The invention concerns nanocapsules, in particular with an average size less than 50 nm, consisting of an essentially lipid core liquid or semiliquid at room temperature, coated with an essentially lipid film solid at room temperature having a thickness of 2-10 nm. The invention also concerns a method for preparing same which consists in producing a reverse phase of an aqueous emulsion brought about by several temperature raising and lowering cycles. Said lipid nanocapsules are particularly designed for producing a medicine.

Description of the Invention

The present invention relates to lipid nanocapsules, to a process for preparing them and to their use for manufacturing a medicament intended especially to be administered by injection, orally or nasally.

In recent years, many groups have developed the formulation of solid lipid nanoparticles or lipid nanospheres (Muller, R. H. and Mehnert, European Journal of Pharmaceutics and Biopharmaceutics, 41(1): 62-69, 1995; W., Gasco, M. R., Pharmaceutical Technology Europe: 52-57, December 1997; EP 605 497). This is an alternative to the use of liposomes or polymer particles. These lipid particles have the advantage of being formulated in the absence of solvent. They allow the encapsulation of both lipophilic and hydrophilic products in the form of ion pairs, for example (Cavalli, R. et al., S.T.P. Pharma Sciences, 2(6): 514-518, 1992; and Cavalli, R. et al., International Journal of Pharmaceutics, 117: 243-246, 1995). These particles may be stable for several years in the absence of light, at 8.degree. 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 of a cold emulsion (Zur Muhlen, A. and Mehnert W., Pharmazie, 53: 552-555, 1998; EP 605 497), or the quench 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): 110-115, 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 nontoxic bile salt, taurodeoxycholate, by injection for the formation of nanospheres greater than or equal to 55 nm in size.

The present invention relates to nanocapsules rather than nanospheres. The term "nanocapsules" means particles consisting of a core that is liquid or semiliquid at room temperature, coated with a film that is solid at room temperature, as opposed to nanospheres, which are matrix particles, ie particles whose entire mass is solid. When the nanospheres contain a pharmaceutically active principle, this active principle is finely dispersed in the solid matrix.

In the context of the present invention, the term "room temperature" means a temperature between 15 and 25.degree. C.

One subject of the present invention is nanocapsules with an average size of less than 150 nm, preferably less than 100 nm and more preferably less than 50 nm. The nanocapsules each consist of an essentially lipid core that is liquid or semiliquid at room temperature, coated with an essentially lipid film that is solid at room temperature.

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%.

The thickness of the solid film is advantageously between 2 and 10 nm. It is also about one tenth of the diameter of the particles.

The core of the nanocapsules consists essentially of a fatty substance that is liquid or semiliquid at room temperature, for example a triglyceride or a fatty acid ester, representing 20% to 60% and preferably 25% to 50% by weight of the nanocapsules.

The solid film coating the nanocapsules preferably consists essentially of a lipophilic surfactant, for example a lecithin whose proportion of phosphatidylcholine is between 40% and 80%. The solid film may also contain a hydrophilic surfactant, for example Solutol.RTM. HS 15.

The hydrophilic surfactant contained in the solid film coating the nanocapsules preferably represents between 2% and 10% by weight of the nanocapsules, preferably about 8%.

The triglyceride constituting the core of the nanocapsules is chosen especially from C.sub.8 to C.sub.12 triglycerides, for example capric and caprylic acid triglycerides and mixtures thereof.

The fatty acid ester is chosen from C.sub.8 to C.sub.18 fatty acid esters, for example ethyl palmitate, ethyl oleate, ethyl myristate, isopropyl myristate, octyldodecyl myristate, and mixtures thereof. The fatty acid ester is preferably C.sub.8 to C.sub.12.

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

The amount of lipophilic surfactant contained in the solid film coating the nanocapsules is set such that the liquid fatty substance/solid surfactant compound mass ratio is chosen between 1 and 15, preferably between 1.5 and 13 and more preferably between 3 and 8.

A subject of the present invention is also a process for preparing the nanocapsules described above.

The process of the invention is based on the phase inversion of an oil/water emulsion brought about by several cycles of raising and lowering temperature.

The process of the invention consists in a) preparing an oil/water emulsion containing an oily fatty phase, a nonionic hydrophilic surfactant, a lipophilic surfactant that is solid at 20.degree. C. and optionally a pharmaceutically active principle that is soluble or dispersible in the oily fatty phase, or a pharmaceutically active principle that is soluble or dispersible in the aqueous phase, bringing about the phase inversion of said oil/water emulsion by increasing the temperature up to a temperature T.sub.2 above the phase inversion temperature (PIT) to obtain a water/oil emulsion, followed by a reduction in the temperature down to a temperature T.sub.1, T.sub.1<PIT<T.sub.2, carrying out at least one or more temperature cycles around the phase inversion zone between T.sub.1 and T.sub.2, until a translucent suspension is observed, b) quenching the oil/water emulsion at a temperature in the region of T.sub.1, preferably greater than T.sub.1, to obtain stable nanocapsules.

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

The number of cycles applied to the emulsion depends on the amount of energy required to form the nanocapsules.

The phase inversion may be visualized by canceling out the conductivity of the formation when the water/oil emulsion is formed.

The process of the invention comprises two steps.

The first step consists in weighing all the constituents, heating them above a temperature T.sub.2 with gentle stirring (for example magnetic stirring) and then optionally cooling them to a temperature T.sub.1 (T.sub.1<T.sub.2). After a certain number of temperature cycles, a water/oil emulsion is obtained.

The phase inversion between the oil/water emulsion and the water/oil emulsion is reflected by a reduction in the conductivity when the temperature increases until it is canceled out. The average temperature of the phase inversion zone corresponds to the phase inversion temperature (PIT). The organization of the system in the form of nanocapsules is reflected visually by a change in the appearance of the initial system, which changes from opaque-white to translucent-white. This change takes place at a temperature below the PIT. This temperature is generally between 6 and 15.degree. C. below the PIT.

T.sub.1 is a temperature at which the conductivity is at least equal to 90-95% of the conductivity measured at 20.degree. C.

T.sub.2 is the temperature at which the conductivity becomes canceled out.

The second step consists of a sudden cooling (or quench) of the oil/water emulsion to a temperature in the region of T.sub.1, preferably above T.sub.1, with magnetic stirring, by diluting it between threefold and tenfold using deionized water at 2.degree. C..+-.1.degree. C. added to the fine emulsion. The particles obtained are kept stirring for 5 minutes.

In one preferred embodiment, the fatty phase is a fatty acid triglyceride, the solid lipophilic surfactant is a lecithin and the hydrophilic surfactant is Solutol.RTM. HS15. Under these conditions, T.sub.1=60.degree. C., T.sub.2=85.degree. C. and the number of cycles is equal to 3.

The liquid substance/solid surfactant compound ratio is chosen between 1 and 15, preferably between 1.5 and 13 and more preferably between 3 and 8.

The oil/water emulsion advantageously contains 1% to 3% of lipophilic surfactant, 5% to 15% of hydrophilic surfactant, 5% to 15% of oily fatty substance and 64% to 89% of water (the percentages are expressed on a weight basis).

The higher the HLB value of the liquid fatty substance, the higher the phase inversion temperature. On the other hand, the HLB value of the fatty substance does not appear to have an influence on the size of the nanocapsules.

Thus, when the size of the triglyceride end groups increases, their HLB value decreases and the phase inversion temperature decreases.

The HLB value, or hydrophilic/lipophilic balance, is as defined by C. Larpent in Treatise K.342 of Editions TECHNIQUES DE L'INGENIEUR.

The particle size decreases when the proportion of hydrophilic surfactant increases and when the proportion of surfactants (hydrophilic and lipophilic) increases. Specifically, the surfactant brings about a decrease in the interface tension and thus a stabilization of the system, which promotes the production of small particles.

Moreover, the particle size increases when the proportion of oil increases.

According to one preferred embodiment, the fatty phase is Labrafac.RTM. WL 1349, the lipophilic surfactant is Lipoid.RTM. S 75-3 and the nonionic hydrophilic surfactant is Solutol.RTM. HS 15. These compounds have the following characteristics: Labrafac.RTM. lipophile WL 1349 (Gattefosse, Saint-Priest, France). This is an oil composed of caprylic and capric acid (C.sub.8 and C.sub.10) medium-chain triglycerides.

Its density is from 0.930 to 0.960 at 20.degree. C. Its HLB value is about 1. Lipoid.RTM. S 75-3 (Lipoid GmbH, Ludwigshafen, Germany). Lipoid.RTM. S 75-3 corresponds to soybean lecithin. Soybean lecithin contains about 69% phosphatidylcholine and 9% phosphatidylethanolamine. They are thus surfactant compounds. This constituent is the only constituent that is solid at 37.degree. C. and at room temperature in the formulation. It is commonly used for the formulation of injectable particles. Solutol.RTM. HS 15 (BASF, Ludwigshafen, Germany). This is a polyethylene glycol-660 2-hydroxystearate. It thus acts as a nonionic hydrophilic surfactant in the formulation. It may be used by injection (via the iv route in mice LD.sub.50>3.16 g/kg, in rats 1.0<LD.sub.50<1.47 g/kg).

The aqueous phase of the oil/water emulsion may also contain 1% to 4% of a salt, for instance sodium chloride. Changing the salt concentration brings about a shift in the phase inversion zone. The higher the salt concentration, the lower the phase inversion temperature. This phenomenon will be advantageous for encapsulating hydrophobic heat-sensitive active principles. Their incorporation may be performed at a lower temperature.

The nanocapsules of the invention may advantageously contain an active principle and may form part of the composition of a medicament to be administered by injection, especially intravenous injection, orally or nasally.

When the active principle is sparingly soluble in the oily phase, a cosolvent is added, for example N,N-dimethylacetamide.

The nanocapsules of the invention are more particularly suitable for the administration of the following active principles: antiinfectious agents, including antimycotic agents and antibiotics, anticancer agents, active principles intended for the Central Nervous System, which must cross the blood-brain barrier, such as antiparkinson agents and more generally active principles for treating neurodegenerative diseases.

The pharmaceutically active principle may be firstly soluble or dispersible in an oily fatty phase, and in this case it will be incorporated in the core of the nanocapsule. To do this, it is incorporated at the stage of the first step of preparing the oil/water emulsion which also contains the oily fatty phase, a nonionic hydrophilic surfactant and a lipophilic surfactant that is solid at 20.degree. C.

The pharmaceutically active principle may also be of water-soluble nature or dispersible in an aqueous phase, and in such a case it will be bound to the surface of the nanocapsules only after the final phase of preparing the stable nanocapsules. Such a water-soluble active principle may be of any nature, including proteins, peptides, oligonucleotides and DNA plasmids. Such an active principle is attached to the surface of the nanocapsules by introducing said active principle into the solution in which are dispersed stable nanocapsules obtained after the process according to the invention. The presence of a nonionic hydrophilic surfactant promotes the interaction bonds between the water-soluble active principle and the free surface of the nanocapsules.

The water-soluble active principle may also be introduced into the aqueous phase during the first step of initial oil/water preparation.
 

Claim 1 of 18 Claims

1. A process for preparing nanocapsules comprising: (a) preparing a mixture in the form of an oil/water emulsion comprising an aqueous phase, an oily fatty phase comprising a fatty substance that is liquid or semiliquid at room temperature, and a lipophilic surfactant that is solid at 20.degree. C., (b) bringing about the phase inversion of said oil/water emulsion by at least two or more temperature cycles, wherein each temperature cycle comprises increasing the temperature up to a temperature T.sub.2 above the phase inversion temperature (PIT) of the mixture to obtain a water/oil emulsion, followed by reducing the temperature to a temperature T.sub.1, T.sub.1<PIT<T.sub.2, to yield an oil/water emulsion as a translucent suspension, and (c) quenching the oil/water emulsion at approximately T.sub.1 to obtain stable nanocapsules.

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