Improvements in or relating to organic compounds

Abstract

The present invention provides hydroxypropyltrialkylammonium hyaluronate and / or salts thereof having a degree of cationization greater than 1.4.

Description

[Technical field]

[0001] The present invention relates to hydroxypropyltrialkylammonium hyaluronate and / or salts thereof having a degree of cationization greater than 1.4. The desire to look attractive is ingrained in the modern consumer, and although ideals of attractiveness change over time, it is universally accepted that the condition and appearance of hair and skin contribute greatly to an attractive appearance.

[0002] The structural framework of skin is called its extracellular matrix. It comprises a network of mesh-like polymers such as collagen and elastin, within which skin cells are contained. It is responsible for the mechanical properties of skin, including firmness, strength, suppleness, and elasticity. The physical signs of skin aging are a reflection of the condition of the skin matrix. More specifically, the weaker and less regular the matrix, the more prone the skin is to have wrinkles, roughness, and sagging.

[0003] Skin hydration, which is essential to ensure the suppleness, softness, tone and appearance of the skin, is a complex phenomenon. Water is the main constituent of our body and accounts for 60% of the body weight of an adult. In the skin, water is distributed mainly in the dermis, where it forms a semi-fluid gel with the different structural proteins of the extracellular matrix. The epidermis and stratum corneum contain very little water.

[0004] The epidermis is the outer structure of the skin, whose role is to ensure protection and exchange with the environment. The stratum corneum is the outermost layer of the epidermis. It is composed of a stack of several layers (15-20) of keratinocytes and is the end product of the epidermal keratinization process. Schematically, the stratum corneum contains keratinocytes, which are protein-rich and hydrophilic, and interkeratinocyte spaces, which are lipid-rich and hydrophobic. Keratinocytes are anucleated cells that have lost their intracytoplasmic organelles. Inside the keratinocytes, a dense network of keratin filaments is dispersed in a matrix composed of another protein, filaggrin. The whole is surrounded by an extremely resistant envelope made of a protein wall lined with a lipid envelope.

[0005] Two layers of keratinocytes are clearly distinguished: the stratum corneum compactum, which lies deeper and where the keratinocytes are connected to each other by corneodesmosomes, providing a barrier function, and the desquamating layer, called the stratum corneum detaches. The keratinocytes can be thought of as bricks that form a wall, and are connected to each other by a small number of desmosomes, which are protein-rich appendages of the cell membrane. In between the keratinocytes creep epidermal lipids synthesized by keratinocytes of the spinous and granular layers, creating a "bricks and cement" model. Epidermal lipids make up 10-30% of the volume of the stratum corneum. They are formed in the Golgi apparatus of the keratinocytes and then exocytosis into the extracellular space. Ceramides make up the majority of lipids in the stratum corneum (40%). They are a group of sphingolipids that essentially contain sphingosine and various fatty acids, such as linoleic acid. Ceramides bind the aqueous components of this complex lipid mixture. The primary function of the intercellular lipids of the stratum corneum is to confer on this barrier layer a relatively impermeable nature to water.

[0006] The level of skin hydration is expressed on the surface, however, the water mainly comes from the dermis, which contains a large amount of water derived from plasma. The dermis is composed of connective tissue characterized by an abundant extracellular matrix (ECM) located between the specialized cells that synthesize it: fibroblasts and fibrocytes. The ECM consists of elastic fibers and collagen and amorphous interstitial material. This interstitial material essentially contains hyaluronic acid (a non-sulfated glycosaminoglycan) and sulfated glycosaminoglycans that associate with protein axes to form proteoglycans. The whole forms a compressible gel that retains water like a sponge and also allows the circulation of water and dissolved molecules. 20% to 40% of the total water in the body is contained in the extracellular matrix. The essential molecule for skin hydration is therefore hyaluronic acid, which allows it to draw in and fix up to 1000 times its weight in water. Understanding the metabolism of hyaluronan, its role in the skin, and its interactions with other skin components is a more logical approach to consider the regulation of skin hydration.

[0007] Hyaluronan is a high molecular weight anionic polysaccharide with the ability to adopt various shapes and configurations depending on the pH, salt content of the medium and associated cations. Its role is essential in cell motility, attachment, proliferation and organization. It is a major component of the extracellular matrix, a vital element in skin hydration. The functions of hyaluronan are multiple in the body. Within the extracellular matrix, hyaluronan is not just a passive structural component, but an intracellular regulator that plays a role in cell metabolism. For example, there are receptors for hyaluronan on the surface of cells: CD44 and the receptor for mobility mediated by hyaluronic acid (RHAMM) are the two most important ones. They can induce cell mobilization after a cascade of intracellular signals and are themselves substrates for many phosphokinases.

[0008] The presence of hyaluronic acid was thought to be limited to the dermis, but very recently histological techniques have made it possible to demonstrate its presence in the epidermis. The granular and spinous layers of the epidermis are the most abundant in hyaluronic acid in the extracellular compartment. The basal layer also contains hyaluronic acid, but in smaller amounts and this is intracellular. The hyaluronic acid contained in the basal layer of the epidermis is involved in the regulation of the cell cycle. The hyaluronic acid contained in the granular and spinous layers participates in skin hydration by retaining water molecules coming from the dermis, contained in the extracellular matrix. Water is fixed by hyaluronic acid and is retained by the hydrophilic lipid membrane.

[0009] Before reaching the superficial layers of the epidermis, water is bound to the macromolecules of the interstitial substance of the dermis: mucopolysaccharides, hyaluronic acid and proteoglycans. Only a small fraction of this dermal water is free. Water diffuses from the dermis through the epidermal junction to the deeper layers of the epidermis. This movement of water from the deeper to the superficial layers then requires the actual water transport system, the aquaporins, located in the cell membrane. In the skin, it is almost exclusively type 3 aquaporins.

[0010] Water then acquires the intercellular spaces held by hyaluronic acid, and also penetrates into the interior of the keratinocyte, plasticizing keratin. Water is drawn into and held in the keratinocyte by the double phenomenon of penetration and attraction by intracellular hygroscopic elements grouped under the name of NMF (natural moisturizing factors). These natural moisturizing factors are agents naturally synthesized by the skin to capture water in the stratum corneum. They result from the decomposition of filaggrin into amino acids under the action of intracellular proteases, which, together with the exfoliated and compacted keratin filaments, constitute a highly hydrophilic intracellular matrix.

[0011] Free amino acids, pyrrolidone carboxylic acids, urea, lactate, sugars, trace elements and chlorides are part of the NMF. Pyrrolidone acids, urea and lactate have a hygroscopic effect such that they can retain up to 70% of their weight in water, hence their strong moisturizing power. Water cannot leave the corneocytes due to the hydrophobic nature of the lipids in the intercellular spaces. Finally, the surface hydrophilic lipid membrane, a natural emulsion formed from water and lipids, keeps water on the surface of the skin and prevents insensible water loss (PIE). The aqueous part, the water-soluble fraction, comes from skin perspiration and sweat secretion. The lipid part, the liposoluble fraction, comes from sebum and epidermal synthesis of lipids. The hydrophilic lipid membrane, essential for skin suppleness, also helps to prevent dehydration.

[0012] Hyaluronic acid plays a major role in skin hydration due to its moisture absorbing ability and its high concentration in the dermis. It is present in the dermis but also in the epidermis, where it holds water in the interkeratinocyte spaces. It is found essentially in the extracellular space of the upper layer of the epidermis; there it is held to the surface by a hydrophilic lipid membrane that fixes the water molecules and limits evaporation.

[0013] Mammalian and especially human hair generally consists of three main components: the cuticle (the outer protective layer), the cortex (the large core of the hair), and the medulla (the central soft protein core, which is more common in thicker hairs and especially in grey hairs). The main constituents of these structures are sulfur-rich proteins, lipids, water, melanin, and trace elements.

[0014] The cuticle is composed of keratin and usually consists of 6-8 layers of flattened overlapping cells. Each cell contains multiple layers. The topmost structure of each cuticle cell contains a thin protein membrane, the epicuticle or f-layer, which is covered with a lipid layer. This lipid layer is covalently attached to the surface of the fiber. The epicuticle is hydrophobic. The complex structure of the cuticle makes the hair slippery when it swells, and the f-layer provides a large degree of water resistance. It is essential in protecting the hair and making it resistant to the inflow and outflow of moisture.

[0015] A normal cuticle has a smooth appearance, allows light reflection, and limits friction between hair shafts. It is primarily responsible for the shine and texture of hair. The cuticle is a chemically resistant region that surrounds the cortex in mammalian hair fibers. The cuticle can be damaged by environmental, mechanical, chemical, and thermal sources. Chemical removal of the f-layer, especially by oxidation during bleaching or perming, annihilates the initial hydrophobic defense and leaves the hair more porous and fragile. When the cuticle is damaged, there is little change in the tensile properties of the hair; however, its protective function is weakened.

[0016] The cortex contributes almost all of the mechanical properties of hair, especially its strength and elasticity. It consists of tightly packed, spindle-shaped cells rich in keratin filaments, which contain 400-500 amino acid residues that pair to form protofilaments that make up the keratin chains. They are oriented parallel to the long axis of the hair shaft and are embedded in an amorphous matrix of high sulfur-containing proteins. Keratin chains have numerous sulfur-containing cysteine ​​bonds, which create strong crosslinks between adjacent chains. These so-called disulfide bonds are essential in giving the hair shaft its shape, stability, and resilience, and can only be broken by external oxidizing chemicals, such as those used in perms and relaxers. Weak hydrogen bonds link the keratin polypeptide chains together. These weak bonds are easily overwhelmed by water, temporarily straightening curly hair. Strong disulfide bonds and weak hydrogen bonds are crucial for hair health. The cortex also contains melanin granules, which are responsible for the color of the fiber.

[0017] The medulla is a soft protein core present in thicker and white hairs. It has no known function in humans. Today's consumers are offered a large number of cosmetic products for hair and skin care. Generally, these products are in the form of leave-on or rinse-off formulations depending on the intended application. Skin care products include, for example, creams and lotions, which contain water to moisturize the skin and fats and lipids to re-lubricate it. Hair care products include, for example, shampoos and conditioners to cleanse, moisturize and UV protect hair.

[0018] As we age, we naturally lose collagen and hyaluronic acid, which makes the skin more easily dehydrated. Also, harsh weather, heaters during the winter, certain skin care products, and underlying skin conditions can cause tiny breaks in the protective skin barrier that allow water to escape. This is why a skin care regimen with moisturizing products can be extra beneficial.

[0019] Hydrating skin care ingredients including hyaluronic acid, glycerin, colloidal oatmeal, urea, propylene glycol, and sorbitol all act as humectants that draw water to the skin to provide moisture. These ingredients are widely used in products such as moisturizers, eye creams, and serums.

[0020] Hyaluronic acid is a sugar molecule that occurs naturally in the skin, and it helps bind water to collagen and trap it in the skin so that the skin appears plumper, hydrated, and more hydrated. It penetrates easily, which is why it works so well when applied topically. Additional benefits of hyaluronic acid include its light weight, water-rich nature, and ability to hydrate the skin well by trapping moisture from the environment and the deep dermis. Hyaluronic acid is not a moisturizer (it is a humectant), but it does help by drawing moisture from the environment.

[0021] Hyaluronic acid is a compound that is naturally produced in the dermis of skin, and is constantly degraded by an enzyme called hyaluronidase.Therefore, with age, the ratio between the hyaluronic acid that cells synthesize and the hyaluronic acid that cells degrade decreases, causing the reduction of dermal moisture and progressive sagging, leading to the appearance of wrinkles.The skin softening and moisturizing effect of hyaluronic acid is known in the art.

[0022] Hair is often subjected to a wide variety of insults that cause damage, including shampooing, rinsing, drying, heating, combing, styling, perming, coloring, exposure to the elements, etc. Thus, due to abrasion of the hair surface and removal of the hair's natural oils and other natural conditioning and moisturizing properties, hair can often become dry, coarse, dull, or frizzy.

[0023] Hyaluronic acid is also beneficial for hair: it hydrates hair, reduces frizz, plumps hair, and hydrates the scalp. The humectant binding properties of hyaluronic acid perform similarly to hair as it does to skin, allowing the hair fiber to retain and seal in moisture from products. It also helps seal the cuticle, which prevents unwanted moisture from getting in there, leading to frizzy hair, and frizzy hair with a curly texture.

[0024] However, there are important drawbacks: The surface of hair and skin is usually negatively charged.Hyaluronic acid is also usually negatively charged due to the presence of anionic functional groups (particularly carboxyl groups).Therefore, when treating hair or skin, hyaluronic acid and hair / skin repel each other because their surfaces are negatively charged. Thus, hyaluronic acid usually adheres to the surface of the hair or skin to a lesser extent, making the treatment less effective.

[0025] To overcome this difficulty, several groups have previously proposed attaching cationic groups to the hyaluronic acid backbone, thereby producing cationized hyaluronic acid or its salts. Such cationized hyaluronic acid derivatives and their salts include, but are not limited to, hydroxypropyltrimonium hyaluronate and its salts. For example, US 2009 / 0281056 discloses a method for preparing cationized hyaluronic acid, for example hydroxypropyltrimonium hyaluronate, in which at least a part of the hydroxyl hydrogen atoms of hyaluronic acid is replaced by a group having a quaternary ammonium cationic group.To this end, hyaluronic acid is reacted with a cationizing agent such as glycidyltrialkylammonium halide.

[0026] For example, US 8,410,076 relates to cationized hyaluronic acid and / or its salts, such as hydroxypropyltrimonium hyaluronate, which contain groups containing quaternary ammonium groups and have a cationization degree of 0.15 to 0.6. This document further explains that if the cationized hyaluronic acid and / or its salts have a cationization degree of less than 0.15, the adhesion of the cationized hyaluronic acid and / or its salts to hair or skin is greatly reduced and sufficient moisturizing effect may not be obtained, and if the cationization degree is more than 0.6, the cationized hyaluronic acid and / or its salts may adhere to hair or skin, but sufficient moisturizing effect and smoothness may not be achieved. Summary of the Invention

[0027] However, the above cationized hyaluronic acid derivatives and salts fail to provide long-lasting moisturizing benefits, even in rinse-off formulations. It is therefore an object of the present invention to provide a cosmetic moisturizer which is also effective in rinse-off applications. This problem is solved by the products, compositions and methods of the present invention as described below.

[0028] In a first aspect, the present invention relates to hydroxypropyltrialkylammonium hyaluronate and / or salts thereof having a degree of cationization greater than 1.4. In a second aspect, the present invention relates to a method for preparing said hydroxypropyltrialkylammonium hyaluronate and / or salts thereof.

[0029] In a third aspect, the present invention relates to a cosmetic composition comprising said hydroxypropyltrialkylammonium hyaluronate and / or its salt. In a fourth aspect, the present invention relates to the use of said hydroxypropyltrialkylammonium hyaluronate and / or its salts for moisture retention and / or UV protection and / or hair repair. [Brief description of the drawings]

[0030] [Figure 1] FIG. 1 shows SEM images of hair fibres either washed with one of compositions P and Q or untreated, prior to UV treatment. [Diagram 2] FIG. 2 shows SEM images of hair fibres washed with either one of compositions P and Q or untreated after UV treatment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0031] The invention is explained in more detail below. The hydroxypropyltrialkylammonium hyaluronate and / or its salt of the present invention has a degree of cationization greater than 1.4. It has been found that this cationized hyaluronic acid derivative can provide long-lasting moisturizing benefits to both skin and hair, and even in rinse-off applications, and is therefore a highly effective cosmetic active.

[0032] In particular, it has been found to have better adhesion to hair and to skin than commercially available hyaluronic acid derivatives having a lower degree of cationization, thereby providing a longer lasting moisturizing effect. It is the first time that hyaluronic acid derivatives have been found to be highly effective in rinse-off applications as well, allowing for much wider formulation options than conventional materials.

[0033] Furthermore, it has been found that the hydroxypropyltrialkylammonium hyaluronate and / or its salts of the present invention have UV protection benefits, again not only in leave-on but also in rinse-off applications. Furthermore, it has been found that the hydroxypropyltrialkylammonium hyaluronate and / or its salts of the present invention have hair repair effects after chemical treatment.

[0034] The term "hydroxypropyltrialkylammonium" refers to a group or substituent having the following structure: [ka] R in the formula 1 , R 2 and R 3 are, independently of each other, linear or branched alkyl groups having 1 to 4 carbon atoms. R 1 , R 2 and R 3 can be the same or different (for example, R 1 =R 2 =R 3 or R 1 =R 2 ≠R 3 or R 1 ≠R 2 ≠R 3 ), preferably they are all the same.

[0035] For example, R 1 , R 2 and R 3 are each independently selected from the group consisting of methyl, ethyl, propyl, iso-propyl, and butyl. "Degree of cationization" as used throughout this application corresponds to the average number of hydroxypropyltrialkylammonium groups attached to hyaluronic acid per unit.

[0036] Hyaluronic acid is a polymer in which N-acetyl-D-glucosamine and D-glucoronic acid are linked together to form a unit. Its general structure is thus (n represents the number of units): [ka]

[0037] As can be seen from the above structure, each unit contains several hydroxyl groups, including primary and secondary alcohols, and a carboxylic acid group. In addition, it also contains an amide group. The hydroxypropyltrialkylammonium group may be attached to any of these groups, i.e., replace the respective hydrogen atom. The degree of cationization may be determined by means of NMR, IR and / or conductivity measurements. Further details are provided below in the Examples section.

[0038] In one embodiment, hydroxypropyltrialkylammonium hyaluronate and / or its salt is selected from the group consisting of hydroxypropyltrimonium hyaluronate and / or its salt;hydroxypropyltriethylammonium hyaluronate and / or its salt;hydroxypropyltripropylammonium hyaluronate and / or its salt;and hydroxypropyltributylammonium hyaluronate and / or its salt.Preferably, hydroxypropyltrialkylammonium hyaluronate and / or its salt is hydroxypropyltrimonium hyaluronate and / or its salt.

[0039] The term "hydroxypropyltrimonium" is a contraction of "hydroxypropyltrimethylammonium" and refers to a group or substituent having the following structure: [ka]

[0040] In one embodiment, hydroxypropyltrialkylammonium hyaluronate and / or its salt has a cationization degree of at least 1.5, more preferably at least 1.6, even more preferably at least 1.7, and most preferably at least 1.8.Cationization degree can also be higher, for example 1.9 or more, or 2.0 or more, or even 2.1 or more, or 2.2 or more. It has been found that a higher degree of cationization can lead to better adhesion to hair and / or skin, thereby improving the deposition and substantivity of cosmetic actives.

[0041] In theory, the degree of cationization may be up to 6.0. However, for cosmetic applications, a degree of cationization of up to 3.0, more preferably up to 2.8, and most preferably up to 2.6 has been found to be most advantageous. By way of illustration, the hydroxypropyltrialkylammonium hyaluronate and / or salt thereof may have a degree of cationization between 1.4 and 3.0, more preferably between 1.6 and 2.4, and most preferably between 1.8 and 2.0.

[0042] In one embodiment, the hydroxypropyltrialkylammonium hyaluronate and / or its salt is prepared from hyaluronic acid or its salt having an average molecular weight of about 10 kDa to about 200 kDa, more preferably about 15 kDa to about 150 kDa, even more preferably about 20 kDa to about 100 kDa, and most preferably about 20 kDa to about 80 kDa.

[0043] Depending on the exact degree of cationization, the average molecular weight of hydroxypropyltrialkylammonium hyaluronate and / or its salts may vary, so it seems more appropriate to define the weight of the hyaluronic acid or its salts from which hydroxypropyltrialkylammonium hyaluronate and / or its salts are derived.Those skilled in the art can easily calculate the average molecular weight of hydroxypropyltrialkylammonium hyaluronate and / or its salts based on the average molecular weight of the original hyaluronic acid or its salts and the analytically determined degree of cationization as follows: MW catHA = MW HA + n*(CatDeg * MW HPT ) During the ceremony, MW catHA is the average molecular weight of hydroxypropyltrialkylammonium hyaluronate and / or its salts; MW HA is the average molecular weight of the hyaluronic acid or salt thereof from which the hydroxypropyltrialkylammonium hyaluronate and / or salt thereof is derived; n is the number of units of hyaluronic acid or its salt from which the hydroxypropyltrialkylammonium hyaluronate and / or its salt is derived; CatDeg is the analytically determined degree of cationization; MW HPT is the molecular weight of the hydroxypropyltrialkylammonium group and / or its salt incorporated in the hydroxypropyltrialkylammonium hyaluronate and / or its salt; and n is M HA It can be calculated by dividing by the molecular weight of one unit of hyaluronic acid or a salt thereof.

[0044] By way of illustration, the chloride salt of hydroxypropyltrimonium hyaluronate may be prepared from sodium monohyaluronate. In this case, n=MW HA / 401.3 g / mol, so that the molecular weight of 1 unit is 401.3 g / mol; and MWHPT is 151.6 g / mol. Thus, for example, a chloride salt of hydroxypropyltrimonium hyaluronate with a degree of cationization of 1.4 prepared from sodium monohyaluronate with an average molecular weight of 20 kDa will have an average molecular weight of about 30 kDa; and a chloride salt of hydroxypropyltrimonium hyaluronate with a degree of cationization of 2.5 prepared from sodium monohyaluronate with an average molecular weight of 50 kDa will have an average molecular weight of about 100 kDa.

[0045] Surprisingly, it has been found that a relatively low average molecular weight as defined above can provide a particularly effective cosmetic active, for example, which exhibits better adhesion to hair and penetrates deeper into the skin. Furthermore, the relatively low average molecular weight facilitates the synthesis of hydroxypropyltrialkylammonium hyaluronate and / or its salt.It has been found that the hyaluronic acid with lower average molecular weight reacts faster and requires fewer equivalents of reagent to obtain desired cationization degree.Without being bound by theory, it is believed that the reaction site of the hyaluronic acid with higher average molecular weight is less accessible to reagent molecules due to both the reduced flexibility of longer polymer chain and increased steric hindrance.

[0046] Throughout this application, unless otherwise indicated, equivalents of reagents are given relative to one repeat unit of hyaluronic acid or a salt thereof used in the reaction. In a specific embodiment, the hydroxypropyltrialkylammonium hyaluronate and / or its salts of the present invention are prepared from hyaluronic acid or its salts having an average molecular weight between 20 and 40 kDa, resulting in - in the case of hydroxypropyltrimethylammonium - hydroxypropyltrimonium hyaluronate and / or its salts having an average molecular weight between 20 and 80 kDa.

[0047] Hydroxypropyltrialkylammonium hyaluronate as it is has a net positive charge, i.e., is cationic.Therefore, there will typically be an anionic counterion, illustratively chloride or other halide ion (e.g. bromide or iodide), hydroxide, phosphate, acetate, carboxylate (which may optionally be part of the hyaluronic acid backbone) or carbonate.Such counterion may be introduced at any time during the synthesis of hydroxypropyltrialkylammonium hyaluronate and / or its salt.Alternatively or in addition, counterion may also be introduced from ion exchange resin. For example, a chloride counterion may be introduced if a reagent bearing a chloride group is used in the preparation of the hydroxypropyltrialkylammonium hyaluronate, or if a chloride salt (e.g., NaCl) is used during the preparation, for example during a washing or purification step.

[0048] In one embodiment, the hydroxypropyltrialkylammonium hyaluronate and / or salt thereof of the present invention comprises or consists of a chloride salt of hydroxypropyltrialkylammonium hyaluronate. Thus, some or all of the anionic counterions may be chloride, and other anionic counterions may also be present. The hydroxypropyltrialkylammonium hyaluronate and / or salt thereof of the present invention may further comprise an additional cation, illustratively an alkali or alkaline earth metal cation (e.g., Na + , K + , Mg 2+ or Ca 2+ ) may also be included.

[0049] In a further aspect, the present invention provides methods for preparing hydroxypropyltrialkylammonium hyaluronate and / or salts thereof, especially those in the embodiments described above. The method includes reacting hyaluronic acid and / or a salt thereof with a cationizing agent in the presence of a base, the cationizing agent being selected from the group consisting of 2,3-epoxypropyltrialkylammonium chloride, 2-chloro-3-hydroxypropyltrialkylammonium chloride, and mixtures thereof.

[0050] Alternatively, instead of or in addition to the chloride, 2,3-epoxypropyltrialkylammonium bromide, 2-chloro-3-hydroxypropyltrialkylammonium bromide, 2,3-epoxypropyltrialkylammonium iodide, 2-chloro-3-hydroxypropyltrialkylammonium iodide, or any mixture of these reagents can also be used. Further alternatives include 2-chloro-3-hydroxypropyltrialkylammonium fluoride and 2-chloro-3-hydroxypropyltrialkylammonium acetate.

[0051] In one embodiment, the cationizing agent is selected from the group consisting of 2,3-epoxypropyltrimonium chloride, 2-chloro-3-hydroxypropyltrimonium chloride, and mixtures thereof, which leads to the formation of hydroxypropyltrimonium hyaluronate and / or its salts. These cationizing agents are commercially available. 2,3-epoxypropyltrimonium chloride is sometimes also called glycidyltrimethylammonium chloride or (2,3-epoxypropyl)trimethylammonium chloride; thus, each of these terms refers to the same reagent. 2-Chloro-3-hydroxypropyltrimonium chloride is sometimes also called 3-chloro-2-hydroxy-N,N,N-trimethylpropan-1-aminium chloride; each of these terms refers to the same reagent.

[0052] Alternatively, instead of or in addition to the chloride, 2,3-epoxypropyltrimonium bromide, 2-chloro-3-hydroxypropyltrimonium bromide, 2,3-epoxypropyltrimonium iodide, 2-chloro-3-hydroxypropyltrimonium iodide, or any mixture of these reagents can also be used. Further alternatives include 2-chloro-3-hydroxypropyltrimonium fluoride and 2-chloro-3-hydroxypropyltrimonium acetate. Preferably, 2,3-epoxypropyltrimonium chloride is used as the cationizing agent because the reaction requires less base and has been found to run more smoothly.

[0053] Depending on the desired degree of cationization and the cationization agent used, more or less equivalents of the cationization agent should be used. For example, about 5 to about 6 equivalents of 2,3-epoxypropyltrialkylammonium chloride can be used to obtain a degree of cationization of about 2.2; or about 5 to about 7 equivalents of 2-chloro-3-hydroxypropyltrialkylammonium chloride can be used to obtain a degree of cationization of about 1.6.

[0054] Suitable bases include, but are not limited to: inorganic bases such as alkali and alkaline earth metal hydroxides, phosphates, hydrogen phosphates or carbonates (e.g., NaOH, KOH, Ca(OH)2, Mg(OH)2, Na3PO4, K3PO4, Na2HPO4, K2HPO4, Na2CO3 or K2CO3); organic bases such as tributylamine, ethylenediamine, triethylamine, trimethylamine, tetra-n-butylammonium hydroxide or tetraethylammonium hydroxide.

[0055] Depending on the cationizing agent used, about 0.1 to about 15 equivalents of base should be used, more preferably about 1.0 to 5.0 equivalents, for example 1.3 equivalents.The use of more base is disadvantageous, since it has been found that the reaction is less efficient in larger volumes.On the other hand, if a higher concentration of base is used, it may cause partial hydrolysis of hyaluronic acid polymer chains, leading to undesirable by-products and lower yield of desired product.

[0056] Notably, when 2-chloro-3-hydroxypropyltrimonium chloride or another 2-chloro-3-hydroxypropyltrialkylammonium is used, an additional equivalent of base is required and the epoxide is prepared in situ, which makes it possible to avoid handling of toxic epoxides: [ka]

[0057] The cationization reaction is typically carried out at a basic pH, illustratively at a pH of about 8 to about 14. In one embodiment, the cationization reaction is carried out at a pH of about 12 to about 13.

[0058] Suitable solvents include, but are not limited to, water, THF, DMSO, ethanol, methanol, isopropanol, acetone, acetonitrile, and combinations thereof. Preferably, aqueous solvents, and especially water, are used.

[0059] The cationization reaction may be carried out at any suitable concentration that allows obtaining a stirrable mixture, more preferably at a concentration where hyaluronic acid and / or its salt is completely or at least essentially completely dissolved.For example, the reaction may be carried out at a concentration of hyaluronic acid and / or its salt of about 0.01 to about 1.00 g / ml, more preferably about 0.05 to about 0.50 g / ml.

[0060] A suitable reaction temperature may be from about 10° C. to about 80° C., more preferably from about 10° C. to about 40° C., illustratively about 25° C. Higher temperatures may lead to hydrolysis of the hyaluronic acid polymer chains. A suitable reaction time may be from about 1 hour to about 6 days, illustratively about 21 hours.

[0061] Optionally, the reaction may be stopped once the desired degree of cationization is reached or after a certain reaction time. Illustratively, the reaction may be stopped by the addition of a neutralizing agent, such as an acid, to bring the reaction to a neutral pH (e.g., a pH of less than 9, more preferably less than 8, and most preferably about 7). Suitable acids include, but are not limited to, inorganic acids, such as HCl or H2SO4, or organic acids, such as acetic acid, citric acid, or oxalic acid.

[0062] By means of the synthetic procedures described above, a crude reaction mixture containing the desired hydroxypropyltrimonium hyaluronate and / or its salts is obtained, from which the hydroxypropyltrimonium hyaluronate and / or its salts are preferably isolated and purified.

[0063] The product thus obtained may be purified by any suitable method, illustratively by means of dialysis and especially ultrafiltration, which has been found to provide a very gentle and efficient purification with highly reproducible results, which also makes it possible to remove any colored by-products.

[0064] Alternatively or additionally, an ion exchange resin (eg, DOWEX MAC-3) may be used. A further alternative is precipitation of the product, for example by addition of ethanol or acetone to the reaction mixture. The hydroxypropyltrialkylammonium hyaluronate and / or its salts may also be subjected to further processing, such as freeze-drying or spray-drying to obtain a powder. The hydroxypropyltrialkylammonium hyaluronate and / or its salts may also be used in the form of an aqueous solution.

[0065] In a further aspect, the present invention relates to a cosmetic composition comprising a hydroxypropyltrialkylammonium hyaluronate and / or a salt thereof as described above. In particular, the present invention relates to a hair care or skin care composition comprising hydroxypropyltrialkylammonium hyaluronate and / or salts thereof as described above.

[0066] The hydroxypropyltrialkylammonium hyaluronate and / or its salts according to the invention are particularly suitable for cosmetic applications: - It provides long-lasting moisturization, - adheres strongly to hair and skin making it suitable for both leave-on and rinse-off formats; - Provides efficient UV protection, - Provides hair restoration after chemical treatments.

[0067] Hair care compositions have been used for decades and for many different applications. The hydroxypropyltrialkylammonium hyaluronate and / or its salt of the present invention may be used in all kinds of hair care compositions, such as hair cleansing compositions, hair conditioning compositions, and hair styling compositions.Many of these compositions are typically water-based formulations.

[0068] Hair cleansing compositions are generally effective in removing dirt from hair. Dirt includes natural exudation from the scalp, environmental factors, and styling products. Dirt can cover or deposit on hair and scalp. Hair covered with such dirt typically feels and looks greasy, feels heavy, possibly has a foul odor, and generally cannot maintain a desired style. Known cleansing compositions typically include a combination of water and a surface active ingredient such as soap or synthetic surfactant, and may also include a non-aqueous blend of starch. The combination of water and surface active agent emulsifies dirt from hair and scalp, allowing it to be rinsed off.

[0069] The cleansing composition may also contain a conditioning agent that is deposited on the hair and scalp during rinsing with water. Such conditioning agents can include polymers, oils, waxes, protein hydrolysates, silicones, and mixtures and derivatives thereof. In addition, the conditioning composition can be a separate and distinct product from the cleansing composition.

[0070] The conditioning compositions known in the art are typically water-based formulations.However, conditioning compositions are also known that include at least one of silicone; animal, mineral or vegetable oil; wax; petrolatum; and grease.Water-based conditioning compositions typically include substituted cationic waxes, fatty alcohols, cationic polymers, hydrolyzed proteins and their derivatives, and fragrances.Such conditioning formulations impart combability and manageability to treated hair, thereby minimizing damage during styling process, resulting in shiny, healthy, and manageable hair.The conditioning composition can also be effective for moisturizing hair.The subsequent drying and styling process can include air drying or heating.

[0071] Today, many different skin care products are available to consumers. The hydroxypropyltrialkylammonium hyaluronate and / or its salts according to the invention may be used in all kinds of skin care compositions, for example in hydrating and moisturizing compositions, anti-aging compositions, cleansing and refreshing compositions or make-up compositions.

[0072] The skin care composition of the present invention may contain one or more cosmetically acceptable excipients. Any excipient commonly used in the preparation of cosmetic formulations for use on human skin may be employed in the present invention. Suitable excipients include, but are not limited to, ingredients that can affect the sensory properties, skin penetration, and bioavailability of cationized hyaluronic acid and / or its salts. More specifically, they include liquids such as water, oil, or surfactants, including those of petroleum, animal, vegetable, or synthetic origin, such as, but not limited to, peanut oil, soybean oil, mineral oil, sesame oil, castor oil, polysorbates, sorbitan esters, ether sulfates, sulfates, betaines, glycosides, maltosides, fatty alcohols, nonoxynol, poloxamers, polyoxyethylene, polyethylene glycols, dextrose, glycerol, digitonin, etc.

[0073] The skin care composition may be in the form of liposomal compositions, mixed liposomes, oleosomes, niosomes, ethosomes, milliparticles, microparticles, nanoparticles and solid-lipid nanoparticles, vesicles, micelles, surfactant mixed micelles, surfactant-phospholipid mixed micelles, millispheres, microspheres and nanospheres, lipospheres, millicapsules, microcapsules and nanocapsules, and microemulsions and nanoemulsions, which can be added to achieve higher penetration of hydroxypropyltrialkylammonium hyaluronate and / or its salts.

[0074] The skin care composition may be produced in any solid, liquid, or semi-solid form useful for topical or transdermal application to the skin. These topical or transdermal formulations thus include multiple emulsions, such as and without limitation creams, oil-in-water and / or silicone-in-water emulsions, water-in-oil and / or water-in-silicone emulsions, water / oil / water or water / silicone / water type emulsions, and oil / water / oil or silicone / water / silicone type emulsions, microemulsions, emulsions and / or solutions, liquid crystals, anhydrous compositions, aqueous dispersions, oils, milks, balsams, foams, aqueous or oily lotions, aqueous or oily gels, creams, aqueous alcoholic solutions, aqueous glycolic solutions, hydrogels, liniments, sera, soaps, face masks, serums, polysaccharide films, ointments, mousses, pomades, pastes, powders, bars, pencils, and sprays or aerosols (sprays), including leave-on and rinse-off formulations.

[0075] For example, the hydroxypropyltrialkylammonium hyaluronate and / or its salts of the present invention may be used in anti-aging products, moisturizing products, washing gels, lotions, cleansers, masks, hair care or skin care products.

[0076] The cosmetic composition of the present invention may contain hydroxypropyltrialkylammonium hyaluronate and / or its salt at any suitable concentration sufficient to provide the desired effect.For example, it may contain about 0.05% to about 1.0% hydroxypropyltrialkylammonium hyaluronate and / or its salt, for example about 0.1%.It has been found that a concentration of about 0.1% ensures high adhesion to hair and skin and activation of biological effectiveness.However, it is also possible to include higher or lower concentrations.

[0077] The cosmetic compositions of the present invention may further comprise additional cosmetic actives, such as anti-aging and anti-wrinkle actives, moisturizers, cleansers or hair conditioners. For example, the hydroxypropyltrialkylammonium hyaluronate and / or its salts of the present invention may also be used in combination with hyaluronic acid and / or its salts, and / or with other hyaluronic acid derivatives, such as, for example, acetate salts. Alternatively or additionally, multiple hydroxypropyltrialkylammonium hyaluronates and / or salts thereof may be combined in one cosmetic composition. Illustratively, they may have different molecular weights and / or degrees of cationization and / or counterions.

[0078] In a further aspect, the present invention also relates to the use of hydroxypropyltrialkylammonium hyaluronate and / or its salts for moisture retention and / or UV protection and / or hair repair. As has been described above and further shown in the examples below, the hydroxypropyltrialkylammonium hyaluronate and / or its salts of the present invention provide particularly effective water retention or moisturization, as well as UV protection and hair repair, even in rinse-off applications.

[0079] The present invention is further illustrated by the following non-limiting examples: Example 1: Preparation of Hydroxypropyltrimonium Hyaluronate Using (2,3-Epoxypropyl)trimonium Chloride A 350 ml 4-neck sulfonation flask was equipped with a thermometer, condenser, overhead stirrer (Heidolph), bubble counter, and pH meter, and flushed with nitrogen. It was then charged with 30 g of sodium hyaluronate (75 mmol; average MW of 41.5 kDa), 80 ml of deionized water, and 72 ml of 1.32 M aqueous sodium hydroxide (95 mmol). The mixture was stirred at 200 rpm for 1 h until a clear, pale yellow solution was obtained.

[0080] 72 g of (2,3-epoxypropyl)trimethylammonium chloride (449 mmol) was added to the mixture in one portion, and the addition funnel was washed with 10 ml of deionized water. The internal temperature of the pale yellow, cloudy reaction mixture rose from 26° C. to 32° C. within 1 h, then cooled to 25° C. The pale yellow, clear solution was then stirred at room temperature for 19 h.

[0081] The viscous reaction mixture was transferred to a 2 L Schott bottle equipped with a magnetic stirrer and a pH meter. The reaction flask was washed twice with 25 ml of deionized water. The mixture was then neutralized by the dropwise addition of 533 g of aqueous HCl (0.5 wt%) until a pH of 7.07 was reached. A clear yellow solution (825 ml) was obtained.

[0082] The mixture was diluted by adding 1175 ml of deionized water. The conductivity of the solution was 20.5 mS / cm at 22.9°C. The mixture was then ultrafiltered through two filtration VivaFlow200 units (MWCO: 10 kDa; polyethersulfone membrane; Sigma-Aldrich) for 3 days until the conductivity of the filtrate reached 200 μS / cm. The mixture was then concentrated to reach a volume of 500 ml (turbid solution), and then freeze-dried to obtain 35 g of hyaluronic acid hydroxypropyltrimonium chloride salt (48 mmol; yield 64%) as defined above in the form of white chips.

[0083] The degree of cationization was determined by NMR (see Example 3 below) to be 2.18. The molecular weight of the product was calculated based on the degree of cationization and assuming that the product still contains sodium and chloride ions (but the product may in fact also partially contain other counter ions as described above): [401.3] + 2.18 × [151.6] = 731.86g / mol

[0084] Example 2: Preparation of hydroxypropyltrimonium hyaluronate using 2-chloro-3-hydroxypropyltrimonium chloride Using the same equipment as described in Example 1, sodium hyaluronate (10 g, 24.92 mmol, 37.8 kDa) was mixed with 3-chloro-2-hydroxy-N,N,N-trimethylpropan-1-aminium chloride (40.6 ml, 150 mmol, 60% solution in water) at room temperature. Sodium hydroxide (16.77 ml, 181 mmol, 32% aqueous solution) was then added over 15 minutes. A water bath was used to control the exotherm. The mixture was stirred at room temperature for 21 h.

[0085] The yellow mixture was then poured into a closed dialysis bag (30 cm / 76 mm tubing, 14 kDa cutoff) and into a 4.5 l water bath. The water bath was changed regularly over 48 h until a neutral pH was reached. The mixture was filtered through neutralized DOWEX-MAC-3 ion exchange resin and then freeze-dried overnight to obtain 7.5 g of white hyaluronic acid hydroxypropyltrimonium chloride salt as defined above. The degree of cationization is 1 The molecular weight was determined by 1 H-NMR (600 MHz) and found to be 1.47.

[0086] Example 3: Determination of the degree of cationization The degree of cationization was quantitatively determined by integration of the trimonium methyl signal versus the N-acetyl methyl signal. 1 The carbonyl group was determined by H-NMR (600 MHz). In addition, DOSY experiments were performed to confirm that all trimonium groups were chemically bound to hyaluronan by comparing the diffusion constants with those derived from hyaluronate.

[0087] The degree of cationization may also be measured by means of IR chemical analysis during the synthesis of hydroxypropyltrimonium hyaluronate.For this purpose, the partial least squares method is used to correlate the IR spectrum of a reference sample with the degree of cationization measured by NMR.The IR spectrum of a new sample with unknown degree of cationization can then be measured, and the degree of cationization can be assigned using the model described so far.This method is found to be independent of the presence of salt and more robust than conductivity measurement.

[0088] Conductivity measurements offer a further alternative, but they are highly dependent on the work-up procedure, since the presence of other ions can dramatically affect the results. For the conductivity measurements, calibration curves were prepared for different dilutions of samples with known cationization degree. This revealed a linear relationship between the conductivity and concentration of the samples, with higher cationization degree leading to a steeper slope. To avoid interference with other salts present in the solution (e.g. NaCl), hydroxypropyltrimonium hyaluronate must be dialyzed prior to the measurement, and the use of ion exchange resins in the final purification step must be avoided.

[0089] Example 4: Skin Adhesion Test for Rinse-Off Applications Preparation of skin explants Fresh human skin explants from two female donors (aged 35 and 57, respectively) who had undergone breast reduction and breast augmentation surgery, were used in this study. The skin explants were topically treated for 1 hour with one of the following six compositions: Composition A: 1% sodium hyaluronate with a molecular weight of 20 to 40 kDa (comparative example) Composition B: 1% hydroxypropyltrimonium hyaluronate (comparative example) prepared from the same sodium hyaluronate used in composition A, with a degree of cationization of 0.4. Composition C: 1% hydroxypropyltrimonium hyaluronate prepared from the same sodium hyaluronate used in composition A, with a degree of cationization of 1.4. Composition D: 1% hydroxypropyltrimonium hyaluronate prepared from the same sodium hyaluronate used in composition A, with a degree of cationization of 2.4. Composition E: 1% hydroxypropyltrimonium hyaluronate prepared from the same sodium hyaluronate used in composition A, with a degree of cationization of 2.0. Composition F: 1% Hyaloveil®-P (formerly Kewpie; hydroxypropyltrimonium hyaluronate with a molecular weight of 579 kDa and a degree of cationization of 0.6; comparative example)

[0090] The skin explants without any treatment were used as the untreated state. After 1 hour of treatment, the skin explants were rinsed twice with sterile water, and the excess water was gently absorbed with cleaning paper. A portion of the skin explants was embedded in OCT for HABP staining on frozen sections, while the other portion was newly analyzed by Raman spectroscopy.

[0091] HABP staining Deposition was demonstrated by fluorescent staining with hyaluronic acid binding protein (HABP) on 8 μm thick frozen sections. Briefly, specific sites were saturated with successive baths of avidin, biotin and 0.1% bovine serum albumin solutions. Biotinylated HABP was then incubated on the frozen sections for 2 hours at room temperature, followed by rinsing and another incubation with streptavidin coupled to Alexa fluor 568 for 30 minutes at room temperature in the dark. The samples were rinsed and assembled with coverslips and mounting medium. Images were collected with an Axio Observed Inverted fluorescent microscope (Zeiss). The fluorescence intensity specific to the hyaluronic acid deposited on the stratum corneum was quantified.

[0092] Raman spectroscopy analysis Axial Z-profiles were recorded directly on the skin samples. The Z-profile consisted of a detailed scan through the skin. In this study, Raman spectra were collected at different focal points on the skin surface from Z=0 μm to Z=4 μm in 2 μm increments. A total of 35 Raman profiles were recorded (5 profiles per condition, n=5). The average spectrum of the HA product was used as the reference spectrum.

[0093] result In the first study, the skin adhesion properties of compositions A, B and D were compared to an untreated control. The fitting coefficients of hyaluronic acid (derivatives) in the first layer of the stratum corneum were measured by Raman spectroscopy. The results are shown in the table below: [Table 1]

[0094] It was found that the cationized hyaluronic acid samples (Compositions B and D) exhibited significantly higher skin adhesion of +106% and +222%, respectively, compared to non-cationized hyaluronic acid (Composition A). Furthermore, composition D of the present invention exhibited significantly higher skin adhesion than composition B. Thus, a higher degree of cationization leads to better skin adhesion.

[0095] In a second study, the skin deposition properties of compositions A, C, E, and F were compared to an untreated control. Deposition of hyaluronic acid (derivatives) onto the skin surface was revealed using HABP staining. The results are shown in the table below: [Table 2]

[0096] It was found that the cationized hyaluronic acid samples of the present invention (Compositions C and E) exhibited significantly higher skin adhesion of +47% and +121%, respectively, compared to non-cationized hyaluronic acid (Composition A). Also, Composition E, which has a higher degree of cationization, exhibited significantly higher skin adhesion (+50%) than Composition C.

[0097] It was further found that the cationized hyaluronic acid samples of the present invention (Compositions C and E) exhibited significantly higher skin adhesion of +67% and +149%, respectively, compared to hyaluronic acid having a degree of cationization of 0.6 (Composition F), which did not exhibit any skin adhesion properties. In conclusion, it was found that a higher degree of cationization led to better skin adhesion.

[0098] Example 5: Skin hydration test in rinse-off application: Comparison with HA of the same molecular weight Preparation of skin explants Fresh human skin explants from a female donor (age 22) undergoing breast reduction surgery were used in this study. The skin explants were treated topically for 5 minutes with one of the following two compositions: Composition G: 0.1% sodium hyaluronate with a molecular weight of 20 to 40 kDa (comparative example) Composition H: 0.1% hydroxypropyltrimonium hyaluronate prepared from the same sodium hyaluronate used in composition G, with a degree of cationization of 1.9.

[0099] The skin explants without any treatment were used as the untreated condition. After 5 min of treatment, the skin explants were rinsed 5 times with sterile water. These treatments were repeated once daily for three days. Skin hydration was analyzed by Raman spectroscopy on fresh skin explants on day 0, corresponding to skin explants without any treatment, and on days 2 and 3, corresponding to the second and third days following the repeated treatment. Aquaporin and filaggrin immunostaining was performed on formalin-fixed and paraffin-embedded skin explants.

[0100] Raman spectroscopy analysis Axial Z-profiles were recorded directly on the skin samples. The Z-profile consisted of a detailed scan through the skin. Raman spectra were collected at different focal points on the skin surface from Z=0 μm to Z=30 μm in 3 μm increments. A total of 40 Raman profiles were recorded (4 profiles per condition, n=4).

[0101] In a first step, the exact location of the SC surface was determined for each Raman profile. In a second step, the lower limit of the SC was determined based on the water content. This involved calculating the νOH / νCH ratio. This location corresponds to the maximum value of the νOH / νCH ratio. The average spectrum of the SC was calculated considering all spectra acquired on the SC. After data processing (baseline correction, normalization, signal-to-noise ratio for spectral quality tests), water retention parameters were calculated for the average spectrum of the SC for each profile.

[0102] For assessment of skin hydration, the integrated intensity of the OH vibration band on the average SC spectrum was calculated, which represents the water content of the skin. The spectral range used for the calculation was νOH: 3100-3600 cm -1 It is.

[0103] Aquaporin and filaggrin immunostaining Skin explants were cut into 4 μm thick sections, dewaxed, and antigen retrieval was performed overnight at 62° C. for filaggrin and aquaporin-3 in EDTA buffer at pH 8.5 and citrate buffer at pH 6, respectively. Nonspecific sites were saturated with BSA at 2% in Tris buffer, and primary antibodies were then incubated on the skin sections overnight at 4° C. (anti-filaggrin antibody 1:100; anti-aquaporin-3 antibody 1:1000).

[0104] The next day, excess antibodies were washed off three times with Tris buffer and secondary antibodies were incubated for 1 h at room temperature: Hoechst 33342 1:5000 coupled to Alexa fluor 488 anti-mouse 1:100 for filaggrin or Alexa fluor 488 anti-rabbit 1:200 for aquaporin-3. Excess antibodies were washed off three times with Tris buffer and mounting medium without DAPI was added together with a coverslip.

[0105] Photographs of the emitted fluorescent signals were taken with an inverted epifluorescence microscope (Axio Observer, Zeiss). The fluorescence intensity for each condition was measured using ImageJ software, and the results obtained with treatments were compared with the untreated condition considered as the 100% control.

[0106] result The skin hydration properties of compositions G and H were compared to an untreated control after 2 and 3 days of application. The results after 2 and 3 days are shown in the following tables, respectively: [Table 3] [Table 4]

[0107] After 2 and 3 days of application, relative to day 0, the phenomenon of basal skin hydration in the untreated state was observed, indicating that the culture conditions induce a gradual loss of hydration in the skin explants. By applying compositions G and H, respectively, skin hydration was significantly improved relative to the untreated condition. Composition H of the present invention exhibited a significantly higher efficacy of +58% compared to composition G on day 2; and an increase of +45% compared to composition G on day 3.

[0108] To understand the difference in skin hydration between compositions G and H, immunostaining on aquaporin-3, a channel involved in water circulation in the skin and directly linked to skin moisturization, was performed on day 2. It was found that composition G had no significant effect on aquaporin-3 expression relative to the untreated condition, while composition H significantly increased its expression by +16% relative to the untreated condition, and with a significant effect also relative to composition G: [Table 5]

[0109] Furthermore, the effect of each composition on skin barrier function was analyzed through expression.It was found that composition G had no effect on filaggrin expression, while composition H significantly increased its expression by +35% compared to untreated condition and +36% compared to composition G. [Table 6] Thus, it was demonstrated that composition H is capable of improving skin hydration in rinse-off applications due to the biological effects associated with aquaporin-3 and filaggrin upregulation.

[0110] Example 6: Skin hydration test in rinse-off application: Comparison with high molecular weight HA Preparation of skin explants Fresh human skin explants from a female donor (age 40) who had undergone abdominal surgery were used in this study. The skin explants were topically treated for 5 minutes with composition H (see Example 5 above; containing 0.1% hydroxypropyltrimonium hyaluronate) or composition I (containing 0.1% sodium hyaluronate with a molecular weight of 1000-1400 kDa) as a moisturizing control, and compared to the untreated condition. After 5 minutes of treatment, the skin explants were rinsed five times with sterile water. These rinsing treatments were repeated daily for 2 days. Skin hydration was analyzed on fresh skin explants by Raman spectroscopy on day 0, which corresponds to skin explants without any treatment, and on day 2, which corresponds to the second day following the repeated treatment.

[0111] Raman spectroscopy analysis The same analysis was performed as in Example 5 above.

[0112] result It was found that composition I was unable to provide a moisturizing effect to the skin in rinse-off conditions, whereas composition H according to the invention increased skin hydration by +66% compared to the untreated condition and a significant effect compared to composition I. [Table 7]

[0113] Example 7: Skin hydration test for leave-on applications: Comparison with HA of the same molecular weight Preparation of skin explants Fresh human skin explants from a female donor (age 35) undergoing breast reduction surgery were used in this study. The skin explants were treated topically for 8 or 24 hours with one of the following four compositions: Composition G: 0.1% sodium hyaluronate with a molecular weight of 20 to 40 kDa (comparative example) Composition J: 0.1% hydroxypropyltrimonium hyaluronate prepared from the same sodium hyaluronate used in composition G, with a degree of cationization of 1.4%. Composition K: 0.1% hydroxypropyltrimonium hyaluronate prepared from the same sodium hyaluronate used in composition G, with a degree of cationization of 2.0%. Composition L: 0.1% Hyaloveil®-P (ex-Kewpie; hydroxypropyltrimonium hyaluronate with a molecular weight of 579 kDa and a degree of cationization of 0.6; comparative example)

[0114] Skin explants without any treatment were used as the untreated condition. After each incubation period, the excess product was gently absorbed onto cleaning paper and the skin explants were newly analyzed by Raman spectroscopy.

[0115] Raman spectroscopy analysis Axial Z-profiles were recorded directly on the skin samples. The Z-profile consisted of a detailed scan through the skin. Raman spectra were collected at different focal points on the skin surface from Z=0 μm to Z=30 μm in 3 μm increments. A total of 40 Raman profiles were recorded (4 profiles per condition, n=4).

[0116] In a first step, the exact location of the SC surface was determined for each Raman profile. In a second step, the lower limit of the SC was determined based on the water content. This involved calculating the νOH / νCH ratio. This location corresponds to the maximum value of the νOH / νCH ratio. The average spectrum of the SC was calculated considering all spectra acquired on the SC. After data processing (baseline correction, normalization, signal-to-noise ratio for spectral quality tests), water retention parameters were calculated for the average spectrum of the SC for each profile.

[0117] For assessment of skin hydration, the integrated intensity of the OH vibration band on the average SC spectrum was calculated, which represents the water content of the skin. The spectral range used for the calculation was νOH: 3100-3600 cm -1 It is.

[0118] result After 8 and 24 hours of application, the skin hydration properties of compositions G, J, K and L were compared to an untreated control. The results are shown in the table below: [Table 8]

[0119] After 8 hours, composition K showed a significant improvement in skin hydration compared to compositions G, J and L. After 24 hours, compositions G, J and L showed a gradual improvement in skin hydration relative to the untreated condition, however, skin hydration with composition K was still significantly better than with the others. Thus, it was demonstrated that a higher degree of cationization promotes skin hydration and provides sustained hydration.

[0120] Example 8: Skin hydration test for leave-on applications: Comparison with high molecular weight HA Preparation of skin explants Fresh human skin explants from a female donor (age 40) undergoing abdominal surgery were used in this study. The skin explants were topically treated with Composition H (of the invention) or Composition I (control) as described in Example 6 above for 8, 24 or 72 hours.

[0121] Skin explants without any treatment were used as the untreated condition. After each incubation period, the excess product was gently absorbed onto cleaning paper and the skin explants were newly analyzed by Raman spectroscopy.

[0122] Raman spectroscopy analysis The same analysis was performed as in Example 7 above.

[0123] result With regard to skin moisturization in leave-on applications compared to high molecular weight moisturizing hyaluronic acid (composition I), significantly better skin hydration was detected after 8 hours of treatment when using cationized hyaluronic acid (composition H): composition H exhibited +95% higher efficacy than composition I. After 24 and 72 hours, the two compositions were found to have almost the same efficacy. Thus, the composition of the present invention can provide a moisturizing effect faster. [Table 9]

[0124] Example 9: Hair Adhesion Test for Rinse-Off Applications Human hair tresses were immersed in a water bath and then massaged for two minutes with one of the following three shampoo compositions: Composition M: 0.1% sodium hyaluronate with a molecular weight of 20 to 40 kDa (comparative example) Composition N: 0.1% hydroxypropyltrimonium hyaluronate prepared from the same sodium hyaluronate used in composition L, with a degree of cationization of 2.0. Composition O: Placebo (comparative example) without hyaluronic acid or hyaluronic acid derivatives

[0125] The full formulation was as follows: [Table 10] Control tresses were left untreated with any shampoo. After shampooing, the tresses were rinsed three times in a controlled volume of water and dried with a hair dryer for 3 minutes.

[0126] result The hair deposition of M and N was compared with that of the placebo composition O. Alcian Blue staining allowed the visualization of the hyaluronic acid (derivatives) on the hair fibers. The results are shown in the table below: [Table 11]

[0127] It was found that the non-cationized hyaluronic acid of Composition M did not bind to the hair fiber compared to the placebo, while the cationized hyaluronic acid of Composition N showed significantly higher hair attachment of +52% compared to the placebo Composition O and +107% compared to the untreated, respectively.

[0128] Example 10: Hair repair testing in rinse-off applications using Scanning Electron Microscopy (SEM) and Atomic Force Microscopy (AFM) Surface visualization with a scanning electron microscope (SEM) To evaluate whether the deposition on hair fibers can form a protective layer against UV radiation, human hair tresses were immersed in a water bath and then massaged for 2 minutes with one of the following two shampoo compositions prior to UV treatment: Composition P: Placebo (comparative example) without hyaluronic acid or hyaluronic acid derivatives Composition Q: 0.1% hydroxypropyltrimonium hyaluronate prepared from sodium hyaluronate having a molecular weight of 20-40 kDa with a degree of cationization of 1.9.

[0129] The full formulation was as follows: [Table 12]

[0130] The control tresses were left untreated without any shampoo. After shampooing, the tresses were rinsed three times in a controlled volume of water and dried with a hair dryer for 3 minutes. Half of each hair bundle was irradiated with UV light once: 20 J / cm 2 UVA and 0.6J / cm 2 The hair surfaces were then visualized using a scanning electron microscope.

[0131] Malondialdehyde (MDA) and total protein content measurement To quantify the findings made in the SEM, MDA and total protein content were measured: MDA is a marker of lipid peroxidation; and UV irradiation significantly increases the MDA content in hair fibers, which reverses the oxidative stress after UV irradiation. And the total protein content makes it possible to assess hair damage: when a biological sample is damaged, proteins are degraded into shorter proteins, thereby increasing the total protein content in the sample.

[0132] Hair Roughness by Atomic Force Microscopy (AFM) Measurements were performed using a Nanowizard III atomic force microscope with an MLCT tip (Bruker). Three 25 μm x 25 μm acquisitions were made on three hair samples (approximately 50 μm in diameter) from each condition.

[0133] Nanoscale analysis was performed using JPK data processing software. Surface topography, hair roughness and mechanical properties (adhesion and elasticity) were measured. Roughness measurements were made on the altitude maps: curves showing the average roughness as a function of the surface (5 μm×5 μm, 12.5 μm×12.5 μm and 25 μm×25 μm) made it possible to analyze the roughness of each sample and to compare them.

[0134] result The hair surface was visualized before and after UV treatment: Figure 1 shows SEM images of hair fibres washed with either one of compositions P and Q or untreated before UV treatment; and Figure 2 shows SEM images of hair fibres washed with either one of compositions P and Q or untreated after UV treatment.

[0135] As can be seen from Figure 1, in basic conditions, before UV irradiation, the hair fiber treated with the composition Q of the present invention appears slightly smoother than the other three. This is the first confirmation that hydroxypropyltrimonium hyaluronate deposits on the hair fiber and smoothes the surface.

[0136] After UV exposure, the peeling of the keratin scales of the hair is clearly observed in the untreated state on the far left of Figure 2, proving the adverse effect of UV exposure on the keratin structure. Composition P (placebo) showed only a slight improvement compared to the untreated sample, while a clear smoothing effect was observed for composition Q according to the invention. These results suggest that the hydroxypropyltrimonium hyaluronate of the present invention can protect hair fibers against UV radiation.

[0137] The results of the MDA measurements are shown in the table below: [Table 13] As can be seen above, treatment with composition Q significantly reduced the MDA content in the hair fibres, demonstrating the protective effect of this composition against UV radiation.

[0138] The results of the total protein content determination are shown in the table below: [Table 14] As can be seen above, UV irradiation caused a significant increase in total protein content, while treatment with Composition P provided protection to the hair fibre.

[0139] While SEM gave a global view of the hair structure, AFM allowed them to focus on smaller scales, such as the 5 μm x 5 μm scale, which revealed an increase in the thickness of the keratin scales after UV irradiation in comparison to the untreated state, but also a kind of roughness on the surface of the scales.

[0140] Application of a placebo shampoo (Composition P) did not improve the hair surface and left visible roughness on the surface of the scale. However, when the hair tresses were treated with a shampoo containing 0.1% cationized hyaluronic acid (Composition Q), a visible smoothing of the surface of the scale was observed. Measurement of this roughness confirmed that UV irradiation significantly increased hair roughness by +31% compared to the untreated condition, with a similar effect for Composition P, while Composition Q significantly reduced hair roughness by -29% vs. the placebo condition. [Table 15]

[0141] Example 11: Hair protection testing in rinse-off applications using photonic birefringence Photonic birefringence analysis of hair protection from UV radiation Human hair tresses were immersed in a water bath and then massaged for two minutes with either one of the two shampoo compositions described in Example 10 above (Compositions P and Q, respectively). The control tresses were left untreated with any shampoo. After shampooing, the tresses were rinsed three times in a controlled volume of water and dried with a hair dryer for three minutes. This successive wash-dry cycle was repeated three times.

[0142] Then, 1 cm of each hair bundle was cut and spread in a Petri dish and UV irradiation: 9 J / cm 2 UVA and 0.33J / cm 2 The hair was treated with seven cycles of UVB. The three shampooing and seven UV irradiation cycles mimic a week of hair care and comprehensive daily UV exposure.

[0143] The hair modifications were then analyzed using photonic birefringence: the more the hair shaft is modified (by UV, heat, chemicals, etc.), the more the photonic birefringence decreases compared to an untreated hair shaft, whereas if the hair shaft is protected against treatment, the photonic birefringence will increase.

[0144] result The results are shown in the table below: [Table 16]

[0145] As can be seen above, UV irradiation significantly reduced photonic birefringence by -13.5% relative to the non-irradiated state, confirming the detrimental effect of daily UV exposure on hair keratin structure. Treatment with placebo composition P did not provide any significant improvement, while composition Q of the present invention was able to significantly improve photonic birefringence. Thus, the cosmetic composition of the present invention provides efficient UV protection for hair.

[0146] Example 12: Hair restoration testing in rinse-off applications using photonic birefringence Photonic birefringence analysis of hair repair after chemical stress Human hair tresses were chemically treated by applying a bleach solution containing 9% H2O2 and 3% ammonium persulfate three times for 1 hour at 40° C. After this treatment, the tresses were rinsed three times in an aqueous solution (200 ml) and then dried in an oven for 1 hour at 40° C. The tresses were straightened using a hair iron at 220° C. for 1 minute to increase the breaking of disulfide bonds and increase porosity (three passes).

[0147] The hair tresses were then immersed in a water bath and then massaged for two minutes with either one of the two shampoo compositions described in Example 10 above (Compositions P and Q, respectively). The control tresses were left untreated with any shampoo. After shampooing, the tresses were rinsed three times in a controlled volume of water and dried with a hair dryer for three minutes. This successive wash-dry cycle was repeated three times. The hair modifications were then analyzed using photonic birefringence.

[0148] result The results are shown in the table below: [Table 17]

[0149] A slight repair effect was observed with composition P, increasing the photonic birefringence by +5.6% relative to the deteriorated hair. However, this result was negligible in comparison with composition Q of the present invention, which was able to significantly improve the photonic birefringence by +24.9%, and which also had a significant effect on composition P. Thus, the cosmetic composition of the present invention provides efficient hair restoration.

Claims

**Claim 1** 1. Hyaluronic acid hydroxypropyltrialkylammonium and / or its salt having a cationization degree exceeding 1.

4. **Claim 2** 2. The hyaluronic acid hydroxypropyltrialkylammonium and / or its salt according to claim 1, wherein the hyaluronic acid hydroxypropyltrialkylammonium and / or its salt is selected from the group consisting of hyaluronic acid hydroxypropyltrimonium and / or its salt; hyaluronic acid hydroxypropyltriethylammonium and / or its salt; hyaluronic acid hydroxypropyltripropylammonium and / or its salt; and hyaluronic acid hydroxypropyltributylammonium and / or its salt. **Claim 3** 3. The hyaluronic acid hydroxypropyltrialkylammonium and / or its salt according to claim 2, wherein the hyaluronic acid hydroxypropyltrialkylammonium and / or its salt is hyaluronic acid hydroxypropyltrimonium and / or its salt. **Claim 4** 4. The hyaluronic acid hydroxypropyltrialkylammonium and / or its salt according to claim 1, having a cationization degree of at least 1.5, more preferably at least 1.6, still more preferably at least 1.7, and most preferably at least 1.

8. **Claim 5** 5. The hyaluronic acid hydroxypropyltrialkylammonium and / or its salt according to claim 1, wherein the hyaluronic acid hydroxypropyltrialkylammonium and / or its salt is prepared from hyaluronic acid or its salt having an average molecular weight of about 10 kDa to about 200 kDa, more preferably about 15 kDa to about 150 kDa, still more preferably about 20 kDa to about 100 kDa, and most preferably about 20 kDa to about 80 kDa. **Claim 6** 6. The hyaluronic acid hydroxypropyltrialkylammonium and / or its salt according to claim 1, comprising or consisting of a chloride salt of hyaluronic acid hydroxypropyltrialkylammonium. **Claim 7** A method for preparing hyaluronic acid hydroxypropyltrialkylammonium and / or a salt thereof according to any one of claims 1 to 6, comprising the step of reacting hyaluronic acid and / or a salt thereof with a cationizing agent in the presence of a base, wherein the cationizing agent is selected from the group consisting of 2,3-epoxypropyltrialkylammonium chloride, 2-chloro-3-hydroxypropyltrialkylammonium chloride, and mixtures thereof, said method.

8. The method according to claim 7, wherein the cationizing agent is selected from the group consisting of 2,3-epoxypropyltrimonium chloride, 2-chloro-3-hydroxypropyltrimonium chloride, and mixtures thereof.

9. The method according to claim 7, wherein about 1.5 to about 20 equivalents of the cationizing agent are used.

10. The method according to claim 7, wherein the reaction is carried out at a concentration of hyaluronic acid and / or a salt thereof of about 0.01 to about 1.00 g / ml, more preferably about 0.05 to about 0.50 g / ml.

11. A cosmetic composition comprising hyaluronic acid hydroxypropyltrialkylammonium and / or a salt thereof according to any one of claims 1 to 6 and a suitable carrier.

12. The cosmetic composition according to claim 11, which is a hair care or skin care composition.

13. Use of hyaluronic acid hydroxypropyltrialkylammonium and / or a salt thereof according to any one of claims 1 to 6 for water retention and / or UV protection and / or hair repair.

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