A S S E S S M E N T OF T H E ACTIVITY OF ATOVAQUONE-LOADED NANOCAPSULES IN T H E TREATMENT OF ACUTE A N D CHRONIC M U R I N E TOXOPLASMOSIS
SORDET F.*, AUMJAUD Y.**, FESSI H.** & DEROUIN F.*
S u m m a r y :
The aim of this work was to develop a new pharmaceutical form of atovaquone and to study its activity against Toxoplasma gondii in vitro and in vivo. Nanocapsules were chosen as the oral dosage form of administration. An analytical method was developed to determine the drug content in nanocapsules. The stability of these nanocapsules were assessed by following drug content, size, pH and osmolarity for a period of six months. The in vitro activity of atovaquone-loaded nanocapsules against
tachyzoites of T gondii (RH stain) was comparable to its suspension form. In vivo studies were carried out in murine models of acute and chronic toxoplasmosis. Mice acutely infected with the virulent RH strain were orally treated with a dose regimen of
15 mg/kg/day for 10 days, starting from day 1 post-infection.
75 % of the mice receiving atovaquone-loaded nanocapsules survived 3 0 days post-infection, compared to none of untreated controls and none of mice treated with the suspension with the same dose regimen. In mice chronically infected by the COUL or the M E 4 9 strain (Type II strains), then treated for six weeks, treatment with atovaquone (15 mg/kg/d, nanoparticles or suspension) resulted in a decrease of brain parasitic burden, which was significantly more pronounced in ME49-infected mice and in those treated with drug-loaded nanocapsules. These results show that the sensibility of T.gondii to atovaquone is different according to the strains and that the activity of atovaquone in the treatment of toxoplasmosis is enhanced when administered in nanoparticular form.
KEY W O R D S : atovaquone, Toxoplasma gondii, cerebral toxoplasmosis, nanocapsules, murine models.
Résumé : ÉVALUATION D E L'ACTIVITÉ D E NANOCAPSULES D ' A T O V A Q U O N E DANS LE TRAITEMENT D E LA T O X O P L A S M O S E MURINE AIGÜE E T CHRONIQUE
L'objectif de ce travail était de développer une nouvelle forme galénique de l'atovaquone (nanocapsules per os) et d'en étudier l'activité sur Toxoplasma gondii in vitro et in vivo. Une méthode analytique a été mise au point afin de déterminer la quantité d'atovaquone intégrée dans les nanocapsules. La bonne stabilité des nanocapsules a été contrôlée par mesure de leur taille, leur pH, leur osmolarité et leur concentration en atovaquone, sur une période de six mois. L'activité in vitro des nanocapsules ddtovaquone sur les tachyzoites de T . gondii (souche RH) s'est révélée comparable à celle observée avec la forme suspension.
Les études in vivo ont été menées sur des modèles murins de toxoplasmose aiguë et chronique. Des souris infectées par souche virulente RH ont été traitées pendant dix jours par une dose d'atovaquone (suspension ou nanoparticulesj de 15 mg/kg/jour, de J1 à J10 post-infection. 75 % des souris recevant la forme nonoparticule d'atovaquone étaient vivantes 30 jours après l'infection alors qu'aucune n'avait survécu parmi les souris non traitées ou traitées par la suspension d'atovaquone. Chez des souris présentant une toxoplasmose chronique (souche COUL et ME49 de type II), un traitement de six semaines par atovaquone (15 mg/kg/j, suspension ou nanocapsules, a permis une baisse importante de la charge parasitaire cérébrale. Cette baisse fut significativement plus marquée chez les souris infectées par la souche ME49 et chez celles traitées par la forme nanocapsulaire.
Ces résultats montrent donc que la sensibilité à l'atovaquone est variable selon les souches et que la forme nanocapsulaire est d'une efficacité supérieure à la forme suspension dans le traitement de la toxoplasmose.
MOTS CLES : atovaquone, Toxoplasma gondii, toxoplasmose cérébrale, nanoparticules, modèle murin.
INTRODUCTION
D
e s p i t e t h e w i d e s p r e a d u s e o f s p e c i f i c c h e - m o p r o p h y l a x i s , t o x o p l a s m i c e n c e p h a l i t i s is still a f r e q u e n t o p p o r t u n i s t i c i n f e c t i o n o c c u r - ring in p a t i e n t s w i t h A I D S ( K a t l a m a , 1 9 9 6 ) . I n d e e d , d r u g s l i k e c o t r i m o x a z o l e o r c o m b i n a t i o n s o f d a p s o n e* L a b o r a t o i r e d e P a r a s i t o l o g i e - M y c o l o g i e , H ô p i t a l S a i n t - L o u i s , 1, a v e n u e C l a u d e - V e l l e f a u x , 7 5 4 7 5 Paris C e d e x 1 0 , F r a n c e .
** L a b o r a t o i r e d e P h a r m a c i e G a l é n i q u e , I S P B - F a c u l t é d e P h a r m a c i e , 8, a v e n u e R o c k e f e l l e r , 6 9 3 7 3 L y o n C e d e x 0 8 , F r a n c e .
C o r r e s p o n d e n c e : F r a n c i s D e r o u i n .
Tel: 3 3 1 4 2 4 9 9 5 0 3 - F a x : 3 3 1 4 2 4 9 4 8 0 3 .
p l u s p y r i m e t h a m i n e p r o v e d efficient in p r e v e n t i n g r e l a p s e s o f p r e v i o u s l y a c q u i r e d Toxoplasma infection, a n d t h e l a r g e a p p l i c a t i o n o f p r o p h y l a c t i c r e c o m m a n - d a t i o n s h a v e r e s u l t e d in m a r k e d d e c r e a s e o f t h e inci- d e n c e o f t o x o p l a s m i c e n c e p h a l i t i s ( R i c h a r d s et al., 1 9 9 5 ; U S P H S / I D S A , 1 9 9 5 ; Leport et al., 1 9 9 6 ; Girard et al., 1 9 9 6 ) . H o w e v e r , t h e drugs that a r e p r e s e n t l y r e c o m m e n d e d for p r o p h y l a x i s o f t o x o p l a s m o s i s ( a n d p n e u m o c y s t o s i s ) a r e n o t effective against brain cysts, a n d d o n o t e r a d i c a t e t h e parasite from t h e o r g a n i s m . T h i s limit justifies t h e i r l i f e - l o n g a d m i n i s t r a t i o n t o p a t i e n t s at risk (i. e. t h o s e w h o a r e s e r o p o s i t i v e for
T. gondii), b u t a l s o u n d e r l i e s t h e n e e d for n e w c o m - p o u n d s t h a t w o u l d e r a d i c a t e c y s t s in c h r o n i c a l l y
Parasite, 1 9 9 8 , 5, 2 2 3 - 2 2 9
Mémoire 223
Article available athttp://www.parasite-journal.orgorhttp://dx.doi.org/10.1051/parasite/1998053223
SORDET F., AUMJAUD Y , FESSI H. & DEROUIN F.
infected patients and more particularly drugs that could b e given orally.
Atovaquone might b e such a candidate drug, as several studies h a v e s h o w n its activity against Toxoplasma cysts, b o t h in vitro and in vivo (Huskinson et al., 1 9 9 1 ; Araujo et al., 1 9 9 2 ) . H o w e v e r , b o t h clinical and experimental data indicate that t w o main factors c o u l d limit its use for primary prophylaxis. First, the b i o availability o f a t o v a q u o n e is low (47 % in patients with AIDS, w h e n suspension is taken with fatty food) with an important inter-individual variability ( H u g u e s et al., 1 9 9 1 ) . S e c o n d , e x p e r i m e n t a l studies have d e m o n s trated the variable sensitivity o f different strains o f
Toxoplasma gondii to this drug ( T o m a v o et al., 1 9 9 5 ; Araujo et al., 1 9 9 1 ) .
T h u s , i m p r o v e m e n t o f the bioavailability o f atova
q u o n e , through the use o f drug-loaded carriers, could represent a valuable solution in increasing intra-cellular and tissue drug concentrations (Kreuter, 1 9 9 4 ; Fusai et al, 1 9 9 7 ) and thus achieving inhibitory levels effective o n less sensitive strains. B e c a u s e o f the physicochemical characteristics o f the drug, i. e. p o o r water solubility, nanocapsules have b e e n c h o s e n as a colloidal drug car
rier. Nanocapsules have a higher drug loading capa
city than n a n o s p h e r e s and have the advantages over other drug carriers such as liposomes o f being physi
cally and chemically m o r e stable during storage (Pui- sieux et al., 1 9 9 4 ) . Furthermore, they are stable in the p r e s e n c e o f biliary salts (Devissaguet et al., 1 9 9 2 ) , w h i c h is a major requirement for oral administration.
T h e main objective o f this w o r k was to prepare stable drug-loaded nanocapsules and to c o m p a r e the efficacy o f these colloidal drug carriers to the water-based sus
p e n s i o n in the treatment o f acute and c h r o n i c t o x o plasmosis.
MATERIALS AND METHODS
D R U G P R E P A R A T I O N S
A
t o v a q u o n e w a s o b t a i n e d from G l a x o - W e l l - c o m e ( F r a n c e ) u n d e r two forms: a p o w d e r e d form which was used to prepare nanocapsules and for in vitro studies, and a liquid suspension form.T h e p o l y m e r Poly ( D , L-lactide) (PLA) o f molecular weight 8 8 , 0 0 0 w a s p u r c h a s e d from B o e r i n g h e r Ingel- h e i m ( G e r m a n y ) ; o l e i c acid, b e n z y l b e n z o a t e a n d c a p r y l i c / c a p r i c a c i d o r Miglyol 812® from C o o p e r (Melun, F r a n c e ) ; Labrafac® from G a t t e f o s s e (Lyon, F r a n c e ) . T h e surfactant used was S y n p e r o n i c PE/F68®
from ICI (Clamart, France) and a phospholipid mixture, Epikuron 170® was purchased from Lucas Mayer (Ham
burg, G r e m a n y ) . All other solvants w e r e o f appropriate grade and w e r e o b t a i n e d from P r o l a b o (Paris, F r a n c e ) and Aguettant (Lyon, F r a n c e ) .
Prior to the preparation o f nanocapsules, solubility stu
dies w e r e c o n d u c t e d at 4 °C and r o o m temperature to determine the m a x i m u m solubility o f a t o v a q u o n e in e a c h o f the following oils: oleic acid, benzyl b e n z o a t e , Miglyol 812®, or Labrafac®. T h e solubility study has also served to determine storage conditions o f drug-loaded n a n o c a p s u l e s .
Preparation o f n a n o c a p s u l e s
F r e e a n d a t o v a q u o n e - l o a d e d n a n o c a p s u l e s o f PLA were prepared using a modification o f the process des
cribed b y Fessi et al. ( 1 9 8 9 ) . 125 mg o f PLA was dis
solved into an organic p h a s e consisting o f 25 ml a c e t o n e , 100 mg Epikuron 170® and 0.5 ml o f a c h o s e n oil ( b e n z y l b e n z o a t e , Miglyol 812®, Labrafac® or oleic acid) either free or containing a required quantity o f atovaquone. T h e c o m b i n e d organic phase was injected into 5 0 ml o f water containing 1 0 0 mg o f S y n p e r o n i c PE/F68® under moderate magnetic stirring. T h e a c e t o n e and s o m e water w e r e e v a p o r a t e d under r e d u c e d pres
sure. T h e resulting n a n o c a p s u l e preparation was fil
tered o n sintirred glass with a porosity range o f 9- 15 micrometers and the final volume adjusted to 10 ml.
Sufficient quantities o f b o t h free and a t o v a q u o n e - loaded n a n o c a p s u l e s w e r e prepared for stability and in vivo studies.
N a n o c a p s u l e s w e r e k e p t in s e a l e d glass bottles at r o o m temperature and a w a y from light throughout the study.
D o s a g e o f a t o v a q u o n e
Atovaquone was assayed b y high-performance liquid chromatography using a modified method o f DeAngelis et al. ( 1 9 9 4 ) . T h e system consisted o f a LiChospher 100 RP, C18 reverse phase column, fitted with a preco- lumn. T h e autosampler, p u m p and UV detection device w e r e integrated in the Hewlett Packard 1050® system.
T h e mobile phase consisted o f acetonitrile/ water/gla- cial acetic acid (85:15:5 v / v ) . T h e volume o f sample injected was 10 µl and was eluted at a constant flow rate o f 3.0 ml/min. Atovaquone was detected by absorption at 2 5 4 nm. An average retention time o f 1.7- 1.8 minutes was obtained in the linear response range o f 1 2 . 4 - 198.0 ug/ml with a correleation coefficient o f 0.999.
Drug c o n t e n t o f n a n o c a p s u l e s and stability studies Total drug concentration in n a n o c a p s u l e s w a s deter
m i n e d b y dissolving the p o l y m e r m e m b r a n e in m o b i l e p h a s e and dosing for a t o v a q u o n e . Encapsulated drug was calculated from the difference b e t w e e n total drug and that in the a q u e o u s p h a s e . A q u e o u s p h a s e drug c o n t e n t w a s determined in the clear filtrate following separation o f n a n o c a p s u l e s from the a q u e o u s medium by a c o m b i n e d ultrafiltration-centrifugation t e c h n i q u e (Ultrafree MC® 1 0 , 0 0 0 D a l t o n s , Millipore, F r a n c e ) .
224 Mémoire P a r a s i t e , 1 9 9 8 , 5, 223-229
A T O V A Q U O N E - L O A D E D N A N O C A P S U L E S IN T H E T R E A T M E N T O F M U R I N E T O X O P L A S M O S I S
Nanocapsule yield and encapsulation rate were h e n c e calculated and used in stability studies. Nanocapsules with a t o v a q u o n e concentrations o f 1,000 ug/ml w e r e prepared.
Nanocapsule m e a n size was followed by Nanosizer N4® (Coultronics, Margency, F r a n c e ) . Osmolarity and pH m e a s u r e m e n t s w e r e used to follow n a n o c a p s u l e stability over six months.
PARASITES-STRAINS O F TOXOPLASMA GONDII
T h e RH strain o f T. gondii was used for in vitro stu
dies and in vivo e x p e r i m e n t s in the model o f acute toxoplasmosis. This strain was maintained in Swiss m i c e b y 3-days interval intraperitoneal p a s s a g e ; for e a c h experiment, tachyzoites w e r e harvested from the peritoneal cavity o f m i c e infected two clays earlier then centrifuged at 1,500 G. T h e pellet was resus- p e n d e d in sterile p h o s p h a t e buffer solution pH 7.2 ( P B S ) then parasites w e r e adjusted at a concentration o f 1 04/ m l .
T w o different strains o f T. gondii b e l o n g i n g to the type II ( H o w e et al., 1 9 9 5 ) w e r e used for inducing c h r o n i c infections in mice. This type o f strain was selected as representative o f the majority o f strains iso
lated in HIV infected individuals ( H o w e et al., 1 9 9 7 ) , and responsible for chronic t o x o p l a s m i c infection in m i c e . T h e ME 4 9 strain (kindly provided by Prof.
M.L. Dardé, Limoges, F r a n c e ) was used, since previous e x p e r i m e n t s b y Huskinson et al. ( 1 9 9 1 ) s h o w e d that treatment o f chronically-infected mice with atovaquone could significantly reduce the cyst burden in brain and induce ultrastructural alteration o f the cyst structure.
T h e COUL strain ( B e a u v a i s et al., 1 9 8 2 ) , which has b e e n extensively studied in our laboratory, was also s e l e c t e d b e c a u s e it p r o d u c e s c h r o n i c infection with mature cysts and stable brain infection (Derouin et al., 1 9 9 1 ) . T h e s e strains w e r e maintained in mice under a c h r o n i c form b y 6-months interval passages in Swiss W e b s t e r m i c e . F o r e a c h e x p e r i m e n t , c y s t s w e r e o b t a i n e d from the brain o f m i c e infected at least six months previously.
IN VITRO E X P E R I M E N T S
In vitro studies w e r e carried out using MRC5 fibroblast tissue cultures as previously described ( D e r o u i n et al.,
1989). Briefly, confluent m o n o l a y e r s prepared in 96- w e l l t i s s u e c u l t u r e p l a t e s w e r e i n o c u l a t e d w i t h 1,500 tachyzoites o f the RH strain. After four hours, serial dilutions o f e a c h drug preparation w e r e added into the medium and cultures w e r e incubated for an additional 72 hours. Toxoplasma growth was a s s e s s e d b y an enzyme-linked immunoassay performed directly o n the fixed cultures. For e a c h well, results w e r e expressed as optical density values. T e n concentrations
o f a t o v a q u o n e (free drug and n a n o c a p s u l e s ) , ranging b e t w e e n 0.001 and 1 µ g / m l , w e r e tested; e a c h c o n c e n tration was studied in eight replicate wells and in t w o replicate culture plates. Regression models w e r e used to summarize the in vitro dose-effect relationship and to determine the 5 0 % inhibitory concentration ( I C5 0) .
IN VIVO E X P E R I M E N T S
F e m a l e Swiss W e b s t e r m i c e weighing 1 8 - 2 0 grams (Iffa Credo, F r a n c e ) w e r e used for optimum reprodu
cibility and breeding facilities.
Acute infection by the RH strain were induced by intra
peritoneal injection o f 2 . 1 04 freshly harvested tachy
zoites.
Chronic toxoplasmosis w e r e induced by oral infection with ten cysts obtained from the brain o f mice that had b e e n infected for at least six months. Fourty-five mice w e r e infected with e a c h strain; four months after infec
tion, m i c e w e r e allocated in the different treatment groups.
TREATMENT P R O T O C O L S
Treatment o f acute toxoplasmosis
At day 1 ( D 1 ) post infection, mice w e r e allocated in four g r o u p s : o n e c o n t r o l g r o u p o f t e n i n f e c t e d untreated mice and three treatment groups o f ten mice each: o n e group was treated with unloaded n a n o c a p sules, o n e group was treated with 15 mg/kg/day o f ato
v a q u o n e - l o a d e d n a n o c a p s u l e s , and o n e group was treated with 15 m g / k g / d a y o f a t o v a q u o n e suspension.
Treatments were administered daily by tube feeding for a 10-day period. This dosage and duration o f treatment w a s selected as it was previously found only partially effective in mice treated with the liquid suspension o f a t o v a q u o n e (Araujo et al., 1 9 9 1 ; Romand et al., 1 9 9 3 ) and thus would b e appropriate to reveal a potentially higher efficiency o f the treatment with atovaquone- loaded n a n o c a p s u l e s .
Treatment o f c h r o n i c toxoplasmosis
From the results o f treatment o f acute toxoplasmosis, the d o s a g e o f 15 m g / k g / d a y was selected for treatment o f c h r o n i c infection. For e a c h strain o f T. gondii, mice infected for four months w e r e allocated in the follo
w i n g t r e a t m e n t g r o u p s ( 1 0 m i c e in e a c h g r o u p ) : untreated controls, treatment with 15 m g / k g / d a y o f ato
v a q u o n e - l o a d e d n a n o c a p s u l e s , and treatment with 15 m g / k g / d a y o f a t o v a q u o n e suspension. Mice w e r e treated daily by gavage during six w e e k s , five days a w e e k .
ASSESSMENT O F T H E R A P E U T I C E F F I C A C Y
Survival o f mice was recorded in each treatment group.
In acutely infected mice, parasite burdens w e r e deter-
Parasite, 1 9 9 8 , 5, 2 2 3 - 2 2 9
Mémoire 2 2 5
SORDET F., AUMJAUD Y., FESSI H. & DEROUIN F.
m i n e d at D 6 post infection in b l o o d , brain and lung o f t w o m i c e in e a c h treatment group. In chronically- infected mice, brain cyst c o u n t and parasitic burdens w e r e estimated at the begining and at the e n d o f treat
ment. At e a c h date, five mice w e r e sacrificed; their brains w e r e removed, weighed, then h o m o g e n e i z e d in 2 ml o f P B S , using an Ultra Turrax T 2 5 h o m o g e n i z e r (Staufen, G e r m a n y ) . T h e resulting h o m o g e n a t e was e x a m i n e d microscopically for cyst counts and titrated for parasitic burdens. A t o v a q u o n e concentrations have not b e e n determined in b l o o d o r tissue h o m o g e n a t e s . Cysts w e r e c o u n t e d o n four blinded samples o f 50 µl o f e a c h brain h o m o g e n a t e (treated, untreated), and the total n u m b e r o f cyst w a s r e c o r d e d and x 1 0 0 to obtain an estimate o f the total brain cysts count.
Parasitic titration in brain was estimated according to the culture technique described by Piketty et al. ( 1 9 9 0 ) . Briefly, serial fourfold dilutions o f the brain suspen
sion w e r e prepared in basal culture m e d i u m and 4 0 pi o f e a c h dilution w a s inoculated in duplicate in 96-well MRC5 fibroblast tissue culture plates. After 96 hours o f incubation at 37 °C in a moist 5 % C 02- 9 5 % air atmos
phere, plates w e r e fixed with c o l d methanol then air- dried. Cultures w e r e e x a m i n e d for T. gondii b y m e a n s o f an indirect immunofluorescence assay, using a poly
clonal rabbit anti-antibody as first antibody and a fluo- r e s c e i n - l a b e l e d anti-rabbit IgG conjugate as s e c o n d antibody; plates w e r e e x a m i n e d under a f l u o r e s c e n c e m i c r o s c o p e and the p r e s e n c e o f fluorescent parasites w a s r e c o r d e d in e a c h well. For e a c h brain h o m o g e nate, the titer w a s defined as the last dilution at w h i c h the tissue culture c o n t a i n e d at least o n e parasite focus.
T h e parasitic burden per gram o f tissue w a s calculated as the reciprocal titer in tissue culture/weight (milli
grams) x 1,000.
STATISTICS
In m i c e , the survival rates w e r e estimated by the Kaplan-Meier product limit m e t h o d and c o m p a r e d using the log-rank test. T h e m e a n value for the para
site burden from five m i c e ( ± 1 standard error) w a s calculated for e a c h time point. Statistical c o m p a r i s o n o f the parasitic loads w e r e determined according to the Mann-Whitney non-parametric test; p values < 0.05 w e r e c o n s i d e r e d as significant.
RESULTS
PREPARATION OF ATOVAQUONE-LOADED NANOCAPSULES
S
tability studies s h o w e d that the highest possible payload could b e a c h i e v e d b y using benzyl b e n - zoate as the oily c o r e , reaching a concentration o f 24.2 m g / m l w h e r e a s m a x i m u m c o n c e n t r a t i o n s in other solvent oils w e r e < 7.5 m g / m l .With benzyl b e n z o a t e , the m a x i m u m possible c o n c e n tration o f a t o v a q u o n e that c o u l d b e l o a d e d per ml o f n a n o c a p s u l e s u s p e n s i o n w a s estimated to b e 1.2 mg.
Solubility studies also s h o w e d that a t o v a q u o n e formed needle-shape crystals in the oily p h a s e at 4 °C and sto
rage at this temperature w o u l d break the structure o f the n a n o c a p s u l e s . F r o m t h e s e results, a t o v a q u o n e n a n o c a p s u l e s w e r e prepared and stored at r o o m tem
perature until use.
T w o b a t c h e s w e r e prepared at 1 m g / m l ( T a b l e I ) . At this concentration, n a n o c a p s u l e yields w e r e respecti
vely 8 4 . 5 % and 8 2 . 9 % and encapsulation rates w e r e 100 % for b o t h b a t c h e s , s h o w i n g that a t o v a q u o n e c o u l d b e e n c a p s u l a t e d into n a n o c a p s u l e s with a rea
s o n a b l e yield. Stability studies w e r e performed o n the s e c o n d batch, w h i c h w a s also used for experimental studies o n T. gondii. Size distribution and polydisper- sity s h o w e d that the n a n o c a p s u l e s w e r e stable o v e r a period o f six m o n t h s despite a m o d e r a t e and constant decline in size, from a m e a n value o f 2 0 6 n m at the date o f preparation ( t 0 ) to 170 nm at six months. T h e increase in osmolarity ( 6 m O s m at t0 to 21 m O s m at six m o n t h s ) and the d e c r e a s e in pH (pH 4 . 2 3 at t0 to pH 3.10 at six m o n t h s ) that w e r e o b s e r v e d are in favor o f a m o d e r a t e degradation o f the p o l y m e r m e m b r a n e , corresponding to a 10 % biodegradation o f PLA into lactic acid.
IN VITRO EXPERIMENTS ON TOXOPLASMA GONDII T h e in vitro activity o f the s u s p e n s i o n and the n a n o capsule forms o f a t o v a q u o n e o n Toxoplasma gondii (RH strain) w a s estimated in two separate e x p e r i m e n t s for c o n c e n t r a t i o n s ranging b e t w e e n 1 and 1 0- 3 mg/1.
T h e results p r e s e n t e d o n figure 1 for o n e representa
tive e x p e r i m e n t s h o w e d that the inhibitory effect o n Toxoplasma growth according to the c o n c e n t r a t i o n
Batch
C o n c e n t r a t i o n ( m g / m l )
Y i e l d (%)
E n c a p s u l a t i o n rate (%)
A v e r a g e s i z e
( n m ) P o l y d i s p e r s i t y
O s m o l a r i t y
( m O s m ) p H
1 1 8 4 . 5 3 100 2 7 3 0
2 0 = 0 ) 1 8 2 . 9 1 100 2 0 6 0 6 4 . 2 3
2 0=6 m o n t h s ) 1 8 1 . 8 2 1 0 0 1 7 0 2 21 3.1
T a b l e I. - A t o v a q u o n e l o a d e d n a n o c a p s u l e s : yield, e n c a p s u l a t i o n rate, a v e r a g e s i z e , p o l y d i s p e r s i t y , o s m o l a r i t y a n d p H o f t h e p r e p a r e d b a t c h e s .
226 Mémoire P a r a s i t e , 1 9 9 8 , 5, 2 2 3 - 2 2 9
A T O V A Q U O N E - L O A D E D N A N O C A P S U L E S I N T H E T R E A T M E N T O F M U R I N E T O X O P L A S M O S I S
Fig. 1. — In vitro i n h i b i t o r y e f f e c t o f a t o v a q u o n e s u s p e n s i o n a n d n a n o c a p s u l e s o n Toxoplasma gondii g r o w t h . M e a n o p t i c a l d e n s i t y v a l u e s for ELISA v e r s u s c o n c e n t r a t i o n o f a t o v a q u o n e s u s p e n s i o n ( • ) o r n a n o c a p s u l e s ( • ) . F o r each c o n c e n t r a t i o n t h e m e a n o p t i c a l d e n sity v a l u e s ± s t a n d a r d e r r o r w e r e c a l c u l a t e d f r o m 8 r e p l i c a t e c u l t u r e w e l l s . I C 5 0 = 5 0 % i n h i b i t o r y c o n c e n t r a t i o n .
w a s roughly similar for b o t h preparations. From the regression curve analysis, the I C5 0 w e r e estimated at a c o n c e n t r a t i o n o f 0.04 µ g / m l with the suspension and 0.07 p g / m l for a t o v a q u o n e - l o a d e d n a n o c a p s u l e s . TREATMENT OF TOXOPLASMOSIS
Acute t o x o p l a s m o s i s
In this e x p e r i m e n t , assessment o f treatment efficacy w a s b a s e d o n the survival in e a c h group o f m i c e a n d the determination o f parasitic b u r d e n in b l o o d , brain and lungs in two m i c e at D 6 post-infection. Untreated m i c e a n d m i c e treated with u n l o a d e d n a n o c a p s u l e s died within s e v e n days (Fig. 2 ) ; in b o t h groups, m i c e p r e s e n t e d high parasitic b u r d e n s with a m e a n r e s p e c tive value o f 4 . 1 0 a n d 5.01 log parasites/ml in b l o o d , 5.75 a n d 5.33 log parasites/g in the brain a n d 6.87 and 6.80 log parasites/g in the lungs. W h e n c o m p a r e d to the treatment with the s u s p e n s i o n o f a t o v a q u o n e , a delay in time to death w a s noted, but 100 % died by D 1 2 . At D 6 , parasites w e r e not detectable in the blood, w h e r e a s m e a n parasitic burdens w e r e at 3 82 log para-
Fig. 2. - A c u t e t o x o p l a s m o s i s . S u r v i v a l r a t e s for c o n t r o l m i c e ( • ) a n d m i c e t r e a t e d w i t h u n l o a d e d n a n o c a p s u l e s ( ) , a t o v a q u o n e - l o a d e d n a n o c a p s u l e s ( • ) , o r a t o v a q u o n e s u s p e n s i o n ( • ) , f r o m d a y 1 t o d a y 1 0 ( 1 5 m g / k g / d a y ) .
sites/g in the brain a n d 6 . 1 8 log parasites/g in the lungs. In the group o f m i c e treated with a t o v a q u o n e - l o a d e d n a n o c a p s u l e s , 7 5 % o f m i c e survived until D 3 0 ; parasites w e r e u n d e t e c t a b l e in the b l o o d and in the brains at D 6 and w e r e at a l o w value in the lungs ( m e a n = 1.90 log parasites/g). In the surviving m i c e at D 3 0 , n o cyst w e r e found by m i c r o s c o p i c e x a m i n a tion o f serial brains smears.
Chronic t o x o p l a s m o s i s
C h r o n i c a l l y i n f e c t e d m i c e w e r e f o l l o w e d d u r i n g 42 days after initiation o f treatment. No death w a s r e c o r d e d in untreated m i c e infected b y the COUL strain, w h e r e a s 3 0 % o f untreated ME-49 infected m i c e died during the follow-up period.
Assessment o f treatment efficacy w a s b a s e d on the d e t e r m i n a t i o n o f b r a i n cysts c o u n t s a n d parasitic burden at the e n d o f treatment ( D 4 2 ) . Results obtained for both strains according to the treatment administered are p r e s e n t e d in Figure 3a a n d 3b. Overall, the results
(1) p= 0.059 vs controls (2) p<0.05 vs controls
Fig. 3a. - C h r o n i c t o x o p l a s m o s i s . B r a i n c y s t c o u n t v e r s u s t r e a t m e n t ( c o n t r o l , a t o v a q u o n e s u s p e n s i o n o r n a n o p a r t i c l e s ( 1 5 m g / k g / d a y ) a n d p a r a s i t i c strain ( C O U L o r M E 4 9 ) .
Fig. 3b. - C h r o n i c t o x o p l a m o s i s . P a r a s i t i c b u r d e n s v e r s u s t r e a t m e n t ( c o n t r o l , a t o v a q u o n e s u s p e n s i o n , o r n a n o p a r t i c l e s ( 1 5 m g / k g / d a y ) a n d p a r a s i t i c strain ( C O U L o r M E 4 9 ) .
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Mémoire 227
SORDET F., AUMJAUD Y., FESSI H. & DEROUIN F.
s h o w e d a marked efficacy o f a t o v a q u o n e on brain infection, either a s s e s s e d by brain cyst count or titra
tion o f parasitic burdens. However, an important hete
rogeneity was noted following the type o f infecting strain, the drug formulation and the m e t h o d that w a s used to quantify parasitic infection in the brain.
T h e quantification o f brain cysts s h o w e d that all mice w e r e infected but it revealed an important variability in cyst c o u n t s a m o n g control mice, for both strains o f
T. gondii. In treated mice, a marked reduction o f cyst c o u n t s w a s n o t e d in c o m p a r i s o n to the controls but it o n l y r e a c h e d s i g n i f i c a n c e in C O U L - i n f e c t e d m i c e treated with n a n o c a p s u l e s (p = 0 . 0 0 9 ) .
T h e determination o f parasitic burdens s h o w e d a much l o w e r variability a m o n g mice. In ME49-infected mice, both s u s p e n s i o n and nanoparticle treatments resulted in a significant d e c r e a s e o f parasitic burdens (from 1.2 to 3.9 log parasites/g, p = 0.009 and 0.036 respectively), w h e r e a s in COUL-infected mice this decrease was only significant in those treated with nanocapsules (decrease o f 1.2 log parasite/g, p = 0.016).
As a w h o l e , these results s h o w e d that the activity o f a t o v a q u o n e is improved w h e n used in nanoparticular form. Treatment with n a n o c a p s u l e s reduced signifi
cantly the parasitic burden (p= 0.009 for both strains), but not the cyst count, w h e n c o m p a r e d to treatment with the suspension.
DISCUSSION
T
hese results confirm previous studies showing that a t o v a q u o n e is an effective drug against T. gondii both in vitro and in vivo (Araujo et al., 1 9 9 2 ; Romand et al., 1993). In the present study, we focused on the development o f a n e w formulation o f atovaquone in an attempt to obtain higher concentrations o f drug in infected tissue, mainly in the brain, and to improve efficacy o n the resistant cystic form o f the parasite. Our c h o i c e was orientated toward nanocap
sules w h i c h have a higher drug-loading capacity, are stable during storage, and can b e given orally. Atova- quone-loaded nanocapsules w e r e prepared after solu
bilization o f the drug in benzyl benzoate. T h e stability o f the nanocapsules, as assessed by following drug content, size, pH and osmolarity, was found satisfactory.
Experimental assessment o f the activity o f atovaquone- loaded nanoparticules was conducted both in vitro and in vivo, and compared to the suspension form. Both for
mulations w e r e found highly inhibitory for T. gondii in vitro with c o m p a r a b l e 5 0 % inhibitory concentrations.
In a murine model o f acute toxoplasmosis, the treatment with atovaquone-loaded nanocapsules at a low dosage (15 m g / k g / d a y ) was found remarkably efficient, com
pared to the s u s p e n s i o n administered at the s a m e dosage, resulting in the survival o f 7 5 % o f the treated
mice with a negativation o f parasitic burdens in b l o o d and brain and a marked reduction o f lung infection.
In c h r o n i c toxoplasmosis, treatment with both formu
lations o f a t o v a q u o n e resulted in a d e c r e a s e o f brain parasitic loads o f treated m i c e c o m p a r e d to untreated controls. W e noticed that this d e c r e a s e w a s better evi
d e n c e d by titration o f parasitic burden using a tissue culture m e t h o d rather than m i c r o s c o p i c counting o f brain cysts. Since the first m e t h o d better estimates parasite viability, w e b e l i e v e that the cysts that w e r e o b s e r v e d by m i c r o s c o p y m a y have lost part o f their infectiosity potential, possibly in relation to s o m e effi
c a c y o f the drug o n the parasite-containing cysts.
T h e pharmaceutical formulation proved to have an important incidence, as already o b s e r v e d in the m o d e l o f acute infection: for the two strains o f T. gondii used in this study, the d e c r e a s e o f parasitic loads w a s signi
ficantly m o r e important with atovaquone-loaded nano
capsules than with the suspension. Although w e could not determine the concentrations o f a t o v a q u o n e in b l o o d and tissues o f treated mice, w e b e l i e v e that the better results o b t a i n e d with a t o v a q u o n e - l o a d e d n a n o capsules are related to increased bioavailability due to the drug solubilization in the oily core o f nanocapsules.
Finally, w e n o t i c e d important differences in the result o f treatment according to the infecting strain. Atova
q u o n e n a n o c a p s u l e s w e r e found to b e m o r e effective against the ME49 strain inducing a 4 log reduction o f brain parasitic burden o f treated mice, c o m p a r e d to a reduction o f 1.5 log in the brain o f COUL-infected mice. T h e s e results corroborate the observation b y Fer
g u s o n et al. ( 1 9 9 4 ) w h o s h o w e d b y e l e c t r o n micro
s c o p y that brain cysts o f the ME49 strain contain a mix
ture o f non-differentiated parasites, s o m e o f t h e m resembling tachyzoites o n which atovaquone would b e m o r e effective. B y contrast, the COUL strain infection is characterized by a c h r o n i c p h a s e with a b s e n c e o f brain tachyzoites and r e m a r k a b l e stable bradyzoite parasite burdens ( D e r o u i n et al., 1 9 9 1 ) resulting in lesser sensitivity to a t o v a q u o n e .
Such difference in the sensitivity o f strain to atova
q u o n e could b e o f c o n s e q u e n c e s in clinical practice and could partly explain the high variability o f inter- individual r e s p o n s e s noticed in clinical studies (Kat- lama, 1 9 9 6 ) . This w o u l d also confirm the hypothesis o f T o m a v o et al. ( 1 9 9 5 ) a c c o r d i n g to w h o m the sen
sitivity o f the different strains o f T. gondii to anti-para
sitic drugs are mainly correlated to their metabolic cha
racteristics.
ACKNOWLEDGEMENTS
T
his work was supported in part by a grant from the Université Paris 7.W e thank the two reviewers o f the manuscript.
228 Mémoire P a r a s i t e , 1 9 9 8 , 5, 2 2 3 - 2 2 9
A T O V A Q U O N E - L O A D E D N A N O C A P S U L E S I N T H E T R E A T M E N T O F M U R I N E T O X O P L A S M O S I S
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Reçu le 14 Janvier 1998 Accepté le 29 avril 1998
P a r a s i t e , 1 9 9 8 , 5 , 2 2 3 - 2 2 9 229
Mémoire
