EP1578404A2

EP1578404A2 - Method of encapsulating an active lipid-soluble substance by preparing a pit emulsion and emulsion obtained

Info

Publication number: EP1578404A2
Application number: EP03814493A
Authority: EP
Inventors: Luc Jugla
Original assignee: Cosnessens
Current assignee: Cosnessens
Priority date: 2002-12-26
Filing date: 2003-12-24
Publication date: 2005-09-28
Also published as: WO2004060358A2; CA2513273A1; JP2006517141A; FR2849379A1; CN100402019C; WO2004060358A3; CN1731986A; FR2849379B1; US20060134222A1; AU2003303610A1; AU2003303610B2

Abstract

The invention relates to a method of encapsulating an active lipid-soluble substance in nanocapsules, by preparing an emulsion. The inventive method is characterised in that it consists in: (a) obtaining an aqueous phase and an oil phase; (b) raising the temperature of the two phases to a temperature greater than the phase inversion temperature; (c) mixing the two phases; (d) incorporating the active lipid-soluble substance into the lipid-soluble phase; (e) allowing the temperature to decrease to the phase inversion temperature; (f) once the phase inversion is effective and the emulsion is in a continuous aqueous phase, quenching the emulsion obtained in order to lower the temperature thereof. The invention also relates to the emulsion that can be obtained using the inventive method, said emulsion being characterised in that the average nanocapsule size is less than 300 nm.

Description

Method for encapsulating a liposoluble active principle by preparation of a PIT emulsion and emulsion obtained.

The present invention relates to the field of vectorization of active principles.

The effectiveness of a formulation both in pharmacy and in cosmetics depends on the active principles but also on their release system and numerous means of vectorization have been explored either in cosmetics or in pharmacy. Among these, mention may be made of nanoparticles. Nanoparticles are colloidal particles ranging in size from 1 to 1000 nm. They are macromolecules in which the active principle is dissolved, trapped or encapsulated. These nanoparticles refer to very different systems such as nanospheres and nanocapsules which are respectively matrix systems for nanospheres and reservoir systems for nanocapsules.

Nanospheres are solid matrix particles in which the active principle is finely dispersed in the polymer matrix. Nanocapsules are particles made up of a liquid or semi-liquid core at room temperature which contains the active principle, coated with a solid film at room temperature.

The present invention relates more particularly to the field of vectorization of liposoluble active principles in a nanocapsule type reservoir system. Nanocapsules are aqueous suspensions of small vesicles (generally between 100 and 400 nm), whose rigid wall, of small thickness, consists of macromolecules of natural, synthetic or semi-synthetic origin. These systems make it possible to encapsulate in the lipophilic heart relatively large quantities of active principles, most often lipophilic, they can be obtained either by polymerization reactions or from preformed polymers. Numerous methods for formulating nanocapsules by emulsification are described, for example, the methods described in patents US 5079322 or EP0717989 which make it possible to obtain emulsions incorporating liposoluble active principles. By liposoluble active principles we mean in particular any chemical compound or mixture soluble in oily bodies for cosmetic, food, pharmaceutical or veterinary use or any compound of interest due to its properties. The liposoluble active ingredients are, for some, sensitive to exposure to temperatures above 50 ° C, sensitive to light and to oxidation. One of the solutions currently used to vectorize these active principles is their formulation in emulsions. But because of their instability, when these liposoluble active principles are used in emulsified systems, they are introduced at the end of the process in an oil-in-water emulsified system at a temperature below 50 ° C. for example, and these will then distribute randomly, particularly in the aqueous phase and will then be partially destroyed by the surrounding medium.

These methods therefore do not give complete satisfaction either that the quantities of active ingredients incorporated are not sufficient to achieve the desired activities, or that the stability is not correct, or that the processes for obtaining them are difficult to industrialize. To improve these formulations, emulsification processes by phase inversion known as “PIT emulsion” (Phase Temperature Inversion) such as those described for example in patents WO20011975, EP1093795 or WO200071676 making it possible to obtain oil-in-water emulsions containing a active ingredient have been proposed. These methods include the incorporation, for example, of an active principle in an oily phase, the addition of part of the aqueous phase to the mixture obtained, heating with stirring at a temperature above the phase inversion temperature, adding the balance of the aqueous phase and cooling. For example, from WO200164328, a process for the preparation of lipid nanocapsules is known based on the phase inversion of an oil / water emulsion caused by several temperature rise and fall cycles. The emulsions obtained are very fine and do not require homogenization steps. These methods make it possible to obtain very fine dispersions of the emulsion (0.1 to 0.3 μm) and great stability because during the phase inversion, the interfacial tension is minimal and makes it possible to obtain very fine droplets . However, the phases of temperature rise to obtain the phase inversion which can be optionally repeated are not compatible with the formulation of active principles liable to undergo physical or chemical degradation due to too long exposure to a temperature above 50 ° C. In the present invention, the lipid nanocapsules are formulated by a process of emulsification by phase inversion caused by passage of the emulsion beyond the phase inversion temperature but making it possible to preserve the active principle thanks to its incorporation in phase. continuous oily therefore without contact with the aqueous phase, beyond the phase inversion temperature.

Indeed, the incorporation of the liposoluble active principle in the formulation, at a temperature higher than the phase inversion temperature, that is to say when the emulsion is in the oily continuous phase (water-in-oil emulsion), allows to obtain a good distribution of the active principle in the oily phase, limits its contact with the aqueous phase, and surprisingly although the temperature is high, the residence time at this temperature is very short since this incorporation is followed by the quenching of the emulsion, the degradation phenomena are limited or eliminated.

The present invention relates to a process for encapsulating a liposoluble active principle in nanocapsules by preparation of an emulsion characterized in that: a) an aqueous phase and a fatty phase are available, b) the temperature of the two phases up to a temperature higher than the phase inversion temperature, c) the two phases are mixed, d) the liposoluble active principle is incorporated into the liposoluble phase, e) the temperature is left to lower to the phase inversion temperature, f) as soon as the phase inversion is effective and the emulsion is in aqueous continuous phase, the emulsion obtained is quenched to lower its temperature. In a variant after step c) a step c ′) is carried out which consists in lowering the temperature to a temperature immediately above the phase inversion temperature before the incorporation of the active principle.

This lowering of temperature can be caused or occur naturally. Alternatively, the temperature can be allowed to drop naturally or the temperature can be lowered to a desired temperature by quenching. It also relates to a method according to claim characterized in that step c) is carried out before step b). In this variant of the process, the two phases are mixed before the temperature rises or during the temperature rise, but before the latter reaches the phase inversion temperature. The emulsion obtained is then brought to a temperature higher than the phase inversion temperature, then the active principle is incorporated.

In a variant of the process according to the invention, the emulsion obtained is then concentrated by removal of part of the aqueous phase.

Advantageously, this concentration step can be carried out by tangential ultrafitration.

According to the invention, step f), known as quenching, is carried out by adding an additional aqueous phase brought to a temperature at least lower than the phase inversion temperature, and optionally lower than ambient temperature. This brutal and rapid cooling step makes it possible to lower the temperature of the emulsion and reduce the time of exposure of the active principle to a higher high temperature.

This quenching can also be carried out using a cooling system by heat exchange or by adding liquefied gas, for example nitrogen.

By temperature immediately above the phase inversion temperature is meant a temperature a few degrees higher, in practice 1 or 2 ° C than the phase inversion temperature. The phase inversion temperature of the system having been previously determined experimentally by monitoring the conductivity of the system or by visual observation.

Among the active ingredients capable of being encapsulated by this process, mention will be made more particularly of the liposoluble active ingredients called unstable, that is to say capable of degrading if they are exposed to temperatures above 40 ° C. for times greater than 30 min, or sensitive to oxidation due to the presence of water in the formulation, or degraded by pH variations, by UN radiation. or by the presence of products capable of causing parasitic reactions with said active ingredients.

Among the liposoluble principles capable of being encapsulated by this process, there may be mentioned by way of example:

- the fat-soluble vitamins and their derivatives, such as the retinoid family (retinol, retinaldehyde, retinoic acid), carotenoids, tocopherol and its derivatives, - Polyphenols such as flavonoids (ex: isoflavonoids, quercetin), stylbenes (ex : resveratrol), catechins (e.g. epicatechin-3-gallate, epigallocatechine-3-gallate)

- perfume components such as vanillin, indol, more generally essential oils such as essential oils of citrus, lavender

- liposoluble active pharmaceutical ingredients such as: Fluvastatin, Ketoprofen, Verapamil, Atenolol, Griseofulvin, Ranitidine

In the process according to the invention, the emulsion comprises from 5 to 30% of fatty substance constituting the fatty phase and from 45 to 92% of water constituting the aqueous phase. The proportion of the fatty phase relative to the aqueous phase which is associated with it depends on the amount of active principle to be encapsulated and on the type of emulsion. The proportion of fatty phase can also have an influence on the size of the nanocapsules obtained. The constituents of the fatty phase can be chosen from paraffinic derivatives or more or less complex triglycerides. The choice of these constituents will depend on the nature of the lipophilic active principle to be encapsulated but also on their potential influence on the phase inversion temperature or even on their influence on the size of the nanocapsules obtained.

The nature of the active principle to be encapsulated will have an influence on the choice of the constituents of the fatty phase because the constituents will be selected according to:

- the potential solubility of the active principle in this phase, - their neutrality with respect to the active principle, that is to say that they should not be oxidizing with respect to the active principle, it is to say that they will have to have a low acrde number, not be acidic and have a low iodine number, - their compatibility with a phase inversion emulsification technique, - their ability to give the lowest phase inversion temperature possible.

When the phase inversion temperature is too high, ingredients capable of lowering this phase inversion temperature will be added to the medium. Indeed, the more marked lipophilic nature of certain constituents which may be chosen for example because of their ability to dissolve the active ingredients can cause the phase inversion temperature to rise because the accentuation of the hydrophobic bonds between the agent surfactant and oil causes an increase in the energy needed to reverse the system. The polarity of the constituents of the fatty phase also has an influence on the phase inversion temperature, the more the constituents are polar the more the phase inversion temperature is increased. The saturated constituents, with the lowest possible iodine index, on the other hand, are capable of reducing the phase inversion temperature. Although the residence time at a temperature higher than the phase inversion temperature is extremely short, it will nevertheless be sought to formulate emulsions whose phase inversion temperature is as low as possible.

The constituents of the fatty phase will therefore preferably be chosen from mineral oils or mineral oil substitutes such as isohexadecane, silicones, in particular cyclomethicones or polydimethylsiloxane, C8 to C12 triglycerides, for example triglycerides of capric and caprylic acids and their mixtures.

The choice of the emulsifying system is also an important criterion having an influence on the stability of the emulsions obtained and on the size of the particles. Two values characterize an emulsifying system, the ratio of lipophilic surfactant / hydrophilic surfactant (RTL / RTH) and the overall percentage of surfactants.

The emulsifying systems used in the present invention will be chosen from systems whose RTL / RTH ratio is between 1/1 and 1/50. The percentage of water-soluble surfactant will preferably be between 2 and 10% and the percentage of lipophilic surfactant will be between 1 and 5%.

The water-soluble surfactants are especially chosen from esters of glycol, glycerol, itol, sorbitan, polyethylene glycol. Among the polyethylene glycol esters, in particular those whose carbon chain is between 10 and 22 carbon atoms will be chosen, and whose number of polyethylene glycol monomers is between 5 and 30. These water-soluble surfactants may also be chosen from ethers of fatty alcohols and of polyethylene glycol, the fatty alcohol of which is chosen from those comprising from 10 to 22 carbon atoms and the number of monomers of which is between 5 and 30.

Lipophilic surfactants will also be added to the mixture, these surfactants are characterized by their ability to give W / O emulsions when used as emulsifiers alone or mainly. Among these emulsifiers, mention will be made of esters of monoglycerols, esters of polyglycerol and of fatty acids, silicone emulsifiers such as cetyl dimethicone copolyol, esters of polyhydroxystearic acid and of polyethylene glycol.

According to one embodiment of the invention, salt can be added to the aqueous phase. It has been shown that the addition of salt reduces the interaction between the polar groups and water and decreases the hydrophilicity of the surfactant, therefore the CMC, in addition it produces a screen effect facilitating the rapprochement between the polar groups.

On the other hand, studies have revealed that the modification of the salt concentration leads to a displacement of the phase inversion zone. The higher the salt concentration, the lower the phase inversion temperature.

Other constituents may be added in one or other of the phases, for example, preservatives intended to avoid the development in the aqueous phase of certain microorganisms,

Antioxidant agents are added to the system to avoid the alteration of certain easily oxidizable compounds in the lipid phase, they are for example chosen from the group consisting of butylhydroxylanisole (BHA), butylhydroxytoluene (BHT), propyl gallate, α-tocopherol, EDTA. These antioxidants will be used at concentrations varying from 0.01 to 3%, for example BHT will be used at concentrations varying from 0.01 to 1%, α-tocopherol at concentrations varying from 0.1 to 3% and EDTA at concentrations varying from 0.05 to 2%.

In the process according to the invention, the stirring speed will be between 100 and 3000 revolutions / min. In fact during the emulsification there is a dynamic equilibrium between the rupture (high shear zones) and the coalescence (low shear zone ). The speed of agitation affects rupture and coalescence, this speed of agitation will therefore have an influence on the size distribution and on the stability of the emulsion.

In the method according to the invention, the detection of phase inversion is carried out:

- Either by visualizing the formulation, the organization of the system in the form of nanoparticles is visually translated by a change in the appearance of the initial system which changes from white-opaque to white-translucent. For weakly dispersed emulsions, the appearance becomes, sometimes bluish during phase inversion,

- or by measuring the conductivity which increases when the emulsion passes from a Water in Oil system to an Oil in Water system. In fact, the conductivity increases when the emulsion passes from a Water in Oil system to an Oil in Water system. A continuous aqueous phase rich in electrolytes is characterized by a high value of conductivity. The PIT zone is defined as being a zone in which the conductivity of the medium varies from a zero value (characterizing an oily continuous phase) to a value of a few μs / cm. This development takes place over a temperature range called the PIT zone.

The diameter of the particles is measured by an optical method of measuring light called light scattering, which is based on various physical and mathematical laws including SPC (Photon Correlation Spectroscopy). The principle of measurement can be described as the study of the speed of particles subjected to Brownian motion, small particles vibrating a lot and moving quickly while those of a larger diameter vibrate little and move more slowly. The interaction of a light beam with the particles allows, after mathematical modeling, to approach the diameter of the particles. The present invention also relates to the lipid nanocapsules obtained by the method according to the invention whose average size is less than 300 nm and is preferably on average 150 nm.

Emulsions according to the invention are described below.

EXAMPLE 1:

An oily phase containing the following ingredients is formulated:

-tocopheryl acetate (vitamin E acetate) 0.5%

- Glyceryl stearate and ceteareth-12 and ceteareth-20 and cetearyl alcohol

(SEV emulgate) 3%

- ceteareth-20 (Eumulgin B2) 2% - isohexadecane (Arlamol HD) 6%

- cyclomethicone (Dow Corning 345) 3%

- butyl hydroxytoluene (BHT) 0.1%

An aqueous phase containing the following ingredients is formulated:

- sodium salt of EDTA (BASF (disodium EDTA) 0.5% Demineralized water 25%

The two previously formulated phases are heated to a temperature of 85 ° C. The two phases are combined by adding the aqueous phase to the fatty phase with shearing stirring at 700 rpm.

The active ingredient RETINOL, in solution in a triglyceride of caprylic acid, 7% is then incorporated into the emulsion obtained by mixing the aqueous phase and the fatty phase at a temperature in the region of 81 ° C.

The phase inversion occurs at 73 ° C, this phase inversion is identified by an increase in the conductivity greater than 1 μS / cm.

A complementary aqueous phase containing a preservative Glydant Plus Liquid (DMDM hydantoin and iodopropynyl butylcarbamate (sold by the company LONZA Inc. (0.5%) and the water 51, 9% is quickly incorporated into the emulsion containing the RETINOL previously obtained.

The emulsion can then be concentrated by tangential ultrafiltration.

EXAMPLE 2:

According to the same operating protocol as in Example 1, an emulsion is prepared from the following phases. :

Fat phase:

- PEG-30 dipolyhydroxystearate 2%

- PEG-6 stearate and ceteth-20 and steareth-20 6%

- Isohexadecane 6%

- cyclomethicone 3% - Tocopheryl acetate 0.5%

- Butylhydroxytoluene 0.1%

Aqueous phase :

- 0.2% EDTA disodium - 25% demineralized water

Active ingredient :

RETINOL, in solution in a triglyceride of caprylic acid 7%

The phase inversion takes place at 71 ° C.

Complementary aqueous phase:

- Chlorhexidine digluconate 0.5%

- water 49.7%

EXAMPLE 3:

Fat phase: - PEG-30 dipolyhydroxystearate 2%

- PEG-6 stearate and ceteth-20 and steareth-20 6%

- Isohexadecane 6%

- cyclomethicone 3 ù - Tocopheryl acetate 0.5%

- Butylhydroxytoluene 0.1%

- Caprylic / capric triglyceride 6%

Aqueous phase: - 0.2% EDTA disodium

- 25% demineralized water

Active ingredient :

RETINOL, in solution in a triglyceride of caprylic acid 0.33%

The phase inversion takes place at 80 ° C.

Complementary aqueous phase:

- Chlorhexidine digluconate 0.5% - Sodium methylparaben 0.2%

- water 50.17%

Among the advantages of the process according to the invention, there may be mentioned the size of the droplets obtained less than 300 nm, which has the following advantages:

an improved bioavailability of the active ingredient incorporated since the penetration of the emulsion is favored by the minimum size of the particles encapsulating the active principle, this improved bioavailability of the incorporated active principle allows a lower final concentration in the product than with systems conventional encapsulation and the reduction of possible side effects,

- better physical stability of the finished product, in fact the smaller the particle size the more the system is physically stable due to the disappearance of maturation and coalescence phenomena, - obtaining monodispersed systems (polydispersity index <0.25) the size of nanocapsules being homogeneous the Oswald ripening is limited,

- faster and more economical manufacturing processes than conventional emulsification processes due to the reduction in energy required.

Claims

1. Method for encapsulating a liposoluble active principle in nanocapsules by preparation of an emulsion characterized in that: a) an aqueous phase and a fatty phase are available, b) the temperature of the two is raised phases up to a temperature higher than the phase inversion temperature, c) the two phases are mixed, d) the liposoluble active ingredient is incorporated into the liposoluble phase, e) the temperature is allowed to drop to at the phase inversion temperature, f) as soon as the phase inversion is effective and the emulsion is in the continuous aqueous phase, the emulsion obtained is quenched to lower its temperature.

2. Method according to claim 1, characterized in that a step c ') is carried out which consists in lowering the temperature to a temperature immediately above the phase inversion temperature before the incorporation of the active principle

3. Method according to claim 1, characterized in that step c) is carried out before step b).

4. Method according to one of the preceding claims, characterized in that the emulsion obtained is then concentrated by removing part of the aqueous phase.

5. Method according to one of the preceding claims, characterized in that step e) is carried out by adding an additional aqueous phase brought to a temperature below the phase inversion temperature.

6. Method according to any one of the preceding claims, characterized in that the active principle is dissolved in an additional fatty phase before its incorporation into the system.

7. Method according to any one of the preceding claims, characterized in that the active principle is chosen from the group consisting of fat-soluble vitamins such as retinol, retinoids, vitamin E and carotenoids, polyphenols and perfume components .

8. An emulsion capable of being obtained by a process according to any one of the preceding claims, characterized in that the size of the nanocapsules is less on average than 300 nm.