Patent Description:
The present invention relates to crystals of beta-sitosterol, processes for their preparation, and compositions comprising them. It also relates to their non-therapeutic use as a dietary supplement or functional food and to these crystalline forms of the invention for use as a medicament.

Arteriosclerosis is the thickening, hardening and loss of elasticity of the walls of arteries. This process gradually restricts the blood flow to one's organs and tissues and can lead to severe health risks brought on by atherosclerosis. Atherosclerosis (also known as arteriosclerotic vascular disease or ASVD) is a specific form of arteriosclerosis caused by the build-up of an atheroma in and on the artery walls. The atheroma is an accumulation of degenerative material in the inner layer of an artery wall. The degenerative material forming the atheroma consists of mostly macrophage cells, lipids (fatty compounds), and fibrous connective tissue. Although the precise significance of an elevation of one or more specific blood lipid fractions is not known, there have been disclosed sufficient evidences that elevated serum cholesterol levels (also known as hypercholesterolemia) were considered to be the most important factor in the ethiology of the atherosclerotic process. Therefore, intensive efforts have been made to find an effective pharmacologic agent that reduces the serum cholesterol levels. High levels of cholesterol in blood and in particular high levels of cholesterol-LDL and also to considerable extend high ratio of cholesterol-LDL to cholesterol-HDL are considered risk factors for arteriosclerosis, atherosclerosis and other alterations and diseases of the blood vessels and cardiovascular system.

Phytosterols (plant sterols) are plant steroids (or phytosteroids), which encompass both plant sterols and plants stanols (phytostanols) are compounds that have a structure similar to cholesterol. In particular, their structures vary only in the carbon side chains and/or the presence or absence of a double bond (e.g. sitosterol and sitostanol). Phytosterols are widely distributed in the plant kingdom and specially found in vegetable oil, and avocados. However, they are not synthesized by the human body and therefore their presence in the body is the result of their consumption as part of the diet.

Particularly, the β-Sitosterol (or beta-sitosterol) is the International Nonproprietary Name (INN) of (<NUM>-(<NUM>-ethyl-<NUM>-methylheptan-<NUM>-yl)-<NUM>,<NUM>-dimethyl-<NUM>,<NUM>,<NUM>,<NUM>,<NUM>,<NUM>,<NUM>,<NUM>,<NUM>,<NUM>,<NUM>,<NUM>-dodecahydro-<NUM>H-cyclopenta[a]phenanthren-<NUM>-ol. It is the phytosterol more abundant in current diets and in many phytosterol-based functional foods or food supplements. The structure of the beta-sitosterol corresponds to the formula (I):
<CHM>.

Several clinical studies disclosed in the state of the art have demonstrated that phytosterols reduce serum cholesterol levels by inhibiting cholesterol absorption in the intestinal lumen and probably by other less known mechanisms. Particularly, beta-sitosterol is useful for the reduction of serum total and LDL-cholesterol levels because the beta-sitosterol competes with cholesterol for up taking the cholesterol by the cells or by interfering with the esterification of cholesterol. In fact, beta-sitosterol has been approved by the FDA (Food and drug Administration) for that indication.

In addition, regarding arteriosclerosis there is also increasing emphasis in other lipid entities such as triglycerides. Therefore, intensive efforts have been and are being made to find pharmacologic agents that possess hypolipemic properties with varying degrees of effectiveness and specificity to the different lipid fractions.

It is known that different solid forms of an active ingredient can have different characteristics, and offer certain advantages, for example with regard to solubility or bioavailability. Thus, the discovery of new solid forms allows for improving the pharmacokinetic and/or pharmacologic or other physicochemical properties of the active ingredients and as a consequence, the characteristics of the pharmaceutical formulations containing the active ingredients, since some forms are more adequate for one type of formulation, and other forms for other different formulations.

Particularly, in recent years cocrystal formation has emerged as a viable strategy towards improving the pharmacokinetic data of active ingredients. By cocrystalizing an active ingredient or a salt of an active ingredient with at least one coformer (the second component of the cocrystal), a new solid state form of the active ingredient is created having unique properties compared with existing solid forms of the active ingredient or its salts. However, cocrystal formation is not predictable, and in fact is not always possible. Moreover, there is no way to predict the properties of a particular cocrystal of a compound until it is formed. Finding the appropriate coformers and right conditions to obtain a particular cocrystal can take significant time, effort and resources.

Monohydrate and hemihydrate forms of beta-sitosterol have been disclosed in the state of the art. In particular, a monohydrate form of beta-sitosterol was obtained by crystallization of the beta-sitosterol in a mixture of acetone and water, then the corresponding hemihydrate was obtained by submitting the monohydrate in dry atmosphere conditions (cf. Furthermore, other monohydrate and hemihydrate forms of beta-sitosterol have been obtained by crystallization of beta-sitosterol with polysorbate <NUM> (cf. Finally, further monohydrate and hemihydrate forms have been also obtained by crystallization of the beta-sitosterol in oil-suspensions (cf. All the above mentioned hydrate and hemihydrate forms of beta-sitosterol have been characterized by X-ray powder diffraction (XRPD).

From what is known in the art, there is still the need of finding new pharmacologic agents that possess hypolipemic properties with varying degrees of effectiveness and specificity to the different lipid fractions.

The inventors have found that beta-sitosterol can form cocrystal with an organic carboxylic acid coformer as a hydrogen bond donor coformer, the cocrystals being as defined in the appended set of claims. These cocrystals have a high pharmacological activity, for instance contributes to the reduction of high blood lipid levels and/or maintenance of normal plasma or blood lipid levels, including specific fractions of blood cholesterol and of blood triglycerides or triacylglycerols. The provision of cocrystals of beta-sitosterol as defined in the appended set of claims gives a new tool to overcome the problems associated with the normalization or maintenance of lipid levels in blood because the inventors have found that such cocrystals of the invention are much more effective and selective at lowering determinate lipid fractions of circulating lipid levels.

On one hand, the inventors have found that cocrystals of beta-sitosterol having an organic carboxylic acid as a hydrogen bond donor coformer as defined in the appended set of claims are much more effective and selective at lowering circulating cholesterol levels than the known anhydrous form, the monohydrate form and the hemihydrate form as described in the state of the art.

On the other hand, the inventors have also found that cocrystals of beta-sitosterol having an organic alcohol as a hydrogen bond donor coformer as defined in the appended set of claims are much more effective and selective at lowering circulating triglyceride levels than the known anhydrous form, the monohydrate form and the hemihydrate form.

Inventors have also found a hydrate crystal form of beta-sitosterol as defined in claims <NUM> to <NUM> which is effective and selective at lowering circulating triglyceride levels.

Advantageously, a combination of a cocrystal of beta-sitosterol and an organic carboxylic acid, with a cocrystal of beta-sitosterol and an organic alcohol, or alternatively with an hydrate crystal form of beta-sitosterol as defined in the appended set of claims is much more effective at lowering circulating lipid levels. In particular, this combination is especially advantageous because allows lowering both the levels of cholesterol and triglycerides in blood. Furthermore, this combination is also advantageous because allows administering both active ingredients separately, sequentially or simultaneously, provided that such administration comprises separate compositions of the two active ingredients.

Therefore, the crystals of the present invention allows designing a personalized treatment (precision medicine or precision nutrition) of the abnormal serum lipid levels based on the specific conditions of the disease or condition that involves the alteration of lipid metabolism, circulating levels of lipids in the blood and/or lipid composition in tissues and organs and also the specific characteristics of the patient to be treated, including its genomic characteristics.

Thus, a first aspect of the invention relates to a cocrystal of beta-sitosterol which is obtainable by a process which comprises: (aʺʺ) mixing the beta-sitosterol with a water-miscible organic solvent in the presence of propionic acid; and (b"") isolating the compound thus obtained; characterized by having a X-ray diffractogram that comprises characteristic peaks at <NUM> and <NUM> ± <NUM> degrees <NUM> theta at a Cu-Kα radiation, A = <NUM>Å; or alternatively, a cocrystal of beta-sitosterol and gallic acid characterized by having a X-ray diffractogram that comprises characteristic peaks at <NUM> and <NUM> ± <NUM> degrees <NUM> theta (Cu-Kα radiation, A = <NUM>Å).

A second aspect of the invention relates to a combination as defined in claim <NUM> of the appended set of claims.

A third aspect of the invention relates to a compositionas defined in claim <NUM> of the appended set of claims.

A fourth aspect of the invention relates to a cocrystal of beta-sitosterol as defined above; a hydrate crystal form of beta-sitosterol as defined above, or alternatively, a combination as defined above for use as a medicament.

A composition as defined above for use as a medicament is also part of the invention.

And, the fifth aspect of the invention relates to a cocrystal of beta-sitosterol as defined above; a hydrate crystal form of beta-sitosterol as defined above, or alternatively, a combination as defined above, for use in the prophylaxis and/or treatment of a disease or condition which involves an alteration of lipid metabolism, circulating levels of lipids in the blood and/or lipid composition in tissues and organs.

A composition as defined above for use in the prophylaxis and/or treatment of a disease or condition which involves an alteration of lipid metabolism, circulating levels of lipids in the blood and/or lipid composition in tissues and organs is also part of the invention.

All terms as used herein in this application, unless otherwise stated, shall be understood in their ordinary meaning as known in the art. Other more specific definitions for certain terms as used in the present application are as set forth below and are intended to apply uniformly throughout the specification and claims unless an otherwise expressly set out definition provides a broader definition.

For the purposes of the present invention, any ranges given include both the lower and the upper end-points of the range. Ranges given, such as temperatures, times, ratios of cocrystal components and the like, should be considered approximate, unless specifically stated.

For the purposes of the invention, the term "cocrystal" refers herein to a crystalline entity with at least two different components constituting the unit cell at room temperature (<NUM>-<NUM>) and interacting by weak interactions. Thus, in a cocrystal, one component crystallizes with one or more neutral components. The cocrystals may include one or more solvent molecules in the crystal lattice. Thus, the term "cocrystal hydrate" or "hydrate cocrystal" have the same meaning and are used interchangeable. They refer to a cocrystal including water as a solvent in the crystal lattice.

The expression "cocrystal obtainable by" is used here to define each specific cocrystal of the invention by the process for obtaining it and refers to the product obtainable by any of the corresponding processes disclosed herein. For the purposes of the invention the expressions "obtainable", "obtained" and equivalent expressions are used interchangeably and, in any case, the expression "obtainable" encompasses the expression "obtained".

The terms "percentage (%) by volume" or "volume/volume %" or "v/v %" have the same meaning and are used interchangeable. They refer to the amount of a solute in relation to the total solution volume.

When values of characteristic peaks of an X-ray diffractogram are given it is said that these are "approximate" values. It should be understood that the values are the ones shown in the corresponding lists or tables ± <NUM> degrees <NUM> theta measured in an X-ray diffractometer with Cu-Kα radiation A=<NUM>Å.

When a ratio of components of the cocrystals of the invention is specified it refers to the molar ratio between the components that forms the cocrystal. The term "molar ratio" has been used to express the stoichiometric amount in mols of each of the components of a cocrystal. The molar ratio can be determined by <NUM>H NMR (Proton nuclear magnetic resonance), thermogravimetric analysis (TGA) or single crystal X-ray diffraction (SCXRD). When values of molar ratio are given according to NMR or TGA it is said that these are "approximate" values due to the measurement error. It should be understood that when a molar ratio is mentioned, it corresponds to a molar ratio ± <NUM> %. The variability of the results is due to the inherent sensibility of the <NUM>H NMR and the TGA equipment.

The term "slurring" as disclosed herein refers to any process, which employs a solvent to wash or disperse by stirring a suspension of a compound.

The term "room temperature" refers to a temperature of the environment, without heating or cooling, and it is generally comprised from <NUM> to <NUM>.

The term "overnight" refers to a time interval comprised from <NUM> to <NUM>.

The term "one day" refers to a time interval comprised from <NUM> to <NUM>.

The term "miscible organic solvent" refers to an organic solvent that, when combined, form a single phase, which means that the mixture thus obtained is "monophasic" under specified conditions of component concentrations and temperature among others. Further, the term "water-miscible organic solvent" refers to an organic solvent that can form a monophasic solution with water at the temperature at which the reaction is carried out. As used herein, the term "monophasic" refers to a reaction medium that includes only one liquid phase, and also a method employing such a reaction medium. Some examples of monophasic mediums are water, aqueous solutions, and solutions containing aqueous and organic solvents that are miscible with each other.

The term "immiscible organic solvent" refers to an organic solvent that, when combined, form two phases, which means that the mixture thus obtained is "biphasic" under specified conditions of component concentrations and temperature among others. Further, the term "water-immiscible organic solvent" refers to an organic solvent that can form a biphasic phase with water at the temperature at which the reaction is carried out. As used herein, the term "biphasic" refers to a reaction medium that includes two immiscible liquid phases, for example, an aqueous phase and a water-immiscible organic solvent phase. The term "biphasic" can also be used to describe a method employing such a reaction medium.

The term "alkyl" refers to a saturated straight, or branched hydrocarbon chain which contains the number of carbon atoms specified in the description or claims. Examples include, among others, the group methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, and tert-butyl.

The term "(C<NUM>-C<NUM>)aryl" refers to an aromatic known ring system comprising one or more rings and from <NUM> to <NUM> ring members, wherein all the ring members comprise carbon atoms. Examples of (C<NUM>-C<NUM>)aryl include phenyl and naphthalene. The term "known ring system" as used herein refers to a ring system which is chemically feasible and is known in the art and so intends to exclude those ring systems that are not chemically possible.

The groups (C<NUM>-C<NUM>)alkyl and (C<NUM>-C<NUM>)aryl as defined in the present invention may be unsubstituted or substituted as described herein, being the substituents placed on any available position.

The term "alkenyl" refers to a straight or branched hydrocarbon chain which contains the number of carbon atoms specified in the description or claims, and that also contains at least one double bond. Examples include, among others, the ethenyl, <NUM>-propenyl, and <NUM>-propenyl.

The term "alkinyl" refers to a straight or branched hydrocarbon chain which contains the number of carbon atoms specified in the description or claims, and that also contains at least one triple bond.

The term "edible" used herein means non-toxic and suitable for consumption.

As mentioned above, the first aspect of the invention is the provision of a compound as defined in claim <NUM>.

The cocrystal of beta-sitosterol and L-lactic acid characterized by an X-ray diffractogram as in <FIG> is a reference example.

The cocrystal of beta-sitosterol and propionic acid characterized by an X-ray diffractogram as in <FIG> is a reference example.

The cocrystal of beta-sitosterol and zymonic acid characterized by an X-ray diffractogram as in <FIG> is a reference example.

In an embodiment, the cocrystal of the invention is a cocrystal of beta-sitosterol and gallic acid named cocrystal Form <NUM>. For the purposes of the invention, gallic acid is the International Nonproprietary Name (INN) of <NUM>,<NUM>,<NUM>-trihydroxybenzoic acid, and has the <NPL>. The structure of gallic acid is the following:
<CHM>.

The cocrystal of beta-sitosterol and gallic acid of the present invention is characterized by having an X-ray powder diffractogram that comprises characteristic peaks at approximately <NUM> and <NUM> ± <NUM> degrees <NUM> theta (Cu-Kα radiation, A = <NUM>Å). In an embodiment, the cocrystal of beta-sitosterol and gallic acid of the invention is characterized by having an X-ray powder diffractogram that comprises further characteristic peaks at <NUM>, <NUM> and <NUM> ± <NUM> degrees <NUM> theta (Cu-Kα radiation, A = <NUM>Å).

More specifically, the cocrystal of beta-sitosterol and gallic acid of the invention is characterized by exhibiting in the X-ray powder diffractogram a pattern of peaks, expressed in <NUM> theta units in degrees, <NUM> (°), which is shown in Table <NUM>.

The cocrystal of beta-sitosterol and gallic acid of the invention may be further characterized by an X-ray diffractogram as in <FIG>.

The thermogravimetric (TG) analysis of the cocrystal of beta-sitosterol and gallic acid of the invention may also be further characterized by a thermal melting/decomposition phenomenon starting at <NUM> (cf.

The cocrystal of beta-sitosterol and gallic acid of the invention may also be further characterized by an endothermic phenomenon at <NUM> with an associated heat of <NUM> J/g by DSC (Differential scanning calorimetry) analysis (cf.

The cocrystal of beta-sitosterol and <NUM>,<NUM>-dihydroxybenzoic acid characterized by an X-ray diffractogram as in <FIG> is a reference example.

The Form A of the cocrystal of beta-sitosterol and <NUM>,<NUM>-dihydroxybenzoic acid characterized by an X-ray diffractogram as in <FIG> is a reference example.

The Form B of the cocrystal of beta-sitosterol and <NUM>,<NUM>-dihydroxybenzoic acid characterized by an X-ray diffractogram as in <FIG> is a reference example.

The cocrystal of beta-sitosterol and <NUM>-hydroxybenzoic acid characterized by an X-ray diffractogram as in <FIG> is a reference example.

The Form A of the cocrystal of beta-sitosterol and <NUM>-hydroxybenzoic acid characterized by an X-ray diffractogram as in <FIG> is a reference example.

The Form B of the cocrystal of beta-sitosterol and <NUM>-hydroxybenzoic acid characterized by an X-ray diffractogram as in <FIG> is a reference example.

The cocrystal of beta-sitosterol and benzyl alcohol characterized by an X-ray diffractogram as in <FIG> is a reference example.

An aspect of the invention relates to a hydrate crystal form of beta-sitosterol, which is obtainable by a process which comprises: (aʺʺ) mixing the beta-sitosterol with a water-miscible organic solvent in the presence of propionic acid; and (bʺʺ) isolating the compound thus obtained; characterized by having an X-ray powder diffractogram that comprises characteristic peaks at approximately <NUM> and <NUM> ± <NUM> degrees <NUM> theta (Cu-Kα radiation, A = <NUM>Å). In an embodiment, the hydrate crystal form of beta-sitosterol of the invention is characterized by having an X-ray powder diffractogram that comprises further characteristic peaks at <NUM>, <NUM> and <NUM> ± <NUM> degrees <NUM> theta (Cu-Kα radiation, A = <NUM>Å).

More specifically, the hydrate crystal form of beta-sitosterol of the invention is characterized by exhibiting in the X-ray powder diffractogram a pattern of peaks, expressed in <NUM> theta units in degrees, <NUM> (°), which is shown in Table <NUM>.

The hydrate crystal form of beta-sitosterol of the invention may be further characterized by an X-ray diffractogram as in <FIG>.

The thermogravimetric (TG) analysis of the hydrate crystal form of beta-sitosterol of the invention may also be further characterized by first weight loss of <NUM>% from <NUM> to <NUM> and a second weight loss of <NUM>% from <NUM> to <NUM> (cf.

The hydrate crystal form of beta-sitosterol of the invention may also be further characterized by a first wide endothermic phenomenon at <NUM> with an associated heat of <NUM> J/g and a second endothermic phenomenon at <NUM> with an associated heat of <NUM> J/g by DSC (Differential scanning calorimetry) analysis (cf.

It is also part of the invention the provision of processes for the preparation of the crystal forms of beta-sitosterol of the first aspect of the invention. The crystal forms of beta-sitosterol of the invention may be prepared as a pure form or as a mixture.

The processes for the preparation of the crystal forms of the present disclosure involve contacting the beta-sitosterol or alternatively a mixture of phytosterols/phytostanols) with the corresponding coformer. The beta-sitosterol used as a starting material in the present disclosure is commercially available. Typically, the beta-sitosterol is available as a mixture of crystal forms. This mixture can be formed by anhydrous beta-sitosterol and hydrate beta-sitosterol, which can be in different proportions such as for example from <NUM>:<NUM> to <NUM>:<NUM>. Particularly, the mixture of anhydrous and hydrate form of beta-sitosterol can be <NUM>:<NUM>.

The process for the preparation of the cocrystal of beta-sitosterol and L-lactic acid (cocrystal Form <NUM>) of the reference example comprises: (a) slurring the beta-sitosterol in a mixture of L-lactic acid in a miscible organic solvent; and (b) isolating the cocrystal thus obtained.

The process for the preparation of the cocrystal of beta-sitosterol and propionic acid of the reference example (cocrystal Form <NUM>) comprises: (a') mixing the beta-sitosterol and propionic acid in a molar ratio from <NUM>:<NUM> to <NUM>:<NUM>; and (b') isolating the cocrystal thus obtained.

The process for the preparation of the cocrystal of beta-sitosterol and zymonic acid of the reference example (cocrystal Form <NUM>) comprises: (a") mixing the beta-sitosterol with pyruvic acid in a miscible organic solvent; and (b") isolating the cocrystal thus obtained.

The process for the preparation of the cocrystal of beta-sitosterol and benzyl alcohol of the reference example(cocrystal Form <NUM>) comprises: (a‴) mixing the beta-sitosterol with benzyl alcohol at a temperature comprised from <NUM> to <NUM> followed by cooling down the temperature until <NUM> to <NUM>; and (b‴) isolating the cocrystal thus obtained.

In an embodiment, the process for the preparation of the cocrystal of beta-sitosterol and gallic acid of the present invention (cocrystal Form <NUM>) comprises: (av) mixing the beta-sitosterol with gallic acid in a miscible organic solvent; and (bv) isolating the cocrystal thus obtained.

All the embodiments disclosed above for step (a) also applies to step (av). In an embodiment, step (av) is carried out at room temperature. In an embodiment, step (av) is carried out in the presence of ethyl acetate. All the embodiments disclosed above for step (b) also applies to step (bv).

The cocrystal of beta-sitosterol and gallic acid of the invention may also be defined by its preparation process. Accordingly, this aspect of the invention can be formulated as the cocrystal of beta-sitosterol and gallic acid (cocrystal Form <NUM>) as defined above, obtainable by any of the previous processes, optionally including any preferred or particular embodiment of the processes and possible combinations of some of the process features disclosed above.

The process for the preparation of the cocrystal of beta-sitosterol and <NUM>,<NUM>-dihydroxybenzoic acid of the reference example(cocrystal Form <NUM>) comprises: (avi) mixing the beta-sitosterol with <NUM>,<NUM>-dihydroxybenzoic acid in a miscible organic solvent; and (bvi) isolating the cocrystal thus obtained.

The process for the preparation of the cocrystal of beta-sitosterol and <NUM>,<NUM>-dihydroxybenzoic acid of the reference example(cocrystal Form <NUM>) comprises: (avii) mixing the beta-sitosterol with <NUM>,<NUM>-dihydroxybenzoic acid in a miscible organic solvent; and (bvii) isolating the cocrystal thus obtained.

The process for the preparation of the Form A of the cocrystal of beta-sitosterol and <NUM>,<NUM>-dihydroxybenzoic acid of the present disclosure (cocrystal Form <NUM>) comprises: (aviii) mixing the beta-sitosterol with <NUM>,<NUM>-dihydroxybenzoic acid in a miscible organic solvent; and (bviii) isolating the cocrystal thus obtained.

The process for the preparation of the Form B of the cocrystal of beta-sitosterol and <NUM>,<NUM>-dihydroxybenzoic acid of the reference example(cocrystal Form <NUM>) comprises: (aviiii) mixing the beta-sitosterol with <NUM>,<NUM>-dihydroxybenzoic acid in a miscible organic solvent; and (bviiii) isolating the cocrystal thus obtained.

The process for the preparation of the cocrystal of beta-sitosterol and <NUM>-hydroxybenzoic acid of the reference example(cocrystal Form <NUM>) comprises: (avv) mixing the beta-sitosterol with <NUM>-hydroxybenzoic acid in a miscible organic solvent; and (bvv) isolating the cocrystal thus obtained.

The process for the preparation of the Form A of the cocrystal of beta-sitosterol and <NUM>-hydroxybenzoic acid of the reference example(cocrystal Form <NUM>) comprises: (avvi) mixing the beta-sitosterol with <NUM>-hydroxybenzoic acid in a miscible organic solvent; and (bvvi) isolating the cocrystal thus obtained.

The process for the preparation of the Form B of the cocrystal of beta-sitosterol and <NUM>-hydroxybenzoic acid of the reference example(cocrystal Form <NUM>) comprises: (avvii) mixing the beta-sitosterol with <NUM>-hydroxybenzoic acid in a miscible organic solvent; and (bvvii) isolating the cocrystal thus obtained.

In an embodiment, the process for the preparation of the hydrate crystal form of beta- of the present invention characterized by having an X-ray powder diffractogram that comprises characteristic peaks at approximately <NUM> and <NUM> ± <NUM> degrees <NUM> theta (Cu-Kα radiation, A = <NUM>Å). comprises: (aʺʺ) mixing the beta-sitosterol with a water-miscible organic solvent in the presence of propionic acid; and (bʺʺ) isolating the cocrystal thus obtained.

In an embodiment, the water-miscible organic solvent of step (aʺʺ) is selected from the group consisting of (C<NUM>-C<NUM>) alkyl-CO-(C<NUM>-C<NUM>) alkyl, (C<NUM>-C<NUM>) alkyl-CO-O-(C<NUM>-C<NUM>) alkyl, water, cyclo(C<NUM>-C<NUM>)alkane, phenyl-(C<NUM>-C<NUM>)alkyl, halogen-(C<NUM>-C<NUM>)alkane, and mixtures thereof. In an embodiment, the water-miscible organic solvent of step (aʺʺ) is selected from the group consisting of acetone, ethyl acetate, water, cyclohexane, chloroform, tetrahydrofurane, toluene, dichloromethane and mixture thereof; preferably acetone and ethyl acetate. The term cycloalkane refers to a "cyclic" alkane which contains the number of carbon atoms specified in the description or claims. The term cycloalkane includes carbocyclic alkanes or heterocyclic alkanes. The term "carbocyclic" alkane refers to a cyclic alkane being each member of the cycle a carbon atom. Examples of carbocyclic alkanes include cyclopentane and cyclohexane. The term "heterocyclic" alkane refers to a "carbocyclic" compound in that at least one carbon atom is substituted by a N, NH, O, or S atom. Examples of heterocyclic alkane include tetrahydrofurane and tetrahydropirane. The term "halogen-alkane" refers to an alkane in that at least one hydrogen atom is substituted by an halogen atom and which contains the number of carbon atoms specified in the description or claims. Examples of halogen-alkane include chloroform, trichloroethane and dichloroethane.

All the embodiments disclosed above for step (a) also applies to step (aʺʺ). In an embodiment, step (aʺʺ) is carried out at room temperature. In an embodiment, step (aʺʺ) is carried out in the presence of acetone. All the embodiments disclosed above for step (b) also applies to step (bʺʺ).

The hydrate crystal form of beta-sitosterol of the present invention may also be defined by its preparation process. Accordingly, this aspect of the invention can be formulated as the hydrate crystal form of beta-sitosterol as defined above, obtainable by any of the previous processes, optionally including any preferred or particular embodiment of the processes and possible combinations of some of the process features disclosed above.

The processes for the preparation of the crystals of beta-sitosterol of the present invention are advantageous because allow obtaining the crystals in a high chemical yield and high polymorphic purity. Typically, the crystals obtained by the processes as mentioned above has a chemical purity equal to or higher than <NUM> area %; preferably, equal to or higher than <NUM> area %. Typically, the crystals obtained by the processes as mentioned above has such a polymorphic purity that no other crystalline forms of the beta-sitosterol is detectable by X-ray powder diffraction measurement when using a X-ray diffractometer with Cu-Kα radiation A=<NUM>Å.

The second aspect of the invention relates to a combination comprising a cocrystal of beta-sitosterol and gallic acid as defined above; and a crystal of beta-sitosterol selected from the group consisting of a cocrystal of beta-sitosterol and organic alcohol as defined above, and a hydrate crystal form of beta-sitosterol as defined above.

All the embodiments disclosed above for crystals of beta-sitosterol as defined above also apply for the combination of the present invention.

As it is mentioned above, the third aspect of the invention relates to a composition comprising an effective amount of a compound as defined in any of the claims <NUM>-<NUM>; or alternatively a combination as defined in claim <NUM> together with one or more appropriate acceptable excipients or carriers.

The term "effective amount" refers to the amount of the crystal of beta-sitosterol or alternatively of the combination of crystals of beta-sitosterol of the present invention which provides a therapeutic effect after its application.

In an embodiment, the composition of the third aspect of the invention is a pharmaceutical composition comprising a pharmaceutically effective amount of a compound as defined in any of the claims <NUM>-<NUM>, or a combination as defined in claim <NUM> together with one or more appropriate pharmaceutically acceptable excipients or carriers. The term "pharmaceutical composition" refers to a mixture of the compound as defined in any of the claims <NUM>-<NUM> or the combination as defined in claim <NUM> with other chemical components, such as diluents or carriers. The pharmaceutical composition facilitates administration of the cocrystal to an organism.

In an embodiment, the composition of the third aspect of the invention is an edible composition comprising an effective amount of a compound as defined in any of the claims <NUM>-<NUM> or a combination as defined in claim <NUM> together with one or more appropriate edible acceptable excipients or carriers.

The edible composition includes dietary supplement or functional food. In an embodiment, the edible composition is a dietary supplement. The term "dietary supplement", "food supplement" or "nutritional supplement" as used herein interchangeably refers to a preparation intended to supplement the diet and provide nutrients, such as vitamins, minerals, fibre, fatty acids, or amino acids, that may be missing or may not be consumed in sufficient quantity in a person's diet, as well as bioactive compounds with health beneficial effects. Dietary supplements can be in form of tablets, capsules, softgels, gelcaps, liquids, powders, bars, drinks, shakes and other food products. The terms "acceptable excipients or carriers" refers to acceptable material, composition or vehicle, which include without limitation fillers, diluents, binders, lubricants and disintegrants. Each component must be acceptable in the sense of being compatible with the other ingredients of the composition. It must also be suitable for use in contact with the tissue or organ of humans and animals without excessive toxicity, irritation, allergic response, immunogenicity or other problems or complications commensurate with a reasonable benefit/risk ratio.

In an embodiment, the edible composition is a functional food. The term "functional food" as used herein refers to any healthy or functional food which helps to maintain the body functions beyond the basic role of supplying nutrients. They can be used as a food additive to produce a functional food. Thus, they can be added to semisolid products, solid products, or liquid products, or their derivatives such as concentrates or powders. When the food additive is added to a liquid product the resulting product is known as functional beverage. The term "functional beverage" refers to drinks that have been enhanced with added ingredients which help to maintain the body functions beyond basic nutrition. Examples of food products are selected from the list consisting of milk and derivatives such as yoghurts or cheese; beverages including juices, soft drinks, sport drinks, or other beverages such as distilled and fermented beverages; salad dressing; yellow fat spread; mayonnaise; confectionary such as chocolates, candies, or jellies; pasta; cereals; and bakery.

The compositions of the present invention can be prepared according to methods well known in the state of the art. The appropriate excipients and/or carriers, and their amounts, can readily be determined by those skilled in the art according to the type of formulation being prepared.

All the embodiments disclosed above for the compound as defined in any of the claims <NUM>-<NUM>, as well as the combination as defined in claim <NUM> also applies for the compositions of the invention.

The forth aspect of the invention relates to a cocrystal of beta-sitosterol as defined above, a hydrate crystal form as defined above, or a combination as defined above for use as a medicament. Furthermore, a composition as defined above for use as a medicament is also part of the invention.

As mentioned above, the sixth aspect of the invention relates to a compound as defined in any of the claims <NUM>-<NUM> or a combination as defined in claim <NUM> for use in the prophylaxis and/or treatment of a disease or condition which involves an alteration of lipid metabolism, circulating levels of lipids in the blood and/or lipid composition in tissues and organs. This aspect could be also formulated as the use of a compound as defined in any of the claims <NUM>-<NUM> or a combination as defined in claim <NUM> for the preparation of a medicament or a dietary ingredient or supplement for the prophylaxis and/or treatment of a disease or condition which involves an alteration of lipid metabolism, circulating levels of lipids in the blood and/or lipid composition in tissues and organs. It also relates to a method for the prophylaxis and/or treatment of a mammal suffering, or susceptible to suffer, from a disease or condition which involves an alteration of lipid metabolism, circulating levels of lipids in the blood and/or lipid composition in tissues and organs, wherein the method comprises administering to said mammal an effective amount of a compound as defined in any of the claims <NUM>-<NUM> or a combination as defined in claim <NUM>, together with one or more acceptable excipients or carriers.

In an embodiment, the disease or condition which involves an alteration of lipid metabolism, circulating levels of lipids in the blood and/or lipid composition in tissues and organs is a pathologic, phisiologic or functional alteration selected from the group consisting of hypercholesterolemia, hypertriglyceridemia, sitosterolemia and mixed dyslipidaemia, cardiovascular diseases and diabetes. In an embodiment, the disease or condition which involves an alteration of lipid metabolism, circulating levels of lipids in the blood and/or lipid composition in tissues and organs is selected form the group consisting of hypercholesterolemia, hypertriglyceridemia, sitosterolemia and mixed dyslipidaemia.

Furthermore, a composition as defined above for use in the prophylaxis and/or treatment of a disease or condition which involves an alteration of lipid metabolism, circulating levels of lipids in the blood and/or lipid composition in tissues and organs is also part of the invention.

The term "hypercholesterolemia" refers to those diseases or conditions that involves an excess of cholesterol in the blood, a phenomenon that occurs when total cholesterol levels are higher than the standard levels, and in particular when associated to higher LDL-cholesterol. The term "hypertriglyceridemia" refers to those diseases or conditions that involve an excess of triglycerides in the blood. The term "mixed dyslipidaemia" refers to those diseases or conditions that involve an excess of cholesterol and triglyceride levels in blood. The term "sitosterolemia" refers to those diseases or conditions that involve higher intestinal absorption and lower biliary excretion of dietary sterols (including plant sterols and cholesterol); this inherited metabolic disorder is associated with hypercholesterolemia.

In an embodiment, wherein the disease or condition is a hypercholesterolemia, then the prophylaxis and/or treatment comprises administering the cocrystal of beta-sitosterol and an organic alcohol as defined above; preferably selected from benzyl alcohol, ethanol, and isopropanol; more preferably benzyl alcohol; more much preferably the cocrystal of Form <NUM>; or alternatively the hydrate crystal form of beta-sitosterol as defined above.

In an embodiment, wherein the disease or condition is hypertriglyceridemia, then the prophylaxis and/or treatment comprises administering the compound as defined in claim <NUM>.

In an embodiment, wherein the disease or condition is a mixed dyslipidaemia, then the prophylaxis and/or treatment comprise administering the combination as defined above.

In an embodiment, the combination for use as defined above, wherein the treatment comprises the simultaneously, separately or sequentially administration of the cocrystal of beta-sitosterol and gallic acid as defined above; and a crystal of beta-sitosterol selected from the group consisting of a cocrystal of beta-sitosterol and organic alcohol as defined above, and the hydrate crystal form as defined above. It means that the treatment comprises the administration of two separate compositions containing each composition one of the crystals of the present invention.

All the embodiments disclosed above for the cocrystals, the hydrate and the combination, as well as the compositions as defined above also applies for the cocrystals, hydrate, combination or composition for use.

It is also part of the invention a food ingredient which comprises a compound as defined in any of the claims <NUM>-<NUM>, a combination as defined in claim <NUM>. In an embodiment, the food ingredient is selected from food supplement and functional food. The term "food supplement" refers to a concentrated source of nutrients or other substances with a nutritional or physiological effect whose purpose is to supplement the normal diet. In other terms food supplement means any food the purpose of which is to supplement the normal diet and which is a concentrated source of a vitamin or mineral or other substance with a nutritional or physiological effect, alone or in combination. The term "functional food" refers to a food or ingredient of a food that provides an additional physiological benefit beyond their basic nutritional needs.

It is also part of the invention a package comprising a compound as defined in any of the claims <NUM>-<NUM>, a combination as defined in claim <NUM> and instructions for use in the prophylaxis and/or treatment of a disease or condition which involves an alteration of lipid metabolism, circulating levels of lipids in the blood and/or lipid composition in tissues and organs.

Furthermore, a package comprising a composition as defined above and instructions for use in the prophylaxis and/or treatment of a disease or condition which involves an alteration of lipid metabolism, circulating levels of lipids in the blood and/or lipid composition in tissues and organs is also part of the invention.

It is also part of the invention a compound as defined in any of the claims <NUM>-<NUM> or a combination as defined in claim <NUM> for use in the treatment of a disease or condition selected from the group consisting of gallstones, cold, flu, acquired immunodeficiency syndrome (AIDS), rheumatoid arthritis, tuberculosis, psoriasis, allergies, cervical cancer, fibromyalgia, systemic lupus erythematosus (SLE), asthma, hair loss, bronchitis, migraine headache, benign prostatic hyperplasia (BPH), menopause, pain, chronic fatigue syndrome, swelling and sexual dysfunction.

It is also part of the invention a compound as defined in any of the claims <NUM>-<NUM> or a combination as defined in claim <NUM> for use as a wound healing agent. Furthermore, a composition as defined above for use as a wound healing agent is also part of the invention. The expression "wound healing" relates to an intricate process in which the skin (or some other organ) repairs itself after injury wound healing of any kind and at any site. It can be normal and impaired wound healing. The latter is found in particular in the case of diseases, such as diabetes mellitus, vasculitis, arterial occlusive disease, chronic venous and/or infected ulcer as well as poorly healing gastric ulcer. Impaired wound healing is also found in the case of innervations impairment such as paraplegia, leprosy, neuropathy, and decubital gangrene of persons in need of care. Impaired wound healing will also be given if weak sutures and impaired healing occur after operations, particularly of the intestines and transplantations of skin and other organs, respectively. Impaired wound healing is also found in the case of bone fractures, burns, and treatments using steroids.

As used herein, the term "wound" includes an injury to any tissue, including for example, delayed or difficult to heal wounds, and chronic wounds. Examples of wounds may include both open and closed wounds. The term "wound" may also include for example, injuries to the skin and subcutaneous tissue initiated in different ways (e.g., pressure sores from extended bed rest and wounds induced by trauma) and with varying characteristics. Wounds may be classified into one of four grades depending on the depth of the wound: i) Grade I wounds limited to the epithelium; ii) Grade II wounds extending into the dermis; iii) Grade III wounds extending into the subcutaneous tissue; and iv) Grade IV (or full-thickness wounds) wounds.

Throughout the description and claims the word "comprise" and variations of the word, are not intended to exclude other technical features, additives, components, or steps. Furthermore, the word "comprise" encompasses the case of "consisting of". The following examples and drawings are provided by way of illustration, and they are not intended to be limiting of the present invention. Reference signs related to drawings and placed in parentheses in a claim, are solely for attempting to increase the intelligibility of the claim, and shall not be construed as limiting the scope of the claim. Furthermore, the present invention covers all possible combinations of particular and preferred embodiments described herein. In the following examples, only examples <NUM>. <NUM> and <NUM> relate to examples of crystalline forms of the present invention. All other crystalline forms of the following examples are reference examples.

Beta-sitosterol, benzylic acid, lactic acid, propionic acid and piruvic acid used for the preparation of the crystalls of the present disclosure (cf. experimental section <NUM>) are commercially available by Sigma-Aldrich with a chemical purity of ≥ <NUM>%, <NUM>%, ≥ <NUM>%, <NUM>%, and <NUM>% respectively. Beta-sitosterol used as a starting material in the present disclosure is commercially available by Sigma-Aldrich (batch number BCB50067V and BCBM5699V). having a chemical purity equal to or higher than <NUM>%. The PXRD of the commercial beta-sitosterol showed that beta-sitosterol was a mixture of anydrous beta-sitosterol (whose PXRD corresponds to the "LOFFET" form disclosed in <NPL>) and monohydrate beta-sitosterol (whose PXRD corresponds to the "TEXQOC" disclosed in <NPL>) in a weight ratio <NUM>:<NUM>. Furthermore, the GC-MS analysis of the commercially beta-sitosterol chowed that beta-sitosterol was a mixture of beta-sitosterol (<NUM>-<NUM> %), stigmasterol (<NUM>-<NUM>%) and campesterol (<NUM>-<NUM>%) (cf. section <NUM> of the experimental part).

Powder X-Ray diffraction (PXRD) analyses were performed by sandwiching the powder samples between polyester films of <NUM> micrometres of thickness or polyamide (kapton) films of <NUM> micrometres of thickness and analysed in a PANalytical X'Pert PRO MPD q/q powder diffractometer of <NUM> millimetres of radius, in a configuration of convergent beam with a focalizing mirror and a flat sample transmission geometry, in the following experimental conditions: Cu Ko radiation (A = <NUM>Å); Work power: <NUM> kV and <NUM> mA; Incident beam slits defining a beam height of <NUM> millimetres; Incident and diffracted beam <NUM> radians Soller slits; PIXcel detector: Active length = <NUM> °; 2θ/θ scans from <NUM> to <NUM> °2θ with a step size of <NUM> °2θ and a measuring time of <NUM> seconds per step. The X-ray diffractogram shows the powder X-ray diffraction pattern (intensity (counts) vs. <NUM>-theta angle (°)) of the crystal form of beta-sitosterol of the present disclosure.

Single crystal X-Ray diffraction (SCXRD) structures were solved on a D8 Venture system equipped with a multilayer monochromator and a Mo microfocus (A = <NUM>Å) has been used too. Frames were integrated with the Bruker SAINT software package using a SAINT algorithm. Data were corrected for absorption effects using the multi-scan method (SADABS). The structures were solved and refined using the Bruker SHELXTL Software Package, a computer program for automatic solution of crystal structure and refined by full-matrix least-squares method with ShelXle Version <NUM>. <NUM>, a Qt graphical user interface for SHELXL computer program.

Differential Scanning Calorimetry (DSC) analyses were carried out by means of a Mettler-Toledo DSC-822e calorimeter. Experimental conditions: aluminium crucibles of <NUM>µL volume, atmosphere of dry nitrogen with <NUM>/min flow rate, heating rate of <NUM>/min. The calorimeter was calibrated with indium of <NUM>% purity.

Thermogravimetric analysis (TGA) was performed on a Mettler-Toledo TGA-851e thermobalance. Experimental conditions: alumina crucibles of <NUM>µL volume, atmosphere of dry nitrogen with <NUM>/min flow rate, heating rate of <NUM>/min.

The HPLC-MS analysis of the cocrystals of beta-sitosterol and the hydrate crystal of the present invention showed that they comprises a mixture of beta-sitosterol (<NUM>-<NUM> %), stigmasterol (<NUM>-<NUM>%) and campesterol (<NUM>-<NUM>%) (cf. section <NUM> of the experimental part). In particular, the cocrystals of beta-sitosterol and an organic carboxylic acid selected from the group consisting of L-lactic acid, propionic acid, zymonic acid, succinic acid, ascorbic acid, gallic acid, <NUM>,<NUM>-dihydroxybenzoic acid, <NUM>,<NUM>-dihydroxybenzoic acid, <NUM>-hydroxybenzoic acid, <NUM>-hydroxybenzoic acid and <NUM>,<NUM>-dihydroxybenzoic acid; the cocrystals of beta-sitosterol and an organic alcohol selected from the group consisting of benzyl alcohol, ethanol and isopropanol; and hydrate crystal form of beta-sitosterol of the present invention comprises a mixture of beta-sitosterol (<NUM>-<NUM> %), stigmasterol (<NUM>-<NUM>%) and campesterol (<NUM>-<NUM>%).

A suspension of beta-sitosterol (<NUM>, <NUM> mmol), L-lactic acid (<NUM>) and ethyl acetate (<NUM>) was stirred at room temperature for <NUM> days. The crystal thus obtained was filtered and dried under vacuum.

A solution of beta-sitosterol (<NUM>, <NUM> mmol) and propionic acid (<NUM>) was stirred at room temperature for one day open to air. The crystal thus obtained was filtered and dried under vacuum.

Beta-Sitosterol (<NUM>) with propionic acid (<NUM>) was suspended until total dissolution (<NUM> hour). Water (<NUM>) was added at room temperature and a white solid precipitated with a gentle stirring. The crystal thus obtained was filtered and dried under vacuum (about <NUM> Hg) for <NUM> hours. The crystal was finally dried by passing an air flow through the filtered crystal for <NUM> hours.

A solution of beta-sitosterol (<NUM>, <NUM> mol), pyruvic acid (<NUM>) and acetone (<NUM>) was stirred at room temperature for <NUM> day. The crystal thus obtained was filtered and dried under vacuum.

Beta-sitosterol (<NUM>, <NUM> mmol) and gallic acid (<NUM>, <NUM> mmol) were suspended in ethyl acetate (<NUM>) at room temperature and stirred overnight. The crystal thus obtained was filtered and dried under vacuum.

Beta-sitosterol (<NUM>, <NUM> mmol) and <NUM>,<NUM>-dihydroxybenzoic acid (<NUM>, <NUM> mmol) were suspended in ethyl acetate (<NUM>) at room temperature and stirred overnight. The crystal thus obtained was filtered and dried under vacuum.

<NUM>,<NUM>-dihydroxybenzoic acid (<NUM>, <NUM> mmol) was stirred in ethyl acetate (<NUM>) during <NUM> hours. The suspension was filtered and beta-sitosterol (<NUM>, <NUM> mmol) was added and it was stirred at room temperature overnight. The crystal thus obtained was filtered and dried under vacuum.

Beta-sitosterol (<NUM>, <NUM> mmol) and <NUM>,<NUM>-dihydroxybenzoic acid (<NUM>, <NUM> mmol) were dissolved in ethyl acetate (<NUM>) at room temperature. The solution was stirred overnight opened to the air. The crystal thus obtained was filtered and dried under vacuum.

Beta-sitosterol (<NUM>, <NUM> mmol) and <NUM>-hydroxybenzoic acid (<NUM>, <NUM> mmol) were suspended in ethyl acetate (<NUM>) at room temperature and stirred overnight. The crystal thus obtained was filtered and dried under vacuum.

<NUM>-hydroxybenzoic acid (<NUM>, <NUM> mmol) was stirred in ethyl acetate (<NUM>) during <NUM> hours. The suspension was filtered and beta-sitosterol (<NUM>, <NUM> mmol) was added and the mixture was stirred at room temperature overnight. The crystal thus obtained was filtered and dried under vacuum.

Beta-sitosterol (<NUM>, <NUM> mmol) and <NUM>-hydroxybenzoic acid (<NUM>, <NUM> mmol) were dissolved in ethyl acetate (<NUM>) at room temperature and stirred opened to the air overnight. The crystal thus obtained was filtered and dried under vacuum.

A solution of beta-sitosterol (<NUM>, <NUM> mmol) in benzyl alcohol (<NUM>) was heated to <NUM> and it was slowly cooled down to room temperature in <NUM> hour. Then, the temperature was cooled down to <NUM>-<NUM> and was kept for <NUM> day until a gel was precipitated. The solution was decanted and pentane was added until clear solution and then the solution was evaporation to dryness. After that, diethyl ether was added until clear solution and it was also evaporated to dryness to obtain a gel, which evolved to a solid overnight. The solid thus obtained was filtered and dried under vacuum to obtain the cocrystal of beta-sitosterol and benzyl alcohol.

A solution of beta-sitosterol (<NUM>, <NUM> mmol), propionic acid (<NUM>) and acetone (<NUM>) was stirred at room temperature for one day. The crystal thus obtained was filtered and dried under vacuum to obtain the hydrate crystal form of the present invention.

The activity test is focused on determining the percentage of reduction of the circulating levels of glucose, cholesterol, triglycerides and beta-sitosterol after oral ingestion of lipids or an oral ingestion of lipids supplemented with either the crystals of beta-sitosterol of the present disclosure or a comparative authorized beta-sitosterol which is outside the scope of the present invention.

The comparative beta-sitosterol used in the activity test corresponds to the compound authorized in the Commission Decision of <NUM> March <NUM> authorising the placing on the market of yellow fat spreads, salad dressings, milk type products, fermented milk type products, soya drinks and cheese type products with added phytosterols/phytostanols as novel foods or novel food ingredients under Regulation (EC) No <NUM>/<NUM> of the European Parliament and of the Council (notified under document number C(<NUM>) <NUM>)- <NUM>/<NUM>/EC (cf. The authorized beta-sitosterol has the following composition (measured by Gas Chromatography - Flame Ionization Detector -GC-FID): < <NUM>% beta-sitosterol, <<NUM>% beta-sitostanol, <<NUM>% campesterol, <<NUM>% campestanol, <<NUM>% stigmasterol, <<NUM>% brassicasterol, <<NUM>% other esterols/stanols.

The animals used in the activity test were Golden Syrian male Hamsters of <NUM> weeks of age. Five experimental groups of <NUM> animals per group were established. The experimental groups were the following:.

The test was performed to animals subjected to a caloric restriction of <NUM>% during the previous <NUM> hours. In all experimental groups, the lard was supplemented by <NUM>/kg BW of comparative beta-sitosterol or crystals of the present disclosure, which is equivalent to a <NUM> times the dose recommended for human use and adapted for use in hamster (Cf.

Before the test and at initial time (T0), blood was collected from the saphenous vein with heparinized capillaries. Then, at <NUM> hours after receiving the oral load of lard (T5), the animals received an intraperitoneal injection of lethal anaesthesia based on the active pentobarbital sodium (lethal dose at <NUM>/mL of pentobarbital, Vetoquinol). Amounts of administration of the lethal dose of sodium pentobarbital (pre-diluted with saline at <NUM> / mL) were calculated based on the individual weight of each animal.

After verifying that the animal was sedated, it was ex-sanguinated by making a small incision at the level of the rib cage, followed by collection of blood by cardiac puncture (<NUM> syringe with <NUM> needles). The interior of the needle and syringe were previously soaked with filtered EDTA (<NUM>, pH = <NUM>) to prevent blood clotting.

Blood was centrifuged at <NUM> rpm for <NUM> minutes at <NUM>° C to obtain the plasma, which was frozen at -<NUM> until use.

Plasma cholesterol levels have been determined with the total cholesterol kit supplied by Biosystems (ref: M11505c-<NUM>). Plasma triglyceride levels have been determined with the Sigma Triglyceride determination kit (ref. Blood glucose concentrations were measured by means of test strips from the Accu-check Aviva glucometer (Roche, ref.

Determination of phytosterols in plasma was performed after saponification and derivatization by gas chromatography (GC-7890A, Agilent Technologies) combined with ionisable flame detector (GS-FID using a HP-<NUM> column (length <NUM>, diameter <NUM>) and a helium mobile phase flow of <NUM>·min-<NUM>. <NPL>; and <NPL>).

Data results were presented as the mean ± error of mean (SEM). Statistical analysis was performed using SPSS <NUM> software (Chicago, IL, USA). The comparison between groups was performed by analysis of variance (ANOVA one-way) followed by Fisher's post-hoc analysis (LSD, of the least significant difference). The comparison between T0 and T5 of the different groups was performed using the ANOVA test of repeated measurements (ANOVA), followed by LSD analysis and t-test of paired means.

No significant differences were observed in the circulating glucose levels between all experimental groups at the times analysed.

As it is shown in <FIG>, plasma cholesterol levels at initial time (T0) did not differ between all experimental groups. Then, the administration of the cocrystal of beta-sitosterol and propionic acid (Test sample C) of the present invention decreased the plasmatic cholesterol levels (<NUM>%), measured at <NUM>, in the same way as the comparative samples A and B (<NUM>% and <NUM>% respectively).

Nevertheless, the administration of the hydrate crystal form of beta-sitosterol of the invention (test sample D) and the cocrystal of beta-sitosterol and benzyl alcohol (Test sample E) significantly lowered the plasmatic cholesterol concentrations (in absolute value) an in a percentage of <NUM>% and <NUM>% respectively (p = <NUM> and p = <NUM> respectively vs. comparative sample control A).

As it is shown in <FIG>, plasma triglyceride levels at initial time (T0) did not differ between all experimental groups.

Nevertheless, the administration of the comparative samples A and B led to a slight increase in triglyceridemia at <NUM> (T5) of <NUM>% and <NUM>% respectively. In comparison, the administration of hydrate crystal form of beta-sitosterol of the invention (test sample D) and the cocrystal of beta-sitosterol and benzyl alcohol (test sample E) attenuated the increase in the triglyceridemia and caused a reduction of <NUM>% and <NUM>% of the triglyceride concentration respectively.

Furthermore, the administration of the cocrystal of beta-sitosterol and propionic acid (test sample C) of the reference example significantly lowered in a <NUM>% the plasmatic triglyceride concentration (p=<NUM> T0 vs. T5 and p = <NUM> vs. comparative sample B).

As it is shown in <FIG>, just after the administration of the comparative sample A circulating β-sitosterol levels were not modified.

Meanwhile, the administration of the comparative sample B promoted a decrease in circulating levels of <NUM>% with respect to the values at initial time (T0) (p = <NUM> vs. comparative sample A-control). In the same way, the administration of the cocrystal of beta-sitosterol and propionic acid (test sample C) and the cocrystal of beta-sitosterol and benzyl alcohol (test sample E) of the reference example also decreased in a similar way the circulating levels of β-sitosterol (<NUM>% and <NUM>%, respectively vs. their T0 value).

Nevertheless, a significantly marked effect in lowering the circulating levels of β-sitosterol for hydrate crystal form of beta-sitosterol (test sample D) was observed. Particularly, a reduction of <NUM>% in relation to its T0 value was observed (p = <NUM> vs. comparative sample A-control).

This test is focused on determining the percentage of stigmasterol and campesterol present in the commercially available beta-sitosterol used as starting material in the preparation of the cocrystals and hydrate of the present invention. Furthermore, this test is also focused on determining the percentage of stigmasterol and campesterol that is still present in the cocrystals and hydrate of the present invention.

Three significant peaks have been observed during the HPLC analysis of the standard β-Sitosterol, Ph. <FIG>) at <NUM>, <NUM> and <NUM>. They were assigned to Stigmasterol, Campesterol and β-Sitosterol respectively according to HRMS analysis having the following structures:
<CHM>.

The molecular weight of each compound were the following:.

The same peaks have been observed in all the tested samples as defined above. The results are summarized in the following Table <NUM>.

Tabla <NUM> represents the HPLC quantification of stigmasterol (peak <NUM>), campesterol (peak <NUM>) and beta-sitosterol in the tested samples. The abbreviature RT corresponds to the retention time (RT) expressed in minutes (min) and the area is expressed in percentage (WAN).

Three significant peaks have been observed during GS-MS of both trimethylsilyl derivative samples of the cocrystal of beta-sitosterol and gallic acid Form <NUM> and commercially available beta-sitosterol of sigma-aldrich (Batch number BCB50067V), which confirm with high precision the identity of the three phytosterols present in all the solid forms of this invention.

The above results show that both the beta-sitosterol used as starting material as well as the obtained cocrystals and the hydrate form of beta-sitosterol of the present invention comprises from <NUM> to <NUM> area/area measured by HPLC of stigmasterol, from <NUM> to <NUM> area/area measured by HPLC of campesterol and from <NUM> to <NUM> area/area measured by HPLC beta-sitosterol.

This test is focused on evaluating the solubility in Fasted State Simulated Intestinal Fluid (FaSSIF-V2).

Solubility is determined in FaSSIF-V2 media, prepared as stated by Biorelevant. com using maleic acid buffer (pH = <NUM>).

All experiments are done under temperature control at <NUM>±<NUM>. The weight of tested samples expressed in mg, and FaSSIF final volume expressed in mL are as defined in Table below:.

Shaking time: <NUM> in a rotational stirrer. After <NUM>-<NUM> of shaking, pH is measured and readjusted to the initial value (<NUM>) in case it is needed.

For sample B quantification: Mobile phase composed of <NUM> formic acid at pH = <NUM> (A) and methanol (B). Gradient conditions: <NUM>-<NUM>, <NUM>% B; <NUM>, <NUM>% B; <NUM>, <NUM>% B; <NUM>, <NUM>% B; <NUM>, <NUM>% B. Flow: <NUM>/min. Injection volume: <NUM>µL.

Quantification wavelength: <NUM> for comparative sample A and <NUM> for sample A.

Table below provides the information related to the standards of the tested samples and coformers used for the calibration, as well as the average quality parameters obtained from the calibration curves.

LCD and LOQ for comparative sample A have been determined as follows:.

The amounts of comparative sample A, sample A and sample B in the filtered solutions coming from Shake-Flask (SF) experiments have been quantified according to the described methodology.

Solubility of the samples A and B has been calculated through the equilibrium concentrations in the eutectic point as described in "<NPL>", taking into account the stoichiometry of each sample.

Table below shows the results obtained for the different tested samples, where log S is the logarithm of S in mol/L, [API] is the concentration of beta-sitosterol in the solutions coming from SF experiments, n is the number of replicates, and PXRD analysis indicates the solid/s form/s obtained after the SF experiments.

The obtained results point out that the cocrystal of beta-sitosterol and gallic acid of the present invention is more soluble than the remaining tested samples. The hydrate crystal of beta-sitosterol from of the present invention is more soluble than the commercially available beta-sitosterol (comparative sample A).

Without being bound to any theory, it seems that the amount of beta-sitosterol in solution depends on the solid form in equilibrium with the solution. This is in accordance with PXRD results, which indicate that in the cocrystal of beta-sitosterol and gallic acid, the amount of beta-sitosterol in solution is in equilibria with the monohydrate crystal of beta-sitosterol disclosed in the art.

The bioavailability assessment of the crystals of beta-sitosterol of the present invention has been carried out by determining the beta-sitosterol levels in plasma samples from animals (hamsters) submitted during three weeks with a high-fat diet supplemented with either the crystals of beta-sitosterol of the present invention (or of a reference example) or a comparative standard sample of beta-Sitosterol available from the Ph. Reference Standard, ref. (Y0001615), which is outside the scope of the present invention.

The animals used in this bioavailability assessment test were Golden Syrian male Hamsters of <NUM> months of age. Six experimental groups of <NUM> animals per group were established. The experimental groups were the following:.

The test was performed to animals maintained with ad libitum feeding of each sample during <NUM> days. In all experimental groups, the diet was supplemented with comparative beta-sitosterol or the crystals of the present disclosure, in a range dose that goes up to <NUM> times the dose recommended for human use (<NUM>/day) and adapted for use in hamster (Cf.

After <NUM> days of feeding with the tested samples, the animals received an intraperitoneal injection of lethal anaesthesia based on the active pentobarbital sodium (lethal dose at <NUM>/mL of pentobarbital, Dolethal, Vetoquinol) under ad libitum feeding conditions. Amounts of administration of the lethal dose of sodium pentobarbital (pre-diluted with saline at <NUM> / mL) were calculated based on the individual weight of each animal.

After verifying that the animal was sedated, it was ex-sanguinated by making a small incision at the level of the rib cage, followed by collection of blood by cardiac puncture (<NUM> syringe with <NUM> needles). The interior of the needle and syringe were previously soaked with filtered EDTA (<NUM>, pH = <NUM>) to prevent blood clotting. Blood was centrifuged at <NUM> for <NUM> minutes at <NUM>° C to obtain the plasma, which was frozen at -<NUM> until use.

Data results were presented as the mean ± standard error of mean (SEM). Statistical analysis was performed using IBM SPSS Statistics <NUM> (Chicago, IL, USA). The comparison between groups was performed by analysis of variance (ANOVA one-way) followed by Fisher's post-hoc analysis (LSD, of the least significant difference). Student's t test was also used to make single comparison between <NUM> groups. The level of significance was set at p≤<NUM>.

Concerning the impact of treatments on sitosterolemia, data are shown as the ratio of variation respect to the control group (comparative group A), which was set to <NUM>. Administration of an oral dose of commercial beta-sitosterol during <NUM> days resulted in increased circulating levels of the compound at both doses, showing a dose-dependent response; the administration of reference sample B1 resulted in a Fold-change (FC) of increase of <NUM> with respect to controls (comparative group A), and the administration of reference sample B2 resulted in a FC of increase of <NUM> with respect to controls (comparative group A). The administration of the cocrystal of beta-sitosterol and propionic acid of the reference example (test sample C) and the cocrystal of beta-sitosterol and gallic acid of the present invention (test samples G1 and G2) also promoted an increase in the circulating levels of beta-sitosterol, FC of increase of <NUM>, <NUM> and <NUM> respectively, with respect to the comparative group A.

Nevertheless, a significantly marked effect in increasing the circulating levels of beta-sitosterol for cocrystal of beta-sitosterol and propionic acid of the reference example (test sample C) was observed in comparison to the reference group treated with the same dose of commercial beta-sitosterol (comparative group B1). Particularly, a FC increment of <NUM> in relation to reference sample B1 value was observed (p = <NUM> vs comparative sample B1, Student t test).

This test provides the particle size of the co-crystals of the present disclosure in comparison with the comparative Standard sample of beta-Sitosterol from the Ph. Reference Standard, ref. (Y0001615).

The particle size was performed by laser diffraction. For this purpose, a Beckman-Coulter model. LS13320 laser diffractometer (Fullerton, CA, USA) was equipped with a Micro. Liquid Module (MLM) wet dispersion modul, an an optic model (Fraunhofer. Rdf, PIDS). The measurement range is <NUM>-<NUM>.

Table below shows the mean, the surface weighted mean diameter (D(<NUM>,<NUM>)), the standard deviation (S. D) and the D10, D50 and D90 of the tested samples.

Claim 1:
A hydrate crystal form of beta-sitosterol, which is obtainable by a process which comprises: (aʺʺ) mixing the beta-sitosterol with a water-miscible organic solvent in the presence of propionic acid; and (bʺʺ) isolating the compound thus obtained;
characterized by having a X-ray diffractogram that comprises characteristic peaks at <NUM> and <NUM> ± <NUM> degrees <NUM> theta at a Cu-Kα radiation, A = <NUM> P,;
or alternatively,
a cocrystal of beta-sitosterol and gallic acid characterized by having a X-ray diffractogram that comprises characteristic peaks at <NUM> and <NUM> ± <NUM> degrees <NUM> theta (Cu-Kα radiation, A = <NUM>Å).