Activity of pentamidine-loaded methacrylate nanoparticles against Leishmania infantum in a mouse model

Pergamon PII:

002cL7519/97

SOO2@-7519(97)00124-O

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Activity of Pentamidine-loaded Methacrylate Nanoparticles Against Leishmania infantum in a Mouse Model RkMY

DURAND,*$

*Laboratoire j-Laboratoire

MURIEL PAUL,? DANIGLE ALAIN ASTIER? and MICHCLE

RIVOLLET,* DENIAU*

RENk

HOUIN,*

de Parasitologie,

FacultP de MPdecine de CrPteil, 8 avenue du GMral Sarrail. 94010 Crkteil, France de Phmnacotechnie, Service Pharmucie, CHU Henri Mondor, 94010 CrPteil, France (Received 3 January 1997; accepted 24 June 1997)

Abstract-Dnrand R., Paul M., RivoIlet D., Houin R., Astier A. & Deniau M. 1997. Activity of pentamidineloaded methacrylate nanoparticles against Leishmonia infanturn in a mouse model. Internarional Journalfor Parasitology 27: 1361-1367. The use of drug delivery systems may reduce the toxicity and improve the activity of antileishmanial compounds. In view of such a strategy, we loaded the antileishmanial agent pentamidine on polymethacrylate nanoparticles. The activity of pentamidine-loaded nanoparticles was compared with that of free pentamidine in a BALB/c mice model of visceral leishmaniasis induced by Leishmania infanturn. On day 0, mice were infected intravenously with 10’ promastigotes and then treated via the tail vein on days 14, 16 and 18 with bound pentamidine, free drug or isotonic saline (control group). On day 21, liver parasite burdens were evaluated using the Stanber method. Livers and spleens were removed and weighed. Effective doses (ED) were determined using the Michael&Menten representation relating the percentage of parasite suppression to the dose. The ED, of bound pentamidine was six times lower than that of free pentamidine (0.17 mg kg-’ vs l.O6mgkg-‘). The ED, value calculated for bound pentamidine was 1 mgkg-‘. It was not possible to obtain the ED, for free pentamidine because the dose-response curve reached a plateau near 60% of parasite suppression. A significant decrease in liver and spleen weights, probably reflecting the leishmanicidal activity, was observed for treated mice with bound pentamidine. These results showed that bound pentamidine was more potent than the free drug against L. infanturn in our BALB/c mice model. 0 1997 Australian Society for Parasitology. Published by Elsevier Science Ltd.

Kq words: pentamidine; methacrylate nanopartlcles; mouse model; effective doses; Lcishmania infar~tum; visceral leishmaniasis.

(Desjeux, 1996). Leis/znrcmia/HfV co-infections are regarded as emerging diseases, especially in southern Europe, where 25570% of adult visceral leishmaniasis cases are related to HIV infection. and where 1 S-9% of AIDS cases suffer from newly acquired or reactivated visceral leishtndniasis (Gradoni et al., 1995). In the Mediterranean basin, Leishmania iqfanturnusually causes the visceral type of infection. which is fatal if not treated (Gradon; et al., 1995; Rosenthal EI al., 1995). Pentavalent antimonials, available as sodium stibogluconate (Pentostam,K’) or N-methylglucamine anti-

1NTRODUCTION

Leishnlania are pathogenic protozoa that cause a wide spectrum of infectious diseases in mammalian hosts, ranging from self-healing cutaneous ulceration to progressive and lethal visceral infection. The estimated prevalence in the world is 12 million cases. with 400000-2 000 000 new cases reported per year $To whom correspondence should be addressed. Tel: (33) 01 49 81 36 31; Fax: (33) 01 49 81 36 01; e-mail: rjdurand

(nbworldnet.fr.

1361

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R. Durand

moniate (Glucantime’“), have been the standard firstline treatment of visceral leishmaniasis for over 40 years (Neal, 1987). Today, unresponsiveness to antimonial compounds for Leishmaniu species such as Leishnmrziu dono~lani is increasingly reported (Modabber, 1992). Moreover, clinical relapses occur regularly in HIV patients treated with pentavalent antimonials (WHO, 1990). Therefore, alternative therapies are required. Pentamidine, a diamidine compound, has long been the second-line treatment of leishmaniasis after antimony failure or intolerance. Amphotericin B is also used in the same cases. The use of these drugs was limited by their major side effects (Davidson & Croft, 1993). Drug delivery systems may reduce the toxicity and improve the activity of antileishmanial compounds. This concept was first applied to antimonial compounds (Alving et al., 1978). Liposomes loaded with antimonial drugs led to an improved anti-leishmanial activity. but the commercial production of antimonial liposomes was abandoned because of their toxicity in monkeys (New & Chance, 1980). Otherwise, two lipid-amphotericin B formulations. Ambisome”‘(Vestar Inc., U.S.A.) and Amphocil’ (Liposome Technology Ltd. U.S.A.), developed earlier for the treatment of fungal infections, have been used in animal and human visceral leishmaniasis (Davidson et al., 1991). Preliminary results of clinical trials with Ambisome’* suggest that short courses and low doses may reliably cure immunocompetent VL patients (Jha et al., 1995), whereas patients co-infected with HIV may require maintenance therapy (Russo et al., 1996). In immunocompromised patients, Ambisome’* led to a period of clinical recovery for several months followed by relapse (Davidson et al., 1994; Russo et al., 1996). Pentamidine was rarely used alternatively in HIV patients because of its toxicity. This toxicity may be reduced by loading pentamidine on drug carriers (Berman et al., 1986). The ability of methacrylate nanoparticles to be taken up by the mononuclear phagocyte system cells makes them ideal carriers for the selective transport of drugs to target tissues in diseases such as leishmaniasis where phagocytic cells are involved (Paul et al., in press). Previous studies have demonstrated that pentamidine loaded on polymethacrylate nanoparticles was more active than free drug against intracellular Leishmuniu major in an in vitro model (Deniau et al., 1993). Preliminary studies in BALB/c mice demonstrated that these loaded nanoparticles

were

well

tolerated

and

were

more

active

than free pentamidine against Leishmaniu nmjor (Fusai’ et al., 1994). The aim of the present study was to compare the activity of pentamidine-bound methacrylate nanoparticles versus free pentamidine against L. infanturn in a mouse model.

et ul MATERIALS

AND

METHODS

Pentamidine-loaded methacrylate nanoparticles. Unloaded methacrylate nanoparticles were prepared by emulsion polymerisation, using a mixture of acrylic copolymers (Hansen & Ugelstad. 1982). The median lethal dose (LD,,)determined in mice was 720mg kg-‘, demonstrating good tolerance in comparison with polyisobutylcyanoacrylate (Kante et al., 1982). The nanoparticles were Loaded with a 1 mM penlamidine methane sulfonate solution (Rhone Poulenc Rorer, Antony, France) in phosphate saline buffer (200mM, pH7.5). at room temperature, with magnetic stirring to obtain a 100pM final concentration (34pgml. ‘). Under these conditions, a 100% binding was obtained immediately. This concentration corresponded to a dose of 0.17 mg kg- ‘. One millilitre of suspension contained 2.4 x 10” nanoparticles. The pentamidine was bound to the nanoparticles by an ionic process involving the free carboxylic acid groups of polymer (Paul et a/., in press). Pentamidine isethionatc. Pentamidine tacarinat’. Bellon, France) was used to tamidine group. Concentrations were which represented 57% of salt molecular ent dilutions were made in 5% dextrose stability of pentamidine in normal saline.

isethionate (Pentreat the free penexpressed in base weight. The differbecause of the low

Leishmania strain. The strain of Lrishmania was identified by the Reference Center of WHO (Montpellier) as L. igfhntum MON 1 (MHOM/PT/93/CRE 41). The zymodeme of this strain is usually responsible for visceral leishmaniasis. The parasites were injected in the hamster by the intraperitoneal route to increase its virulence and maintained in McNeal, Novy & Nicolle (NNN) medium at 27’C for 8 days. Bulk culture of the infectious promastigote forms was initiated and propagated in RPM1 1640 medium (Eurobio. France) supplemented with 0.15% sodium bicarbonate (BiomCrieux. France), 15% heat-inactivated foetal calf serum (D.A.P.. France), 15% Schneider-medium (Gibco Ltd, United Kingdom), 1% L-glutamrne (Biomerieux, France) and 0.66% gentamicin (Pharmacie Centrale des HGpitaux, Paris, France). The promastigotes reached their infectious metacychc phase after an g-day period at 27°C. Animals. Male adult BALB/c mice (5 weeks old, 20 * 2 g) were purchased from IFFA CREDO, (L’arbresle, France). The infection model used was modified from Neal et al., 1985. The mam difference was the use of stationary promastigotes instead of amastigotes obtained from hamster spleen. Promastigotes were obtained after centrifugation and resuspended in normal saline solution. On day 0, mice were infected by i.v. injection (tail vein) with 10’ infective L. injbtunt promastigotes in a 0.1 ml volume. This procedure induced a heavy Leishmania liver burden after 12 days. The mice were divided randomly into three different groups: the control group (normal saline), the group treated with free pentamidine and the group treated with pentamidine-loaded methacrylate nanoparticles. Groups. In the control group, 12 mice received normal saline solution. The different doses were chosen according to our prevtous studies to obtain the median effective dose (ED,,} for each regimen (Fusai; et ai., 1994). In the bound pentamldine-treated groups, six mice received 0.05,0.09,0.17 or 0.24 mg kg- I of pentamidine-loaded nanoparticles on days 14, 16 and 18. In the free pentamidine base-treated groups, six mice received 0.57, 1.14 or 2.28 mg kg-’ of the free drug on days 14, 16 and 18. Except for the 0.24mgkgg’ dose (0.14 ml). 0.1 ml was injected via the tail vein.

Pentamidine

nanoparticles

in Leiskwaniu

Sacrifice. Twenty-one days after the initial infection, the animals were killed by cervical dislocation. The Guiding Principles for Biomedical Research involving animals were followed during all procedures (CIOMS, 1985). Treatment eficacr. The following to assess treatment efficacy:

parameters

burden

Control

=

were used

in livers

Parasite

of parasite

suppression

mean of Stauber count of the treated group mean of Stauber count of control group

burden

experiments

In infected mice treated with normal saline solution, there was a mean of 3.5 (range =3.3-3.7, N= 12) L. infanturn amastigotes per liver cell nucleus and a mean of 0.76 (range 0.74-0.79) amastigotes per spleen cell nucleus at the end of the 21-day period of experimentation. The average liver parasite burden reached was 12.7 x 10’ amastigotes (Stauber count) for the control group with lo7 promastigotes inoculated 21 days before as described in Table 1.

The number of amastigotes per 500 hepatocytes was calculated and related to the liver weight (mg), following the Stauber formula (Stauber et al., 1958). The percentage of parasite suppression was calculated as follows: Percentage

1363

mice

RESULTS

l Purasitr burdens: the liver and spleen parasite burdens were evaluated after Giemsa staining of the smears.

Parasite

infected

I

x 100.

Treatment

suppression in livers. The maximal percentage of parasite suppression was obtained with 0.24 mg kg-’ of pentamidine-loaded nanoparticles (63.7 +2.18%) (Table 1). Parasite suppression of 57.7+ 2.06% was obtained with 2.28 mg kg-’ of pentamidine isethionate. This result was not significantly different from that observed with a dose of bound pentamidine 10 times lower. The relationship between the percentage of parasite suppression and the dose is represented in Fig. 1. The MichaelisMenten model was used to fit the data obtained from groups treated with pentamidine-loaded nanoparticles (r = 0.94) or with isethionate pentamidine groups (r=O.999). The maximal effect (E,,,,,) calculated with pentamidine-loaded nanoparticles was significantly higher than that calculated with pentamidine isethionate (108.7% vs 65.8%).

in spleens

The number of amastigotes per 500 host cell nuclei was calculated and related to the spleen weight (g) following the Bradley & Kirkley formula (Bradley & Kirkley, 1977). Spleen parasite burdens were expressed ctL. infanturn units~ (LIU); LIU = amastigotes number xspleen weight (g).

per

1000

host

cell

nuclei-

Median effectioe doses: the ED,,, ED,, and ED,, (dosages of drug calculated to eliminate 25%. 50% and 90% of organisms compared with controls) were determined using a Michaelis-Menten model. . Weight of Ihlers and spleens: immediately after sacrifice, the livers and the spleens were removed and weighed. l

Stnristirul uzalwis. were expressed as Results mean+S.E.M. A one-way analysis of variance or a C/-test was performed to compare the influence of the various parameters. A P value lower than 0.05 was considered as statistically significant.

Table

l-Liver

Group

Amastigotes counted per 500 hepatocytes

Pentamidine 0.57 1.14 2.28

nanoparticle

in spleens.

count

10’

The maximal

with pen-

Percentage

of

suppression (%)

12.75+0.65

dose (mg kg-‘)

1600+52 1475+89 1139k42 801k55

isethionate

suppression

leishmaniasis in mice treated and free pentamidine”

Stauber

1745*90

Pentamidine-loaded 0.05 0.09 0.17 0.24

Parasite

suppression of experimental tamidine-loaded nanoparticles

Control

groups

Parasite

9.66i0.46 8.26+0.45 7.51+0.26 4.63+0.27

24.3+ 3.68b" 35.2f3.51b.' 41.7T2.49c 63.7k2.18'

7.39kO.43 6.22k0.67 5.39i0.26

42.1k3.37b 51.255.28 57.7F2.06

dose (mg kg-‘)

1053+24 864k87 758+21

“The suppression percentage was calculated using Stauber count vs untreated mice. Data are expressed as mean & S.E. for six mice per group, except the control group (n= 12). U-test was used to compare the different groups. ~P<0.01;~P<0.001. b0.05 vfr 0.17 and 0.57; ‘vs 0.24, 1.14 and 2.28. ‘0.09 vs 1.14; ‘vs 0.24 and 2.28. ‘0.17 vs 0.24 and 2.28. ‘0.24 vs 0.57. b0.57 vs 2.28.

per-

1364

R. Durand

et 01

pentamidine groups, no significant observed between livers and spleens.

difference

was

Evaluation of the eifective doses The pentamidine-loaded nanoparticles were sixfold more active than the free drug. The median effective doses of free and bound pentamidine were, respectively, l.O6mgkgand O.l7mgkg-‘. The other values of the effective doses are presented in Table 3.

-

Influence of treatment on the weights of livers and spleens The weights of livers and spleens treated by pentamidine-loaded nanoparticles were significantly lower than those of untreated mice (Table 4). In the isethionate pentamidine group, the liver weights were significantly lower than those of untreated mice. whereas there was no difference in spleen weights.

I 0.01

Dose (mgikg) Fig. 1. Semilogarithmic plot of suppression of experimental leishmaniasis in mice treated with free pentamidine (0, doses ranging from 0.57 to 2.28mg kg-‘) or with pentamidineloaded methacrylate nanoparticles (0, doses ranging from 0.05 to 0.24mg kg- ‘). The suppression percentage was calculated using the Stauber count vs untreated mice as described in Materials and methods. Suppression was expressed as the percentage of the maximal infestation. Data were fitted to a Michaelis-Menten model (r=0.94) for pentamidine-loaded nanoparticles and (r> 0.999) for isethionate pentamidine. Each point represents the mean + S.E. for six mice per group, except the control group (n = 12).

DISCUSSION

The results showed that bound pentamidine was more potent than free drug against L. infanturn in our BALB/c mice model. The dramatically low value obtained for the bound pentamidine ED,, (0.17 mg kg-‘) demonstrated the ability of methacrylate carriers to increase the activity and decrease the toxicity of pentamidine for the treatment of visceral leishmaniasis. It was not possible to calculate the EDgo for free pentamidine because the dose-response curve reached a plateau near 60% of parasite suppression. The extrapolated value of ED,, for nano-

centage of parasite suppression was obtained with 0.24 mg kg- ’ of pentamidine-loaded nanoparticles (79.0+2.7%) (Table 2). The percentages of spleen parasite suppression were significantly higher than those in livers with bound-pentamidine at the doses of 0.09, 0.17 and 0.24 mg kg-’ liver (PC 0.01). In free Table

2-Spleen

Group

suppression of experimental tamidine-loaded nanoparticles Amastigotes counted per 500 nuclei cells

Control

382+

Pentamidine-loaded 0.05 0.09 0.17 0.24 Pentamidine 0.57 1.14 2.28

Leishmania inf’ntum unit (LIU)

12

nanoparticle 232k9.4 189k9.2 141*4.8 75k4.2

leishmaniasis in mice treated and free pentamidined

with pen-

Percentage suppression

of (X)

183.1+4.6 dose (mg kg-‘)

isethionate dose (mg kg-‘) 215k8.6 149k5.2 119k4.5

11 1 * 7.4 91+6.3 68k4.2 38+4.7 82k5.7 66 k 6.2 52+3.8

39.4 + 3.$=d 50.3 +2.8b.d 62.9z3.1” 79.0+2.7’.’ 55.3k4.7 64.2k3.8 71.5k6.1

“The suppression percentage was calculated using the Leishmania i@nfum untreated mice. Data are expressed as mean f S.E. for six mice per group, control group (n= 12). CT-test was used to compare the different groups. bP<0.05; “Pt0.01; d~
count vs except the 1.14and

Pentamidine Table

3-Efficacy

nanoparticles

of pentamidine

(F) nanoparticles

“The effective

doses (ED)

of treatment spleen”

on the weight Organ

Group Control

(n = 12)

Pentamidine-loaded 0.05 0.09 0.17 0.24 Pentamidine 0.57 1.14 2.28

(B)

were calculated of liver and

(wet)

Liver

Spleen

1.84*0.04

0.24+0.01

nanoparticle dose (mg kg- ‘) (n = 24) 1.63k0.06b 0.17+0.02’ 1.53*0.036 0.18+0.01’ l.68+0.05b 0.18+0.02c 1.59 f 0.07’ 0.16f0.01d

isethionate

in

ED,, (w kg-‘)

Free pentamidine Pentamidine-loaded Ratio ED (F/B)

&Influence

Leishmania infected mice

(bound or free) mice”

Treatment

Table

in

dose (mg kg-‘) 1.58&-0.041’ 1.72+0.072 1.65+0.075”

(n=

18) 0.19 + 0.09 0.22+0.13 0.2250.04

“Mice were treated at day 14, day 16 and day 18 and sacrificed 3 days later. Organs were weighed immediately after sacrifice. The results are expressed as mean + SE. U-test was used to compare the control group to other groups of treatment. “P
particle-bound pentamidine was 1 mg kg- ’ This dose would not be expected to be toxic. Therefore, the use of slightly higher doses of nanoparticle-bound pentamidine may totally clear parasites from the liver without major toxicity for the mice. Higher doses of bound pentamidine were not tested, as 100pM was found to be the optimal loading pentamidine concentration. Higher concentrations induce free pentamidine in the medium which might hinder the interpretation of results (Paul et aI., in press). Therefore, at the same concentration, administration of higher doses would imply more than three injections modifying the animal model. Unloaded nanoparticles were not tested in this work because their lack of efficacy has been described previously (Fusdi et al., 1994). Red cell ghosts containing pentamidine in a L. donorantSyrian hamster model led to similar results, with a reported ED,, of 0.38 mg kg-‘. Immunoglobulin ghost red cells had a better efficacy, but their delicate use had limited their further development (Berman et al., 1986). These authors did not report a ratio value (free pentamidine vs bound pentamidine) because the free drug did not reach 50% of parasite suppression (ED,,> 50mg kg-‘). In our model, 50% of parasite

0.201 0.063 3.19

1365

Leishmaniu infanturn infected

(rni$

‘)

(rni$

1.06 0.17 6.24

using a Michaelis-Menten

‘) 1

model.

suppression was obtained for 1.06mg kg-’ with free pentamidine. The ratio value (free pentamidine vs bound pentamidine) was 6.25, which represented a major enhancement of activity. Other antileishmanial drugs such as amphotericin B were targeted and tested in viva. In a L. donvani-BALB/c mice model, the ratio value (ED, Ambisome@ vs ED, conventional amphotericin) was 3.7 after 3 injections of drugs (Croft ef al., 1991). The ED,,, value of liposomal amphotericin B was 0.256 mg kg-‘, which is comparable with the ED,, value obtained for the pentamidine-loaded nanoparticles (Table 3). Other authors showed a 3.3-fold improvement of the efficacy of primaquine, an antimalarial compound, by targeting it on poly (D, L-lactide) (PLA) nanoparticles against I,. donovani in a BALB/c mice model. However, these authors reported a relatively high ED,, value (6.6 mg kg-‘) for bound primaquine (Rodrigues et al., 1994). These findings may be due to the low intrinsic activity of primaquine against L. donovani. The better activity of pentamidine-loaded nanoparticles vs free pentamidine can be explained by the efficacy of the drug delivery system. The decrease of liver weights probably reflected its leishmanicidal activity. Previous studies have shown that methacrylate nanoparticles were cleared from the blood stream shortly after their i.v. administration in mice and that they concentrated mainly in the mononuclear phagocyte system, particularly in Ktipffer cells (A. Rolland, 1987. Mise au point et applications de nanospheres a base de copolymeres mtthacryliques. InttrCt pour la vectorisation d’agents cytostatiques (anthracyclines). Ph.D. thesis, Universite de Rennes, France). The significant decrease of spleen parasite burden and of the spleen weight observed in mice treated with bound pentamidine suggested that bound pentamidine could concentrate in the spleen as well as in the liver. The percentages of parasite suppression in spleens were higher than those in livers in bound-pentamidine groups. However, the interpretation of spleen parasite burdens may be critical as the distribution of splenic cells may vary following immunological events. In that way, the determination of parasite burden in the liver by the Stauber method appeared more accurate. The

R. Durand et al.

1366

access to the spleen was probably due to iterative regimens saturating the liver (Carter et al., 1989). Indeed, a single dose of colloidal carrier did not allow access to the spleen because all the carrier was entrapped at the first passage in the liver (Carter et al., 1988). Moreover, ultrastructural studies have shown that these nanoparticles reached the parasitophorous vacuole and were located close to Leishmania (Fusai’ et al., 1997). Physicochemical studies have demonstrated that pentamidine was bound on methacrylate nanoparticles by ionic interaction (Paul et al., in press) and that release of pentamidine reached 50% of the loaded drug at pH 5. Thus, the acidic pH found in the parasitophorous vacuole (Antoine et al., 1990) may allow the in situ release of pentamidine from its carrier. These results obtained with L. infanturn paralleled those obtained with L. major and confirm the interest in methacrylate nanoparticles in enhancing the activity of pentamidine. Methacrylate polymers are known to be slowly biodegradable (Rolland, 1987. Ph.D. thesis), but these polymers are well tolerated in implants in humans for many years (Danilewicz-Stysiak, 1980) and were tested in humans for the treatment of hepatocarcinoma (Rolland, 1989). Drug targeting arises as a new tool in the fight against visceral leishmaniasis at a time when new anti-leishmanial drugs are still needed. Results obtained on L. infanturn are of particular interest, because L. infanturn is the Leishmania species most frequently isolated from HIV patients in Mediterranean countries. Bound pentamidine-methacrylate nanoparticles now require further investigation, particularly in immunodepressed mice models in order to evaluate their activity in a model more related to the co-infected HIV-Leishmania patient status.

Acknowledgements-We thank Julie Ducharme and Christine Fernandez for their linguistic assistance. This investigation received financial support from the UNDPjWORLD BANK/ WHO special Programme for Research and Training in Tropical Diseases and the ctAgence Nationale de Recherche co&e le Sida (ANRS))). We thank Prof. Leverge for providing us with unloaded polymethacrylate nanoparticles.

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in Leishmaniu

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