EP2355801A1

EP2355801A1 - Statin nanoparticles

Info

Publication number: EP2355801A1
Application number: EP09759783A
Authority: EP
Inventors: Patrick Couvreur; Didier Desmaele; Fatima Zouhiri; Harivardhan Reddy Lakkireddy
Original assignee: Centre National de la Recherche Scientifique CNRS; Universite Paris Sud Paris 11
Current assignee: Centre National de la Recherche Scientifique CNRS; Universite Paris Sud Paris 11
Priority date: 2008-10-29
Filing date: 2009-10-28
Publication date: 2011-08-17
Also published as: FR2937537A1; JP2012507503A; US20110269830A1; CA2742181A1; WO2010049900A1; US8748414B2

Abstract

The present invention relates to a complex made up of at least one molecule of statin or a derivative thereof, covalently bonded to at least one hydrocarbon radical including at least 18 carbon atoms and containing at least one 2-methyl-buta-2-ene unit, to nanoparticles of such a complex, and to a method for preparing same, said complex and/or said nanoparticles optionally being in the form of a lyophilisate. The present invention also relates to a pharmaceutical composition including at least one complex and/or nanoparticles such as previously defined. The invention finally relates to said complex and/or to said nanoparticles for the treatment and/or prevention of hyperlipemia and hypercholesterolemia.

Description

Nanoparticles of statin

The present invention aims at providing novel statin derivatives, in particular in a water-dispersible nanoparticulate form, compositions containing them and their therapeutic uses. Statins belong to the therapeutic class of cholesterol-lowering drugs. In particular, they lower LDL-cholesterol (for "Low Density Lipoprotein Cholesterol") and are particularly useful for treating or preventing certain cardiovascular diseases (Journal of the American Medical Association, 251, No.3, 351-374). (1984)). Indeed, a large number of medical studies have shown that LDL-cholesterol is best correlated with the occurrence of cardiovascular events. In addition, LDL-cholesterol represents the type of lipids most responsible for the formation of atheroma plaques.

Specifically, statins decrease the biosynthesis of cholesterol in the liver by inhibiting the enzyme HMG-CoA reductase which controls the limiting step in mevalonic cholesterol synthesis by transforming 3-hydroxy-3-methylglutaryl-coenzyme A in mevalonic acid, a precursor of sterols. The use of statins is prescribed if LDL-cholesterol levels exceed 2.4 mmol / l in coronary patients.

The medical treatment of cardiovascular diseases has been disrupted by the arrival of statins whose effect on the lowering of blood cholesterol is often spectacular. Indeed, statins can lower by 20 to 50%, the total cholesterol concentration, by 25 to 60%, the concentration of LDL cholesterol and by 15 to 30%, the concentration of triglycerides.

However, the contraindications and side effects of statins are often heavy and may for example manifest as hypersensitivity to one of the constituents of the drug, myopathy, which can sometimes lead to severe renal failure, progressive liver disease and / or or a prolonged elevation of transaminases (Armitage J., Lancet 2007; 370: 1781-1790). These effects are notably due to inadequate tissue and / or cell distribution. It is therefore necessary to adjust at best the dose and the mode of administration of the statins. In the pharmaceutical industry, improving the bioavailability of active ingredients, intended to be administered orally is a major concern for galenists.

In particular, the formulation of statins, molecules, generally lipophilic, poses real problems, due mainly to their low solubility in aqueous liquid pharmaceutical excipients, their propensity to precipitate or recrystallize in aqueous solution and their low solubility in liquids. gastrointestinal tract from which they must be absorbed.

The bioavailability of an active ingredient is furthermore a function of its concentration in the gastrointestinal fluid, which itself depends on the release of the active ingredient. In particular, the more lipophilic an active ingredient, the less it tends to migrate into the digestive fluids.

In order to increase the bioavailability of the lipophilic active principles, it has been envisaged in WO 95/24893 to formulate them using digestible oils and hydrophilic and lipophilic surfactants. This type of formulation makes it possible to maintain the active ingredient in solution during its passage in the digestive tract and until its intestinal absorption.

The digestion of the oily ingredients of this type of formulation often has the advantage of solubilizing the active ingredient in mixed micelles consisting of bile salts and triglyceride lipolysis products of the digestible oil used. However, the presence of surfactants can inhibit lipolysis, which requires the prior in vitro evaluation of the digestibility of the oils of a given formulation.

On the other hand, the amounts of digestible oils that must sometimes be used to avoid recrystallization of the active ingredient in vivo are too important to allow the manufacture of a marketable capsule. The present invention aims precisely to overcome the aforementioned drawbacks and to provide formulations adapted to statins.

The inventors of the present invention have demonstrated that it was possible to formulate the statins in the form of nanoparticles in suspension in an aqueous medium and of reduced size, in particular compatible for administration by injection or oral route, under reserve to couple them covalently to at least one hydrocarbon radical of squalene nature. Thus, according to a first aspect, the present invention relates to a complex formed of at least one statin molecule or derivative, covalently coupled to at least one hydrocarbon radical comprising at least 18 carbon atoms and containing at least one unit represented by the following formula:

also called 2-methyl-buta-2-ene.

According to another object, the invention provides a complex as defined above, in which the hydrocarbon compound comprises from 18 to 40 carbon atoms, preferably from 18 to 32 carbon atoms.

Advantageously, the two entities forming the complex defined above are coupled by a covalent bond of the ester, ether, thioether, disulfide, phosphate or amide type and preferably ester.

Another object of the present invention is nanoparticles of a complex as described above.

Admittedly, WO 2006/090029 and Couvreur et al., Nano Lett. 2006, 6, pp. 2544-25 48, already describe the ability of a molecule containing at least one hydrocarbon radical as defined above, such as for example squalene, to spontaneously form nanoparticles of a hundred nanometers in an aqueous medium, when is covalently coupled to a nucleoside derivative such as gemcitabine. However, for obvious reasons, gemcitabine is very different from the derivatives considered according to the invention.

It is also known that the administration of squalene, as such, leads to its concentration after oral or intravenous administration, in LDL and VLDL (for "very low density lipoprotein") (Strandberg et al, J Lipid Research, 31, 1637, 1999 and H. Relas et al., J Lipid Research, 42, 988, 2001).

As indicated above, the formulation of the therapeutic active agents considered according to the present invention in the form of nanoparticles according to the present invention constitutes an advantageous alternative with regard to already existing formulations, for several reasons. Firstly, the nanoparticulate state of the statins advantageously makes it possible to improve the tissue and / or cell distribution, making it possible in particular to overcome the side effects of statins and thus to adapt the modes and doses of administration as well as possible. Advantageously, the average size of these nanoparticles varies from 30 to

500 nm, in particular 50 to 250 nm, or even 100 to 400 nm.

The present invention also relates to a process for the preparation of said nanoparticles comprising at least the dispersion of the complex according to the present invention, in at least one organic solvent, at a concentration which is sufficient, when the resulting mixture is added, to stir, to an aqueous phase, the instantaneous formation of nanoparticles of said complex in suspension in said aqueous phase, and, where appropriate, the isolation of said nanoparticles.

Advantageously, said method may further comprise a lyophilization step, particularly suitable for accessing solid forming. Thus, the present invention further relates to a lyophilizate comprising at least one complex and / or at least nanoparticles as described above.

According to another subject, the present invention aims at a complex and / or nanoparticles as defined above, optionally in the form of a lyophilizate as defined above, for the preparation of a pharmaceutical composition intended for the treatment and / or the prevention of hyperlipemia, hypercholesterolemia, and in particular for the treatment and / or prevention of cardiovascular diseases, obesity, dyslipidemia and / or the prevention of a cardio vascular accident e.

In other words, the subject of the present invention is a complex and / or nanoparticles, optionally in the form of a lyophilizate, as defined according to the present invention, for the treatment and / or prevention of hyperlipemia, hypercholesterolemia, and in particular for the treatment and / or prevention of cardiovascular disease, obesity, dyslipidemia and / or the prevention of a cardiovascular event.

The term "cardiovascular diseases", atherosclerosis (atheroma plaques), cerebral or stroke attacks or cardiac (infarction), arteritis of the lower limbs, coronary diseases such as angina (or angina of chest) or heart attacks such as myocardial infarction, ischemia, thromboses and thromboembolic diseases, diseases of the vessels, such as aneurysms, arteriopathy obliterans of the lower limbs, acute aortic dissection, pulmonary arterial hypertension, thromboembolic diseases, heart attacks, congenital heart disease. Hypercholesterolemia usually results from disruption of cholesterol biosynthesis and / or is due to abnormal circulating cholesterol levels.

In addition, the role of statins in the prevention of cancers with various localizations (lung, prostate, breast, pancreas, esophagus or colon cancer) has recently been demonstrated in Therapeutic Medicine, Corcos L. et al., Vol. 13, No. 1, 22-9, January-February 2007. Statins are also useful as adjuvants of current cancer therapies.

Thus, the subject of the present invention is also the complexes and / or nanoparticles according to the invention, optionally in the form of a lyophilisate, for the treatment and / or prevention of cancers, in particular of the lung, prostate, breast, pancreas , esophagus or colon or as adjuncts to current cancer therapies.

The present invention also extends to a pharmaceutical composition, in particular a medicament, comprising at least one complex and / or nanoparticles, said complexes and / or nanoparticles being optionally in the form of a lyophilizate, as described above, in association with at least one pharmaceutically acceptable carrier.

The present invention also relates to said composition for use as a medicament for the treatment and / or prevention of the aforementioned diseases and / or disorders. Such a composition may be particularly useful as a medicament for the treatment and / or prevention of the aforementioned diseases and disorders in so-called "at risk" patients, that is to say in patients having a high blood pressure and with other cardiovascular risk factors such as smoking, overweight, a family history of heart disease or diabetes, including type II diabetes In fact, the presence of such risk factors in a patient increases his risk of occurrence cardiovascular events. Compound or hydrocarbon radical with a squalene structure

For the purposes of the present invention, a compound or radical with a squalene or squalenoyl structure is a compound or radical comprising at least one 2-methyl-buta-2-ene unit, as defined above.

More specifically, a compound or hydrocarbon radical with squalene or squalenoyl structure comprises at least 18 carbon atoms and containing at least one 2-methyl-buta-2-ene unit, like a squalene radical.

It should be noted that in the present invention, the term "compound" or "radical" with a squalene or squalenoyl structure is used as the case may be. The term "compound" is more specifically intended to define a compound with a squalene or squalenoyl structure, which, when reacted with an active molecule, forms a complex, whereas the term "radical" defines more precisely the squalene or squalenoyl of the complex formed.

For the purposes of the present invention, a hydrocarbon radical with a squalene structure may be represented by the following formula (I):

in which :

- mi = 1, 2, 3, 4, 5 or 6;

m ₂ = 0, 1, 2, 3, 4, 5 or 6; and represents the binding to the statin molecule or derivative, it being understood that when m ₂ is 0, then mi is at least 2.

More specifically, when speaking of the squalenoyl compound or one of its derivatives, the starting entity used for the coupling, this compound or one of its derivatives can be represented by the compound of formula (Ibis):

in which : Y represents a hydrogen atom or a group -L ₂ -X 'in which X' represents a function of alcohol, carboxylic acid, thiol, phosphate, amine, carboxamide or ketone type and L ₂ represents a single covalent bond or a group C 1 -C ₄ alkylene; and mi and m ₂ are as defined for the radical of formula (I).

The hydrocarbon radical comprises at least 18 carbon atoms, in particular from 18 to 40 carbon atoms and preferably from 18 to 32 carbon atoms.

More specifically, a compound useful for the formation of a complex according to the present invention is squalene (also called spiracene or sirprene), which is an essential intermediate of cholesterol biosynthesis. Chemically, it is also called (E) 2, 6, 10, 15, 19, 23-Hexamethyl-2, 6, 10, 14, 18, 22-tetracosahexene) of the following formula:

According to a preferred embodiment of the invention, the squalene derivative present in a complex according to the present invention is a radical of formula (I) in which mi = 1 and m ₂ = 2.

Advantageously, said complex is a radical of formula (I) in which mi = 1 and m ₂ = 3.

According to a preferred embodiment of the present invention, the squalene derivative present in a complex according to the present invention is a radical of formula (V) which follows, corresponding to a radical of formula (I) above in which m ₂ = 0:

(I ¹ )

In this case, mi = 2, 3, 4, 5 or 6.

As an illustration of hydrocarbon compounds capable of forming a complex according to the present invention, mention may be made more particularly of squalene acid and its derivatives such as 1, 1 ', 2-tris-norsqualenic acid, 1, 1', 2-tris-norsqualenamine, 1,1 ', 2-tris-norsqualenol, 1,1', 2-tris-norsqualethiol, squalenacetic acid, squalenylethanol, squalenylethanethiol, squalenylethylamine A complex according to the present invention comprises at least one hydrocarbon radical represented by a radical of formula (I) as defined above.

In particular, a complex according to the present invention may contain at least one radical derived from a molecule of 1, Y, 2-tris-norsqualenic acid. Alternatively, a complex according to the present invention comprises at least two hydrocarbon radicals as defined according to the present invention and in particular, represented by the compound of formula (I) above.

As the inventors have found, a hydrocarbon compound as defined above spontaneously shows, when it is placed in the presence of a polar medium and more particularly water, a compacted conformation.

Unexpectedly, the inventors have found that this ability remains when such a radical is associated and in particular covalently linked to a molecule of statin or one of its derivatives. It follows the generation of a compacted architecture in the state of nanoparticles in which at least partially include a molecule entity of statin and at least one hydro-carbon radical.

The statin molecule can indeed be only partially or totally in the compacted state in the nanoparticles formed.

Generally, at least one hydrocarbon radical mentioned above is covalently bound to a statin molecule. However, the number of hydrocarbon derivative molecules capable of interacting with said molecule may be greater than 1.

statin

Structurally, the statins or derivatives have in common a 4-hydroxy-6-oxo-2H-pyran system, which can also be in dihydroxy acid form which interacts with the active site of the HMG-CoA reductase enzyme involved in the synthesis of cholesterol and a lipophilic moiety presenting itself in particular as a polysubstituted hexahydronaphthalenic system but may also be replaced by a polysubstituted heteroaromatic system as in atorvastatin or fluvastatin.

The main known statins are: simvastatin, lovastatin, pravastatin, atorvastatin, fluvastatin and rosuvastatin.

These statins are generally of a lipophilic nature (WO 02/053131). For example, simvastatin has a log P = 4.68 and lovastatin a log P = 4.04. The lipophilicity of an active principle can be determined according to its partition coefficient (P) between octanol and water, which corresponds to the ratio of concentration of the active ingredient in octanol (C _oct ) / concentration of the active principle in water (CEau).

When the ratio P is greater than 1, it means that C _oct is greater than CEau, and that consequently the active ingredient is lipophilic (log P> 0). It can therefore be deduced that the higher the log P of an active ingredient, the more pronounced this lipophilic character is.

More specifically, within the meaning of the present invention, the expression statin or derivative is understood to mean a compound represented by the formula (IIa), (Hb) or (IIc) which follows:

Mc

in which :

- Ra represents an aryl group, heteroaryl, optionally substituted with one or more group (s) R;

- R independently represents a hydroxyl group, a Ci-C ₆ alkyl,

Alkoxy Ci-C _6, an -0-C (O) Ci-C ₆ alkyl, phenyl, -NRiR ₂ group, a -C (O) NRiR _2, -C (O) OR3, said alkyl and phenyl groups being optionally substituted with one or more halogen atoms or with one or more hydroxyl groups;

R 1 and R ₂ represent, independently of one another, a hydrogen atom, a C ₁ -C ₆ alkyl group, -SO ₂ -C ₁ -C ₆ alkyl, a phenyl, said groups C ₁ -C ₆ alkyl and phenyl being optionally substituted with one or more halogen atoms or with one or more hydroxyl groups;

- R3 represents a hydrogen atom, a Ci-C ₆ alkyl optionally substituted by one or more halogen atoms or by one or more hydroxyl groups.

For the purposes of the present invention, the following terms mean:

a halogen atom: a fluorine atom, a chlorine atom, a bromine atom or an iodine atom;

a hydroxyl group: an -OH group; an alkyl: a saturated, linear or branched aliphatic group. By way of example, mention may be made of methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl and tert-butyl;

an alkoxy: an -O-alkyl radical where the alkyl group is as defined previously, for example methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, tert -butoxy;

an aryl group: an aromatic group which may be partially unsaturated monocyclic or bicyclic comprising from 6 to 10 carbon atoms. As an example of a unicycle, mention may be made of phenyl. By way of example of a bicycle, there may be mentioned naphthyl, said naphthyl may be partially unsaturated, such as 1,2,6,7,8,8a-hexahydronaphthyl;

- A heteroaryl group: a said aryl group further comprising at least one heteroatom selected from nitrogen, sulfur or oxygen. As an example of a monocycle, mention may be made of furanyl, thiophenyl, thienyl, pyrrolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, thiadiazolyl, pyridinyl, pyrimidyl and pyrazinyl. By way of example of a bicycle, mention may be made of indolyl, isoindolyl, indolizinyl, benzofuranyl, benzimidazolyl, quinolyl, isoquinolyl and phthalazyl.

The compounds of general formula (IIa), (Hb) or (IIc) may comprise one or more asymmetric carbons. They can therefore exist as enantiomers or diastereoisomers. These enantiomers, diastereoisomers, as well as their mixtures, including the racemic mixtures, form part of the invention. The compounds of the aforementioned formulas may exist in the form of bases or addition salts with acids. Such addition salts are part of the invention.

These salts are advantageously prepared with pharmaceutically acceptable acids, but the salts of other acids that are useful, for example, for the purification or the separation of the compounds of the abovementioned formulas also form part of the invention.

The compounds of general formula (IIa), (Hb) or (IIc) may, in addition, be in the form of hydrates or solvates, namely in the form of associations or combinations with one or more water molecules or with a solvent. Such hydrates and solvates are also part of the invention. Amongst the statins of formula (IIa) that are suitable for the present invention, mention may be made, for example, of pravastatin and atorvastatin.

Amongst the statins of formula (Hb) that are suitable for the present invention, mention may be made, for example, of simvastatin and lovastatin.

Amongst the statins of formula (IIc) that are suitable for the present invention, mention may be made, for example, of fluvastatin and rosuvastatin.

A statin particularly suitable for the implementation of the present invention is represented by a compound of formula (IIa).

Advantageously, according to the present invention, it is possible to use in the complexes and / or nanoparticles according to the invention simvastatin, lovastatin, pravastatin, atorvastatin, fluvastatin or rosuvastatin, and most particularly pravastatin.

Hydrocarbon radical complex / statin

The conjugation of a molecule of statin with a hydrocarbon derivative according to the invention, and more particularly with 1,1 ', 2-tris-norsqualenol, confers on the molecule of statin physico-chemical characteristics which are sufficient to confer on it ability to form particles by nanoprecipitation, particles whose size is compatible with any mode of administration, in particular intravenous and oral.

For the purposes of the present invention, such conjugation leads to the formation of a complex or conjugate statin / hydrocarbon radical according to the present invention, that is to say an entity comprising a radical derived from a molecule of statin, bound covalently, to a hydrocarbon radical as defined above. For the purposes of the present invention, the terms "complex" or "conjugate" will thus be used indifferently to designate such an entity.

Thus, the present invention relates to a complex characterized in that it has the ability to spontaneously organize in the state of nanoparticles when in the presence of an aqueous medium.

The formation of the statin / hydrocarbon radical complex according to the invention requires that the two entities of the complex carry functions capable of forming a covalent bond and / or a linker, as defined below. These functions may or may not be present on the two starting entities. In the negative, the 171st entity (s) of departure will have to undergo a modification, prior to the coupling reaction.

More specifically, the hydrocarbon compound according to the invention is generally carrying a function capable of reacting with a function present on the molecule of said statin, so as to establish a covalent bond between the two entities, for example ester, ether , thioether, disulfide, phosphate or amide, thereby forming a covalent complex.

Advantageously, it may be an ester function. In which case, the terpene-containing hydrocarbon compound is 1,1 ', 2-tris-norsqualenol or a derivative thereof and in particular 1,1', 2-tris-norsqualenol bromoacetate. According to an alternative embodiment, the covalent link existing between the two types of molecules can be represented by a spacer or linker. Such an arm may in particular be useful for increasing the strength of the statin / hydrocarbon radical interaction according to the invention or to facilitate the activation of the conjugate after its administration by the enzymes of the organism (esterases, cathepsins, for example ) and thus allow the controlled release of the statin molecule.

Such an arm makes it possible precisely to introduce, via each of the two ends of its skeleton, the appropriate functions, that is to say respectively possessing the expected reaction affinity, one for the function present on the derivative with hydrocarbon structure according to US Pat. invention and the other for the function present on the relevant statin molecule.

It can also be envisaged that this linker also has at its skeleton a labile function, which is favorable later to the separation of the compound from hydrocarbon structure of the statin molecule considered. It may for example be a peptide motif recognizable by an enzyme.

The linkage type patterns are well known to those skilled in the art and their implementation clearly falls within its competence. As a representative of the linking arms that can be envisaged according to the invention, mention may be made in particular of the alkylene chains as defined above, the (poly) amino acid, polyol, saccharide and polyethylene glycol (polyetheroxidic) units. For the purposes of the present invention, the following terms mean:

"Saccharide unit", a radical comprising at least one radical chosen from trioses (glyceraldehyde, dihydroxyacetone), tetroses (erythrose, threose, erythrulose), pentoses (arabinose, lyxose, ribose, deoxyribose, xylose, ribulose, xylulose), hexoses (allose, altrose, galactose, glucose, gulose, idose, mannose, talose, fructose, psicose, sorbose, tagatose, heptoses (mannoheptulose, sedoheptulose), octose (octolose, 2-keto-3-deoxy-manno-octonate), isonoses (sialose), and - "(poly) amino acid motif", a unit having at least one unit:

in which n is greater than or equal to 1, and R 'represents a hydrogen atom, a C 1-6 alkyl group, optionally substituted by one or more hydroxyls, a C 1-6 alkoxy group.

Thus, within the meaning of the present invention, a "covalent bond" preferably represents a covalent bond especially as specified above, but also covers a covalent bond represented by a linker as defined above.

Thus, the covalent complex according to the present invention can be represented by the compound of formula (III) which follows:

(III)

in which X represents a single covalent bond or a function of ester, ether, thioether, disulfide, phosphate or amide type;

Li and L ₂ represent, independently of each other, a single covalent bond or a C 1 -C ₄ alkylene group;

Ra is as defined for the compound of formula (IIa) (Hb) or (IIc); and mi and m ₂ are as defined above for the compound of formula (I); and

- '^' represents the possible presence of a center of unsaturation.

For the purposes of the present invention, the term Ci-C ₄ alkylene group, a divalent alkyl group may include 1 to 4 carbon atoms. By way of example, mention may be made of methylene, propylene, isopropylene and butylene.

The present invention relates more particularly to a complex, represented by the compound of formula (HIa) or (HIb) which follows:

IIa)

where mi and m ₂ are as defined for the compound of formula (I) and L ₂ is as defined for the compound of formula (III). In particular, it is implemented according to the present invention a complex of formula (HIa) or (HIb), preferably the complex of formula (HIa).

An object of the present invention is therefore a complex according to the invention, which can be represented by formulas (III), (HIa) or (HIb), as defined above.

Process for preparing the complex

The reaction necessary for the establishment of at least one covalent bond between at least one molecule of said statin and at least one hydrocarbon-based radical according to the present invention can be carried out according to standard conditions and its realization therefore clearly falls within the knowledge of the skilled person.

More particularly, the bond between the statin and the hydrocarbon radical is produced by nucleophilic substitution with 1,1 ', 2-tris-nor-squalenyl bromoacetate. This reaction is generally carried out in solution in a polar solvent in the presence and in excess of at least one hydrocarbon compound considered according to the present invention with respect to the statin molecule implemented according to the invention, for example at the rate of two equivalents. , according to the standard conditions required to make the two specific functions carried by each of the two entities interact.

As indicated above, the establishment of the covalent bond between the two entities to be considered according to the invention requires, where appropriate, that each of the groups to be reacted carries functions capable of reacting with each other such that by for example a carboxyl function with a hydroxyl function to form an ester bond or an amino function with a carboxyl function to form an amide bond. Thus, if necessary, one or both entities, the statin molecule on the one hand, and the hydrocarbon compound on the other hand, are modified prior to the coupling reaction in order to provide them with the appropriate function to give them the reactivity necessary for the formation of a covalent bond between them. Preferably, each of the two molecules is modified in order to establish an amide or ester bond between them. Preferably, a hydrocarbon compound starting for the synthesis of a complex according to the invention is a squalene derivative, such as, for example, 1, 1 ', 2-tris-norsqualenol, as illustrated in step 1.1 of Example 1 Nanoparticles according to the invention

As specified above, the covalent coupling of at least one molecule of statin considered according to the invention with at least one hydrocarbon compound according to the invention is such as to confer on the molecule of statin thus complexed, an ability to organize under a compacted form in a polar solvent medium, thus leading to the formation of nanoparticles.

In general, the nanoparticles thus obtained have an average size ranging from 30 to 500 nm, and in particular from 50 to 250 nm, or even from 100 to 400 nm, measured by light scattering using the Coulter ^® nanosizer. N4MD, Coulter Electronics, Hialeah, USA.

An object of the invention is nanoparticles according to the invention, the mean size of which varies from 30 to 500 nm, in particular from 50 to 250 nm, or even from 100 to 400 nm.

Another object of the present invention relates to 4- (N) - squalenoylpravastatin nanoparticles. Obtaining such nanoparticles is illustrated in Example 2.

Advantageously, the nanoparticles according to the present invention, in particular in the form of lyophilizate, are particularly advantageous for administration intended for the oral route.

Process for preparing nanoparticles

The formation of nanoparticles from the complex described above can be carried out according to conventional techniques insofar as they involve bringing into the presence of a complex with an aqueous medium under conditions conducive to its agglomeration in the state of nanoparticles. It may in particular be methods known as nanoprecipitation or emulsion / solvent evaporation.

The nanoparticles according to the present invention can preferably be obtained in the following manner.

Preliminarily, a statin / hydrocarbon compound complex is formed, by coupling at least one hydrocarbon compound according to the invention to at least one statin molecule according to the invention, as described above.

Said complex obtained is then dispersed in at least one organic solvent (for example an alcohol such as ethanol or acetone) at a concentration of sufficient to obtain, during the addition of the resulting mixture, with stirring, and generally at drip, with an aqueous phase, the instantaneous formation of nanoparticles according to the invention in suspension in said aqueous phase. If necessary, the nanoparticles are isolated according to the techniques well known to those skilled in the art.

The reaction can generally be carried out at room temperature. Whatever it is, the reaction temperature must not affect the activity of the statin molecule under consideration. The process for preparing the nanoparticles according to the invention is particularly advantageous insofar as it does not necessarily require the presence of surfactants, but may nevertheless be necessary in the case, for example, where the use of pravastatin.

This property is particularly appreciable insofar as a large number of surfactants do not prove to be compatible with an application in vivo.

However, it is understood that the use of surfactants, generally advantageously devoid of any toxicity, is conceivable within the scope of the invention. This type of surfactant can also provide access to even smaller sizes during the formation of nanoparticles. By way of non-limiting illustration of this type of surfactant which may be used in the present invention, polyoxyethylene-polyoxypropylene copolymers, phospholipid derivatives and lipophilic derivatives of polyethylene glycol may be mentioned.

As the lipophilic derivative of polyethylene glycol, mention may be made, for example, of polyethylene glycol cholesterol and polyethylene glycol squalene. As examples of polyoxyethylene-polyoxypropylene block copolymers, there may be mentioned polyoxyethylene-polyoxypropylene-polyoxyethylene triblock copolymers, also called poloxamers ^® , pluronics ^® or synperonics and which are sold in particular by the company BASF.

Related to these families of copolymers, poloxamines, which consist of hydrophobic segments (based on polyoxypropylene), hydrophilic segments (based on polyoxyethylene) and a central portion derived from the ethylene diamine unit can also be used.

The nanoparticles according to the invention are of course capable of carrying on the surface a multitude of reactive functions, such as hydroxyl functions or amine for example. It is therefore conceivable to fix these functions all kinds of molecules, including covalent bonds.

By way of non-limiting illustration of this type of molecule capable of being associated with the nanoparticles, mention may be made especially of marker-type molecules, compounds capable of providing a targeting function, as well as any compound capable of conferring on them characteristics. specific pharmacokinetics. As regards the latter aspect, it is thus possible to envisage fixing on the surface of these nanoparticles lipophilic derivatives of polyethylene glycol, such as, for example, the polyethylene glycol / cholesterol conjugate, polyethylene glycol-phosphatidylethanolamine or better still polyethylene glycol / squalene. . Indeed, given the natural affinity of the squalene residues with each other, the polyethylene glycol / squalene conjugate is associated, in this case, with the nanoparticles according to the invention, and thus leads to the formation of coated nanoparticles. on the surface of polyethylene glycol. Moreover, and as mentioned above, the polyethylene glycol / squalene conjugate advantageously acts during the process of forming the nanoparticles according to the invention as a surfactant because of its amphiphilic behavior and thus stabilizes the colloidal solution, thus reducing the size of the nanoparticles formed. . A surface coating based on such compounds and in particular polyethylene glycol or the polyethylene glycol / cholesterol conjugate or the polyethylene glycol / squalene conjugate, is in fact advantageous for conferring increased vascular remanence because of a significant reduction in the capture of nanoparticles by hepatic macrophages.

According to an advantageous embodiment, the nanoparticles according to the invention are formulated in the form of an aqueous dispersion.

According to another particular embodiment, this aqueous dispersion contains less than 5% by weight, or even less than 2% by weight and more particularly is devoid of surfactant or the like such as, for example, polyethylene glycols, polyglycerol and their derivatives, such as esters for example.

According to another advantageous embodiment, this aqueous dispersion contains less than 5% by weight, or even less than 2% by weight of C ₂ to C ₄ alcohol such as, for example, ethanol.

Thus, the formulation in an aqueous medium of the statin in question using squalenic acid in the form of water-dispersible nanoparticles makes it possible advantageously to to obtain a suspension of nanoparticles with no other additive than the 5% dextrose necessary to obtain the isotonicity of the injectable suspension.

According to another advantageous embodiment, the nanoparticles according to the invention are in the form of lyophilisate. As indicated above, the present invention also relates to the use of at least one nanoparticle according to the invention in pharmaceutical compositions.

Another aspect of the invention therefore relates to a pharmaceutical composition comprising at least, as an active ingredient, a complex according to the present invention, in particular in the form of nanoparticles. The complexes according to the present invention can be associated with at least one pharmaceutically acceptable vehicle.

As examples of pharmaceutical formulations compatible with the compositions according to the invention, mention may be made in particular of: intravenous injections or infusions; saline or purified water solutions; inhalation compositions; capsules, sugar-coated tablets, cachets and syrups incorporating, in particular as a vehicle, water, calcium phosphate, sugars, such as lactose, dextrose or mannitol, talc, stearic acid, starch, sodium bicarbonate and / or gelatin.

When the complexes and / or nanoparticles are used in dispersion in an aqueous solution, they can be combined with sequestering or chelating agent excipients, antioxidant, pH modifying agents and / or buffering agents. In addition to the abovementioned compounds, the pharmaceutical compositions according to the invention may contain preserving agents, wetting agents, solubilizing agents and coloring agents.

However, it may contain other assets that may benefit from therapeutic benefit, in addition to the effect of statins. As representative of these active substances that can be combined with the complexes and / or nanoparticles according to the present invention, mention may be made in particular of active agents intended to reduce arterial hypertension, such as antihypertensives or hypotensives or other compounds with lipid-lowering activity. Among them are diuretics, such as thiazides, diuretics of Henle's loop such as furozemide, anti-aldosterones, or β-blockers such as acebutolol, atenolol, betaxolol, bisoprolol, esmolol, metoprolol, nebivolol, nadolol, oxprenolol, propranolol, pindolol, sotalol, carvedilol, labetalol, levobunolol, timolol, angiotensin converting enzyme inhibitors ( IECA, IEC) such as benazepril, captopril, cilazapril, enalapril, fosinopril, imidapril, lisinopril, moexipril, perindopril, quinapril, ramipril, spirapril, trandolapril, angiotensin II receptor antagonists such as candesartan, candesartan cilexetil, eprosartan , irbesartan, losartan and its potassium salt, olmesartan, olmesartan medoxomil, telmisartan, valsartan, calcium channel blockers such as amlodipine, gallopamil, verapamil, amlodipine, barnidipine, felodipine, isradipine, lacidipine, lercanidipine, nicardipine, nifedipine, nimodipine, nisoldipine, nitrendipine, diltiazem, central antihypertensives, alpha-stimulants such as prazosin, terazosin, alfuzosin, doxazosin, tamsulosin, peripheral alpha-blockers, vasodilators.

Mention may also be made of other lipid-lowering agents chosen from: fenofibrate, bezafibrate, cipofibrate, colestipol, ezetimibe, tiadenol, gemfibrozil, omega-3 polyunsaturated fatty acids, triglycerides of omega-3 acids, benfluorex, alpha-tocopherol (vitamin E), colestyramine. The complexes and / or nanoparticles according to the present invention can be administered by any conventional route. However, as previously stated, given the small size of their particles, they can be administered in the form of an aqueous suspension intravenously and therefore compatible with vascular microcirculation. For obvious reasons, the quantities of the complex and / or nanoparticle according to the invention that may be used may vary significantly depending on the mode of use and the route chosen for their administration.

On the other hand, for topical administration, it is conceivable to formulate at least one complex and / or nanoparticle conforming to the present invention in a proportion of 0.1 to 10% by weight, or even more, relative to the total weight of the pharmaceutical formulation considered. The following examples illustrate the present invention without being limited thereto.

Infrared spectra are obtained by measurement on a solid or a pure liquid using a Fourier spectrometer (Transform Bruker Vector ^® 22). Only significant absorptions are noted. Optical rotations were measured using a Perkin-Elmer ^® 241, at a wavelength of 589 nm. The ¹ H and ¹³ C NMR spectra were recorded using a Bruker ARX ^® 400 spectrometer (at 400 MHz and 100 MHz, respectively for ¹ H and ¹³ C) or Bruker Avance ^® 300 (at 300 MHz and 75 MHz, respectively for ¹ H and ¹³ C). Mass spectra were recorded using a Bruker Esquire-LC ^® instrument. Thin layer chromatography analysis was performed on 6OF254 silica plates (0.25 mm layer). The chromatography column was carried out on silica gel 60 (Merck, 230-400 mesh ASTM). All reactions involving compounds sensitive to air or water were conducted under a nitrogen atmosphere. IPD = Polydispersity Index

Example 1 Preparation of the complex (7V) -squalenoylpravastatin (SQprava)

Step 1.1: (4 _J E, 8 _J E, 12 _J E, 16 _J E) -4,8,13,17,21-Pentamethyldocosa-4,8,12,16,20-pentaen-1-ol:

Squalene aldehyde is reduced to squalenol. To do this, 1.15 g (3 mmol) of squalene aldehyde dissolved in 6 mL of ethanol are added, at 0 ^° C., in small portions to 106 mg (0.9 equiv., 2.7 mmol) of sodium borohydride. The mixture is stirred under nitrogen at room temperature for 15 min, then the reaction mixture is neutralized with a solution of 2N HCl. Then, the solvent is distilled under reduced pressure. The residue is dissolved in 10 ml of water and extracted with ethyl acetate (3 times 20 ml) and the combined organic phases are washed with a saturated aqueous solution of NaCl (10 ml), dried over MgSO ₄ and then the solvent is distilled under reduced pressure to yield a pale yellow oil. IR (neat, cm ^-1 ) v: 3060-2840, 1667 (weak), 1445, 1381.

¹ H NMR (300 MHz, CDCl ₃ ) δ: 5.16-5.09 (m, 5H), 3.62 (t, J = 6.4, 2H), 2.13-1.04 (m, 21H), 1.61 (s, 3H), 1.53 (s, 15H). MS (APCI +, MeOH), m / z (%): 387 ([M + H] ⁺ (100)).

Step 1.2. Bromoacetate (4 _J , 8 _J E, 12 _J E, 16 _J E) -4,8,13,17,21-pentamethyldocosa-4,8,12,16,20-pentaen-1-yl. 500 mg (1.3 mmol) of squalenol dissolved in 6 ml of anhydrous CH ₂ Cl ₂ are added, 216 mg (1.55 equiv., 2.01 mmol) of bromoacetic acid and a few mg of DMAP. The mixture is cooled to 0 ^° C., then 317 mg (1.5 equiv, 1.95 mmol) of DCC dissolved in 2 ml of CH ₂ Cl ₂ are added in small portions. After total addition, the mixture is stirred under nitrogen at ambient temperature for 18 h and then filtered through celite. The filtrate is concentrated under reduced pressure. The residue is chromatographed on silica gel using an AcOEt / Cyclohexane: 1/4 mixture to give 55 mg of a colorless oil.

IR (neat, cm ^-1 ) v: 2960-2850, 1737, 1668 (weak), 1450, 1382, 1276, 1381 ¹ H NMR (300 MHz, CDCl ₃ ) δ: 5.18-5.07 (m, 5H), 4.14 (t, J = 6.4, 2H), 3.82 (s, 2H), 2.16-1.98 (m, 18H), 1.81-1.71 (m, 2H), 1.68 (s, 3H), 1.53 (s, 15H), ¹³ C (75 MHz, CDCl ₃ ) δ: 167.2 (C), 135.0 (C), 134.8 ( 2 C), 133.2 (C), 131.2 (C), 125.3 (CH), 124.4 (2 CH), 124.2 (2 CH), 65.9 (CH ₂ ), 39, 7 (2 CH ₂ ), 39.6 (CH ₂ ), 35.5 (CH ₂ ), 28.2 (2 CH ₂ ), 28.7 (CH ₂ ), 26.6 (CH ₂ ), 26, 5 (2 CH ₂ ), 25.6 (CH ₂ ), 25.6 (CH ₃ ), 17.6 (CH ₃ ), 16.0 (3 CH ₃ ), 15.8 (CH ₃ ).

Step 1.3: 2-Oxo-2 - {[(4E, 8E, 12E, 16E) -4,8,13,17,21-pentamethyldocosa-

4,8,12,16,20-pentaen-1-yl] oxy} ethyl (3R, 5R) -3,5-dihydroxy-7 - ((1S, 2S, 6R, 8S, 8aR) -6-hydroxy- 2-methyl-8 - {[(2S) -2-methylbutanoyl] oxy} -1,2,6,7,8,8a-hexahydronaphthhalen-1-yl) heptanoate 50 mg (0.11 mmol) dissolved pravastatin in 0.4 ml of anhydrous DMSO is added 111 mg (2 equiv, 0.22 mmol) of squalenyl bromoacetate. The mixture is heated under a nitrogen atmosphere at 40 ^° C. for 5 h, then the solvent is distilled under reduced pressure. The residue is chromatographed on silica gel using an AcOEt / cyclohexane: 1/1 mixture to give 55 mg of pravastatin-SQ in the form of an amorphous white solid.

[α] _D = 77.7 (c = 2.4, CHCl ₃ );

IR (mp ₁₅ Cm +) V: 3500-3200, 2924, 1728, 1448, 1375, 1264, 1182, 1151. ¹ H NMR (CDCl ₃ , 400 MHz) δ: 5.99 (d, J = 9.7 Hz, 1H, H-4), 5.89 (dd, J = 9.7, 6.0 Hz, 1H). , H-3), 5.56 (s, 1H, H-5), 5.42 (s, 1H, H-8), 5.20-5.05 (m, 5H, HC = C (Me) ), 4.72 (d, J = 15.8 Hz, 1H, OCH ₂ OCO), 4.61 (d, J = 15.8 Hz, 1H, OCH ₂ OCO), 4.46-4.36 ( m, 1H, H-6), 4.38-4.22 (m, 1H, H-3 '), 4.15 (t, J = 6.9 Hz, 2H, OCH ₂ CH ₂ CH ₂ C ( Me)), 3.91 (brs, 1H, OH), 3.83-3.78 (m, 1H, H-5 '), 3.53 (brs, 1H, OH), 2, 65-2.55 (m, 3H, 2H-2 ', HI), 2.46-2.35 (m, 1H, H-2), 2.35-2.28 (m, 2H, HC (M. ) (Et) CO ₂ ), 2.12-1.92 (m, 18 Η, = C (Me) CH ₂ CH ₂ ), 1.75 (quint, J = 8.0 Hz, 2H, OCH ₂ CH); ₂ CH ₂ C (Me)), 1.68 (s, 3H, = C (CH ₃ ) ₂ ), 1.69-1.50 (m, 21H, 5 = C (CH ₃ ), H-7, H-4 ', 1 H CH ₃ CH ₂ CH (me) CO ₂ H 6 1'), 1.49 to 1.35 (m, 2H, H 1 CH ₃ CH ₂ CH (me) CO _2, 1H-7 '), 1.16 (m, 1H, 1H-6'), 1.11 (d, J = 6.8Hz, 3H, (CH ₃ ) (Et) CHCO ₂ ), 0, 90 (d, J = 7.6 Hz, 3H, C-2 (CH ₃ )), 0.88 (d, J = 7.6 Hz, 3H, (CH ₃ ) (CH ₂ CH ₃ ) CHCO ₂ ) ;

¹³ C NMR (CDCl ₃ , 100 MHz) δ: 176.3 (CO), 171.3 (CO), 168.3 (CO), 136.1 (CH), 135.6 (C), 135.1 (C), 134.9 (2C), 133.3 (C), 131.2 (C), 127.3 (CH), 125.9 (CH), 125.4 (CH), 124.4 (CH), 2CH), 124.3 (2CH), 72.2 (CH), 69.5 (CH), 69.2 (CH), 65.6 (CH ₂ ), 65.1 (CH), 60.6 (CH ₂ ), CH ₂ ), 42.35 (CH ₂ ), 42.30 (CH ₂ ), 41.6 (CH), 39.7 (CH ₂ ), 39.6 (CH ₂ ), 37.7 (CH), 36.8 (CH ₂ ), 36.6 (CH), 35.6 (CH ₂ ), 34.6 (CH ₂ ), 30.9 (CH), 28.3 (2CH ₂ ), 26.8 (CH ₂ ), CH ₂ ), 26.65 (3CH ₂ ), 26.60 (2CH ₂ ), 25.7 (CH ₃ ), 23.8 (CH ₂ ), 16.8 (CH ₃ ), 16.0 (4CH ₃₎ ), 15.8 (CH ₃ ), 13.6 (CH ₃ ), 11.8 (CH ₃ );

MS (ESI +, MeOH, DMSO), m / z (%): 874 ([M + Na] ⁺ (100)).

Example 2 Preparation of Pravastatin Nanoparticles

Preparation of nanoparticles consisting of 4- (N) -saualenoylpravastatin

The nanoparticles are obtained by the precipitation / solvent evaporation method, by analogy with the method described in Fessi H. et al, Int. J. Pharm., 55; 1989, R1-R4.

4 mg of SQpravastatin are dissolved in 0.5 mL of ethanol in a pillbox. In another vial, 2 mg of SQ-PEG is dissolved in 0.2 ml of acetone and 0.1 ml of ethanol, in the order mentioned. The two solutions are then mixed and the solution obtained is added dropwise, with stirring (500 rpm) to 1 ml of a 5% aqueous dextrose solution. The precipitation of the nanoparticles is immediate. The vial containing the SQpravastatin solution is rinsed and the rinse solution added to the nanoparticle suspension. After 2 or 3 minutes of agitation, the suspension of nanoparticles is transferred to a calibrated flask and concentrated under reduced pressure in a rotary evaporator (50-100 mbar at 20 ^° C. for 10 min and then at 37 ^° C. for approximately 3-5 minutes) to a weight of from 0.8 to 0.degree. 9 g. The solution is then supplemented to 1 g using either a 5% dextrose solution or sterile water. The size of the nanoparticles obtained, measured with a Malvern nanosizer (Zetasizer) is 146 nm.

The nanoparticles have an average size, compatible with any mode of administration, as well as a good stability in aqueous solution.

They have a polydispersity index of 0.08.

The polydispersity index was determined according to the methods well known to those skilled in the art (for example, by analogy with the method described in Couvreur et al., Nanoletters, Vol 6, No. 1, pages 2544-2548). , 2006).

Claims

A complex formed of at least one statin molecule or derivative, covalently coupled to at least one hydrocarbon radical comprising at least 18 carbon atoms and containing at least one unit represented by the following formula:

2. Complex according to the preceding claim, wherein the hydrocarbon compound comprises from 18 to 40 carbon atoms, preferably from 18 to 32 carbon atoms.

3. Complex according to any one of the preceding claims, wherein the hydrocarbon radical is represented by the radical of formula (I) which follows:

wherein: mi = 1, 2, 3, 4, 5 or 6; m ₂ = 0, 1, 2, 3, 4, 5 or 6; and

represents the bond to the statin molecule or derivative, it being understood that when m ₂ is 0 then mi is at least 2.

4. Complex according to any one of the preceding claims, wherein the hydrocarbon radical is the radical of formula (I) in which mi is 1 and m ₂ is 2.

5. Complex according to any one of the preceding claims, in which the statin or derivative molecule comprises a 4-hydroxy-6-oxo-2H-pyran system or its dihydroxy acid form and a lipophilic part that is in particular a polysubstituted hexahydronaphthalenic system. or polysubstituted heteroaromatic.

A complex according to any one of the preceding claims, wherein the molecule derived from statin is represented by the formula (IIa), (Hb) or (IIc) which follows:

Mc

in which :

- R independently represents a hydroxyl group, a Ci-C ₆ alkyl,

R 1 and R ₂ represent, independently of one another, a hydrogen atom, a C ₁ -C ₆ alkyl group, a -SO ₂ -C ₁ -C ₆ alkyl, a phenyl, said groups Ci-C ₆ alkyl and phenyl being optionally substituted with one or more halogen atoms or with one or more hydroxyl groups;

- R3 represents a hydrogen atom, a Ci-C ₆ alkyl optionally substituted by one or more halogen atoms or by one or more hydroxyl groups; in the form of a base or an acid addition salt, as well as a hydrate or solvate state, as well as its enantiomers, diastereoisomers, and their mixture.

7. Complex according to any one of the preceding claims, wherein the molecule, derived from statin, is selected from atorvastine, lovastatin, simvastatin, pravastatin, fluvastatin and rosuvastatin.

A complex according to any one of the preceding claims, wherein the two entities forming said complex are coupled by a covalent bond of ester, ether, thioether, disulfide, phosphate or amide type.

9. Complex according to any one of the preceding claims, represented by the compound of formula (III) which follows:

(III) in which

X represents a single covalent bond or a function of ester, ether, thioether, disulfide, phosphate or amide type; Li and L ₂ represent, independently of each other, a single covalent bond or a C 1 -C ₄ alkylene group;

Ra is as defined for the compound of formula (IIa) (Hb) or (IIc); and mi and m ₂ are as defined for the compound of formula (I); and

- '^' represents the possible presence of a center of unsaturation; in the form of a base or an acid addition salt, as well as a hydrate or solvate state, as well as its enantiomers, diastereoisomers, and their mixture.

10. Complex according to any one of the preceding claims, characterized in that it has the ability to spontaneously organize in the state of nanoparticles when in the presence of an aqueous medium.

11. Nanoparticles of a complex as described in any one of claims 1 to 10.

12. Nanoparticles according to the preceding claim, the average size varies from 30 to 500 nm, in particular from 50 to 250 nm, or even from 100 to 400 nm.

13. Nanoparticles according to any one of claims 11 or 12, of A- (N) -squalenoylpravastatin.

14. Process for the preparation of nanoparticles according to any one of claims 11 to 13, characterized in that it comprises at least: the dispersion of a complex according to any one of claims 1 to 10, in at least one organic solvent, at a concentration sufficient to obtain, upon addition of the resulting mixture, with stirring, to an aqueous phase, the formation instantaneous nanoparticles of said complex suspended in said aqueous phase, and - if appropriate, the isolation of said nanoparticles.

15. Preparation process according to the preceding claim further comprising a lyophilization step.

16. Lyophilizate comprising at least one complex as defined according to one of claims 1 to 10 and / or at least nanoparticles as defined according to one of claims 11 to 13.

17. A pharmaceutical composition comprising at least one complex as defined according to one of claims 1 to 10 and / or at least nanoparticles as defined in one of claims 11 to 13, said complex and / or said nanoparticles being optionally in the form of a lyophilisate as defined in claim 16, in association with at least one acceptable pharmaceutical vehicle.

18. Complex as defined according to one of claims 1 to 10 and / or nanoparticles as defined in one of claims 11 to 13, said complex and / or said nanoparticles being optionally in the form of a lyophilisate as defined according to claim 16, for the treatment and / or prevention of hyperlipemia, hypercholesterolemia, and in particular for the treatment and / or prevention of cardiovascular diseases, obesity, dyslipidemia.