoparticles Liver Spleen Liver Spleen

(pg/g) (fig/g) <l'g/g) Dose

(mg/kg)

0.055 — — 0.30 ±0.040 0.20±0.042 0.11 — — 0.42 ±0.060 ().25±0.042

0.22 — — 0.73±0.055 0.32±0.065

0.44 — — 1.29±0.078 0.56 ±0.036

0.57 0.48±0.043 0.24±0.031 — — 1.14 0.82 ±0.065 0.32±0.046 — — 2.28 1.40 ±0.072 0.54±0.110 — — Table III. — Pentamidine levels in liver and spleen.

Mice were killed 3 days after cessation of treatment. Each mouse received 3 doses of free pentamidine or pentamidine loaded on PLA on days 14, 16 and 18.

The values are expressed as means ± standard errors for 5 mice per group.

TREATMENT GROUPS

The maximal percentage of parasite suppression was obtained with 0.44 mg/kg of pentamidine-loaded nano- particles (58.2 ± 3.5 %) (Table I). This result was not significantly different from the one observed with 2.28 mg/kg of pentamidine isethionate (57.7 ±2.1 %).

The relationship between the percentage of parasite suppression and the dose is represented in figure 1.

The Michaelis-Menten model was used to fit the data obtained from groups treated with pentamidine-loaded nanoparticles (r= 0.963) or with isethionate pentami- dine ( r = 0.999)· The maximal effect (E

m ; l x

) calculated with pentamidine-loaded nanoparticles was not signi- ficantly different from that calculated with pentamidine isethionate (70.4 % vs 65.8 %).

EVALUATION OF THE EFFECTIVE DOSES

Pentamidine-loaded nanoparticles were 3-3 fold more active than free dmg: ED

50

=1.05 mg/kg (pentamidine ise- thionate) vs 0.32 mg/kg (pentamidine loaded nanoparti- cles). The EDçx, values were not detemiined as a plateau was reached for 70 % of parasite suppression. The other values of the effective doses are presented in table II.

PENTAMIDINE LEVELS

Administration of pentamidine-loaded nanoparticles (0.44 mg/kg) led to similar drug levels, in liver and spleen, to that reached with 2.28 mg/kg of isethionate pentamidine (Table III). The maximal concentrations obtained in liver and in spleen were 1.4 pg/g and 0.56 pg/g, respectively. For all dose tested, dnigs levels were higher in liver than in spleen. The drug levels in the liver were 2 fold higher than those in the spleen.

The relationship between dose and concentration was linear for pentamidine-loaded nanoparticles (Fig. 2) and for pentamidine isethionate.

Fig. 2. — Intratissular concentrations of pentamidine in liver and spleen in Leishmania infantum infected mice, 3 days after the last injection. Mice were treated on days 14, 16 and 18 with pentamidine- loaded (D,L-lactide) nanoparticles (doses ranging from 0.054 to 0.44 mg/kg). Each point represents the mean ± standard error (n = 5).

334 Mémoire Parasite, 1997, 4, 331-336

Dose (mg/kg)

Dose (mg/kg)

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PENTAMIDINE NANOPARTICLES IN LEISHMANIA INFECTED MICE

DISCUSSION

R

esults s h o w e d that b o u n d pentamidine was m o r e potent than free drug against Leishmania infantum in our B A L B / c m i c e model. T h e ratio o f median effective d o s e s ( E D5 0) for free and b o u n d drug was 3-3· T h e l o w E DW value o b s e r v e d for b o u n d pentamidine ( 0 . 3 2 m g / k g ) demonstrated the ability o f PLA nanoparticles to induce a higher activity and a l o w e r toxicity o f pentamidine for the treatment o f VL.

It was not possible to obtain the E Dy o for free and b o u n d pentamidine as the d o s e r e s p o n s e curves rea­

c h e d a plateau at 7 0 % o f parasite suppression.

Red cell ghosts containing pentamidine tested in a L. donovani Syrian hamster m o d e l led to very similar results with a reported E D „ , o f 0 . 3 8 m g / k g ( B e r m a n et al, 1 9 8 6 ) . Immunoglobulin ghost red cells had a better efficacy, nevertheless the u s e o f such natural materials had limited their further development. T h e e n h a n c e m e n t o f pentamidine activity o b s e r v e d with PLA nanoparticles w a s lower than that o b t a i n e d with polymethacrylate nanoparticles evaluated on the s a m e in vivo m o d e l ( E D5 0 ratio = 3-3 versus 6 ) ( D u r a n d et al, in p r e s s ) . Conversely, the e n h a n c e m e n t o f activity o f primaquine, an antimalarial c o m p o u n d which also has antileishmanial activity, by targeting it o n PLA nanoparticles against L. donovani in a B A L B / c m i c e model, was identical to that o b s e r v e d with pentami- d i n e - b o u n d PLA n a n o p a r t i c l e s . H o w e v e r , a u t h o r s reported a relatively high E D5 0 value ( 6 . 6 m g / k g ) for b o u n d primaquine (Rodrigues et al, 1 9 9 4 ) . T h e s e fin­

dings may b e due to a low intrinsic activity o f prima­

q u i n e against L. donovani.

In our model, the parasite load increased to 13 x 1 08

amastigotes per liver after 12 days and remained at that level for at least 5 w e e k s allowing time to test for drug suppression o f parasites. T h e different d o s e s w e r e c h o s e n according to our previous studies to obtain the E D5 0 for e a c h regimen. T h e unloaded nanoparticles w e r e not tested in this work as their lack o f activity has already b e e n reported in a m o d e l associating L. donovani and B A L B / c mice (Rodrigues et al, 1 9 9 4 ) . Determination o f drug levels 3 days after the last injec­

tion s h o w e d a w e a k d a i g accumulation in hepatic and splenic tissues following PLA b o u n d pentamidine admi­

nistration c o m p a r e d to free drug. Pentamidine carried o n h u m a n red cell ghosts o r l i p o s o m e s c o n c e n t r a t e d m o r e markedly than pentamidine carried o n PLA in the liver and in the spleen in rodents ( B e r m a n et al, 1986; D e b s et al, 1 9 8 7 ) . P e n t a m i d i n e levels w e r e l o w e r in the s p l e e n than in the liver as it was pre­

viously described by authors ( D e b s et al, 1987). A daig accumulation in hepatic and splenic tissues following the u s e o f drug carriers was already reported for other antileishmanial c o m p o u n d s ( G a n g n e u x et al, 1 9 9 6 ) .

The drug accumulation in tissues and the drug targe­

ting mainly in the m o n o n u c l e a r p h a g o c y t e s led to an increase in d a i g delivery to intracellular leishmania and e x p l a i n e d probably most o f the e n h a n c e m e n t o f acti­

vity observed.

T h e l o w e r activity o b s e r v e d with pentamidine-loaded PLA nanoparticles compared to pentamidine-loaded PM nanoparticles may b e due to dissimilar kinetics o f drug release. Actually, m e c h a n i s m s o f pentamidine binding to PM and PLA carriers are different. Penta­

midine was b o u n d b y ionic interaction to PM (Paul et al, in p r e s s ) w h e r e a s it was b o u n d mainly by adsorption to PLA (data not s h o w n ) . P e n t a m i d i n e release from PLA may b e e a s y and therefore, may o c c u r not in close contact to leishmania. In contrast, pentamidine release from PM, which requires a pH value near 5 (found in the parasitophorous v a c u o l e 4 8 h after the leishmania phagocytosis (Antoine et al, 1 9 9 0 ) , m a y b e m o r e finely c o n t r o l l e d . T h e major advantage o f PLA c o m p a r e d to PM nanoparticles is their biodegradability. PLA nanoparticles are b i o c o m ­ patible and w e r e approved by the F o o d and Drug Administration for human administration (Makino et al, 1985). PLA w e r e well tolerated in m i c e at the maximal d o s e tested o f 1,000 m g / k g and n o sign o f acute toxi­

city was o b s e r v e d (Rodrigues et al, 1 9 9 5 ) .

Results obtained with L. infantum are o f particular interest b e c a u s e it is the most frequently Leishmania s p e c i e s isolated from HIV patients in mediterranean c o u n t r i e s . B o u n d - p e n t a m i d i n e PLA n a n o p a r t i c l e s require n o w further investigations particularly in immu- n o d e p r e s s e d mice models in order to evaluate m o r e accurately their interest for HIV-Leishmania co-infected patients.

ACKNOWLEDGEMENTS

W

e thank Christine Fernandez for her lin­

guistic assistance.

This w o r k received the financial support o f A g e n c e Nationale d e R e c h e r c h e contre le SIDA for animal studies and that o f B a x t e r Dubernard Hospital Foundation for pharmaceutical studies.

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Reçu le 1 0 avril 199V Accepté le 2 7 juin 1 9 9 7

336 Mémoire Parasite, 1997, 4, 331-336

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