EP2480219A1 - Lipid nanocapsules, method for preparing same, and use thereof as a drug - Google Patents

Abstract

The present invention relates to nanocapsules, including: a core essentially consisting of a fatty substance, which is liquid or semi-liquid at ambient temperature, and including a hydrophobic active principle and a diethylene glycol ether; an outer lipid shell which is solid at ambient temperature. The lipid nanocapsules of the invention are intended in particular for the manufacture of a drug.

Description

 LIPID NANOCAPSULES, PREPARATION METHOD AND USE AS MEDICAMENT

The present invention relates to lipid nanocapsules (NCLs), a process for their preparation and their use for the manufacture of a medicament, intended in particular to be administered orally.

 In recent years, many groups have developed lipid solid nanoparticle formulations or lipid nanospheres (Muller, RH and Mehnert, European Journal of Pharmaceutics and Biopharmaceutics, 41 (1): 62-69, 1995; W., Gasco, MR, Pharmaceutical Technology Europe: 52-57, December 1997, EP 605,497). This is an alternative to using liposomes or polymer particles. These lipid particles have the advantage of being formulated in the absence of a solvent. They have made it possible to encapsulate both lipophilic and hydrophilic products in the form of ion pairs, for example (Cavalli, R. et al, ST Pharma Sciences, 2 (6): 514-518, 1992; Cavalli, R. et al., International Journal of Pharmaceutics, 17: 243-246, 1995). These particles can be stable for several years away from light, at 8 ° C (Freitas, C. and Muller, R.H., Journal of Microencapsulation, 1 (16): 59-71, 1999).

 Two techniques are commonly used to prepare lipid nanoparticles:

 homogenization of a hot emulsion (Schwarz, C. et al, Journal of Controlled Release, 30: 83-96, 1994, Muller, R.H. et al, European

Journal of Pharmaceutics and Biopharmaceutics, 41 (1): 62-69, 1995) or cold (Zur Muhlen, A. and Mehnert W., Pharmazie, 53: 552-555, 1998; EP 605 497), or

quenching of a microemulsion in the presence of co-surfactants such as butanol. The size of the nanoparticles obtained is generally greater than 100 nm (Cavalli, R. et al, European Journal of Pharmaceutics and Biopharmaceutics, 43 (2): 1-10-1-15, 1996; Morel, S. et al, International Journal of Pharmaceutics, 132: 259-261, 1996). Cavalli et al. (International Journal of Pharmaceutics, 2 (6): 514-518, 1992 and Pharmazie, 53: 392-396, 1998) describe the use of a bile salt, taurodeoxycholate, which is nontoxic by injection for the formation of nanospheres larger than or equal to 55 nm.

 The Applicant in the international application WO01 / 64328 discloses nanocapsules consisting of a liquid or semi-liquid core at room temperature, coated with a solid film at room temperature. In particular, the core of these nanocapsules consists essentially of a liquid or semi-liquid fatty substance at room temperature, for example a triglyceride or a fatty acid ester, and the solid film coating the nanocapsules essentially consists of a lipophilic surfactant, for example a lecithin whose proportion of phosphatidylcholine is between 40 and 80%. The preparation process is based on a phase inversion step of the oil / water emulsion formed by the constituents of the nanocapsules.

 However, this method requires solubilizing the active ingredient. However, some active ingredients are insufficiently soluble in the fatty phase to be encapsulated according to this method.

 The technical problem solved by the present invention consists in proposing a process for preparing nanocapsules which allows encapsulation in nanocapsules in sufficient quantity of such active principles.

 Unexpectedly, the Applicant has developed such a method. The inventors have in particular observed that the solubilization of such an active ingredient in a diethylene glycol ether is necessary to solve the technical problem.

The present invention therefore relates to nanocapsules comprising: a core consisting essentially of a liquid or semi-liquid fatty substance at ambient temperature, and comprising a hydrophobic active ingredient and a diethylene glycol ether,

a solid outer lipid shell at room temperature. Nanocapsules are understood to mean particles consisting of a liquid or semi-liquid core at ambient temperature, coated with a solid film at ambient temperature, as opposed to nanospheres which are matrix particles, ie whose whole mass is solid. . When the nanospheres contain a pharmaceutically active ingredient, it is finely dispersed in the solid matrix.

 In the context of the present invention, ambient temperature is understood to mean a temperature of between 15 and 25 ° C.

 The present invention relates to nanocapsules of average size less than 150 nm, preferably less than 100 nm, more preferably less than 50 nm.

 Given their size, the nanocapsules of the invention are colloidal lipid particles.

 The polydispersity index of the nanocapsules of the invention is advantageously between 5 and 15%.

 Preferably, the diethylene glycol ether used is chosen from the group consisting of diethylene glycol monoethyl ether, diethylene glycol monomethyl ether and diethylene glycol mono-n-butyl ether.

 Particularly preferably, diethylene glycol monoethyl ether, for example Transcutol® HP, is used as the diethylene glycol ether.

 The active ingredient is not or only slightly soluble or dispersible in oily fat phase and is preferably not soluble in most pharmaceutically acceptable solvents.

 Advantageously, the active ingredient is SN38.

SN38, 7-ethyl-10-hydroxycamptothecin, is the active metabolite of Irinotecan (CPT1 1), an inhibitor of topoisomerase I. SN 38 is 200 to 2000 times more toxic than CPT1 1. However, SN38 has not been used as an anticancer because of its low solubility in pharmaceutically acceptable solvents.

 Advantageously, the nanocapsules according to the invention contain more than 0.1 mg, preferably 0.3 mg, particularly preferably 0.4 mg of active ingredient per gram of nanocapsules.

 Preferably, the fatty body of the core consists essentially of at least one triglyceride, a fatty acid ester, a polyethoxylated glyceride, or a mixture thereof.

 The fatty body of the core represents 20 to 60%, preferably 25 to 50% by weight of the nanocapsules.

 The triglycerides used may be synthetic triglycerides or triglycerides of natural origin. Natural sources may include animal fats or vegetable oils eg soybean oils or sources of long chain triglycerides (TCL).

 Other triglycerides of interest are composed mainly of medium length fatty acids also called medium chain triglycerides (MCTs). A medium chain triglyceride oil (MCT) is a triglyceride in which the hydrocarbon chain has 8 to 12 carbon atoms (C 8 to C 12). Such TCM oils are commercially available.

 By way of example of these TCM oils, mention may be made of the TCRs (commercial name of the industrial oilseed company, France, for a mixture of triglycerides in which about 95% of the fatty acid chains have 8 or 10 carbon atoms. ) and Miglyol 812 (triglyceride marketed by the companies Dynamit Nobel and Sasol Condea Chemie, for a mixture of triesters of glycerides of caprylic acid and capric).

The fatty acid units of these triglycerides may be unsaturated, monounsaturated or polyunsaturated. Mixtures of triglycerides with variable fatty acid units are also acceptable. The triglyceride constituting the core of the nanocapsules is in particular chosen from C 8 to C 12 triglycerides, for example triglycerides of capric and caprylic acids, and mixtures thereof.

 The fatty acid ester is chosen from Cs to C18 fatty acid esters, for example ethyl palmitate, ethyl oleate, ethyl myristate, isopropyl myristate and myristate. octyldodecyl, and mixtures thereof. The fatty acid ester is preferably C 8 to C 12.

 The polyethoxylated glyceride is chosen from a mixture of glycerides and polyethylene glycol, a PEG-6 ester and apricot kernel oil, olive oil, hydrogenated palm oil, for example Labrafil® M 1944 CS Labrafil® M 1969 or Labrafil® M 1980 (Gattefossé, Saint Priest, France).

 Advantageously, the outer shell of the nanocapsules according to the invention consists essentially of a lipophilic surfactant and a hydrophilic surfactant.

 Preferably, the lipophilic surfactant is a phospholipid such as lecithins, phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, phosphatidic acid and phosphatidylethanolamine.

 Phospholipids are advantageous because of their biocompatible nature.

 As commercial products derived from phospholipids, mention may be made more particularly of:

 - EPICURON 120 (Lukas Meyer, Germany) which is a mixture of about 70% phosphatidylcholine, 12% phosphatidylethanolamine, and about 15% other phospholipids;

 OVOTINE 160 (Lukas Meyer, Germany) which is a mixture comprising approximately 60% of phosphatidylcholine, 18% of phosphatidylethanolamine, and 12% of other phospholipids,

 a mixture of purified phospholipids in the image of the products

Lipoid E75 or Lipoids E-80 (Lipoid, Germany) which is a mixture of phospholipids comprising about 80% by weight of phosphatidylcholine, 8% by weight of phosphatidylethanolamine, 3.6% by weight of non-polar lipids and 2% of sphingomyelin.

 According to a preferred embodiment, the lipophilic surfactant is a lecithin whose proportion of phosphatidylcholine ranges from 40 to 80% by weight. Particularly suitable as a source of phosphatidylcholine is Lipoid S75-3 (Lipoid GmbH, Germany). This is soy lecithin. The latter contains about 69% phosphatidylcholine and 9% phosphatidylethanolamine. This component is the only component solid at 37 ° C and at room temperature in the formulation. It is also possible to use polyglyceryl-6-dioleate (Plural®). The hydrophilic surfactant used according to the present invention is advantageously an amphiphilic hydrophilic surfactant.

 The emulsifying surfactants of the oil-in-water type usually used have an HLB (HLB = Hydrophilic Lipophilic Balance) ranging from 8 to 18. These emulsifiers, thanks to their amphiphilic structure, are placed at the oily phase / aqueous phase interface, and thus stabilize the dispersed oil droplets.

Thus, the surfactant system used in the microemulsion may comprise one or more surfactants whose solubility in the oil increases with increasing temperature. The HLB of these surfactants can vary from 8 to 18 and preferably from 10 to 16, and these surfactants can be chosen from ethoxylated fatty alcohols, ethoxylated fatty acids, partial glycerides of ethoxylated fatty acids, and triglycerides of acids. polyethoxylated fatty acids and mixtures thereof. As ethoxylated fatty alcohols, mention may be made, for example, of ethylene oxide adducts with lauryl alcohol, especially those containing from 9 to 50 oxyethylenated groups (Laureth-9 to Laureth-50 in CTFA names); adducts of ethylene oxide with behenyl alcohol, in particular those containing from 9 to 50 oxyethylenated groups (Beheneth-9 to Beheneth-50 in CTFA names); adducts of ethylene oxide with keto-stearyl alcohol (mixture of cetyl alcohol and stearyl alcohol), in particular those containing from 9 to 30 oxyethylenated groups (Ceteareth-9 to Ceteareth-30 in CTFA names); adducts of ethylene oxide with cetyl alcohol, especially those containing from 9 to 30 oxyethylenated groups (Ceteth-9 to Ceteth- in CTFA names); ethylene oxide adducts with stearyl alcohol, especially those having from 9 to 30 oxyethylenated groups (Steareth-9 to Steareth-CTFA names); adducts of ethylene oxide with isostearyl alcohol, especially those containing from 9 to 50 oxyethylenated groups (lsosteareth-9 to lsosteareth-50 in CTFA names); and their mixtures.

 As ethoxylated fatty acids, mention may be made, for example, of ethylene oxide adducts with lauric, palmitic, stearic or behenic acids, and mixtures thereof, in particular those comprising from 9 to 50 oxyethylenated groups such as laurates of PEG-9 to PEG-50 (CTFA names: PEG-9 laurate to PEG-50 laurate); palmitates of PEG-9 to PEG-50 (in CTFA names: PEG-9 palmitate to PEG-50 palmitate); stearates from PEG-9 to PEG-50 (CTFA names: PEG-9 stearate PEG-50 stearate); palmitostearates from PEG-9 to PEG-50; behenates from PEG-9 to PEG-50 (in CTFA names: PEG-9 behenate to PEG-50 behenate); and their mixtures.

Mixtures of these oxyethylenated derivatives of fatty alcohols and fatty acids can also be used. These surfactants may also be either natural compounds like the phospholipids écholates or synthetic compounds such as polysorbates, which are fatty acid esters of polyethoxylated sorbitol (Tween ^®), polyethylene glycol esters of fatty acid from e.g. castor oil (Cremophor ^®), polyethoxylated fatty acids, eg stearic acid (Simulsol M-53 ^®), fatty alcohol ethers polyoxyethylene (Brij ^®), polyoxyethylene nonphenyl ethers (Triton ^® N), polyoxyethylenated hydroxylphenyl ethers (Triton ^® X).

It may especially be a 2-hydroxystearate polyethylene glycol and in particular that marketed under the name Solutol ^® HS15 by BASF (Germany). The hydrophilic surfactant contained in the solid film coating the nanocapsules is preferably between 20 to 50% by weight of the nanocapsules, preferably about 30%.

 The amount of lipophilic surfactant contained in the solid film coating the nanocapsules of the invention is preferably set so that the weight ratio of liquid fatty substance / solid surfactant compound is chosen between 1 and 15, preferably between 1 and 15. , 5 and 13, more preferably between 3 and 8.

 The nanocapsules of the invention are particularly suitable for the formulation of pharmaceutical active principles. In this case, the lipophilic surfactant may be advantageously solid at 20 ° C. and liquid at about 37 ° C.

 Advantageously, the nanocapsules of the invention have a ratio in the core lipids / [active principle + diethylene glycol ether] of between 0.5: 1 and 1: 2.

 Advantageously, the nanocapsules of the invention comprise

a core consisting of SN38, diethylene glycol monoethyl ether, a capric and caprylic acid triglyceride and a PEG-6 ester of apricot kernel oil,

 an outer shell made of lecithin, preferably a lecithin of which the proportion of phosphatidylcholine is between 40 and 90%, and 2-hydroxystearate polyethylene glycol in a ratio of 1: 0.09 to 0.15: 1.

 Advantageously, the core of the nanocapsules of the invention consists of

 a central core consisting of the hydrophobic active ingredient and a diethylene glycol ether,

 a lipid layer consisting of the fatty substance surrounding said core.

The present invention also relates to a process for preparing the nanocapsules described above. The method of the invention is based on the phase inversion of an oil / water emulsion caused by several cycles of rise and fall in temperature.

 The preparation process of the invention comprises the following steps:

 a) solubilization of the active ingredient in a solution of a diethylene glycol ether,

 b) preparing an oil / water emulsion by adding to the solution of step a) at least one triglyceride, a polyethoxylated glyceride, a solid lipophilic surfactant at 20 ° C, a nonionic hydrophilic surfactant, an aqueous phase and a salt,

 c) effecting the phase inversion of said oil / water emulsion by increasing the phase inversion temperature (TIP) with stirring, to obtain a water / oil emulsion, followed by a decrease in the temperature up to at a temperature T1, T1 <TIP <T2,

 d) carrying out one or more temperature cycles with stirring, preferably at least 3, around the phase inversion zone between T1 and T2, until a translucent suspension is observed,

 e) performing quenching with an aqueous solution of the oil / water emulsion at a temperature close to T1, preferably greater than T1, to obtain stable nanocapsules

 This step stabilizes the nanocapsules formed. It consists of a rapid cooling, with magnetic stirring, by diluting the emulsion between 3 and 10 times with deionized water or an acid buffer at 2 ° C. +/- 1 ° C., which is thrown into the emulsion. fine.

Thus, all the constituents intended to form the emulsion are weighed in a container. The mixture is homogenized and heated, for example by means of stirring on a hot plate, until a temperature greater than or equal to the phase inversion temperature T2, that is to say until a white phase is obtained which indicates the obtaining of the inverse emulsion (W / O). The heating is then stopped and the stirring maintained until it returns to ambient temperature, passing through the phase inversion temperature T1, that is to say the temperature at which the expected oil emulsion is formed. water, in the form of a transparent or translucent phase. When the temperature has fallen below the phase inversion temperature (TIP), an oil / water emulsion is obtained. More specifically, the phase inversion between the oil / water emulsion and the water / oil emulsion results in a decrease in the conductivity when the temperature increases. Thus, T1 is a temperature at which the conductivity is at least 90 - 95% of the measured conductivity and T2 is the temperature at which the conductivity decreases and the water-in-oil emulsion is formed. The average temperature of the phase inversion zone corresponds to the phase inversion temperature (TIP). The organization of the system in the form of nanocapsules visually translates into a change in appearance of the initial system that changes from white-opaque to white-translucent. This change occurs at a temperature below the TIP. This temperature is generally between 6 to 15 ° C below the TIP. In the formation zone of an oil / water emulsion (translucent mixture), the hydrophilic and hydrophobic interactions are balanced. By heating beyond this zone, an W / O emulsion (opaque white mixture) is formed, since the surfactant promotes the formation of a water-in-oil emulsion. Then, during cooling below the phase inversion zone, the emulsion becomes an O / W emulsion.

 The temperature T1 is between 55 and 70 ° C, preferably between 60 and 70 ° C. Particularly preferably T1 is 65 ° C.

The temperature T2 is between 85 and 100 ° C, preferably between 85 and 95 ° C. Particularly preferably T2 is 90 ° C. The number of cycles applied to the emulsion depends on the amount of energy required to form the nanocapsules. In a particularly preferred manner 3.

 Advantageously, step b) is broken down as follows:

 b1) adding to the solution of step a) at least one triglyceride, a polyethoxylated glyceride and a lipophilic surfactant, b2) heating until solubilization of the lipophilic surfactant, b3) cooling,

 b4) addition of the hydrophilic surfactant, the aqueous phase and the salt.

 The heating step b2) is carried out at a temperature between 50 and 70 ° C, preferably 70 ° C, in particular when the lipophilic surfactant is a lecithin.

 When the active principle is SN38, a basic buffer is added in step b4) to transform SN38 in free lactone form into SN38 in carboxylate form and step e) of the process of the invention is carried out with an aqueous solution acid. When the active ingredient is Sn38, the quenching is carried out with an acid buffer in order to retransform Sn38 in lactone form at the time of its encapsulation, preferably with an acid buffer at 2 ° C. ± 1 ° C.

 The particles obtained are maintained after quenching with stirring for 5 minutes.

The nanocapsules obtained according to the process of the invention are advantageously free of co-surfactants, such as C ₁ -C ₄ alcohols.

 The oil / water emulsion advantageously contains 1 to 3% of lipophilic surfactant, 5 to 15% of hydrophilic surfactant, 5 to 10% of cosurfactant (diethylene glycol ether), 5 to 15% of oily fatty substance, 40 to 65% of water (the percentages are expressed by weight).

The higher the HLB index of the liquid or semi-liquid fatty substance, the higher the phase inversion temperature. On the other hand, The value of the HLB index of the fatty substance does not seem to have any influence on the size of the nanocapsules.

 Thus, as the size of the end groups of the triglycerides increases, their HLB index decreases and the phase inversion temperature decreases.

 The HLB index or hydrophilic-lipophilic balance is as defined by C. Larpent in the K.342 Treaty of TECHNIQUES DE L'INGENIEUR.

 The size of the particles decreases as the proportion of hydrophilic surfactant increases and the proportion of surfactants (hydrophilic and lipophilic) increases. Indeed, the surfactant causes a decrease in the interfacial tension and thus a stabilization of the system which promotes the obtaining of small particles.

 On the other hand, particle size increases as the proportion of oil increases.

According to a preferred embodiment, the fatty substance consists of Labrafac ^® WL 1349 and Labrafil ^® M1944CS, the lipophilic surfactant is Lipoid ^® S 75-3 and the nonionic hydrophilic surfactant is Solutol ^® HS 15 and the co-surfactant or solubilizer is Transcutol ^® HP. These compounds have the following characteristics:

- Lipophilic Labrafac ^® WL1349 (Gattefossé, Saint-Priest, France). It is an oil composed of medium chain triglycerides of caprylic and capric acids (Cs and C10). Its density is 0.930 to 0.960 at 20 ° C. Its HLB index is of the order of 1.

 - The Labrafil® M1944CS (Gattefossé, Saint-Priest, France).

 It is an oleic macrogolglyceride (hydrophilic oil) composed of mono-, di-, triglycerides and mono- and di-esters of polyethylene glycol and fatty acid. Its density is 0.935 to 0.955 at 20 ° C. It can be used orally (in rats LD 50> 20ml / kg).

- Lipoid ^® S 75-3 (Lipoid GmbH, Ludwigshafen, Germany). The

Lipoid ^® S 75-3 corresponds to soy lecithin. This last contains about 69% phosphatidylcholine and 9% phosphatidyl ethanolamine. They are therefore surfactant compounds. This component is the only component solid at 37 ° C and at room temperature in the formulation. It is commonly used for the formulation of injectable particles.

- Solutol ^® HS 15 (Basf, Ludwigshafen, Germany). It is a 2-hydroxystearate of polyethylene glycol-660. It therefore plays the role of nonionic hydrophilic surfactant in the formulation. It is usable by injection (in the mouse in IV LD50> 3.16 g / kg, in the rat 1, 0 <LD 50 <1, 47 g / kg).

- Transcutol ^® HP (Gattefossé, Saint-Priest, France). It is a monoethyl ether of diethylene glycol. Its density is 0.985 to 0.991 at 20 ° C. It acts as a solubilizing or co-surfactant agent. It can be used orally (in rats LD 50> 5g / kg).

 The aqueous phase of the oil / water emulsion may also contain

1 to 4% of a salt such as sodium chloride. Changing the salt concentration causes a shift in the phase inversion zone. The higher the salt concentration, the lower the phase inversion temperature. This phenomenon will be interesting for the encapsulation of hydrophobic thermosensitive active ingredients. Their incorporation can be done at a lower temperature.

 The present invention also relates to the nanocapsules of the invention for their use as a medicament.

 The subject of the present invention is also the nanocapsules of the invention loaded with Sn38 for treating cancer.

 The present invention therefore also relates to the use of the nanocapsules of the invention for the manufacture of a medicament.

The present invention therefore also relates to the use of the nanocapsules of the invention loaded with Sn38 for the manufacture of a medicament for the treatment of cancer. The present invention also relates to a method of treatment comprising administering an effective amount of the nanocapsules of the invention to a patient in need thereof.

 The present invention also relates to a method of treatment comprising administering an effective amount of the nanocapsules of the invention loaded with Sn38 to a patient with cancer.

 The present invention also relates to a pharmaceutical composition comprising nanocapsules of the invention and at least one pharmaceutically acceptable vehicle.

 In the present invention, the term "pharmaceutically acceptable" is intended to mean that which is useful in the preparation of a pharmaceutical composition which is generally safe, non-toxic and neither biologically nor otherwise undesirable and which is acceptable for veterinary use as well as human pharmaceutical.

 The pharmaceutical compositions according to the invention can be formulated for oral, sublingual, subcutaneous, intramuscular, intravenous, intrathecal, epidural, transdermal, local or rectal administration, preferably oral, intended for mammals, including humans.

 The nanocapsules of the invention can be lyophilized. In this case a cryoprotectant such as trehalose may be added to the formulation to prevent the aggregation of the nanoparticles and maintain their redispersion. Lyophilization can make it possible to improve the stability of the nanocapsules over time, and also to envisage a dry formulation of these particles.

The nanocapsules of the invention may be administered in unit dosage forms, in admixture with conventional pharmaceutical carriers, to animals or humans. Suitable unit dosage forms include oral forms such as tablets, capsules, powders, granules and oral solutions or suspensions, administration forms. sublingual and oral, subcutaneous, intramuscular, intravenous, intranasal or intraocular administration forms and rectal administration forms.

 The present invention also relates to a pharmaceutical composition according to the invention as defined above for its use as a medicament.

 The nanocapsules of the invention are more particularly suitable for the administration of the following active ingredients:

 anti-infectives including antimycotics, antibiotics,

 - anticancer drugs,

 the active principles intended for the Central Nervous System which must pass the blood-brain barrier, such as anti-Parkinson's and more generally the active principles for treating neurodegenerative diseases.

 The present invention is illustrated by the following examples with reference to Figures 1 to 7.

 FIG. 1 represents the evolution of the conductivity as a function of the temperature of the oil / water emulsion described in Example 2.

 FIG. 2 represents the evolution of the Sn38 encapsulation rate as a function of time for different pH. The 100% corresponds to the initial encapsulation rate of the Sn38 formulation.

 Figure 3 shows the evolution of the release percentage of Sn38 from the initial encapsulation rate as a function of time after storage of the formulation at 2-8 ° C.

 FIG. 4 represents the percentage of cellular survival (HT-29 cells) as a function of the concentration of uncharged NCLs-Sn38, Sn38 or NCLs (white LNC)

FIG. 5 represents the rate of Sn38 encapsulation as a function of time after incubation of NCLs-Sn38 in a simulated gas medium. The 100% corresponds to the initial encapsulation rate of the Sn38 formulation. FIG. 6 represents the rate of Sn38 encapsulation as a function of time after incubation of NCLs-Sn38 in a fasting intestinal medium or a simulated fed intestinal medium. The 100% corresponds to the initial encapsulation rate of the Sn38 formulation.

Figure 7 shows the apparent permeability in cm. s ^"1 as a function of time for the dispersion of free Sn38 and Sn38 encapsulated in the NCLs.

EXAMPLE 1 Lipid Nanocapsules Not Loaded with Active Ingredient (White NCLs)

 A) Preparation

7% w / w of Transcutol ^® HP, 9.8% w / w of Labrafil ^® M 1944 CS, 3.9% w / w of Labrafac ^® and 1.5% w / w of Lipoid ^® S75-3 were mixed and heated at 85 ° C to solubilize Lipoid ^® . After cooling, Solutol ^® HS15 (9.8% w / w) NaCl (1, 0% w / w) and water (17.7%) were added and homogenized under magnetic stirring. Three progressive heating / cooling cycles between 65 and 90 ° C were then performed and at 70 ° C during the last cycle, an irreversible shock was induced by dilution with water at 2 ° C (49.3%). / w). Then, the NCLs suspension was mixed gently with magnetic stirring for 5 min at room temperature.

 B) Characterization

 Table I below shows the average size of the nanocapsules obtained under the conditions described above, after three temperature cycles, their polydispersity and their zeta potential and the pH of the dispersion obtained.

Size index

 potential

 average Polydispersity PH

 Zeta (mV)

 (nm) (PDI)

 NCLs

white 39 ± 3 0.210 ± 0.078 -8 ± 1 7.4 ± 0.2

(N = 12)

 TABLE I

 Example 2: Lipid nanocapsules loaded with Sn38

 A) Preparation

Sn38 was first solubilized in Transcutol ^® HP (0.5% w / _w ). A 7% w / w of the solution, 9.8% w / w Labrafil ^® M 1944 CS 3.9% w / w Labrafac ^® and 1.5% w / w Lipoid ^® S75-3 were added and the mixture was heated to 85 ° C to solubilize Lipoid ^® . After cooling, Solutol ^® HS15 (9.8% w / w) NaCl (1, 0% w / w) and the basic buffer (17.7%) were added and homogenized under magnetic stirring. The basic buffer was added to convert Sn38 free lactone to Sn38 carboxylate. Three progressive heating / cooling cycles between 65 and 90 ° C were then carried out and at 70 ° C during the last cycle, an irreversible shock was induced by dilution with acid buffer at 2 ° C (49.3% w / w). p). This quenching in acid buffer makes it possible to retransform the Sn38 in lactone form at the time of its encapsulation. Then, the NCLs suspension was mixed gently with magnetic stirring for 5 min at room temperature.

 B) Characterization

 Table II below shows the average size of the nanocapsules obtained under the conditions described above, after three temperature cycles, their polydispersity and their zeta potential and the pH of the dispersion obtained.

Size index

 potential

 average Polydispersity PH

 Zeta (mV)

 (nm) (PDI)

 NCLs

charged in 38 ± 2 0.133 ± 0.043 -8 ± 1 7.4 ± 0.1

Sn38 (n = 30)

 TABLE II

Figure 1 shows the evolution of the conductivity as a function of the temperature of the oil / water emulsion described in Example 2, during the 3 cycles of rise and fall in temperature between 50 and 95 ° C. The phase inversion zone starts at 70 ° C; at this temperature the system is translucent. Therefore, quenching which causes the irreversible shock is performed at a temperature <70 ° C or 68 ° C.

The encapsulation rate of Sn38 in the NCLs was carried out by UV spectrometry after filtration. It is 0.38 ± 0.06 mg / g of dispersion of NCLs, an encapsulation efficiency compared to the initial weighed quantity of 96 ± 8%

Example 3 Formulation of Lipid Nanocapsules Loaded with Sn38 in Greater Volume

It is possible to formulate LNCs of Sn38 by quadrupling the quantities.

 A) Preparation

Sn38 was first solubilized in Transcutol ^® HP (0.5% w / _w ). A 2.8 g of this solution, 4 g of Labrafil ^® M 1944 CS, 1, 6 g of Labrafac ^® and 0.6 g of Lipoid S75-3 ^® were added and the mixture was heated at 85 ° C for solubilize Lipoid ^® . After cooling, Solutol ^® HS15 (4 g), NaCl (0.4 g), the basic buffer (1, 7 g) and water (7.2 g) were added and homogenized under magnetic stirring. Three cycles of progressive heating / cooling between 65 and 90 ° C were then carried out and at 70 ° C during the last cycle, an irreversible shock was induced by dilution with acid buffer at 2 ° C (20 g) at room temperature. using a syringe. Then, the NCLs suspension was mixed gently with magnetic stirring for 5 min at room temperature. A final amount of the dispersion of

LNCs of Sn38 of about 40 g is obtained.

 B) Characterization

Table III below shows the average size of the nanocapsules obtained under the conditions described above, after three temperature cycles, their polydispersity and their zeta potential and the pH of the dispersion obtained. Rate

Size index

 average encapsulation potential Polydispersity

 Zeta (mV) (mg / g of

(nm) (PDI)

 dispersion)

NCLs

charged 7.3 ±

 35.1 ± 0.8 0.069 ± 0.014 -8.7 ± 1 .9 0.45 ± 0.03 in Sn38 0.1

(N = 5)

 TABLE III

Example 4: Short-term Stability at Different pHs

A stability study was conducted over 6h at 25 ° C at 3pH different. The pH of the dispersion of Sn38 NCLs was adjusted to 3, 7 or 10. The Sn38 loading rate of NCLs was measured after acidification of the sample taken in order to precipitate Sn38 and filtered with a filter.

Minisart ^® 0.2 μιτι (Sartorius, Goettingen, Germany).

FIG. 2 represents the rate of encapsulation with Sn38 as a function of time. The 100% corresponds to the initial encapsulation rate of the Sn38 formulation.

 At pH 3 a rapid release of Sn38 is observed. At pH7, this release is less important and is of the order of 40% after 6h. At pH 10, the encapsulation rate remains stable.

Example 5: Long-term stability at 4 ° C.

 The stability of NCLs loaded with Sn38 was evaluated after storage at

2-8 ° C. The pH, the particle size distribution, the zeta potential and the Sn38 loading of the sample were determined after filtering the sample using a Minisart ^® 0.2μηη filter (Sartorius, Goettingen,

Germany). Time Rate Yield Size Polydispersity PH Potential (days) encapsulation encapsulation (nm) zeta

 (mg / g) (%) (mV)

0 0.43 ± 0.06 89 ± 10 38.2 ± 1, 1 0.141 ± 0.042 7.3 ± 0.1 -7.7 ± 1, 0

14 0.41 ± 0.08 85 ± 13 39.0 ± 1, 1 0.147 ± 0.055 7.6 ± 0.08 -7.6 ± 0.3

28 0.34 ± 0.04 72 ± 16 39.3 ± 1, 2 0.141 ± 0.037 7.4 ± 0.03 -8.5 ± 0.4

84 0.33 ± 0.06 68 ± 12 40.1 ± 1, 1 0.135 ± 0.037 7.5 ± 0.03 -8.6 ± 0.7

At 4 ° C, there is no change in size, zeta potential and polydispersity nanocapsules. A decrease in the rate of encapsulation

 30% is observed after 1 month.

 5

 Example 6 Study of Release of Encapsulated Sn38

 A release study of Sn38 encapsulated in NCLs was

 performed. NCLs loaded with Sn38 were diluted 1: 200 (v / v) in

 phosphate buffered saline (PBS, pH = 7.4) and placed at 37 ° C under

 150rpm. A 0.5 mL sample was collected and replaced with PBS

 different time intervals. Samples were acidified and filtered

using a Minisart ^® 0.2 μιτι filter to remove free precipitated Sn38.

 Then, the load was measured by LC-MS / MS. The release was

 calculated by difference with initial load and profiles (percentage

 15 release time) were established.

 Figure 3 shows the percentage release of Sn38 from

 initial encapsulation rate as a function of time.

 The percentage of release of the active ingredient of the NCLs at pH 7.4 is approximately 8% after 3 days.

 20

 Example 7: Study of the in vitro toxicity of NCLs-SN38

 Cytotoxicity of NCLs-SN38, free SN38 and uncharged NCLs

was determined by an MTS test (CalITiter 96 ^® AQ _ue0 us non-radioactive

cell proliferation assay kit (Promega, Charbonnières, France)) on HT-29 cells (human cancer cell line colorectal human cells). The value IC ₅ o was calculated as the concentration of NCLs-SN38, SN38 or uncharged NCLs causing 50% cell death.

FIG. 4 represents the percentage of cell survival as a function of the concentration of uncharged NCLs-Sn38, Sn38 or NCLs (white LNCs). The IC ₅ o obtained with the NCLs of Sn38 and the free SN38 is less than 0.1 μΜ. This result is approximately 250 times greater than the cytotoxicity obtained with CPT1 1.

Example 8: Gastrointestinal Stability Study

The stability of Sn38 loaded NCLs was evaluated in various gastrointestinal media simulated at 37 ° C at 150rpm. A sample of 0.5 mL was analyzed at different time intervals. Samples were acidified and filtered using a Minisart ^® 0.2μηη filter to remove free precipitated Sn38. Then, the Sn38 loading was measured by LC-MS / MS. The study was performed in a simulated gastric medium described by the European Pharmacopoeia (1), in a simulated intestinal medium under fasting conditions and a simulated intestinal medium (2).

FIG. 5 represents the Sn38 encapsulation rate as a function of time after incubation in a simulated gas medium. The 100% corresponds to the initial encapsulation rate of the Sn38 formulation.

FIG. 6 represents the Sn38 encapsulation rate as a function of time after incubation in a fasting intestinal medium or a simulated fed intestinal medium. The 100% corresponds to the initial encapsulation rate of the Sn38 formulation.

Example 9 Study of the In Vitro Intestinal Permeability of NCLs-Sn38

The apparent permeability of free or encapsulated Sn38 in NCLs was studied on an in vitro intestinal cell model (Caco-2 model). The study is carried out on a system of culture chamber of the type ^® Transwell (Corning Costar, Cambridge, MA) at 37 ° C, 5% CO2. A dispersion of free Sn38 or Sn38 NCLs at the 5μΜ concentration was deposited at the apical level of the culture chambers. Samples of 0.05 ml and 0.150 ml respectively were taken apical and basolateral respectively and replaced with HBSS at different time intervals. Sn38 was measured by LC-MS / MS. The apparent permeability was measured according to the following formula (3, 4): P _e p = dQ / df x MACO (dQ / df = quantity in SN38 to basolateral level (gs ^"1), Co = initial concentration of SN-38 in apical level (pg.mL ^"1 ) and A = surface of the cell monolayer (cm ² ).

Figure 7 shows the apparent permeability in cm. s ^"1 in function of time for the dispersion of free SN38 and SN38 encapsulated in NCLs. A Papp 1, 63 ± 0,56.10 ⁶ cm ^{s" 1} is obtained after 6 h of incubation at 2h and increases to 5 , 69 ± 0.87.10 ⁶ cm.s ^"1 to ⁶ h for the dispersion of NCLs-Sn38 In the presence of free Sn38, a Papp of 0.31 ± 0.02 × 10 ⁶ cm.s ^-1 is obtained after ⁶ h of incubation.

References

1. Pharmacopeia U. Rockville, MD; 2006.

 2. Jantratid E, Janssen N, Reppas C, Dressman JB. Dissolution of media simulating conditions in the proximal human gastrointestinal tract: an update. Pharm Res 2008 Jul; 25 (7): 1663-76.

 3. Artursson P, Borchardt RT. Intestinal drug absorption and metabolism in cell cultures: Caco-2 and beyond. Pharm Res 1997

December; 14 (12): 1655-8.

4. Artursson P, Karlsson J. Correlation between oral drug uptake in humans and apparent drug permeability coefficients in human intestinal epithelial (Caco-2) cells. Biochem Biophys Res Commun 1991 Mar 29; 175 (3): 880-5.

Claims

1. Nanocapsules comprising:

 a core consisting essentially of a liquid or semi-liquid fatty substance at ambient temperature, and comprising a hydrophobic active ingredient and a diethylene glycol ether,

a solid outer lipid shell at room temperature.

2. Nanocapsules according to claim 1, characterized in that the diethylene glycol ether is diethylene glycol monoethyl ether for example Transcutol ^® HP

 3. Nanocapsules according to claim 1 or 2, characterized in that the active ingredient is SN38.

 4. Nanocapsules according to claim 3, characterized in that they contain more than 0.3 mg of SN38 per gram of nanocapsules.

 Nanocapsules according to any one of claims 1 to 4, characterized in that the fatty substance of the core consists essentially of at least one triglyceride, a fatty acid ester, a polyethoxylated glyceride, or a mixture thereof.

 6. Nanocapsules according to claim 5, characterized in that the triglyceride is a Cs to C12 triglyceride, for example a capric and caprylic acid triglyceride.

7. Nanocapsules according to claim 5, characterized in that the polyethoxylated glyceride is a PEG-6 ester of the apricot kernel oil, for example Labrafil ^® M1944CS

 8. Nanocapsules according to any one of claims 1 to 7, characterized in that the outer shell consists essentially of a lipophilic surfactant and a hydrophilic surfactant.

9. Nanocapsules according to claim 8, characterized in that the lipophilic surfactant is a phospholipid, for example a lecithin whose proportion phosphotidylcholine ranges from 40 to 90% by weight, for example Lipoid ^® S75.

10. Nanocapsules according to claim 8, characterized in that the nonionic hydrophilic surfactant is a 2-hydroxystearate polyethylene glycol, for example Solutol ^® HS15.

 1 1. Nanocapsules according to any one of the preceding claims, characterized in that the ratio in the lipid core / [active ingredient + diethylene glycol ether] is between 0.5: 1 and 1: 2.

 12. Nanocapsules according to any one of claims 1 to 11, comprising

 a core consisting of SN38, diethylene glycol monoethyl ether, a capric and caprylic acid triglyceride and a PEG-6 ester of apricot kernel oil,

 a hull made of lecithin, the proportion of phosphatidylcholine and 2-hydroxystearate polyethylene glycol in a ratio of 1: 0.09 to 0.15: 1.

 13. Nanocapsules according to any one of the preceding claims, characterized in that said core consists of

 a central core consisting of the hydrophobic active ingredient and a diethylene glycol ether,

 a lipid layer surrounding said core.

 14. Process for the preparation of nanocapsules according to any one of the preceding claims, comprising the following steps: a) solubilization of the active principle in a solution of a diethylene glycol ether,

 b) preparing an oil / water emulsion by adding to the solution of step a) at least one triglyceride, a polyethoxylated glyceride, a solid lipophilic surfactant at 20 ° C, a hydrophilic nonionic surfactant and a salt,

c) effecting the phase inversion of said oil / water emulsion by increasing the phase inversion temperature (TIP) under stirring, to obtain a water / oil emulsion, followed by a decrease in temperature to a temperature T1, TKTIP <T2,

 d) carrying out one or more temperature cycles with stirring, preferably at least 3, around the phase inversion zone between T1 and T2, until a translucent suspension is observed, e) carrying out the quenching with an aqueous solution acid of the oil / water emulsion at a temperature close to T1, preferably greater than T1, to obtain stable nanocapsules.

15. Preparation process according to claim 14, characterized in that step b) is decomposed as follows:

 b1) adding to the solution of step a) at least one triglyceride, a polyethoxylated glyceride and a lipophilic surfactant, b2) heating until solubilization of the lipophilic surfactant, b3) cooling,

 b4) addition of the hydrophilic surfactant and salt.

 16. Preparation process according to claim 14 or 15, characterized in that:

 the active ingredient is SN38,

 a basic buffer is added in step b4) to transform the

SN38 in lactone free form in SN38 in carboxylate form,

 the quenching is carried out by dilution in step e) with an acid buffer at 2 ° C. ± 1 ° C.

 17. Method according to one of claims 14 to 16, characterized in that the oil / water emulsion contains:

 - 1 to 3% of lipophilic surfactant

 - 5 to 15% hydrophilic surfactant

 - 5 to 15% oily fatty substance

 - 5 to 10% of diethylene glycol ether

 - 40 to 65% water

Percentages being expressed by weight

18. Method according to one of claims 14 to 16, characterized in that the aqueous phase of the oil / water emulsion further contains from 1 to 4% of a salt, such as sodium chloride

 19. Use of the nanocapsules according to any one of claims 1 to 13, for the manufacture of an orally administered drug, sublingual, subcutaneous, intramuscular, intravenous, intrathecal, epidural, transdermal, local or rectal.