United States Patent:  6,013,284

Assignee:  Biovector Therapeutics S.A. (Ramonvillesainte-Agne, FR)

Filed:  May 1, 1996

A synthetic particulate vector comprising a non-liquid hydrophilic nucleus and an outer layer at least partially consisting of amphiphilic compounds, which is combined with the nucleus by hydrophobic interactions and/or ionic bonds. A process for preparing a particulate vector by encapsulating an ionizable active principle, vectors obtained according to such a process, and pharmaceutical, cosmeticological and food compositions comprising such vectors are also disclosed.

Description of the Invention

The present invention relates to new types of particles which can be used alone or as vectors for various compounds. It also relates to a process for the preparation of particulate vectors which makes possible improved control of the active principle charging.

Supramolecular biovectors or SMBVs are particles which are biomimetic of the endogenous vectors of the body and which are capable of encapsulating and of carrying a large number of active principles for, in particular, pharmaceutical, cosmetic or agribusiness use.

A first type of SMBV was described in Application EP 344,040. Their structure is very well suited to the role of vector, in particular as a result of the possibility of modifying their size and their composition according to the molecule or molecules transported and their use.

SMBVs are synthesized in three successive steps:

synthesis of a central core composed, for example, of crosslinked natural polysaccharide, which can be derived by ionic groups and brought, in particular by ultramilling, to the desired size (between 10 nanometers and a few microns, according to the desired use)

establishment of a ring of fatty acids grafted covalently solely at the periphery of the central core, in order to confer a peripheral hydrophobic nature on the latter while retaining its internal hydrophilic nature

stabilization of one or of a number of external lipid lamellae, composed in particular of phospholipids or of ceramides, sometimes with the addition of other constituents, for example of constituents of biological membranes.

The active principles, according to their physicochemical characteristics, can be transported either in the external lipid lamellae (in the case of lipophilic or amphiphilic compounds) or within the hydrophilic core (in the case of polar compounds).

Encapsulation of active principles of polar nature can take place, according to the structure of the latter, either before formation of the fatty acid ring or between this step and stabilization of the external lamella.

Despite their suitability for many uses, the synthesis of SMBVs can sometimes cause problems and in particular:

it requires a step which is problematic to control in grafting the fatty acid ring;

this grafting, carried out solely at the periphery of the core, must be carried out homogeneously, which requires in particular a prior drying step, under very specific conditions;

if the active principle is encapsulated before the grafting of the fatty acid ring, some of these molecules, localized, after their encapsulation, at the periphery of the core, can be derived by the fatty acid, leading to modification of the properties of this active principle;

if the active principle is encapsulated after the grafting of the fatty acid ring, the latter can be detrimental to the penetration of the active principle into the hydrophilic core.

The Applicant Company has shown that, surprisingly, in certain applications, it was possible to scale down the reaction scheme by not grafting the ring of fatty acids to the periphery of the crosslinked hydrophilic core.

The Applicant Company has shown that the polysaccharide particles thus obtained could be used as is. They are then named PS-type SMBVs, by analogy with supramolecular biovectors.

The Applicant Company has indeed shown that the polysaccharide particles, even of small size, could be used provided that suitable charging protocols are adopted.

They can also be used in combination with natural amphiphilic compounds, in particular phospholipids, and the Applicant Company has shown that the external lipid lamellae can possibly be arranged around this core in the absence of a grafted fatty acid ring as in the case of SMBVs. These particles have a supramolecular nature and are known as L-type SMBVs.

This is why the subject of the present invention is a synthetic particulate vector, characterized in that it comprises:

a non-liquid hydrophilic core,

an external layer composed at least in part either of amphiphilic compounds, combined with the core via hydrophobic interactions and/or ionic bonds, or by the external ring of the hydrophilic core, by using a special process which avoids encapsulation of the active principle in this ring and which concentrates the active principle in the internal part of the core.

The notion of vector must, in this instance, be understood within the broad meaning, that is to say that it comprises particles having a support role, for example when they are incorporated in a composition, either as such or for the transportation, the presentation and/or the stabilization of active compounds.

A non-liquid hydrophilic core (or matrix) can be a hydrophilic polymer. The hydrophilic matrix can in particular be composed of polysaccharides or oligosaccharides which are naturally or chemically crosslinked. The polysaccharide is preferably chosen from dextran, starch, cellulose and their derivatives.

Two types of interactions can explain the stabilization of phospholipids, for example, on a core composed of crosslinked polysaccharide which is derived throughout its bulk by ionic groups: these are either interactions of hydrophobic type or bonds of ionic type, it being possible for the two modes to collaborate in the stabilization of these lamellae.

In fact, phospholipids are composed, on the basis of a glycerol unit, of a polar head comprising a phosphate group, with a strong anionic charge, which is optionally derived by various polar groups, and of two fatty acids constituting the hydrophobic tail.

The polar head has the ability to bind itself by ionic interaction with the ionic groups grafted into the polysaccharide matrix, either via the phosphate or via the ionic groups grafted onto the phosphate of the phospholipid (quaternary ammonium phosphatidylcholines, for example).

Moreover, it is known that polysaccharides, while they have an overall hydrophilic nature, have a hydrophobic groove, which is there because the polar hydroxyl groups are directed in a given direction, allowing access to the base structure of the sugars, composed of carbon-carbon bonds, of hydrophobic nature.

In the case of polysaccharide particles constituting the core of L-type SMBVs, stabilization of compounds such as phospholipids can be due to a collaborative phenomenon between the two binding modes.

The phospholipids/polysaccharide cores combination could be demonstrated by fluorescence energy transfer techniques. The theory of energy transfer rests on the interactions which exist between two fluorophores when the emission band of the first fluorophore (F1) overlays the excitation band of the second fluorophore (F2). If the two components are close, the energy of a photon absorbed by the fluorophore F1 can be transferred to the fluorophore F2, which will then fluoresce as well as if it had been excited directly. The fluorescence of F1 can then decrease until totally extinct. The efficiency of the energy transfer between the two fluorophores is thus dependant on their spatial separation. After having labeled the polysaccharide cores using rhodamine isothiocyanate and the phospholipids using nitrobenzodiazole (NBD), an energy transfer could be demonstrated between the two fluorophores, whether in the case of L-type SMBVs of 1 .mu.m, 200 nm or 20 nm. This transfer remains stable after incubations at 4^oC., 37^oC. and even at 100^oC.

The hydrophilic core can be obtained by various methods and in particular, if it is a core of polysaccharide nature, by using a branched or linear biodegradable polysaccharide. This polysaccharide can be, for example, starch or one of its derivatives. Crosslinking processes are known to a person skilled in the art and can be carried out by means of bi- or trifunctional agents, such as epichlorohydrin or phosphorus oxychloride.

The properties of the polysaccharide can be modified by substituting the sugars by acidic or basic ionic functional groups which are important in the stabilization of the external lipid lamella and for the encapsulation of ionic active principles.

Encapsulation of the hydrophilic active principles can be carried out at this stage of the synthesis. The gel obtained during the synthetic step is then washed and partially dehydrated by means, for example, of centrifugation techniques, then brought into the presence of the active principle and slowly rehydrated. As the gel has the ability to swell with water, the active is carried within the polysaccharide network where it can be bound by ionic bonds with the groups grafted within the gel.

The gel obtained, whether it contains or does not contain an encapsulated compound, must be mechanically ground for the purpose of obtaining particles of desired size. The ultramilling methods are known in the state of the art and can in particular involve a high pressure extrusion using a homogenizer.

The encapsulation of hydrophilic compounds within the L-type SMBVs is generally carried out at this step. For this, the particle suspension is dried beforehand by using drying methods, such as lyophilization or atomization.

The dried particles are, for example, mixed with the active principle, which is also in the dry form. Progressive rehydration makes it possible, as in the case of the gel, to dissolve the active principle and then to carry it within the particle where it is bound by ionic bonds.

The external lipid lamella of the L-type SMBVs can be produced with various types of natural or synthetic lipids, including phospholipids and ceramides, but also ionic or nonionic surface-active agents capable of being arranged as micelles, to which other compounds, either lipid compounds or amphiphilic compounds, such as cholesterol, fatty acids or fat-soluble vitamins, can also be added.

This lamella is preferably obtained by using methods which are used for the preparation of liposomes, that is to say reversed-phase preparation, detergent dialysis or high pressure extrusion. The active principles which have to be carried or presented at the surface of the L-type SMBVs can be introduced during this step, mixed with the surface-active agents.

Another subject of the present invention is a process for the preparation of a particulate vector, characterized in that:

a) at least one basic ionizable active principle is encapsulated in a crosslinked hydrophilic matrix grafted by acid ligands, at a pH below the pK_a of the active principle,

b) the pH of the medium is increased to a value above the pK_a of the active principle,

c) the hydrophilic matrix, containing the active principle localized mainly at the center of the said matrix, is recovered.

In fact, the adoption of a suitable protocol for the charging of hydrophilic cores makes it possible to control the topology of the charging.

The hydrophilic matrix is preferably composed of polysaccharides or of oligosaccharides, which are naturally or chemically crosslinked.

This process, which can be used with SMBVs, is more particularly important with particles in which the external lipid lamellae have been reduced (L-type SMBVs) or eliminated (PS-type SMBVs) with respect to the method described above. The Applicant Company has observed that it is difficult to use such SMBVs containing reduced lipid lamellae as vectors for the encapsulation of ionic active principles with conventional charging methods.

In fact, if molecules of the active principle are bound with the polysaccharide particle of the core while being maintained at the periphery of the core, this can result in an instability in the particle suspension, it being possible for the particles to aggregate with one another by virtue of interparticulate bonds due to the active principle. This phenomenon is relatively minor for low levels of charging of active principles, whereas it becomes very important with high levels of charging of active principles. Likewise, the size of the particles is extremely important. With particles of large size (for example greater than 100 nanometers), the ratio of the surface area to the internal volume of the particle is very low; for this reason, in comparison with the total amount of active principle encapsulated, the amount of active principle bound at the periphery of the particles is very low, thus limiting the possibilities of interparticulate bonds. In contrast, when the particles are very small in size, this aggregation phenomenon is very noticeable. It should also be noted that this phenomenon is not very marked with SMBVs which have a layer of fatty acids grafted onto the core, which then serves to isolate from interparticulate interactions.

In order to overcome this, the Applicant Company has shown that it is possible to control topologically the penetration of the active principle within the particles by controlling the ionic conditions of the encapsulation.

The polysaccharide cores can be regarded as polyelectrolyte matrices and, as such, they have a pH differential between the inside and the outside of the particle. This phenomenon is due to the more or less significant dissociation of the counterions and to the immobilization of the ionic functional groups on the polysaccharide network. This property makes it possible to control the localization of the active principle to be incorporated, by causing a solely internal encapsulation or an encapsulation solely at the surface or alternatively in the periphery of the core.

When the active principle to be incorporated is basic in nature, its solubilization in a medium with a pH below its pK_a leads it to exist in the ionized form; it can then become attached to the anionic groups grafted onto the polysaccharide core. When the pH rises above the pK_a, the active principle is in a deionized form, which does not allow it to interact with the matrix. In order to control the localization of the encapsulated active principle, use is therefore made of the pH differential which exists between the inside and the outside of the particle: if the external medium has an excessively high pH, the active principle cannot interact with the ionic groups placed at the periphery of the cores. The internal pH of the cores derived by acidic ligands being lower than the external pH, the active principle, which has entered the particle in the deionized form, becomes ionic again and thus is bound to the anionic groups of the L-type SMBVs. In this specific case, the active principle will be localized solely in the core of the particle, to the exclusion of the peripheral region. This type of encapsulation is thus very favorable to an optimum dispersion of the particles.

In the case of acidic active principles, it is possible symmetrically to apply the process with cores derived by basic ligands, according to the following steps:

a) at least one acidic ionizable active principle is encapsulated in a crosslinked hydrophilic matrix grafted by basic ionic ligands, at a pH above the pK_a of the active principle,

b) the pH of the medium is decreased to a value below the pK_a of the active principle,

c) the hydrophilic matrix, containing the active principle localized mainly at the center of the said matrix, is recovered.

This type of charging with topological control of the localization of the active principle in the polysaccharide core is particularly advantageous for vectorization applications with SMBVs in which the external lipid lamellae have been reduced (L-type SMBVs) or eliminated (PS-type SMBVs) but it is also suitable and desirable for SMBVs already described in the above patents, in order to increase the degree of charging or to minimize the disturbances caused to the structure of the external phospholipid lamella in the case of external attachment of macromolecules, for example of recognition units and in particular of an apoprotein.

Another subject of the invention is a particulate vector composed of a crosslinked hydrophilic core grafted by ionic groups and here called PS-type SMBV. The ionic groups can be anionic groups, such as for example phosphates, succinates or carboxymethylates, or cationic groups, for example quaternary ammoniums or amines. The size of the PS-type SMBVs is preferably between 20 nm and 200 nm.

The crosslinked hydrophilic core can be composed of natural or synthetic polymers which are naturally or chemically crosslinked. Use is in particular made of polysaccharides or oligosaccharides, such as starch, dextran, cellulose and their derivatives.

Advantageously, an active principle is encapsulated in the PS-type SMBV mainly at the center of the matrix; the external part of the core is virtually devoid of active principle, which makes it possible to avoid the aggregation phenomena which generally occur for particles of small size.

One of the subjects of the invention is therefore a particulate vector composed of a crosslinked polysaccharide matrix containing an active principle, the said active principle preferably being localized mainly at the center of the matrix.

According to yet another aspect, a subject of the invention is a process for charging which makes it possible to encapsulate the active principle in a complete SMBV, a PS-type SMBV or an L-type SMBV, which can be in the form of a suspension.

The charging is carried out on the particulate vector on which the various lipid and/or amphiphilic layers are, if appropriate, fixed. In order to do this, the hydrophilic core must contain ionic groups. The process thus requires the following steps:

a) a crosslinked hydrophilic core is prepared in which ionic groups are fixed,

b) optionally, lipid compounds, such as, for example, fatty acids, are fixed on the crosslinked core and/or an amphiphilic lamella (a phospholipid lamella, for example) is formed on the core which is optionally acylated,

c) the active principle is charged within the vector at a pH suitable for the active principle and while supplying energy,

d) having incorporated the active principle, the vector is recovered.

In the case of PS-type SMBVs, it is difficult to use conventional charging methods for incorporating ionic active principles. It is true that methods with topological control make it possible to overcome this problem. However, topological control requires precise adjustment of the pH which must be compatible with the active principle and the vector.

In the case of normal or of L-type SMBVs, the methods developed until now comprise incorporation of the active principle in the cores and the subsequent formation of the phospholipid lamella. These methods certainly produce very advantageous incorporation results but require the active principle to be handled at all steps of the preparation process. In the case of very toxic or very expensive active principles, these methods cannot be recommended for reasons of safety or of cost.

The Applicant Company has therefore developed an alternative method to overcome these problems. The ionic ligands grafted into the polysaccharide network of the vectors result in a significant affinity for the ionic active principles of opposite charge. However, this affinity, during the incorporation, must be controlled in order to avoid aggregation of the vectors and to make it possible to localize the active principle mainly within the particles. For the precharging, this control requires precise adjustment of the pH or a low level of incorporation. This aggregation is mostly due to localization of the active principle at the surface, which localization is itself due to the presence of ligands at the surface of the particles.

In order to effect this new type of charging, three factors come into play:

a) a significant affinity of the active principle for the vector in order to provide for incorporation of the active principle: this affinity is created by acidic or basic ionic ligands which are grafted into the crosslinked polymer; the density and the strength of the ligands can be adjusted according to the active principle,

b) a significant dispersion of the vectors during the incorporation in order to avoid the interactions between particles which promote aggregation: this dispersion can be provided for by the dilution of the vectors in the reaction medium at a concentration which is sufficient to decrease the interparticulate interactions but also at a concentration which is compatible with pharmaceutical applications,

c) the use of any means for promoting entry of the active principle within the vector: the contribution of energy, in the form, for example, of stirring or of heat, will accelerate the kinetics of entry of the active principle but will also promote dispersion of the vectors; the appropriate form of the active principle, which must be sufficiently ionic to make it possible to attach the active principle but also the least charged, in order to avoid surface interactions.

For SMBVs or L-type or PS-type SMBVs, it is possible to use incorporation protocols corresponding to these requirements. The presence of grafted ionic ligands in the crosslinked polymer provides for attachment of the active principles for the three species. Dispersion of the vectors can be carried out by suspending PS-type SMBVs in water. SMBVs or L-type SMBVs are prepared from acylated or polysaccharide cores and from phospholipids dispersed beforehand in aqueous medium and are thus suspended in water. The contribution of energy, for example in the form of stirring or of heat, does not damage the SMBVs. It is possible to vary the pHs and to define pH ranges which are compatible with this type of charging.

In the case of SMBVs and of L-type SMBVs, the lipid and/or phospholipid lamellae represent a barrier which must be crossed by the active principles. However, in the charging process described, two additional factors intervene to promote this crossing:

a) the contribution of energy which can fluidify the lipid and phospholipid lamellae and increase the kinetics of entry of the active principle within the vectors,

b) the form of the active principle which can be very weakly ionic due to the existence of the gradient existing between the interior and the exterior of the vector.

In addition, the strong affinity between the active principle and the crosslinked polyelectrolyte polymer provides for its attachment and the strong dispersion prevents aggregation of the vectors with one another.

This new process makes it possible to prepare SMBVs of any type which are charged with active principle, while retaining the size of the base vectors, and has many advantages. This method consists in preparing the blank vectors, without active principle, before the incorporation, which makes it possible to process the blank vectors, according to conditions which are suitable for the vectors and which do not depend on the active principle to be encapsulated, and subsequently to charge. These conditions can therefore be more or less drastic. They also make it possible to be able to characterize the blank vector as base entity.

The incorporation step is the final step of the process, which results in the active principle, which is capable of being toxic and expensive, being handled during only one step of the process. This process thus reduces the handlings and the possible losses of the active principle. It therefore makes it possible to be more certain as regards safety but also more profitable.

In addition, for some active principles, the incorporation conditions can be relatively simple, which makes it possible to envisage charging the vectors with the active principle at the time of use. This method of preparation at the time of use can eliminate the problems of storage in the liquid state.

This new method of charging is based on the significant affinity between the vectors and the ionic active principles but also on the simple control of the incorporation by the dispersion of the vectors and the ionic form of the active principle. It has very worthwhile advantages: preparation of the blank vector independent of the active principle, handling of the active principle in a single step and the possibility of preparation at the time of use.

Another subject of the invention is a particulate vector which contains, from the inside towards the outside, a crosslinked polysaccharide matrix containing an ionizable active principle, a first lipid layer fixed to the matrix by covalent bonds and a second layer of amphiphilic compounds on which protein or peptide molecules are optionally grafted.

The particulate vectors according to the invention preferably have a diameter of between 10 nm and 5 .mu.m and more preferably between 20 and 70 nm.

These particulate vectors are intended to carry or to present at their surface one or a number of molecules possessing biological activity. Mention must be made, among these molecules, without this list being limiting, of:

antibiotics and antivirals,

proteins, proteoglycans, peptides,

polysaccharides, lipopolysaccharides,

antibodies,

antigens,

insecticides and fungicides,

compounds which act on the cardiovascular system,

anticancers,

antimalarials,

antiasthmatics,

compounds having an effect on the skin,

constituents of dairy fat globules.

In the examples below, a description will be given of the charging of various products according to their characteristics, and in particular:

a hydrophilic product of small size intended for systemic administration,

of an active principle possessing anticancer activity,

of two enzymes possessing antibacterial activity, lactoperoxidase and glucose oxidase,

of a plant extract composed of procyanidol oligomers possessing an antioxidant activity,

of constituents of the fat globule of milk.

Just like SMBVs having a ring of grafted fatty acids, L-type SMBVs can be sterilized either by filtration or by autoclaving. They can also be frozen or lyophilized, for example in the presence of an additive, or alternatively atomized.

The advantages of L-type or of PS-type SMBVs are in particular:

a modular construction which allows adaptation to the product to be carried or to be presented

a biomimicry with respect to natural structures, such as lipoproteins or dairy fat globules

a greater ease of synthesis and of industrialization with respect to SMBVs containing a fatty acid ring

great chemical and thermal stability due to the polymeric structure of the core

a defined size and the possibility of homogeneously obtaining very small sizes (20 nm)

their ability to stabilize the compounds encapsulated within the core and in particular enzymes or antioxidant compounds.

The subject of the invention is therefore a pharmaceutical composition, characterized in that it contains a particulate vector according to the invention and a pharmaceutically acceptable support for its administration. The vectors according to the invention are in particular useful for therapeutic and immunological applications.

Another subject of the invention is a cosmetological composition, characterized in that it contains a particulate vector as described above and cosmetologically acceptable excipients.

Finally, food compositions containing particulate vectors according to the invention form parts of the invention.

Claim 1 of 17 Claims

1. A synthetic particulate vector, having a diameter of 10 nm to 5 .mu.m comprising a non-liquid non active principle hydrophilic core, and an external layer which comprises amphiphilic compounds, wherein said external layer is combined with the core via hydrophobic interactions and/or ionic bonds.

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