FR3005858A1 - USE OF NANOPARTICLES FOR THE PREPARATION OF WATER-SOLUBLE, INJECTABLE ANTIPALUDE COMPOSITIONS - Google Patents

Abstract

The invention relates to the use of nanoparticles for the preparation of soluble antimalarial compositions, the pharmaceutical compositions comprising or consisting of said nanoparticles, and a process for preparing the nanoparticles.

Description

The invention relates to the use of nanoparticles for the preparation of soluble antimalarial compositions, the pharmaceutical compositions comprising or consisting of said nanoparticles, and a preparation method. nanoparticles.

Malaria, or malaria, threatens half of the world's population. More than 200 million cases of malaria are reported annually leading to about one million deaths annually. This disease is still a major public health problem. It is an infectious disease caused by a parasite, belonging to the genus Plasmodium (mainly P. falciparum), transmitted to humans through infected Anopheles mosquitoes. The life cycle of the parasite includes a sexual phase (sporogony), taking place in the anopheles, and an asexual phase (schizogony), taking place in humans. This asexual phase, causing the disease in humans, has itself two stages: a pre-erythrocytic stage (hepatic), followed by a stage erythrocyte (blood). The parasite transforms into the hepatocyte into hepatic schizonts (merosomes) containing several thousand merozoites. After this stage of maturation that lasts about two weeks, the merosomes burst and release thousands of merozoites that invade the red blood cells. In the erythrocytic stage, the merozoite multiplies. The parasite degrades hemoglobin and digests 30% or more of this protein to recover the amino acids needed to synthesize its own proteins. During the bursting of the red blood cell, the parasites continue their replicative cycle but some merozoites are differentiated into gametocytes. The erythrocyte stage is the symptomatic stage of malaria: fever. Severe malaria (coma, altered brain function) is due to obstruction of blood capillaries of the brain by parasitized red blood cells and can be lethal in 15 to 20% of infections.

In the face of the scourge of malaria, the most critical problem is the development of parasite resistance to classical quinoline / quinoline antimalarials. Other drugs such as artemisinin and its semisynthetic derivatives (ARTs), however, are effective against multi-resistant strains of P. falciparum, and also have broad specificity against asexual and sexual blood stages. WHO recommends therapeutic combinations based on artemisinin derivatives to fight against non-severe cases of malaria. The combination of several antimalarial molecules with different mechanisms of action makes it possible to combat the effects of resistance. ARTs induce a rapid reduction of parasite biomass, which results in rapid clinical and parasitological responses following treatment. No other antimalarial molecule currently offers the same level of efficacy and tolerance as artemisinin, making it indispensable for the treatment of severe malaria. Despite being the most effective antimalarial drug against all erythrocyte stages of Plasmodium to date, artemisinin has a very low water solubility, short half-life, significant first-pass liver. In addition, the parasite has also developed resistance to artemisinin in Asia. The molecules of the indolone-N-oxide family also constitute a new chemical archetype with antimalarial properties (F. Nepveu et al., J. Med Chem., 2010, 53 (2), 699-714, F.Nepveu et al. ., "Derivatives for the treatment of malaria", patent n ° 07/06425 of 13-09-2007) based on new mechanisms of action. Indeed, the indolone-N-oxides have an equipotent anti-parasitic activity against strains of P. falciparum sensitive or resistant to chloroquine. Similarly, the indolone-N-oxides have the advantage of having a moderate affinity for serum human albumin (HSA), thus allowing their transport by this protein (N. Ibrahim et al., Biomacromolecules 2010, 11, 3341- 3351). However, the very low aqueous solubility of indolone-N-oxides is a limiting factor in determining a suitable dosage form for the clinical evaluation of these molecules.

Plasma proteins can serve as carriers for drugs. The affinity of an active substance for plasma proteins is an important physico-chemical characteristic determining its pharmacokinetics, also known as ADMET: Abbreviation for "Absorption, Distribution, Metabolism, Excretion, Toxicity".

Human serum albumin (HSA) is the major extracellular protein in blood (60%), and the major carrier of blood medications as well as endogenous compounds. The binding of a drug to HSA has two important consequences: (i) the solubilization of hydrophobic molecules allowing their transport to the cells in vitro and in vivo, and (ii) the decrease in the free molecule concentration, which is the pharmacologically active form. For example, a number of active molecules in vitro have been rendered ineffective in vivo due to an excessive affinity for albumin. Other highly active molecules in vitro are ineffective in vivo because of their insolubility in aqueous medium. It is therefore generally necessary to study the interaction of a future drug with albumin.

Currently, because of the resistance effects of the parasite Plasmodium to many drugs, the number of cases of severe malaria is increasing. The impact of malaria on infant mortality remains high, especially in Africa where 1/5 of infant deaths are due to malaria. Severe malaria and treatment of children require hospitalization and rapid-acting formulations such as intravenous injections. There is, to date, a real lack of formulations of antimalarials soluble in aqueous medium and therefore injectable. Artemisinin is not soluble in water and does not exist in an injectable formulation. The pre-clinical and clinical trials of the "leads" of the indolone-N-oxide series are not feasible because of their insolubility in an aqueous medium. One of the aims of the invention is to provide novel soluble formulations of antimalarial compositions that can be used for intravenous administration. Another object of the invention is to provide novel formulations of antimalarial compositions targeting the parasitized red blood cell. Another object of the invention is also to provide a process for the preparation of nanoparticles used in the constitution of new antimalarial compositions.

The invention thus relates to a composition comprising an antimalarial agent as an active substance and human albumin as a carrier for use as an injectable drug in the form of nanoparticles in the treatment of malaria.

The invention is based on the surprising observation made by the inventors that albumin can solubilize the antimalarial agent without losing its therapeutic properties.

In addition, the albumin retains its function as a vector for targeting red blood cells and makes it possible to deliver the antimalarial agent preferentially to the parasitized red blood cells, thereby promoting the therapeutic activity of the antimalarial agent.

In the invention, the term "transporter" refers to an agent used to solubilize an active substance. This term corresponds in particular to the human serum albumin used to solubilize an antimalarial agent. In the invention, the term "targeting vector" refers to an agent used to transport an active substance to specific cells. This term corresponds in particular to the human serum albumin used to transport an antimalarial agent preferentially to parasitized red blood cells. In the invention, the term "injectable" is used to indicate that a solution is parenterally administrable. In the case of treatment of complicated malaria cases, or cerebral or infant, an antimalarial drug is preferentially injected into the bloodstream intravenously to deliver the drug directly to the site of infection, that is to say the blood to obtain a rapid action and to avoid the first-pass effect (metabolization in the liver during enteral administration).

"Nanoparticles" correspond to particles having a size of 10 to 1000 nm. In the invention, the nanoparticles are composed of a carrier and an antimalarial agent.

The compositions are soluble in water, in a phosphate buffer and in physiological fluids, especially blood. In addition, nanoformulation has many advantages over traditional formulations. In fact, the nanoparticles make it possible to improve the dissolution rate, the saturation solubility, the bioavailability and therefore the effectiveness of the active substances. Similarly, the nanoparticles make it possible to obtain an effect that eliminates fluctuations in the plasma concentrations of the drug, avoids the sub-therapeutic concentrations that cause the development of resistance phenomena and prolongs the life of the active substances.

A particular embodiment of the invention relates to a composition comprising an antimalarial agent as an active substance and human albumin as a carrier, the antimalarial agent and the human albumin not being bound to each other. other by a chemical bond, and especially by a covalent bond, for its use as injectable drug in the form of nanoparticles in the treatment of malaria. The term "antimalarial agent and human albumin not being bound together by a chemical bond, and especially by a covalent bond" means that the antimalarial agent and albumin are found together. in the composition as two separate substances. Another particular embodiment of the invention relates to a composition comprising an antimalarial agent as the active substance and human albumin as a carrier, the antimalarial agent and the human albumin being linked to each other. by a covalent bond, for its use as injectable drug in the form of nanoparticles in the treatment of malaria.

The term "anti-malarial agent and human albumin being covalently bound to one another" means that the antimalarial agent and albumin are present in the composition as a single substance. that is, albumin is functionalized with the antimalarial compound. In one embodiment, the invention relates to a composition comprising an antimalarial agent as the active ingredient and purified human albumin as a carrier for use as an injectable drug in the form of nanoparticles in the treatment of malaria. Purified human albumin corresponds to albumin essentially free of fatty acids, that is to say less than 0.005%. In a particular embodiment, the invention relates to a composition defined above, wherein the active substance is selected from indolone-N-oxide and artemisinin derivatives. The invention thus has the advantage of making the indolone-N20 oxides derivatives soluble in an aqueous medium or a buffered aqueous medium. Similarly, it also has the advantage of providing a new soluble formulation of artemisinin, which is made more effective. The artemisinin molecule can be represented by the following formula: By way of example, artemisinin in the form of nanosuspensions has a longer half-life and can be used at lower doses and therefore at a lower toxicity.

In a particular embodiment, the invention relates to a composition defined above, in which the indolone-N-oxide derivative and albumin are not bound together by a chemical bond, and in particular by a binding of covalently. The expression "the indolone-N-oxide derivative and albumin are not bound together by chemical bonding, and especially by a covalent bond" means that the indolone-N-oxide derivative and the albumin are found in the composition as two distinct substances. In another particular embodiment, the invention relates to a composition defined above wherein an indolone-N-oxide derivative is covalently attached to albumin. It is indeed possible to covalently bond an indolone-N-oxide to albumin. The binding is from an indolone-N-oxide bearing an amine group. This group can be linked to the carboxylic residues of albumin by using activation protocols under mild conditions (aqueous medium). For example, it is possible to activate the carboxylic groups of the protein with N-hydroxysuccinimide, and then add the indolone-N-oxide carrying an NI-12 group according to the following reaction (reactions with N-hydroxysuccinimide esters). and amino-indolone-N-oxide derivatives): 0 DCCI, DMF, 0 NOH + RN 0 HO rt, Ar Synthesis of N-hydroxysuccinimide N-O esters This covalent linkage between the indolone-N derivative -oxides and albumin is such that the pharmacokinetic properties are substantially preserved. In a particular embodiment, the invention relates to a composition defined above, in which artemisinin and albumin are not bound together by a chemical bond, and especially by a covalent bond. The expression "artemisinin and albumin are not bound together by a chemical bond, and especially by a covalent bond" means that artemisinin and albumin are found in the composition in the form of two substances. distinct. In one embodiment, the invention relates to a composition defined above, wherein the active substance is selected from the compounds of formula la in which: the substituent (s) R 1 and R 2 are chosen indifferently from group comprising the hydrogen atom, a halogen, especially chlorine and fluorine, the CF 3 group, the alkyl groups of 1 to 3 carbons, in particular methyl, the alkyloxy groups of 1 to 6 carbons, in particular methoxy, benzyloxy, the -COCH3 groups, -COOCH3, -OCF3, NO2, the NH2 group and alkylamino groups, - the two adjacent substituents R1 and R2 that can form the methylenedioxy-O (CH2) O- ring; Y represents either: a branched phenyl group of formula IIa IIa in which the substituent or substituents R3, R4 and R5 are chosen indifferently from the group comprising hydrogen, a halogen, in particular F, Cl, the alkylfluorinated groups in particular CF3; , alkyl groups having 1 to 3 carbons, alkyloxy groups, in particular OCH 3, OCF 3, phenyloxy, alkylamino, dialkyl or monoalkyl groups, in particular (CH 3) 2 N, (C 2 H 5) 2 N, (C 3 H 7) 2 N, (C 3 E 17) NH and their salts, the groups NO 2, NH 2, CH 3 CONH, CH 3 OCO, 4-chlorophenyl, tert-butyl, isopropoxy, hydroxymethyl, morpholin-4-yl, butylamino, a pyren-1-yl group, a 6'-group, methoxy-naphthalen-2-yl, - a 2'-pyridinyl group, - a 3'-pyridinyl group, or a thiophen-3-yl group, - Z represents either: - 1 \ 1 + 0-, and - corresponds to a covalent double bond, or - NH or NOH, and - corresponds to a single covalent bond. In another embodiment, the invention relates to a composition defined above, wherein the active substance is selected from the following compounds: ## STR2 ## 5. 8. O CF3 02N 11 N 10. N 11. 11. NO 2 HAN 12. NH 2 13. 9.

O-0 16. 12 14. 15. CI 17. 18. 19. 5. In one embodiment, the invention relates to a composition defined above, wherein the active substance is artemisinin. In one embodiment, the invention relates to a composition defined above wherein the active ingredient and human albumin are present in a weight ratio of 1: 5 to 1:20. In one embodiment, The invention relates to a composition as defined above, in which the active substance and human albumin can be used in unit dose form, in which the active substance is present from 375 to 1125 mg. In one embodiment, the invention relates to a composition defined above in which the active substance is an indolone-N-oxide derivative used in unit dose form of 375 to 1125 mg. In one embodiment, the invention relates to a composition defined above wherein the active substance is an indolone-N-oxide derivative used as a unit dose of 375 to 1125 mg. per day. In one embodiment, the invention relates to a composition defined above in which the active substance is an indolone-N-oxide derivative used in unit dose form of 5 to 15 mg / kg, at the rate of one daily catch

In one embodiment, the invention relates to a composition defined above wherein the active substance is artemisinin used as a unit dose of 375 to 750 mg.

In one embodiment, the invention relates to a composition defined above in which the active substance is artemisinin used in unit dose form of 375 to 750 mg, one dose per day. In one embodiment, the invention relates to a composition defined above wherein the active substance is artemisinin used as a unit dose of 5 to 10 mg / kg, once daily. In the compositions defined above comprising unit doses of active substance of 375 to 1125 mg, the albumin is present in a proportion of 1.8 to 22.5 g. In one embodiment, the invention relates to a composition defined above, in which the nanoparticles have a size ranging from 200 to 400 nm. In one embodiment, the invention relates to a composition defined above comprising nanoparticles in which the active substance is an indolone-N-oxide derivative and nanoparticles in which the active substance is artemisinin. Another aspect of the invention relates to a pharmaceutical composition comprising an antimalarial agent as active substance and human albumin carrying the active principle to the parasitized red blood cell, in particular in the form of nanoparticles of active substance and albumin. In one embodiment, the invention relates to a pharmaceutical composition defined above comprising an antimalarial agent as an active substance and human albumin acting both as a carrier of the antimalarial agent for solubilizing and as a targeting vector. to deliver the antimalarial agent to parasitized red blood cells. In one embodiment, the invention relates to a pharmaceutical composition defined above comprising an antimalarial agent and albumin, as well as an excipient. In a particular embodiment, the invention relates to a pharmaceutical composition defined above, containing as active substance a molecule chosen from indolone-N-oxide derivatives, or artemisinin, in association with a vector. targeting the erythrocyte stage of the parasite, human albumin. In one embodiment, the invention relates to a pharmaceutical composition defined above, wherein the active substance is selected from compounds of formula la wherein: - the substituent (s) R 1 and R 2 are independently selected in the group comprising the hydrogen atom, a halogen, especially chlorine and fluorine, the CF 3 group, the alkyl groups of 1 to 3 carbons, in particular methyl, the alkyloxy groups of 1 to 6 carbons, especially methoxy, benzyloxy, the groups -COCH3, -COOCH3, -OCF3, NO2, the NH2 group and the alkylamino groups, - the two adjacent substituents R1 and R2 that can form the methylenedioxy-O (CH2) O- ring; Y represents either: a branched phenyl group of formula IIa IIa in which the substituent or substituents R3, R4 and R5 are chosen indifferently from the group comprising hydrogen, a halogen, in particular F, Cl, the alkylfluorinated groups in particular CF3; , alkyl groups having 1 to 3 carbons, alkyloxy groups, in particular OCH 3, OCF 3, phenyloxy, alkylamino, dialkyl or monoalkyl groups, in particular (CH 3) 2 N, (C 2 H 5) 2 N, (C 3 H 7) 2 N, (C 3 E 17) NH and their salts, the groups NO 2, NH 2, CH 3 CONH, CH 3 OCO, 4-chlorophenyl, tert-butyl, isopropoxy, hydroxymethyl, morpholin-4-yl, butylamino, a pyren-1-yl group, a 6'-methoxy group; -naphthalen-2-yl, - a 2'-pyridinyl group, - a 3'-pyridinyl group, or - a thiophen-3-yl group, - Z represents either: - N + 0-, and - corresponds to a double covalent bond, or - NH or NOH, and - corresponds to a single covalent bond. In another embodiment, the invention relates to a pharmaceutical composition defined above, wherein the active substance is selected from the following compounds: ## STR1 ## FC 5.

O CF 3 O 2 N 8. N 10. N 11. 11. NO 2 HAN 12. N H 2 13. 9.

O-0 16. 20 14. 15. CI 17. 18. 19. 5. 22. In a particular embodiment, the invention relates to a pharmaceutical composition defined above, in which the active substance is artemisinin.

In a particular embodiment, the invention relates to a pharmaceutical composition defined above, in which the active substance and human albumin are present in a weight ratio of 1/5 to 1/20.

In one embodiment, the invention relates to a pharmaceutical composition defined above, in which the active substance and human albumin can be used in unit dose form, in which the active substance is present from 375 to 1125 mg.

In one embodiment, the invention relates to a pharmaceutical composition defined above wherein the active substance is an indolone-N-oxide derivative used as a unit dose of 375 to 1125 mg. In one embodiment, the invention relates to a pharmaceutical composition defined above wherein the active substance is an indolone-N-oxide derivative used as a unit dose of 375 to 1125 mg, taken daily. In one embodiment, the invention relates to a pharmaceutical composition defined above in which the active substance is an indolone-N-oxide derivative used in unit dose form of 5 to 15 mg / kg, at a rate of one daily catch

In one embodiment, the invention relates to a pharmaceutical composition defined above wherein the active substance is artemisinin used as a unit dose of 375 to 750 mg.

In one embodiment, the invention relates to a pharmaceutical composition defined above in which the active substance is artemisinin used in unit dose form of 375 to 750 mg, one dose per day. In one embodiment, the invention relates to a pharmaceutical composition defined above wherein the active substance is artemisinin used in unit dose form of 5 to 10 mg / kg, once daily. In the pharmaceutical compositions defined above comprising unit doses of active substance of 375 to 1125 mg, albumin is present in a proportion of 1.8 to 22.5 g. In a particular embodiment, the invention relates to a pharmaceutical composition defined above, in the form of injectable solution intravenously. In a particular embodiment, the invention relates to a pharmaceutical composition defined above, in the form of lyophilisate. In one embodiment, the invention relates to a pharmaceutical composition defined above comprising nanoparticles in which the active substance is an indolone-N-oxide derivative and nanoparticles in which the active substance is artemisinin. Another aspect of the invention relates to nanoparticles comprising an anti-malarial agent as active substance and human albumin.

The nanoparticles were prepared from human serum albumin (HSA). This protein has several advantages for an injectable formulation of nanoparticles for treating blood-stage malaria: (i) HSA has surfactant properties that improve the wettability of the drug, (ii) the bioavailability of the drug is maximal (100%), (iii) HSA targets the parasitized red blood cells because it is selectively picked up and degraded, (iv) the drug retains its rapid activity, (v) hepatic metabolism is avoided, (vi) the therapeutic doses are reduced, which reduces the toxicity of the drug, (vii) HSA is biocompatible, biodegradable and non-immunogenic.

In a particular embodiment, the invention relates to nanoparticles defined above, in which the active substance is chosen from indolone-N-oxide derivatives or artemisinin.

In one embodiment, the invention relates to nanoparticles defined above, in which the active substance is chosen from the compounds of formula la in which: in which: the substituent (s) R 1 and R 2 are chosen indifferently from the group comprising the hydrogen atom, a halogen, especially chlorine and fluorine, the CF 3 group, the alkyl groups of 1 to 3 carbons, in particular methyl, the alkyloxy groups of 1 to 6 carbons, in particular methoxy, benzyloxy, the -COCH3, -COOCH3, -OCF3, NO2 groups, the NH2 group and the alkylamino groups, - the two adjacent substituents R1 and R2 that can form the methylenedioxy-O (CH2) O- ring; Y represents either: a branched phenyl group of formula IIa in which the substituent (s) R3, R4 and R5 are chosen indifferently from the group comprising hydrogen, a halogen, in particular F, Cl, the alkylfluorinated groups in particular CF3, alkyl groups having 1 to 3 carbons, alkyloxy groups, in particular OCH 3, OCF 3, phenyloxy, alkylamino, dialkyl or monoalkyl groups, in particular (CH 3) 2 N, (C 2 H 5) 2 N, (C 3 H 7) 2 N, (C 3 E 17) NH and their salts, the groups NO 2, NH 2, CH 3 CONH, CH 3 OCO, 4-chlorophenyl, tert-butyl, isopropoxy, hydroxymethyl, morpholin-4-yl, butylamino, a pyren-1-yl group, a 6'-methoxy group; naphthalen-2-yl, - a 2'-pyridinyl group, - a 3'-pyridinyl group, or - a thiophen-3-yl group, - Z represents either: - N + O-, and - corresponds to a double bond covalent, or - NH or NOH, and - corresponds to a single covalent bond. In another embodiment, the invention relates to nanoparticles as defined above, wherein the active substance is selected from the following compounds: Cl 3. 6. o- 7. o CI CI CF3 cIN + 0- 4. FC CF3 5. 2. 26 O CF3 02N 8. N 10. N \ _ 11. NO2 HAN 12. NH 2 13. 9.

O-0 16. 28 14. 15. CI 17. 18. 19. 5. In a particular embodiment, the invention relates to nanoparticles as defined above, in which the active substance is artemisinin. In a particular embodiment, the invention relates to nanoparticles defined above, in which the active substance (indolone-N-oxide) and human albumin are present in a substance: albumin concentration ratio ranging from 10: 5 to 1: 20 to obtain a particle diameter of less than 330 nm with satisfactory homogeneity, ie a polydispersity index of less than 0.25. In a particular embodiment, the invention relates to nanoparticles defined above, in which the active substance, artemisinin and human albumin are present in an artemisinin concentration ratio: albumin ranging from 1: 5 to 1 Resulting in particle sizes of less than 350 nm and a polydispersity index of less than 0.25. In a particular embodiment, the invention relates to nanoparticles defined above, in which the active substance is capable of being used in unit dose form of 375 to 1125 mg. In a particular embodiment, the invention relates to nanoparticles defined above, having a size ranging from 200 to 400 nm. Another aspect of the invention relates to a method for preparing nanoparticles comprising: a step of evaporation of the organic solvent contained in the nanosuspension, in order to obtain a nanosuspension devoid of organic solvent; a step of homogenization at a pressure ranging from from 15,000 (103,421.35 kPa) to 30,000 psi (206,842.71 kPa), a mixture of an antimalarial agent and dissolved albumin, respectively, in an organic solvent and an aqueous solvent, to obtain an nanosuspension, and a lyophilization step of the nanosuspension devoid of organic solvent, to obtain nanoparticles.

The homogenization step is carried out for 10 to 40 cycles, which cycles can be carried out in one or two stages. One embodiment of the invention relates to a process for the preparation of nanoparticles defined above, wherein the antimalarial agent is selected from indolone-N-oxide derivatives and their pharmaceutically acceptable salts. In one embodiment, the invention relates to a method for preparing nanoparticles defined above, wherein the antimalarial agent is artemisinin. In another embodiment, the invention relates to a process for the preparation of nanoparticles defined above wherein the indolone-N-oxide derivatives are dissolved in acetone. In another particular embodiment, the invention relates to a nanoparticle preparation method defined above, wherein the homogenization step is carried out from 15,000 psi (103,421.35 kPa) to 30,000 psi ( 206,842.71 kPa) for 10 to 20 cycles. In another particular embodiment, the invention relates to a nanoparticle preparation method defined above, wherein the homogenization step is carried out at 25,000 psi (172,368.93 kPa) for 15 cycles.

In another particular embodiment, the invention relates to a process for the preparation of nanoparticles defined above, wherein the artemisinin is dissolved in ethanol.

In another more particular embodiment, the invention relates to a process for the preparation of nanoparticles defined above, wherein the homogenization step is carried out at 15,000 psi (103 421.35 l (Pa) for 5 cycles, followed by 35 cycles at 25,000 psi (172,368.93 L (13a).

In another more particular embodiment, the invention relates to a process for the preparation of nanoparticles defined above, wherein the albumin is dissolved in water or in a phosphate buffer. Solvent removal is generally relatively easy on a laboratory scale, but becomes more problematic when producing in large quantities. Preliminary tests were therefore carried out to determine the appropriate organic solvent. The solvent selection criterion was the best possible solubility with the active substance and the best possible volatility. Based on these criteria, acetone / water (14%, v / v) was selected as the solvent / antisolvent system. Water acts as a miscible antisolvent of the hydrophobic substance. While albumin incorporates the active substance into its matrix and increases its wettability. It is important to note that in the absence of active substance, an aqueous solution of HSA can not be transformed into nanoparticles when it is mixed with acetone under the same experimental conditions. The presence of the active substance is therefore a prerequisite for the formation of stable nanoparticles according to this process. This can be explained by the precipitation of the active substance in contact with water which would lead to its inclusion in the HSA matrix, favored by the affinity between the active substance and HSA. In addition, the addition of active substance in acetone and in water without HSA produces macroscopic aggregates of active substance.

The method of the invention has therefore been optimized in terms of the ratio of active substance / HSA, concentration of active substance, ionic strength and homogenization parameters (homogenization pressure and number of cycles).

The physical stability of the nanoparticles is mainly represented by the absence of aggregates and the formation of particles. The appearance of such particles is explained by Ostwald maturation, a phenomenon that results from the difference in saturation solubility between small particles of a few nm to 500 nm and large particles, of the order of a micrometer. The small particles have a high degree of curvature and therefore have greater saturation solubility compared to larger particles. As a result, small particles dissolve and deposit on the surface of large particles, which are less soluble. In this way, the small particles disappear while the larger ones continue to grow in microparticles.

In general, the larger the PI, the more pronounced the Ostwald maturation phenomenon. To overcome this problem, additional homogenization cycles have been carried out after the particles have reached their minimum size in order to improve the homogeneity of the population and thus limit the Ostwald maturation.

In one embodiment, the invention relates to a method for preparing nanoparticles comprising: a step of evaporation of the organic solvent contained in the nanosuspension, in order to obtain a nanosuspension devoid of organic solvent; a 15-cycle homogenization step; at 25,000 psi (172,368.93 l (Pa) of a mixture of indolone-N-oxide derivative and dissolved albumin, respectively, in acetone and in water, to obtain nanosuspension, - and a freeze-drying step of the nanosuspension devoid of organic solvent, to obtain nanoparticles. In one embodiment, the invention relates to a process for preparing nanoparticles comprising: a step of evaporation of the organic solvent contained in the nanosuspension to obtain a nanosuspension free of organic solvent, - a homogenization step of 5 cycles at 15,000 psi (103,421.35 kPa), then 35 cycles at 25,000 psi (172,368.93 l (Pa) of one mel artemisinin angel and dissolved albumin, respectively, in ethanol and in water, to obtain a nanosuspension, and a lyophilization step of the nanosuspension without organic solvent, to obtain nanoparticles. In another more particular embodiment, the invention relates to a process for the preparation of nanoparticles defined above, comprising a cryoprotection agent used before the lyophilization step, to avoid the aggregation of the nanoparticles. The following figures and examples will better illustrate the invention without limiting its scope. FIGURE LEGENDS FIG. 1. Optimization of the preparation conditions of nanoparticles of active substance (human 6- (4-chlorophenyl) -7H-11,31dioxolo 14,5-indol-7-one-5-oxide) / human serum albumin (HSA). (A) Effects of the ratio [active substance] / [HSA] on the average diameter in nm (histograms) and the polydispersity index (curves) of the nanoparticles. The average diameter is measured before lyophilization (left bars) and after lyophilization (right bars). The polydispersity index is measured before (diamonds) and after (squares) lyophilization. (B) Effects of the concentration of active substance on the average diameter in nm (histogram) and the polydispersity index (curve) of the nanoparticles. (C) Effects of the ionic strength (NaCl concentration) on the average diameter in nm (histograms) and the Zeta potential in mV (curves) of the nanoparticles. The average diameter is measured before (left bars) and after (right bars) lyophilization. The Zeta potential is measured before (diamonds) and after (square) lyophilization. (D) Effects of homogenization pressure in psi on the average diameter in nm (histogram) and the polydispersity index (curve) of the nanoparticles. (NH: Not homogenized). (E) Effects of the number of cycles on the average diameter in nm (histogram) and the polydispersity index (curve) of the nanoparticles.

Figure 2. Observation and distribution of nanoparticles of active substance (6- (4-chlorophenyl) -7H-11,31dioxolo 14,5-indol-7-one-5-oxide) / human serum albumin (HSA). The active substance and the nanoparticles of active substance / HSA were examined by transmission electron microscopy (TEM) and analyzed by photon correlation spectroscopy (PCS) at different stages of nanoparticle preparation. Figure a) shows the crystals of pure active substance whose size exceeds the limit measurable by PCS (3! Am). Figure b) corresponds to the initial suspension, obtained after addition of the active substance and evaporation of the solvent and before homogenization. It has rectangular particles with an average diameter of 786 nm and a polydispersity index of the population of 0.325. Figure c) corresponds to the suspension after homogenization and shows particles with an average diameter of 222.2 nm and a polydispersity index of the population of 0.166. Figure d) corresponds to the suspension after lyophilization and shows particles with an average diameter of 367.8 nm and a polydispersity index of the population of 0.268. Figure 3. Kinetics of dissolution (release) of the active substance (6- (4-chlorophenyl) -7H- [1,3] dioxolo-14,5-flindol-7-one-5-oxide) in PBS.

Dissolution of the active substance, for the pure active substance (triangles) and for nanoparticles of active substance / human serum albumin (squares), was measured as a function of time. The ordinate axis corresponds to the percentage of active substance released, and the abscissa axis corresponds to the time in hours.

The dissolution of the pure active substance is relatively slow (100% in 3 hours) with an initial offset of about 45 minutes due to a lack of wettability. In contrast, homogenized active substance / human serum albumin nanoparticles exhibit rapid dissolution of the active substance (100% in 30 minutes, compared with only 1.5% of pure active substance during the same period). Figure 4. Evolution of the mean parasitaemia of Plasmodium berghei ANKA in infected mice given different doses of nanoparticles of active substance (6- (4-chlorophenyl) -7H-11,31dioxolo 14,5-11indol-7-one-5 -oxide) / human serum albumin (HSA). The mice were infected at day 0, then received one injection per day of loaded nanoparticles, the day of infection (OD) and for the next 3 days (D1, D2 and D4). Parasitaemia (%) is then measured daily from the 4th day after infection (D4). The ordinate axis corresponds to parasitaemia in%, and the axis of abscissa corresponds to time in days. The curves correspond to mouse samples injected with nanoparticles at 0 mg / kg (diamond, control), 6.25 mg / kg (squares), 12.5 mg / kg (triangles) or 25 mg / kg (cross ). The dashed line corresponds to the theoretical median effective dose (ED50). Figure 5. Evolution of parasitaemia (a) and survival (b) in humanized mice infected with Plasmodium falciparum, whether or not treated with nanoparticles of active substance (artemisinin) / human serum albumin (HSA). Humanized mice correspond to immunodeficient mice that have been transplanted with human red blood cells. Mice were infected at day 0, then parasitaemia and survival were measured every other day. The y-axis is parasitaemia in% (a) or survival (b), and the x-axis is time in days (a, b). The curves correspond to the mice not receiving treatment (rhombi) or having been treated with nanoparticles from day 7 (injection at 10 mg / kg for 4 days) (round). EXAMPLES Example 1 Preparation of Nanoparticles Comprising an Indolone-N10 Oxide Derivative and Human Albumin The Indolone-N-oxide Derivative (6- (4-chlorophenyl) -7H41,3] dioxolo [4,5- f] indol-7-one-5-oxide) is prepared according to the protocol described in the following article: F. Nepveu et al., J. Med. Chem., 2010, 53, 699-714. (Molecular weight = 301.7 g.mo1-1, Log P = 2.09 calculated with the computer tool 15 VCCLAB (www.virtuallaboratory.org)). The preparation requires acetone (HPLC grade), PBS (phosphate buffered saline) and human albumin (HSA), (essentially free of fatty acids, molecular weight = 66,478 g.mo1-1), purchased at Sigma-Aldrich (St. Quentin, France). All aqueous solutions are prepared using high purity distilled water obtained via a Milli-Q® water purification system (Millipore, St. Quentin, France). The dissolution of the indolone-N-oxide derivatives is done by sonication in an ultrasonic bath (Elma®, Germany). The mixture of the indolone-N-oxide derivative and human albumin is prepared in a ratio of 1: 5 (mass: mass) by geometric dilution. 10 mg of the Indolone-Noxyde derivative is first mixed with 10 mg of human albumin, then 20 mg of human albumin are added and mixed and the remaining 20 mg of albumin are added at the end. The nanosuspensions containing the indolone-N-oxide derivative and human albumin contain a compound concentration of 0.5, 1, 2.5 or 10 mg / ml. They are prepared by precipitation followed by homogenization under high pressure.

For precipitation, the indolone-N-oxide derivative is dissolved in acetone (3.3 mg / mL). Human albumin is dissolved separately in 0.01 M PBS (or highly purified water) at a concentration of 2.5, 5, 12.5 or 50 mg / mL for nanosuspensions containing 0.5, 1, and 1 respectively. , 2.5 or 10 mg / mL of indolone-N-oxide derivative. The solution of indolone-N-oxide derivative in acetone is added dropwise into the human albumin solution with magnetic stirring (addition rate: 1 mL / min). The water acts as a miscible antisolvent for the hydrophobic indolone-N-oxide derivative. Human albumin incorporates the indolone N-oxide derivative into its matrix and increases its wettability. The initial suspension is then evaporated under reduced pressure at 36 ° C for 15 minutes to remove acetone. For the high pressure homogenization phase, the initial suspension is homogenized for 15 cycles at 25,000 psi (172368.93 kPa) using a high pressure Emulsiflex-C3 homogenizer (SODEXIM S.A.S., France). During homogenization, the temperature increases sharply. A heat exchanger is then used to prevent the degradation of the thermolabile indolone-N-oxide derivative and to ensure that the recycled solution is sufficiently cold. The obtained nanosuspension is dried to increase the chemical stability of the formulation but also the physical stability of the nanoparticles. Lyophilization is used as a drying method in this process. A cryoprotectant, such as mannitol or trehalose, is usually added prior to lyophilization to prevent aggregation of the particles after reconstitution. In the process, albumin serves as a cryoprotectant. So no other cryoprotectant is added. Then, 10 mL of the nanosuspension: human indolone-N-oxide / albumin derivative, is frozen at -80 ° C for 24 h, then freeze-dried for 48 h at -55 ° C, 6 μM Hg, using a control system. drying marketed by Labconco. The particle powder is stored at 4 ° C until use. For reconstitution, 10 mL of highly purified distilled water is added to the freeze-dried powder with gentle agitation to avoid foaming with albumin. EXAMPLE 2 Size Analysis of Nanoparticles Containing an Indolone-N-Oxide Derivative and Human Albumin and Measurement of Their Zeta Potential Measurement of Nanoparticle Size and Potential Zeta is Performed Using an Analyzer of particles DelsaNano C (Beckman Coulter). The measurements are made in triplicate at 25 ° C. Polydispersity and mean diameter analyzes are based on a photon correlation spectroscopy (PCS) technique, also known as dynamic light scattering (DLS). Before the measurements, the nanosuspension respectively composed of indolone-N-oxide derivative and albumin is diluted in MilliQ water until an appropriate diffusion intensity (1: 100) is obtained. The diffusion angle is 165 °. The size of the particles is expressed in intensity-weighted mean diameter in nanometers, and the particle size distribution is expressed in polydispersity index (PI). Zeta potential is a measure of the electrical charge on the surface of particles. High Zeta potentials (> or = at 30 mV) indicate a physically stable colloid system, whereas nanosuspensions with a low Zeta potential tend to flocculate. The Zeta potential measurements are based on the electrophoretic light scattering (ELS) technique. The sample preparation is identical to that for measuring particle sizes, using a 15 ° scattering angle and the applied magnetic field is 5 V. Standardization is done to have a conductivity of 10 mS / cm using an aqueous solution of KCl (0.582% w / v). Example 3: Analysis of the stability of the nanoparticles comprising an indolone-N-oxide derivative and human albumin The physical stability of the nanosuspensions was examined for the potential growth of the particles or their aggregation after storage for 4 days at 4 ° C, using a DelsaNano C particle analyzer (Beckman Coulter, France). The chemical stability of the indolone-N-oxide derivative in the nanoparticles was evaluated after storage of nanosuspensions at 4 ° C. for 4 days and freeze-dried powders for 1 month. 1 mg of nanoparticles is reconstituted in 1 mL of Milli-Q water. 4 mL of acetonitrile are then added and the mixture is sonicated and then centrifuged. The amount of compound in the supernatant is analyzed by HPLC-UV. No significant particle growth was observed in nanosuspensions after 4 days of storage at 4 ° C. The increase in the average diameter is 4.3% with good homogeneity (PI <0.2). The physical stability of nanosuspensions is mainly reflected by the absence of particle growth or aggregation. Indolones-N-oxides show good chemical stability in albumin nanoparticles as in powder form (freeze-dried) or in aqueous forms. In the case of the freeze-dried form, the percentage of indolone-N-oxide derivative remaining in the particles after storage for 1 month at 4 ° C is 97.65% (± 0.66). The good stability of the compounds is also observed in aqueous suspension after 4 days of storage at 4 ° C., with an average of 99.73% (± 0.24). Example 4: In vivo evaluation of the effect of nanoparticles comprising an indolone-N-oxide derivative and human albumin in Plasmodium berghei-infected mice BALB / c female mice, 5 to 6 weeks old , 16 to 18 g (Charles River, France) are used in a Peter test, with 4 groups of 5 mice. Peter Test Procedure: The parasitized RBCs are removed from infected BALB / c donor mice and diluted in saline at 5 * 107 parasitized RBCs / mL. Day 0: The mice are infected intraperitoneally with a 0.2 mL aliquot of suspension. The lyophilized nanoparticles are reconstituted in sterile physiological serum and the resulting nanosuspension is filtered through a 5 μm filter (low protein uptake). Two hours after infection, the mice are treated intravenously with 0.1 ml of the nanosuspension at different doses: 6.25, 12.5, 25 mg / kg of body weight. The control group receives 0.1 mL of NaCl (0.9%) instead. 30 Days 1 to 3: 24, 48, 72 hours post infection, the experimental groups of mice are reprocessed with the same dose and by the same route of administration as day 0 (recommendation of Peter's test).

Day 4: From day 4 (96 hours post-infection), blood smears are prepared daily from the tail vein and are labeled with eosin - methylene blue. Parasitaemia is determined microscopically by counting the number of red blood cells infected per 3000 erythrocytes (if the parasitaemia is> 1%). For low parasitaemias (<1%), 10,000 erythrocytes are counted. Clinical signs and weight loss of animals are monitored daily. The statistical analysis is carried out with the R version 2.4.1 software (www.r-project.org). The analysis of survival is done by the logarithmic Kaplan-Meier test. A difference is considered significant when the P value is <0.05.

A significant difference in survival time (p = 0.00236) was observed between mice treated with nanoparticles: indolone-N-oxide / human albumin derivative versus untreated mice (controls). This effect is also significant on the evolution of parasitaemia (Figure 1). The analysis of the data reveals differences between the control group and the groups treated with different doses of nanoparticles: indolone-N-oxide / albumin derivative. In this 4-day trial, 80% of the mice in the control group died with parasitaemia (86.4%) between day 8 and day 13. All control mice died before day 19. The mice treated with lower dose (6.25 mg / kg) and intermediate dose (12.5 mg / kg) nanoparticles of indolone-N-oxide / human albumin derivative later die, respectively 80% and 60% at day 22 with less than 40% parasitaemia. All mice treated with the 25 mg / kg dose of nanoparticles of indolone-N-oxide / human albumin derivative remain alive without parasitaemia until the end of the experiment, day 34. A major difference is observed between control and mouse mice treated with 25 mg / kg of nanoparticles of indolone-N-oxide / albumin derivative (p = 0.0017). Previous in vivo tests in a mouse model infected with Plasmodium berghei ANKA strain reveal that this formulation gives no sign of toxicity or distress at a dose up to 4x100 mg / kg. The results of the present invention show that in this same model, the dose of 25 mg / kg / day is 100% curative (Table 1). Mice without parasitaemia at 30 days are cured. Indolone-N-oxide / human albumin derivative nanoparticles show an ED50 estimated at 7.66 mg / kg with a standard error at 0.96, a value slightly higher than that reported for artesunate (5.9 mg / kg) and chloroquine (1.8 mg / kg). In addition, the ED90 / ED50 is 2.96 which reflects a good response to the activity of the indolone-N-oxide derivative as a function of the dose (ED90 is 22.65 mg / kg with a standard error of 1.19). Interestingly, this formulation is more effective than artesunate and chloroquine in prolonging the survival time (8.2 days and 17 days for artesunate and chloroquine respectively). The results of the anti-malarial activity in vivo of the indolone-N-oxide-formulated (nanoparticles) or non-formulated derivative tested in mice infected with P. berghei according to the Peter test is presented in Table 1 below. Below: Compound Dose Percentage Mean parasitaemia inhibition survival at (days) day 4 Indolone-N-derivative 30 mg / kg / 4 days (oral) 14.5 - unformulated oxide Indolone-N-30 derivative mg / kg / 4 days (intraperitoneal route) 62.1 - unformulated oxide Indolone-N-oxide / HSA nanoparticles (Human Serum Albumin) 25 mg / kg / 4 days (intravenously) 99.1> 34 Chloroquine 10 mg / kg / 4 days 99 17 Artesunate 10 mg / kg / 3 days (99.1 8.2 oral route) Artesunate 3.2 mg / kg / 4 days 58 11.7 (intravenous) Artesunate 6.4 mg / kg / 4 days 87 12.4 (intravenous route) Table 1.

In addition, nanoparticles of indolone-N-oxide / albumin derivative allow intravenous administration of indolones-N-oxides, which was previously impossible due to their limited solubility. Commercial formulations of artesunate resulted in only 58% inhibition of parasitaemia at a dose of 3.2 mg / kg and 87% inhibition of parasitaemia at a dose of 6.4 mg / kg. In addition, recrudescences are observed for both doses and survival does not exceed 20 days. For the nanoparticles of indolone-N-oxide / albumin derivative described, there is no recrudescence and the survival time exceeds 34 days.

Example 5 In Vivo Evaluation of the Effect of Nanoparticles Comprising an Indolone-N-Oxide Derivative and Human Albumin in Plasmodium falciparum-Infected "Humanized" Mice NSG mice 9 to 11 weeks old (Charles River, US) are used to obtain an in vivo P. falciparum infection.

Human red blood cells are obtained from donors without history of malaria (French Blood Establishment, Rungis, France). The human red blood cells are washed twice with RPMI-1640 medium (Gibco / BRL) and centrifuged at 900 rpm, 10 min at 25 ° C. The supernatant is removed and the human red blood cells are resuspended in RPMI-1640 medium and stored at 4 ° C for a maximum of 2 weeks. Before use, the human red blood cells are washed twice again and centrifuged at 900 rpm for 10 min at 25 ° C in RPMI-1640 medium. The "humanized" NSG mice for red blood cells are infected with the P. falciparum strain 3D7 according to the protocol described in the following article: Moreno et al., Int. J. Parasitol. 2006, 36, 361-369.

Tissue depletion in macrophages is induced by clodronate provided by Roche Diagnostic and encapsulated in clodronate liposomes as described in the following article: Van Rooij et al., J. Immunol. Methods, 1994, 174, 83-93. At day 0, each mouse receives an intraperitoneal injection with a dose of 10 mg / kg of this antibody NIMP-R14, mixed with 300 μg of clodronate liposomes.

At day 2, 4 and 6, each mouse receives, intraperitoneally, 0.5 ml of human red blood cells mixed with a dose of 10 mg / kg of NIMP-R14 antibody and 300 μg of clodronate liposomes.

At day 8, each mouse is injected with 0.5 ml of P. falciparum-infected human red blood cells at a parasitaemia of 0.3%, mixed with a dose of 10 mg / kg of NIMP-R14 antibody and 300 μg of liposome of clodronate. After infection with P. falciparum, a dose of 10 mg / kg of NIMP-R14 antibody and 150 μg of clodronate liposomes is injected every 2 to 3 days. Hematocrit and transplantation of human red blood cells into the peripheral blood of the mouse are monitored throughout the test in blood samples taken from the tail. After P. falciparum infection, human red cell transplantation is performed every 2-3 days except for mice with a hematocrit greater than 60% and a% human red blood cell above 60%. These mice only receive immunosuppressive therapy and the transplantation of human red blood cells occurs when the hematocrit decreases to 50%. On day 4, treatment, starting at day 7 post-infection, with a dose of 25 mg / kg / day of indolone-N-oxide / albumin nanoparticles administered intravenously, induced a significant reduction in parasitaemia. The formulation proves to be very effective with a parasitaemia reduction of 97.5% measured the day after the end of the treatment, in all the treated mice. In addition, 67% of the treated mice survive until the end of the experiment.

Table 2 below shows the results obtained for the in vivo anti-malarial activity of indolone-N-oyide / HSA derivative nanoparticles tested in humanized mice infected with P. falciparum compared with the treatment references of the present invention. chloroquine and artesunate. Compound Dose Percentage inhibition of parasitemia Chloroquine 15 mg / kg / 4 days (oral route 100) Artesunate 4 mg / kg / 4 days (oral route) Nanoparticles 25 mg / kg / 4 days (pathway 97.5 derived from Indolone-N-Intravenous) Oxide / HSA (Human Serum Albumin) Table 2. Example 7: Analysis of Release Kinetics of an Indolone-N-Oxide Derivative As shown in Figure 2, the dissolution kinetics of pure indolone-N-oxide derivative is relatively weak (100% dissolved in 3h) with a shift in time of 45 minutes which corresponds to the lack of wettability of the compound. In contrast, the homogenized nanoparticles of the indolone-N-oxide and albumin derivative show a rapid dissolution of the indolone-N-oxide derivative (100% dissolved in 30 minutes compared to 1.5% for the indolone derivative). Pure N-oxide over the same period). Example 8: Preparation of nanoparticles comprising artemisinin and human albumin 30 mg of artemisinin are dissolved in 9 ml of ethanol (3.3 mg / ml), the dissolution being improved in an ultrasonic bath. 300 mg of human albumin are dissolved in 60 ml of Milli-Q water in a suitable flask. The artemisinin / ethanol solution is gently added to the albumin solution, with stirring with a magnetic stirrer, to give a white colloidal solution. This solution is subjected to evaporation by a rotavapor for 30 minutes at 36 ° C. (to eliminate the ethanol). After complete evaporation of the ethanol, the contents of the flask are transferred to a graduated container and the volume is made up to 60 ml with Milli-Q water. The initial suspension is homogenized under a pressure gradient: 5 cycles at 15,000 psi (103421.35 l (13a), then 35 cycles at 25,000 psi (172368.93 l (13a). , 10, 15, 20, 25, 35 and 40 cycles 50 ml are rapidly cooled The sample is immersed in liquid nitrogen for 1.5 hours and immediately bonded to a lyophilizer.

Example 9: Analysis of the Size of the Nanoparticles Produced and Measurement of Their Zeta Potential Measurements of the size of the nanoparticles and their Zeta potential are made using a DelsaNano C particle analyzer (Beckman Coulter). The measurements are made in triplicate at 25 ° C. Polydispersity and mean diameter analyzes are based on a photon correlation spectroscopy (PCS) technique, also known as dynamic light scattering (DLS). Prior to measurements, the nanosuspension of artemisinin and albumin is diluted in Milli-Q water to an appropriate diffusion intensity (1: 100). The diffusion angle is 165 °.

The size of the particles is expressed in intensity-weighted mean diameter in nanometers, and the particle size distribution is expressed in polydispersity index (PI). Zeta potential is a measure of the electrical charge on the surface of particles. High Zeta potentials (> or = at 30 mV) indicate a physically stable colloid system, whereas nanosuspensions with a low Zeta potential tend to flocculate. Zeta potential measurements are based on the electrophoretic light scattering (ELS) technique. The sample preparation is identical to that for measuring particle sizes, using a 15 ° scattering angle and the applied magnetic field is 5 V. Standardization is done to have a conductivity of 10 mS / cm using a aqueous solution of KCl (0.582% w / v). EXAMPLE 10 Analysis of the Stability of Nanoparticles Comprising Artemisinin and Human Albumin The physical stability of the nanosuspensions was examined for the potential growth of the particles or their aggregation after storage of the reconstituted nanoparticles for 4 days at 4 ° C. using a DelsaNano C particle analyzer (Beckman Coulter, France). The chemical stability of artemisinin in the nanoparticles was evaluated after storage of nanosuspensions at 4 ° C. for 4 days and freeze-dried powders for 1 month. 1 mg of nanoparticles is reconstituted in 1 mL of Milli-Q water. 4 mL of acetonitrile are then added and the mixture is sonicated and then centrifuged. The amount of artemisinin in the supernatant is analyzed by HPLC-MS.

The reconstituted nanoparticles show satisfactory physical stability when stored at 4 ° C for 4 days. Particle growth is 5.8% and the polydispersity index remains within an acceptable range (<0.25). High Zeta potential values (-43.8 mV) can be involved in this stability by providing an electrostatic repulsion that prevents aggregation of particles. Artemisinin shows good chemical stability in nanoparticles both in powder form and in reconstituted form. After storage of the nanoparticles for 1 month at 4 ° C., the percentage of artemisinin remaining in the nanoparticles is 98.4%. In the reconstituted aqueous form at 4 ° C for 4 days, artemisinin was detected with an average recovery percentage of 96.6%. Example 11: Anti-malaria activity evaluated in vivo in humanized mice infected with P. falciparum The evaluation of the anti-malaria activity in vivo of the artemisinin nanoparticles is carried out on humanized mice. 9- to 11-week-old NSG mice (Charles River, US) are used to obtain an in vivo P. falciparum infection.

Human red blood cells are obtained from donors with no history of malaria (French Blood Establishment, Rungis, France). The human red blood cells are washed twice with RPMI-1640 medium (Gibco / BRL) and centrifuged at 900 rpm, 10 min at 25 ° C. The supernatant is removed and the human red blood cells are resuspended in RPMI-1640 medium and stored at 4 ° C for a maximum of 2 weeks. Before use, the human red blood cells are washed twice again and centrifuged at 900 rpm for 10 min at 25 ° C in RPMI-1640 medium. The "humanized" NSG mice for red blood cells are infected with the P. falciparum strain 3D7 according to the protocol described in the following article: Moreno et al., Int. I Parasitol., 2006, 36, 361-369.

Tissue depletion in macrophages is induced by clodronate provided by Roche Diagnostic and encapsulated in liposomes as described in the following article: Van Rooijen et al., Immunol. Methods, 1994, 174, 83-93. Neutrophils are monitored using a monoclonal antibody NIMP-R14. The hybridoma that produces this antibody was provided by Dr. M. Strath (National Institute for Medical Research, London, U.K.). At day 0, each mouse is injected intraperitoneally with a dose of 10 mg / kg of this NIMP-R14 antibody, mixed with 300 μg of clodronate liposomes. On day 2, 4 and 6, each mouse receives, intraperitoneally, 0.5 ml of human red blood cells mixed with a dose of 10 mg / kg of NIMP-R14 antibody and 300 μg of clodronate liposome. At day 8, each mouse is injected with 0.5 mL of P. falciparum-infected human red blood cells at a parasitaemia of 0.3%, mixed with a dose of 10 mg / kg of NIMP-R14 antibody and 300 i_tg of clodronate liposome. After infection with P. falciparum, a dose of 10 mg / kg of NIMP-R14 antibody and 150 μg of clodronate liposome is injected every 2 to 3 days. Hematocrit and human red cell transplantation into the peripheral blood of the mouse are monitored throughout the assay in blood samples taken from the tail. After P. falciparum infection, human red cell transplantation is performed every 2-3 days except for mice with a hematocrit greater than 60% and a% human red blood cell above 60%. These mice only receive immunosuppressive therapy and the human red cell transplant is done when the hematocrit decreases to 50%. The formulated compound is administered to mice infected with P. falciparum at a dose of 10 mg / kg every 4 days. Humanized mice are infected intravenously with the human P. falciparum parasite (strain 3D7). At day 4, treatment with 10 mg / kg artemisinin nanoparticles and albumin induced a significant reduction in parasitaemia (96%) measured after the last day of the end of treatment (Figure 5). Mice survive more than 18 days without recrudescence (Table 4). In comparison, artesunate inhibits by itself only 28% parasitaemia at a dose of 4 mg / kg tested in the same model (Coslédan et al., 2008, PNAS 105 (45): 17579-17584).

The in vivo anti-plasmodial activity of the artemisinin / HSA nanoparticles tested in humanized mice infected with P. falciparum is shown in Table 4 below. Compound Dose (mg / kg / day) Percentage inhibition of parasitaemia Nanoparticles (intravenous) 96 artemisinin / HSA Artesunate (Coslédan et al, 2008) 4 (oral) 28 Chloroquine 15 (oral) 100 Table 3. 10 15 20 25

Claims (21)

CLAIMS1- A composition comprising an antimalarial agent as an active ingredient and human albumin as a carrier, for use as an injectable drug in the form of nanoparticles in the treatment of malaria. 2. Composition according to claim 1, in which the active substance is chosen from indolone-N-oxide derivatives or artemisinin. 3- Composition according to one of claims 1 to 2, wherein the active substance is selected from compounds of formula Ia: the wherein: - the substituent or substituents R1 and R2 are chosen indifferently in the group comprising the atom d hydrogen, a halogen, especially chlorine and fluorine, the CF 3 group, the alkyl groups of 1 to 3 carbons, in particular methyl, the alkyloxy groups of 1 to 6 carbons, in particular methoxy, benzyloxy, the -COCH 3 groups, -COOCH 3 groups; , -OCF3, NO2, the NH2 group and the alkylamino groups, - the two adjacent substituents R1 and R2 that can form the methylenedioxy-O (CH2) O- ring; Y represents either: a branched phenyl group of formula IIa in which the substituent or substituents R3, R4 and R5 are chosen indifferently from the group comprising hydrogen, a halogen, in particular F, Cl, the alkylfluorinated groups in particular CF3, the alkyl groups having 1 to 3 carbons, the alkyloxy groups, in particular OCH 3, OCF 3, phenyloxy, the alkylamino, dialkyl or monoalkyl groups, in particular (CH 3) 2 N, (C 2 H 5) 2 N, (C 3 H 7) 2 N, (C 3 H 7) NH and their salts, the groups NO2, NH2, CH3CONH, CH3OCO, 4-chlorophenyl, tert-butyl, isopropoxy, hydroxymethyl, morpholin-4-yl, butylamino, - a pyren-1-yl group, a 6'-methoxy-naphthalen-2-yl group, a 2'-pyridinyl group a 3'-pyridinyl group, or - a thiophen-3-yl group, - Z represents either: - N + O-, and - corresponds to a covalent double bond, or - NH or NOH, and - corresponds to a covalent single bond, and in particular wherein the active substance is selected from the following compounds: 0 Cl 2. 3. 5. F3C CF3 CF3 CIN + 0- 4. F3C. ON + 0- 6. CF3 0 02N7. 8.14. 53 N 10. N \ _ 11. NO2 HAN 12. N H 2 13. 15.20. C. The composition according to one of claims 1 or 2, wherein the active substance is artemisinin. 26. 24. 25. 23. 22.105- Composition according to one of the preceding claims, wherein the active substance and human albumin are present in a weight ratio of 1: 5 to 1: 20. 6- Composition according to the one of the preceding claims, wherein the active substance is capable of being used in unit dose form of 375 to 1125 mg. 7. Composition according to one of the preceding claims, wherein the nanoparticles have a size ranging from 200 to 400 nm. 10 8. A pharmaceutical composition comprising an antimalarial agent as the active substance and human albumin as a carrier of the drug delivering the active ingredient to the parasitized red blood cells, in particular in the form of nanoparticles, and in particular containing as active substance a molecule chosen from indolone-N-oxide derivatives or artemisinin. 9. The pharmaceutical composition as claimed in claim 8, in which the active substance is chosen from the compounds of formula Ia in which: the substituent (s) R1 and R2 are chosen indifferently from the group comprising the hydrogen atom; a halogen, especially chlorine and fluorine, the CF 3 group, the alkyl groups of 1 to 3 carbons, in particular methyl, the alkyloxy groups of 1 to 6 carbons, in particular methoxy, benzyloxy, the -COCH 3 groups, -COOCH 3, OCF 3, NO 2, the NH 2 group and the alkylamino groups, the two adjacent substituents R 1 and R 2 which can form the methylenedioxy-O (CH 2) O- ring; Y represents either: a branched phenyl group of formula IIa IIa in which the substituent or substituents R3, R4 and R5 are chosen indifferently from the group comprising hydrogen, a halogen, in particular F, Cl, the alkylfluorinated groups in particular CF3, alkyl groups having 1 to 3 carbons, alkyloxy groups, in particular OCH 3, OCF 3, phenyloxy, alkylamino, dialkyl or monoalkyl groups, in particular (CH 3) 2 N, (C 2 H 5) 2 N, (C 3 H 7) 2 N, (C 3 E 17) NH and their salts, the groups NO 2, NH 2, CH 3 CONH, CH 3 OCO, 4-chlorophenyl, tert-butyl, isopropoxy, hydroxymethyl, morpholin-4-yl, butylamino, a pyren-1-yl group, a 6'-methoxy group; naphthalen-2-yl, - a 2'-pyridinyl group, - a 3'-pyridinyl group, or - a thiophen-3-yl group, - Z represents either: - N + O-, and - corresponds to a double covalent bond, or - NH or NOH, and - corresponds to a single covalent bond, and in particular in which the active substance is chosen from the compounds following values: 0 20 1o ci Cl 2. Cl 3. o- 6. 7. CF3 4. CF3 F C 5. O CF3 02N8. NOT 10. N \ _ 11. NO2 H N 12. N H 213. 9. O-0 16. 60 14. 15. CI 17. 18. 19.5. 22.- Pharmaceutical composition according to claim 8, in which the active substance chosen is artemisinin. 11- The pharmaceutical composition according to one of claims 8 to 10, wherein the active substance and human albumin are present in a weight ratio of 1: 5 to 1: 20. 12- Pharmaceutical composition according to one of claims 8 at 11, in which the active substance in the presence of human albumin is capable of being used in the form of a unit dose of 375 to 1125 mg. 13- Pharmaceutical composition according to one of claims 8 to 12, in the form of injectable solution or intravenously in the form of lyophilisate. 14. Nanoparticles comprising an anti-malarial agent as active substance and human albumin, and in particular in which the active substance is chosen from indolone-N-oxide derivatives or artemisinin. 15- Nanoparticles according to one of claims 14, wherein the active substance is selected from the compounds of formula Ia: in which: - the substituent or substituents R1 and R2 are chosen indifferently in the group comprising the hydrogen atom, a halogen, especially chlorine and fluorine, the CF 3 group, the alkyl groups of 1 to 3 carbons, in particular methyl, the alkyloxy groups of 1 to 6 carbons, in particular methoxy, benzyloxy, the groups -COCH 3, -COOCH 3, - OCF3, NO2, the NH2 group and the alkylamino groups, - the two adjacent substituents R1 and R2 which can form the methylenedioxy ring O (CH2) O-; Y represents either: a branched phenyl group of formula IIa in which the substituent or substituents R3, R4 and R5 are chosen indifferently from the group comprising hydrogen, a halogen, in particular F, Cl, the alkylfluorinated groups in particular CF3; , alkyl groups having 1 to 3 carbons, alkyloxy groups, in particular OCH 3, OCF 3, phenyloxy, alkylamino, dialkyl or monoalkyl groups, in particular (CH 3) 2 N, (C 2 H 5) 2 N, (C 3 H 7) 2 N, (C 3 H 7) NH and their salts, the groups NO 2, NH 2, CH 3 CONH, CH 3 OCO, 4-chlorophenyl, tert-butyl, isopropoxy, hydroxymethyl, morpholin-4-yl, butylamino, a pyren-1-yl group, a 6 'group; -methoxy-naphthalen-2-yl, - a 2'-pyridinyl group, - a 3'-pyridinyl group, or - a thiophen-3-yl group, - Z represents either: 25 - N + O-, and - corresponds to a covalent double bond, or - NH or NOH, and - corresponds to a single covalent bond, and in particular in which the active substance is chosen from the following compounds: Cl 2. 110 0 4. cIN + 0- 5. F CF3 6. o- 3. CF3 64 1. o CI CI7. CF 3 O 02N 65 8. N 10. N 11. 11. NO 2 HAN 12. 9. O-016. 17. 18. NH 2 13. 14. 15. CI 6623. 67 19. 20. H 21. 22. 24. 25. 26. 68 16. The pharmaceutical composition according to claim 14, wherein the selected active substance is artemisinin. 17- Nanoparticles according to one of claims 14 to 15, wherein the active substance is an indolone-N-oxide derivative, the derivative of indolone-N-oxides and human albumin being present in a concentration ratio active substance: albumin ranging from 1: 5 to 1:20, the nanoparticles forming a substantially homogeneous assembly with a polydispersity index of less than 0.25 and a particle diameter of less than 330 nm. 18- Nanoparticles according to claim 16, wherein the active substance is artemisinin, artemisinin and human albumin being present in an active substance concentration ratio: albumin ranging from 1: 5 to 1: 10 the nanoparticles forming a together substantially homogeneous with a polydispersity index of less than 0.25 and a particle diameter of less than 350 nm. 19. A process for the preparation of nanoparticles comprising: a step of homogenization at a pressure ranging from 15,000 to 30,000 psi, of a mixture of an antimalarial agent and dissolved albumin, respectively, in an organic solvent and in a solvent aqueous, to obtain a nanosuspension, - a step of evaporation of the organic solvent contained in the nanosuspension, to obtain a nanosuspension devoid of organic solvent, - and a lyophilization step of the nanosuspension devoid of organic solvent, to obtain nanoparticles. 5 20. Process for preparing nanoparticles according to claim 19, in which the antimalarial agent is chosen from indolone-N-oxide derivatives, the indolone-N-oxide derivatives being dissolved in acetone, albumin. dissolved in water or phosphate buffer, the homogenization step being carried out at 25,000 psi for 15 cycles. 21. A process for preparing nanoparticles according to claim 19, wherein the antimalarial agent is artemisinin, artemisinin being dissolved in ethanol, the albumin being dissolved in water or phosphate buffer. and the homogenization step being performed at 15,000 psi for 5 cycles, followed by 35 cycles at 25,000 psi. 20