Chitosan derivatives are well-known compounds within the cosmetology field. Mention may be made for example of patent US 3,879, 376, which relates to chitosan derivatives which can be employed particularly as a skin moisturizer or film former in cosmetic skincare or cleansing products.

Mention may also be made of US 4,765, 976, which relates to polymers derived from chitosan which can be used to treat the greasy appearance of hair.

Among the very many derivatives of chitin or of chitosan (partially deacetylated chitin) one of the known classes is that of the N-carboxymethylchitosans (called"N-CMC"hereinafter). These derivatives are generally prepared by reacting a chitosan with a carboxymethylating agent such as glyoxylic acid (HO2CCHO), which allows the reaction to be oriented on the nitrogen atom rather than on the oxygen atom; a second step consists in reducing the imine formed (reductive amination).

Mention may be made in particular of the publication of Muzzarelli et al., Carbohydr. Res.

(1982), 107, 199-214, which describes a process for preparing N-CMC by reacting the free amino groups of

shrimp-extract chitosan with glyoxylic acid to give a soluble imine, which is reduced using cyanoborohydride.

The N-CMC obtained contain free amino moieties, amino moieties which are acetylated, and N-carboxymethyl moieties, in proportions which are controlled, say the authors, as a function of the initial degree of acetylation and of the molecular weight of the chitosan, and of the amount of glyoxylic acid employed.

Mention may also be made of the Muzzarelli et al. publication Carbohydr. Polym. (1988), 8 (1), 1-21, which mentions that the solubility of the N-CMC obtained may vary depending on the nature of the chitosan employed. This document describes the preparation, by reacting chitosan with glyoxylic acid, followed by a reduction with NaBH4, of N-CMC in which all of the free amine moieties have reacted to give, on the one hand, carboxymethyl moieties (-NHCH2-COOH) and, on the other hand, acetamido moieties (-NHCOCH3), the latter moieties being already present in the starting product.

Other chitosan derivatives have been prepared in the Rinaudo et al. publication Int. J. Biol.

Macromol. (1992), 14 (6), 122-128, and are in the form of totally soluble N-CMC in which all of the free amines have reacted, to give rise to monocarboxylated secondary amines and dicarboxylated tertiary amines in a ratio of 70/30.

The Muzzarelli et al. document Int. J. Biol.

Macromol. (1994), 16 (4), 177-180 describes, for its part, water-soluble N-CMC prepared from starting chitosan possessing a weight-average molecular mass (Mw) of 700,000. It is specified in that document, moreover, that an excess of glyoxylic acid in the first reaction step results in an insoluble N-CMC being obtained.

It has been noted, however, that the chitosan derivatives prepared in the literature do not all exhibit very good solubility in water. This is because some of the N-CMC obtained have a fraction which is insoluble in water, and which has to be removed in order for them to be completely water-soluble.

Moreover, the prior-art N-CMC synthesis processes are difficult to reproduce on an industrial scale. In particular the first reaction step (the formation of the imine) must be carried out, according to the literature, on a highly dilute aqueous chitosan solution, containing of the order of 6 g to 14 g of chitosan per litre of water, corresponding to a concentration of 0. 6% to 1.4% by weight. It has been noted, in the prior art, that this dilution of the chitosan is necessary in order to prevent possible problems of gelling of the mixture during the formation of the N-CMC. However, the use of a highly dilute solution of chitosan is not desirable from an industrial standpoint, particularly since it implies,

among other things, the use of large-sized reactors if the desire is to prepare industrial quantities of N-CMC.

The aim of the present invention is to provide new N-carboxymethylchitosan derivatives which are totally soluble in aqueous media within a wide pH range, thereby allowing them in particular to be employed advantageously in the cosmetic and pharmaceutical fields.

Furthermore, another aim of the present invention is to provide a new process for synthesizing N-CMC which is simple to implement and can be transposed to the industrial scale with a substantial productivity.

The present invention accordingly first provides a process for preparing chitosan derivatives, comprising the following steps: in a first step, reacting a chitosan with a carboxymethylating agent to form an imine, in a second step, reducing the imine formed, by using a reducing agent, characterized in that the weight-average molecular mass (Mw) of the starting chitosan is between 50 000 and 160 000, in particular between 60 000 and 150 000, and/or the level or degree of acetylation (DA) of the said chitosan is greater than or equal to 12%, in particular between 12% and 27%, or even 15%-25%,

preferably 20%-24%.

The invention secondly provides a process for preparing chitosan derivatives, comprising the following steps: in a first step, reacting a chitosan with a carboxymethylating agent to form an imine, in a second step, reducing the imine formed, by using a reducing agent, characterized in that the average molecular mass Mw and/or the degree of acetylation of the chitosan are selected such that the viscosity at 25°C of a solution of the said chitosan at 1% by weight in an aqueous solution containing 1% by weight of acetic acid is less than or equal to 4 mm2/s, in particular between 1 and 3.5 mm2/s, especially between 1.5 and 3.2 or even between 2.5 and 3.0 mm2/s.

The invention additionally provides the chitosan derivatives of the formula given below.

The invention further provides a composition, particularly a cosmetic or pharmaceutical composition, comprising the said chitosan derivatives.

It has been found that, surprisingly, the process according to the invention, and especially the selection of the starting chitosan, makes it possible to obtain a chitosan derivative which no longer contains a free amine unit (-NH2) and which is totally soluble in water. Moreover, the process according to

the invention makes it possible to prepare N-CMC without having to carry out substantial dilution in water in order to employ the process, without, moreover, gelling of the mixture being observed.

This has the advantage, in particular, of making the process easier to exploit on the industrial scale, by enhancing the productivity and lowering the industrial cost price of the N-CMC: this is because the process according to the invention is characterized primarily in that the aqueous solution of chitosan is particularly concentrated especially in the first step, and possibly in the second step, of the process, thereby making it possible to reduce the size of the reactors employed, to generate less"waste"for recycling, to make energy savings, to improve the recovery of the end product, by having a more concentrated solution to filter, which also makes it possible to improve the yield.

Moreover, the end product obtained exhibits excellent cosmetic characteristics which are entirely reproducible from one batch to the other; furthermore, the end product is totally water-soluble at 25°C.

According to the process for preparing chitosan derivatives according to the invention the first step consists in reacting, in water, a chitosan with a carboxymethylating agent to form an imine.

The starting chitosan may be of any origin,

especially from crab or from shrimp.

Its weight-average molecular mass (M) is preferably between 50 000 and 160 000, in particular between 60 000 and 150 000. Mw is determined by gel permeation liquid chromatography (solvent: aqueous solution containing 0.3 M acetic acid + 0.1 M ammonium acetate, in 1 litre of water; calibration curve constructed using dextran standards, refractometric detector).

Furthermore, and advantageously, the chitosan employed preferably has a level or degree of acetylation (DA) of greater than or equal to 12%, in particular between 12% and 27%, or even 15%-25%, preferably 20%-24%.

The DA is determined by NMR or from the amine index.

The starting chitosan preferably has an amine index of less than or equal to 5.31 meq/g, in particular between 4.23 and 5.29 meq/g, especially between 4.37 and 5.08 meq/g, or even between 4.44 and 4.72 meq/g.

The amine index is determined by potentiometric assay (back-titration assay with NaOH of an acidic solution of chitosan).

This is because it has been found that the use of an initial chitosan having an average molecular mass, and preferably a degree of acetylation as well, that are selected from within the above ranges makes it

possible advantageously to avoid excessive dilution of the chitosan in water when preparing the N-CMC, hence in the first and second steps of the process, especially in the first step (formation of the imine).

It is therefore possible to work at a chitosan concentration of from 5% to 10% by weight, in particular from 6% to 9% by weight, or even from 7% to 8% by weight, in water, in the 1st reaction step, without observing any increase in the viscosity of the solution or any formation of gel.

The reaction mixture remains continuously fluid, despite the fact that the concentration of chitosan is greater than in the prior-art processes.

When the molecular mass Mw of the chitosan is greater than approximately 150 000 the formation of a gel is generally observed during the reaction.

Accordingly, commonly, the viscosity at 25°C of a solution of chitosan of Mw 100 000 at 1% by weight in an aqueous solution containing 1% by weight of acetic acid is approximately 2.5 cSt (2.5 mm2/s) as measured in an Ubbelohde capillary tube viscometer, at 25°C and 1 atm.

The viscosity of the same solution of chitosan with an initial Mw of the order of 200 000, however, will be approximately 5 cSt (5 mm2/s), which is equivalent to a gelled solution.

The average molecular mass Mw and/or the

degree of acetylation of the chitosan are preferably selected such that the viscosity at 25°C of a solution of the said chitosan at 1% by weight in an aqueous solution containing 1% by weight of acetic acid is less than or equal to 4 cSt (4 mm2/s), in particular between 1 and 3.5 mm2/s, especially between 1.5 and 3.2 mm2/s, or even between 2.5 and 3.0 mm2/s.

The carboxymethylating agent used in the first reaction step may be an oxo acid, and especially oxalacetic acid, pyruvic acid or glyoxylic acid of formula CH (O)-COOH.

The carboxymethylating agent is very preferably present in excess relative to the chitosan, in particular in an amount of from 2 to 4 molar equivalents per equivalent of amine present in the starting chitosan.

Preferably the first reaction step is carried out at a pH of between 2 and 3.2, in particular at a pH of between 2.3 and 3.0, and hence without addition of base such as NaOH, and at ambient temperature (20-25°C).

The second step of the process consists in reducing the imine formed, using a reducing agent, such as sodium borohydride or sodium cyanoborohydride.

It is possible in particular to add a molar amount of sodium (cyano) borohydride which is greater than or equal to the amount of moles of carboxymethylating agent, glyoxylic acid in particular;

preferably the reducing agent, in particular the sodium (cyano) borohydride, is added in an equimolar amount relative to the amount of carboxymethylating agent.

The second reaction step is preferably carried out at a temperature of between 5 and 20°C, preferably between 10 and 15°C.

The N-CMC thus obtained may then be precipitated, in methanol or ethanol for example, and then filtered and dried according to the usual methods.

The reaction scheme may be illustrated as follows:

It has been found that this novel process for preparing chitosan derivatives is particularly advantageous: it allows these derivatives to be

prepared on the industrial scale in so far as it can be conducted in standard light equipment under pH, pressure and temperature conditions which can be employed without danger on the industrial scale.

Furthermore, it does not require large amounts of water for its implementation, particularly in the first step of the process. This is because of the specific choice of the initial chitosan, which makes it possible to operate at a higher concentration of product in water than in the prior art, without any increase in viscosity being noted.

Finally all of the glucosamine units are substituted and the N-CMC are totally water-soluble.

By water-soluble is meant that the N-CMC of formula (I) form a homogeneous aqueous solution, without visually apparent deposition, at a concentration of 10% by weight in water at 25°C.

It has even been noted that the chitosan derivatives of formula (I) according to the invention could advantageously still be soluble in water at 25°C at a concentration which may range up to 20% by weight, or even 25% by weight.

The chitosan derivatives preparable according to the process of the present invention are new products per se. They correspond to the following formula (I):

in which x, y and z are such that x + y + z = 100, with x being between 12 and 27 inclusive, y being between 60 and 81 inclusive and z being between 3 and 17 inclusive.

Preferably x is between 15 and 25 inclusive or even 20-24 inclusive.

Preferably z is between 3 and 10 inclusive, in particular between 4 and 6 inclusive.

Preferably y is between 65 and 77 inclusive, in particular between 70 and 74 inclusive.

The values x, y and z are statistical values and represent the relative molar proportion (molar %) of each of the units; x corresponds to the initial degree of acetylation of the chitosan and can be determined by NMR or from the amine index of the initial chitosan.

The values of y or z can be determined by 13C NMR.

Preferably the chitosan derivatives according to the invention have a viscosity at 25°C and 1 atm in solution at 20% by weight in an aqueous solution of less than or equal to 22 000 mPa. s, in particular between 2000 and 21 600 mPa. s, preferably between 2500 and 18 000 mPa. s, or even between 3000 and 15 500 mPa. s.

The viscosity of the chitosan derivatives is measured at 25°C by means of a Rheomat rheometer at a shear rate (imposed stress) of 200 s-1.

Preferably the chitosan derivatives have a weight-average molecular mass (Mw) of between 200 000 and 600 000, preferably between 400 000 and 550 000; the average molecular mass is measured by gel permeation liquid chromatography, with an aqueous solution containing 0.1 M NaHC03 and 0.1 M Na2CO3 per litre of water, as solvent; the calibration curve is drawn up with dextran standards, and the detector is refractometric.

The chitosan derivatives according to the invention may advantageously be prepared according to the process described above.

These derivatives find particular application in the cosmetic and/or pharmaceutical field, especially in topical application.

The invention therefore likewise provides cosmetic or pharmaceutical compositions comprising, in a cosmetically or pharmaceutically acceptable medium, at least one chitosan derivative according to the invention.

The said derivative may be present in a proportion of from 1% to 20% by weight in the composition, in particular from 2% to 12. 5% by weight or even from 5% to 10% by weight.

In accordance with the desired application the cosmetically or pharmaceutically acceptable medium may comprise the customary adjuvants which are incorporated into cosmetic or pharmaceutical compositions.

Among these adjuvants mention may be made of fatty substances, and particularly hydrocarbon and/or silicone waxes, oils, gums and/or pasty fats, and pulverulent compounds such as pigments, fillers and/or nacres.

Among the waxes that may be present in the composition according to the invention mention may be made, alone or in a mixture, of hydrocarbon waxes such as beeswax; carnauba wax, candelilla wax, ouricury wax, Japan wax, cork fibre wax or sugarcane wax; paraffin wax and lignite wax; microcrystalline waxes; lanolin wax; montan wax; ozokerites; polyethylene waxes; waxes obtained by Fischer-Tropsch synthesis; hydrogenated oils, fatty esters and glycerides which are solid at 25°C. It is also possible to use silicone waxes, among which mention may be made of the alkyls, alkoxys, and/or polymethylsiloxane esters.

Among the oils that may be present in the composition according to the invention mention may be made, alone or in a mixture, of hydrocarbon oils such as liquid paraffin or vaseline; perhydrosqualene; acara oil; sweet almond oil, calophyllum oil, palm oil, castor

oil, avocado oil, jojoba oil, olive oil or cereal germ oil; esters of lanolic acid, of oleic acid, of lauric acid, of stearic acid; alcohols such as oleyl alcohol, linoleyl alcohol or linolenyl alcohol, isostearyl alcohol or octyl dodecanol. Mention may also be made of silicone oils such as PDMS, optionally in phenylated form such as the phenyltrimethicones. Mention may also be made of volatile oils, such as cyclotetradimethyl- siloxane, cyclopentadimethylsiloxane, cyclohexadi- methylsiloxane, methylhexyldimethylsiloxane, hexa- methyldisiloxane or isoparaffins.

The pigments may be white or coloured, mineral and/or organic. Among mineral pigments mention may be made of titanium dioxide, zirconium dioxide or cerium dioxide, and also zinc oxide, iron oxide or chromium oxide, and ferric blue. Among organic pigments mention may be made of carbon black and lakes of barium, strontium, calcium or aluminium.

The nacres may be selected from mica covered with titanium oxide, with iron oxide, with natural pigment or with bismuth oxychloride, and also coloured titanium mica.

The fillers may be mineral or synthetic, lamellar or non-lamellar. Mention may be made of talc, mica, silica, kaolin, nylon powder and polyethylene powder, Teflon, starch, mica-titanium, natural nacre, boron nitride, hollow microspheres such as Expancel (Nobel

Industrie), polytrap (Dow Corning) and silicone resin microbeads (Tospearls from Toshiba, for example).

The medium may further comprise water and/or an organic solvent such as a C1-C8 alcohol or a polyol, for example glycerol.

The composition may further comprise any additive which is commonly used in the cosmetic field, such as antioxidants, perfumes, essential oils, preservatives, cosmetic actives, moisturizers, vitamins, essential fatty acids, sphingolipids, sunscreens, surfactants, fat-soluble polymers such as polyalkylenes, especially polybutene, polyacrylates and silicone polymers which are compatible with the fatty substances. The skilled person will or course take care to select this or these possible complementary compounds, and/or the amount thereof, in such a way that the advantageous properties of the composition according to the invention are not, or not substantially, adversely affected by the contemplated addition.

The compositions according to the invention may be present in any form which is acceptable and customary for a cosmetic or pharmaceutical composition, and particularly in the form of a hair composition, especially a hair washing, hair care or hair shape retention composition; a face or body skin care composition; or a composition for making up the face, body, nails or hair.

The present invention likewise provides a cosmetic treatment process for the care, cleansing and/or making-up of keratin materials such as the skin of the body or face, the scalp, eyebrows, eyelashes, lips and nails, which consists in applying to the said keratin materials a composition as defined above.

The invention is illustrated in greater detail in the examples below.

Example 1 A reactor is charged with 1 kg of shrimp chitosan, with a weight-average molecular mass (Mw) of 148 900 and a degree of acetylation of 23%, 1.14 litres of 50% aqueous glyoxylic acid solution and 9.86 litres of water.

This initial charge is mixed at 20°C for 1 hour until the chitosan has completely dissolved; stirring of the solution is continued at 20°C for 6 hours. The solution has a pH of 3.

A solution is prepared containing 283 g of NaBH4, 910 ml of water and 20 g of 35% sodium hydroxide.

The reaction mixture is cooled to 10°C and then the solution of NaBH4 is run in over 1 hour, during which the temperature is held below 15°C. Stirring is continued for 1 hour after the end of the addition of NaBH4 (the pH is 7.4).

In a second reactor 35 litres of methanol are

cooled to 10°C and then the reaction mixture containing the product is poured on top.

A white precipitate forms which is isolated by filtration, washed with isopropanol and dried under vacuum at 40°C.

This gives 1.15 kg of a light beige powder (yield: 76%).

Characterization of the end product Qualitative 13C NMR (D2O + DCl, 125 MHz) : # (ppm) 29.95 (CH3, Ac), 54.05 (C8, (I)), 58.28 (C2, Ac), 59.08 (C8, (II)), 63. 01-63. 53 (C6), 64. 36 (CH2, sodium glycolate), 65.73 (C2, (I)), 69.79 (C2, (II)), 72.57 (C3, (II)), 74.61-75. 56 (C3, (I)), 77. 40-77.58 (C5), 80.03-82. 14 (C4), 102.16-104. 22 (C1), 176.99 (C=O, Ac), 180.55- 180.88 (C9), 182.08 (C=O, sodium glycolate) Degree of substitution (molar): x = 24%, y = 72%, z = 4% Sodium glycolate content: 6% by mass (determination by qualitative 13 C analysis).

GPC analysis: Mw = 492 400 ; Mn = 67 700 ; Ip (polydispersity index) = 7. 27 Viscosity (at 25°C) of an aqueous solution containing 20% solids of the derivative, Rheomat rheometer, shear rate 200 s-1 : 15 300 mPa. s Turbidity zone (range of insolubility of an aqueous solution containing 2% by weight of the derivative): between a pH of 2.4 and 5.3 Spontaneous pH of an aqueous solution containing 2% by weight of the derivative: 7.9 Solubility: greater than 20% by weight in water at 25°C It is therefore found that, with the process according to the invention, a derivative of chitosan is obtained for which all of the free amine functions are substituted.

Example 2 New derivatives of chitosan according to the invention are prepared in accordance with Example 1, varying the weight-average molecular mass (Mw) and/or the degree of acetylation (DA) of the starting chitosan.

The following different N-CMC derivatives are obtained: Mw of x y z Sodium M ; ; of Viscosity the (DA) glycol-the at 25°C starting ate deri- (mPa. s) chitosan content vative obtained Ex. 2a 140 000 25 64 11 11% 592 800 2950 Ex. 2b 140 000 24 61 15 12% 455 600 3450 Ex. 2c 60 000 22 61 17 9% 186 600 650 Ex. 2d 60 000 20 72 8 5% 179 400 710 Ex. 2e 100 000 16 81 3 7% 410 900 15 300

Example 3 A mascara is prepared comprising the following ingredients (in % by weight): - waxes (beeswax, carnauba wax) 20% - stearic acid 5. 8% - 99% triethanolamine 2. 4% - chitosan derivative according to Example 1 5% - pigment millbase (black iron oxide 50/ propylene glycol 50) 14% - preservative qs - water qs 100% Preparation: The waxes and the stearic acid are melted at around 80-90°C. When melting is complete the pigment millbase and then the aqueous phase and the chitosan derivative are added with vigorous stirring (Moritz stirrer).

Stirring is continued for 20 minutes and then the batch is returned to ambient temperature (25°C) with moderate stirring.