US20210128442A1 - Organic compounds - Google Patents

Organic compounds Download PDF

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US20210128442A1
US20210128442A1 US16/486,710 US201816486710A US2021128442A1 US 20210128442 A1 US20210128442 A1 US 20210128442A1 US 201816486710 A US201816486710 A US 201816486710A US 2021128442 A1 US2021128442 A1 US 2021128442A1
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molecular weight
hyaluronic acid
low molecular
skin
acetylated hyaluronic
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Martin Lovchik
Gerhard Brunner
Romain Reynaud
Amandine SCNDOLERA
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Givaudan SA
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Givaudan SA
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/08—Anti-ageing preparations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
    • A61K8/73—Polysaccharides
    • A61K8/735—Mucopolysaccharides, e.g. hyaluronic acid; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
    • C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
    • C08B37/0072—Hyaluronic acid, i.e. HA or hyaluronan; Derivatives thereof, e.g. crosslinked hyaluronic acid (hylan) or hyaluronates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
    • A61K2800/59—Mixtures
    • A61K2800/594—Mixtures of polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/74—Biological properties of particular ingredients
    • A61K2800/78—Enzyme modulators, e.g. Enzyme agonists
    • A61K2800/782—Enzyme inhibitors; Enzyme antagonists
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
    • A61K2800/84—Products or compounds obtained by lyophilisation, freeze-drying

Definitions

  • the invention relates to active ingredients and methods that are useful to reduce the visible signs of aging on human skin. More specifically, the invention relates to the use of acetylated hyaluronic acid or its sodium salt, to inhibit certain matrix-degrading enzymes that are involved in chronological-induced and UV-induced skin deterioration. The invention also relates to novel forms of acetylated hyaluronic acid useful in reducing the visible signs of aging, and to methods of preparing same.
  • the structural framework of the skin is referred to as its extracellular matrix.
  • This internal framework comprises a network of inter-meshed polymers, such as collagen and elastin, inside which skin cells are contained. It is responsible for the skin's mechanical properties, including firmness, strength, suppleness and elasticity.
  • the physical signs of skin aging are a reflection of the condition of the skin matrix. More particularly, the weaker and less regular the matrix, the more wrinkles, roughness and sag the skin tends to have.
  • the skin's extracellular matrix is a precious resource, which in healthy and youthful skin is both produced and consumed.
  • the smoothness, firmness and youthfulness of human skin depend to a large degree on the condition of its matrix, which, in turn, depends on the balance of matrix synthesis and matrix break-down/recycling.
  • the skin's extracellular matrix is composed of collagens (type I collagen being the most prevalent); elastins; and other components such as glycosaminoglycans (GAGs).
  • GAGs glycosaminoglycans
  • the matrix is synthesized by fibroblasts in the dermis, and it is subject to remodeling by certain specific enzymes (the matrix metalloproteinases, or MMPs).
  • MMPs are usually involved in wound healing, but they are over-expressed during chronological and UV-induced aging, whereupon they promote intense and uncontrolled matrix break-down, which is known to be responsible for the visible signs of aging, such as skin collapse and wrinkle formation.
  • MMPs There are around 20 MMPs that are grouped into different categories according to their substrate specificity. Among them are the collagenases, gelatinases, stromelysins, and membrane-type MMPs.
  • MMP-1 is the most well-known collagenase and is involved in the cleavage of structural collagens, such as the type-1 collagen.
  • MMP-3 is a stromelysin that is involved in the cleavage of basement membrane collagens, such as collagen IV.
  • Matrix proteins are large, structural molecules that have long half-lives (in the order of 70 years in the case of elastin). Consequently, these materials are hardly replenished or renewed over the normal human life-span. As such, there is a progressive alteration and degradation of the matrix that accelerates with age, disrupting and weakening the skin's scaffold and creating the visible signs of aging.
  • Skin aging is the result of the intrinsic chronological aging process superimposed with environmental factors, predominantly consisting of exposure to ultraviolet radiation. MMP levels rise excessively in the course of normal chronological aging, and the environmental factors act to elevate MMP levels still further. Reducing the levels of MMP enzymes and returning them to normal, youthful levels, which are sufficient to alleviate matrix degradation, and preserve a healthy matrix, may help to reduce or eliminate the visible signs of aging on human skin.
  • the invention relates to the use of acetylated hyaluronic acid or its sodium salt (sodium acetyl hyaluronate) to control the balance of matrix-degrading materials in the skin's extra-cellular matrix, reducing the extent of degradation of essential matrix proteins such as collagen, and to reduce the visible signs of aging (chronologically-induced and environmentally-induced), particularly the prevention or reduction of skin wrinkles and to the production and maintenance of youthful looking skin.
  • acetylated hyaluronic acid or its sodium salt sodium acetyl hyaluronate
  • a method of inhibiting the degradation of the skin's extracellular matrix associated with elevated levels of matrix-degrading enzymes comprising the step of applying to the skin an effective amount of acetylated hyaluronic acid or its sodium salt, in particular in the form of the skin care composition of the present invention.
  • the method described above which is a method for reducing the visible signs of skin aging, more particularly a method of preventing or reducing wrinkles.
  • the matrix-degrading enzymes are matrix metalloproteinases, and more particularly the enzymes MMP-1 or MMP-3.
  • acetylated hyaluronic acid or its sodium salt acts to reduce the extent of degradation of collagen in the extra-cellular-matrix, in particular the degradation by metalloproteinases.
  • acetylated hyaluronic acid, or its sodium salt, described above having a weight average molecular weight of about 50 kDa or less, more particularly about 35 kDa or less, and still more particularly about 30 kDa or less.
  • acetylated hyaluronic acid, or its sodium salt, described above having a weight average molecular weight of about 50 kDa or less, more particularly about 35 kDa or less, and still more particularly about 30 kDa or less, and a polydispersity index that is less than 2.3, more particularly less than 2.0, more particularly less than 1.8, more particularly less than 1.7, more particularly less than 1.6 or less, more particularly less than 1.5, more particularly less than 1.4, and more particularly about 1.3 or less.
  • acetylated hyaluronic acid, or its sodium salt, described above having a degree of acetylation that is greater than 3.6, more particularly 3.7 or greater, more particularly 3.8 or greater, more particularly 3.9 or greater, and more particularly still 4.
  • acetylated hyaluronic acid, or its sodium salt, described above having a degree of acetylation that is greater than 3.6, more particularly 3.7 or greater, more particularly 3.8 or greater, more particularly 3.9 or greater, and more particularly still 4 determined using quantitative 2D NMR.
  • a cosmetic preparation comprising an effective amount of the acetylated hyaluronic acid, or its sodium salt, described above.
  • a skin care composition comprising low molecular weight acetylated hyaluronic acid or its sodium salt, characterized in that the low molecular weight acetylated hyaluronic acid or its sodium salt has a weight average molecular weight of 50 kDa or less and an average degree of acetylation of greater than 3.6.
  • the cosmetic preparation and in particularly the skin care composition, described above, comprising at least one cosmetically acceptable excipient.
  • Skin-aging is a term that refers to the changes experienced by the skin with age, whether that is through chronological aging or through exposure to the sun (photo-aging) or through other environmental agents such as tobacco smoke, extreme climatic conditions of cold, heat, or wind, chemical contaminants or pollutants, and includes all the external visible and/or perceptible changes through touch, such as but not restricted to, the development of discontinuities on the skin such as wrinkles, fine lines, furrows, irregularities or roughness, increase in the size of pores, loss of elasticity, loss of firmness, loss of smoothness, loss of the capacity to recover from deformation, sagging of the skin such as sagging cheeks, the appearance of bags under the eyes or the appearance of a double chin, among others, changes to the colour of the skin such as marks, reddening, or the appearance of hyper-pigmented areas such as age spots or freckles among others, anomalous differentiation, hyper-keratinization, elastosis, keratosis, loss of collagen structure and other histological changes of the strat
  • MMPs matrix metalloproteinases
  • acetylated hyaluronic acid or its sodium salt, is able to reduce the levels of MMPs, and in particular MMP-1 and MMP-3. It is believed, although the applicant does not wish to be bound by theory, that the lower level of MMPs is a result of down-regulation of genes encoding for these enzymes.
  • acetylated hyaluronic acid or its sodium salt of the present invention has been demonstrated in certain gene expression studies performed on the three main cell types present in the skin: fibroblasts, keratinocytes and melanocytes.
  • the biological impact of acetylated hyaluronic acid, or its sodium salt, was also determined by in-vitro protein and functional studies. We have found that it presents a new anti-aging activity linked to limiting matrix degradation and protecting the skin against age signs. The studies are described in more detail herein below in the examples.
  • acetylated hyaluronic acid, or its sodium salt when applied to the skin, can elicit a biological effect that is observable as a reduction in the visible signs of skin aging, such as a reduction in skin-wrinkles enables the skilled person in the art to provide cosmetic preparations for application to the skin of a human subject.
  • Cosmetic preparations, and in particular skin care compositions, of the present invention contain a cosmetically acceptable amount of acetylated hyaluronic acid or its sodium salt.
  • a cosmetically effective amount of acetylated hyaluronic acid or its sodium salt is understood to be a non-toxic but sufficient quantity of acetylated hyaluronic acid or the sodium salt to provide the desired effect.
  • the acetylated hyaluronic acid or its sodium salt may be present in amounts of about 0.005 to about 5.0 wt %, and more particularly about 0.05 to about 0.5 wt %.
  • Cosmetic preparations, and in particular skin care compositions, of the present invention may contain one or more cosmetically acceptable excipients. Any excipients commonly used in the preparation of cosmetic preparations for use on the human skin may be employed in the present invention. Suitable excipients include, but are not limited to ingredients that can influence organoleptic properties, penetration of the skin, and the bioavailability of the acetylated hyaluronic acid or it sodium salt.
  • liquids such as water, oils or surfactants, including those of petroleum, animal, plant or synthetic origin, such as and not restricted to, peanut oil, soybean oil, mineral oil, sesame oil, castor oil, polysorbates, sorbitan esters, ether sulfates, sulfates, betaines, glycosides, maltosides, fatty alcohols, nonoxynols, poloxamers, polyoxyethylenes, polyethylene glycols, dextrose, glycerol, digitonin, and the like.
  • the cosmetic preparation may be in the form of a liposome composition, mixed liposomes, oleosomes, niosomes, ethosomes, milliparticles, microparticles, nanoparticles and solid-lipid nanoparticles, vesicles, micelles, mixed micelles of surfactants, surfactant-phospholipid mixed micelles, millispheres, microspheres and nanospheres, lipospheres, millicapsules, microcapsules and nanocapsules, as well as microemulsions and nanoemulsions, which can be added to achieve a greater penetration of the acetylated hyaluronic acid or its sodium salt.
  • the cosmetic preparation may be produced in any solid, liquid, or semi-solid form useful for application to the skin topically or by transdermal application.
  • these preparations of topical or transdermal application include, but are not restricted to, creams, multiple emulsions, such as and not restricted to, oil and/or silicone in water emulsions, water-in-oil and/or silicone emulsions, water/oil/water or water/silicone/water type emulsions, and oil/water/oil or silicone/water/silicone type emulsions, micro-emulsions, emulsions and/or solutions, liquid crystals, anhydrous compositions, aqueous dispersions, oils, milks, balsams, foams, aqueous or oily lotions, aqueous or oily gels, cream, hydro-alcoholic solutions, hydro-glycolic solutions, hydrogels, liniments, sera, soaps, face masks, serums, poly
  • acetylated hyaluronic acid or its sodium salt for use in the present invention are characterized in that they have a very high degree of acetylation. More particularly, preferred forms of acetylated hyaluronic acid are characterized by an average degree of acetylation of greater than 3.6, more particularly 3.7 or higher, more particularly 3.8 or higher, more particularly 3.9 or higher, and more particularly still 4.
  • Hyaluronic acid is a polysaccharide consisting of a repeat unit containing N-acetyl glucosamine and glucuronic acid. It can exist in its free-acid or sodium salt form.
  • degree of acetylation is understood as a measure of the number of hydroxyl groups on the polymer repeat unit that are acetylated.
  • Each repeat unit of native hyaluronic acid/salt polymer contains four hydroxyl groups that can be acetylated and the degree of acetylation is a measure of how many of these groups are substituted by an acetyl group.
  • Measuring the degree of acetylation is particularly challenging using conventional analytical techniques. For example, we found that traditional one dimensional NMR is ineffective because of the coincidence of chemical shifts of the protons that need to be measured.
  • the 2D-NMR method is a hetero-nuclear method (Hetero-nuclear Single Quantum Coherence (HSQC) analysis), measuring a proton signal in one dimension, and the signal from carbon nuclei in the second dimension.
  • HSQC Hetero-nuclear Single Quantum Coherence
  • the 1 H- 13 C coupling constant provides information concerning the connectivity of atoms in a molecule, and in NMR spectroscopy it is responsible for the appearance of many signals in a spectrum.
  • the integration volumes of cross-peaks in a HSQC experiment are modulated by the 1 H- 13 C coupling constant 1 J C,H .
  • cross-peaks arising from different coupling constants usually do not permit quantitative integration.
  • a sufficient relaxation delay may be provided in order to ensure that both N-acetyl and O-acetyl protons have time to relax from one scan to another, in order to stay in quantitative conditions. Additionally, in order to restrain differentiated T 2 relaxation between the N- and O-acetyl species of interest during the sequence, the two delays given by (4 ⁇ J C,H ) ⁇ 1 within the INEPT (Insensitive Nuclei Enhanced by Polarization Transfer) transfer of the HSQC were held as short as possible, e.g. for about 1.5 milliseconds.
  • the 2D-NMR measurements can be carried out on any high-field instrument available in the art.
  • An example of a suitable instrument is a high-field NMR instrument equipped with a Cryoprobe probehead, more particularly a Bruker Avance III 600 MHz, MicroCryoprode TCI 1.7 mm.
  • Samples for analysis may be prepared as 1.2 mg of acetylated hyaluronic acid, or its sodium salt, dissolved in 50 ml D 2 O 99.96% D.
  • the sample solution can be transferred in the NMR tube (1.7 mm, Bruker) for measurement.
  • Software for data acquisition and processing, including cross-peak integration is TopSpin 3.0 (Bruker).
  • this 2D-NMR technique allowed for distinguishing the acetylated hyaluronic acid or sodium salt of the present invention from the product described in EP 0 725 083 and sold by Shiseido: Shiseido's acetyl hyaluronate had an average acetylation degree of only about 3.6. This is important, as Shiseido's product also proved inferior to the product of the present invention with respect to anti-aging activity, as will be shown in Example 5 below.
  • the acetylated hyaluronic acid or sodium salt used in methods and cosmetic preparations, in particular skin care compositions, of the present invention is a low molecular weight acetylated hyaluronic acid or sodium salt thereof.
  • the term “low molecular weight” refers to polymeric material having a weight average molecular weight (Mw) that is 50 kDa or less, more particularly 35 kDa or less, more particularly about 30 kDa or less, more particularly 25 kDa or less, more particularly 20 kDa or less, more particularly 15 kDa or less, and still more particularly about 10 kDa to 15 kDa, for example 13 kDa+/ ⁇ 1 kDa.
  • the molecular mass of the acetylated hyaluronic acid or its sodium salt polymer fractions should be rather narrowly dispersed.
  • the acetylated hyaluronic acid or its sodium salt has a polydispersity index (Mw/Mn) that is less than 2.0, more particularly less than 1.8, more particularly less than 1.7, more particularly less than 1.6 or less, more particularly less than 1.5, and still more particularly about 1.4 or less.
  • the intrinsic viscosity of the acetylated hyaluronic acid or its sodium salt is preferably less than 0.3 dl/g (30 cm 3 /g), and more particularly 0.245 dl/g (24.5 cm 3 /g)+/ ⁇ 0.01 dl/g (1 cm 3 /g).
  • the foregoing physical parameters can be measured by HPLC Size Exclusion Chromatography.
  • a suitable instrument is a Viscotek GPC max VE2001, equipped with Triple Detection Parameters (Viscotek TDA 305): refractometer, capillary viscosimeter and light scattering.
  • Light scattering measurements characterize particle size distribution by measuring the angular variation in intensity of light scattered as a laser beam passes through a dispersed particulate sample. Large particles scatter light at relatively smaller angles relative to the laser beam and small particles scatter light at relatively larger angles. The angular scattering intensity data is then analyzed to calculate the size of the particles responsible for creating the scattering pattern, using the Mie theory of light scattering. The particle size is reported as a volume equivalent sphere diameter. Size Exclusion Chromatography (SEC) is an analytical technique that separates dissolved macromolecules by size based on their elution from columns filled with a porous gel. When SEC is coupled with light scattering, viscometer and concentration detectors (known as triple detection), one can measure molecular weight, molecular size and intrinsic viscosity.
  • SEC Size Exclusion Chromatography
  • the acetylated hyaluronic acid or its sodium salt (0.2 g) is solubilized in TBS buffer (100 ml) by stirring for 1 h at 45° C., before storing the solution for an additional 2 h at room temperature under stirring to fully hydrate the polymer.
  • the solution is filtered through a 0.2 ⁇ m nylon syringe filter.
  • the solution is then injected in HPLC SEC LALS (100 ⁇ l). Analysis is performed at 0.35 ml/min with TBS buffer pH 7 at 35° C. A typical run time is 90 minutes and a typical injection volume 100 ⁇ l.
  • the low molecular weight acetylated hyaluronic acid or its sodium salt is a preferred material for use in methods and cosmetic preparations, in particular skin care compositions, of the present invention.
  • the use of low molecular weight material is believed to be advantageous because of its superior skin penetration properties compared with higher molecular weight fractions.
  • Hyaluronic acid or its sodium salt can be acetylated according to techniques known in the art.
  • hyaluronic acid is dissolved in a mixture of acetic anhydride and acetic acid. Thereafter, a strong acid, such as sulphuric acid is added to the solution, to effect acetylation.
  • a strong acid such as sulphuric acid
  • the mixing ratio (wt/wt) of acetic acid to acetic anhydride may vary within the range of 1:4 to 1:1.
  • the mixing ratio (wt/wt) of strong acid to hyaluronic acid may be 1:5 to 1:1.
  • low molecular weight hyaluronic acid is more easily and more extensively acetylated than high molecular weight starting material because the hydroxyl groups are less sterically hindered in low molecular weight starting material and therefore more easily acetylated.
  • the acetylated hyaluronic acid or its sodium salt of the present invention is prepared using low molecular weight hyaluronic acid or sodium salt as the starting substrate.
  • hyaluronic acid starting material is a low molecular weight fraction with a weight average molecular weight of about 50 kDa or less, more particularly of about 35 kDa or less, and still more particularly of about 30 kDa or less.
  • a hyaluronic acid substrate having the aforementioned low molecular weight By employing a hyaluronic acid substrate having the aforementioned low molecular weight, it is possible to prepare highly acetylated hyaluronic acid, or its sodium salt, having a weight average molecular weight (Mw) of 50 kDa or less, more particularly 35 kDa or less, more particularly about 30 kDa or less, more particularly 25 kDa or less, more particularly 20 kDa or less, more particularly 15 kDa or less, and still more particularly about 10 kDa to 15 kDa, for example 13 kDa+1-1 kDa.
  • Mw weight average molecular weight
  • a method of preparing the low molecular weight acetylated hyaluronic acid or its sodium salt comprising the steps of selecting a low molecular weight hyaluronic acid having a weight average molecular weight of about 50 kDa or less, more particularly about 35 kDa or less, and still more particularly about 30 kDa or less, and reacting it under acetylating conditions, for example, those conditions describe herein, forms yet another aspect of the invention.
  • the invention provides in another of its aspects a method of isolating low molecular weight acetylated hyaluronic acid from a crude reaction mixture containing the low molecular weight acetylated hyaluronic acid in solution.
  • the method of isolating the low molecular weight acetylated hyaluronic acid comprises the step of precipitating it from solution using additive water.
  • the characteristic low molecular weight of the acetylated hyaluronic acid enables the precipitation step to be carried out using additive water, rather than an additive volatile organic solvent. This is contrasted with prior art processes that employ additive volatile organic solvents, such as acetone, to precipitate sodium salts of acetylated hyaluronic acid out of a crude reaction mixture.
  • additive volatile organic solvents such as acetone
  • a significant advantage of the present invention is that low molecular weight acetylated hyaluronic acid can be obtained that is free of volatile organic solvents, such as acetone.
  • the low molecular weight acetylated hyaluronic acid, free of volatile organic solvent can be further converted to its sodium salt, in accordance with a method described herein below, and the sodium salt of the low molecular weight acetylated hyaluronic acid, free of volatile organic solvents, forms an additional aspect of the present invention.
  • Acetic acid is a solvent for the low molecular weight acetylated hyaluronic acid, and its partial removal, along with the acetic anhydride, facilitates the precipitation of the low molecular weight acetylated hyaluronic acid by enabling the precipitation step to be carried out with relatively small volumes of additive water.
  • the acetic acid/acetic anhydride mixture can be removed by distillation, leaving the crude reaction mixture in the form of a viscous paste consisting mainly of the desired low molecular weight acetylated hyaluronic acid. Water is then added to this crude mixture to effect precipitation. The distilled acetic acid/acetic anhydride mixture may be collected and recycled.
  • the amount of additive water is 1 to 5 weight equivalents, more particularly 2 to 4 weight equivalents per one part of the crude reaction mixture containing the desired acetylated hyaluronic acid.
  • any residual acetic anhydride is hydrolyzed.
  • the strongly exothermic hydrolysis is preferably performed in such a manner that the temperature of the reaction mass does not exceed 40° C.
  • Removal of acetic acid/acetic anhydride may be carried out by distillation. Prior to the distillation step, it may be desirable to neutralize any strong acid in the reaction mixture to avoid contacting the acetylated hyaluronic acid with the strong acid under reflux conditions, which may lead to the emanation of undesirable discolouration.
  • the adjustment of the pH of the crude reaction mixture before precipitation is therefore a preferred process step in an isolation and purification process according to the invention.
  • a method of isolating the low molecular weight acetylated hyaluronic acid from a reaction mixture containing the low molecular weight acetylated hyaluronic acid in solution comprising the step of precipitating the low molecular weight acetylated hyaluronic acid from the solution at a pH of 5 to 8 using additive water.
  • a method of isolating the low molecular weight acetylated hyaluronic acid from a reaction mixture containing the low molecular weight acetylated hyaluronic acid in solution comprising the step of adjusting the pH of the reaction mixture to a pH in the range of 5 to 8 and precipitating the low molecular weight acetylated hyaluronic acid from this solution using additive water.
  • pH adjustment may be carried out using a suitable buffering agent, for example phosphates, acetates and citrates known for such purpose, and more particularly, buffering is carried out by the addition of an effective amount of sodium acetate.
  • a suitable buffering agent for example phosphates, acetates and citrates known for such purpose, and more particularly, buffering is carried out by the addition of an effective amount of sodium acetate.
  • the precipitated acetylated hyaluronic acid may be separated and isolated from other components of the reaction mixture by filtration over a suitable filter, such as any fine mesh textile filter known for such purpose.
  • the precipitated low molecular weight acetylated hyaluronic acid may be subjected to additional work-up procedures after precipitation. For example, it may be washed with water in order to remove any traces of water-soluble impurities, before drying the precipitated material to afford the low molecular weight acetylated hyaluronic acid in the form of a bright, white free-flowing powder with no or practically no yellowish hue.
  • the low molecular weight acetylated hyaluronic acid may be used in this form in the methods and cosmetic preparations, in particular skin care compositions, of the present invention.
  • acetylated hyaluronic acid it might also be desirable to subject the acetylated hyaluronic acid to further work-up procedures, such as a bleaching or polishing step to remove any unwanted yellow colouration using, for example, activated carbon.
  • the purified acetylated hyaluronic acid may thereafter be dried, optionally under vacuum, to provide acetylated hyaluronic acid in a form suitable for use in methods and cosmetic preparations of the present invention without further modification.
  • the acetylated hyaluronic acid of the present invention may be converted to its corresponding sodium salt by means of a neutralization step.
  • Neutralization may be carried out by adding an aqueous source of sodium ions, such as sodium hydroxide, sodium acetate or any other suitable sodium-ion-containing base to an aqueous solution or suspension of the low molecular weight acetylated hyaluronic acid.
  • an aqueous source of sodium ions such as sodium hydroxide, sodium acetate or any other suitable sodium-ion-containing base
  • the neutralization step may be carried out at a temperature of 0 to 30° C., and the reaction time may vary within the range of 0.5 to 12 hours.
  • the base for example sodium hydroxide
  • the base is added to the solution or suspension of the low molecular weight acetylated hyaluronic acid slowly in order that appreciable concentrations of the base do not build-up, and the pH does not exceed about 9, more particularly about 8, more particularly about 7.5, more particularly about 7, and more particularly still about 6.5. In this way, no undesirable by-products are created and the emanation of undesirable discolouration is avoided.
  • the course of the neutralization reaction may be conveniently followed by following the pH of the reaction mixture, and a neutralized solution of the corresponding low molecular weight sodium acetyl hyaluronate is obtained when the pH reaches 6.5+/ ⁇ 0.5.
  • the low molecular weight sodium acetyl hyaluronate solution which forms another aspect of the invention, may be used in this form in the methods and cosmetic preparations, in particular skin care compositions, of the present invention without further modification.
  • a suitable antimicrobial agent which agents are well known in the art.
  • the low molecular weight sodium acetyl hyaluronate may be further processed to render it in solid form, more particularly a powder form.
  • Prior art methods for isolating sodium acetyl hyaluronate in its solid form comprises the step of precipitating it from an aqueous acetone solution using neat acetone.
  • isolating and purifying it in this way can leave residues of volatile organic solvent in the finished product, and these solvents should generally be avoided as far as possible (or removed where appropriate) in order to comply with product specifications and other quality characteristics that may typically be required of raw materials intended for use in the cosmetics industry.
  • Another aspect of the present invention is therefore based on the object of providing a method for the production of volatile organic solvent-free low molecular weight sodium acetyl hyaluronate in a solid form, and preferably a powder form, which is bright white, free-flowing and easy to handle.
  • Applicant achieved these objects and provides a method of converting an aqueous solution of the low molecular weight sodium acetyl hyaluronate into powder form, said method comprising the step of dehydrating the low molecular weight sodium acetyl hyaluronate solution.
  • the dehydration step may be carried out by known techniques, such as lyophilization. More preferably, however, the dehydration step is carried out by spray drying.
  • the invention provides in another aspect a method of dehydrating a liquid aqueous medium, and in particular a solution, comprising sodium acetyl hyaluronate, in particular, sodium acetyl hyaluronate formed according to a process described herein.
  • the method described above comprises the steps of atomizing the liquid aqueous medium as droplets into a drying chamber maintained under conditions causing evaporation of the liquid aqueous medium to form particles of sodium acetyl hyaluronate.
  • the inlet temperature of the spray drying chamber is about 150° C.
  • the outlet temperature of the spray-drying chamber is about 70° C. or less.
  • air is pumped into the drying chamber such that the drying chamber outlet temperature is reduced to about 40 to 50° C.
  • Spray-dried sodium acetyl hyaluronate which forms another aspect of the present invention, exhibits desirable properties, such as high bulk density, shape and flowability, which make the product easy to handle transport and use.
  • the avoidance of a low density and dusty product is a particular advantage of spray-dried sodium acetyl hyaluronate.
  • Spray-dried sodium acetyl hyaluronate is formed with sufficient whiteness and clarity that it is eminently suitable for use in products intended for cosmetic applications, and in particular for skin care compositions.
  • Both the low molecular weight acetylated hyaluronic acid and the low molecular weight sodium acetyl hyaluronate are easily incorporated into cosmetic preparations, and in particular skin care compositions. Owing to their very low molecular weight and their powdered physical form, they are not stringy or lumpy, and can be easily dissolved or suspended in aqueous media and mixed into cosmetic bases.
  • both the low molecular weight acetylated hyaluronic acid and the low molecular weight sodium acetyl hyaluronate can be prepared as white powders with no, or substantially no, yellowish hue, and which do not alter or impair the colour tone of any cosmetic preparation into which they might be incorporated, irrespective of the amount of material employed in the cosmetic preparation.
  • a bleaching or polishing step whereby slight discolouration is removed using a suitable bleaching agent, such as a clay or a form of activated carbon.
  • a suitable bleaching agent such as a clay or a form of activated carbon.
  • the methods of preparation and isolation described herein provide the low molecular weight acetylated hyaluronic acid and low molecular weight sodium acetyl hyaluronate as products that are in the form of white powders of sufficient brightness and clarity that, should a bleaching or polishing step be carried out, it is able to effectively decolourize the products.
  • the methods of preparation and isolation describe herein provide low molecular weight acetylated hyaluronic acid and low molecular weight sodium acetyl hyaluronate as products that are in the form of white powders, each having an L* value of 90 or more; and a b* value, which is less than 8, wherein L*and b* values represent the CIELAB system's chromaticity coordinates.
  • the lightness and hue of a powder can be characterized by CIELAB chromaticity coordinates L* a* b* according to colorimetric methods known in the art.
  • the L* value is a value specifying the lightness of a substance and is indicated by a value between 0 and 100.
  • An L* value of 100 indicates the brightest state (completely white), and an L* value of 0 indicates the darkest state (completely black).
  • the b* value specifies the blue-yellow hue of a substance. The larger is the b* value, the higher is the degree of yellowness. The smaller is the b* value, the higher is the degree of blueness.
  • Both the L* value and the b* value may be measured using any suitable commercially available spectrophotometer, such as the Minolta CM3500d.
  • the spectrophotometer should be powered up for at least one hour before making a measurement.
  • a glass container provided therefor should be half filled with the solid product to be measured taking care to ensure that the bottom of the container is fully covered by the product. Thereafter, the filled container should be placed in the sample stand provided therefor.
  • the sample key on the instrument should be pressed and the L* and b* values read off the display panel. Before taking any readings, the instrument should be calibrated for the zero and 100% reflection by placing black and white objects provided therefor on the window of the instruments optical sensor.
  • EXAMPLE 1 METHOD FOR MANUFACTURING SODIUM ACETYL HYALURONATE
  • a 5 l reactor was flushed with nitrogen and charged with hyaluronic acid (RenovhyalTM manufactured by Soliance, molecular weight 10-50 kDa; 300 g, 0.7 mol).
  • Commercial grade acetic anhydride (2160 g, 21.2 mol) and acetic acid (525 g, 8.7 mol) were added and the temperature of the suspension was set to 25° C.
  • Sulfuric acid 96% (194 g, 1.9 mol) was added over the period of 10 minutes.
  • the temperature was maintained at 25° C. by internal control. Stirring was continued for 15 hours.
  • the internal control was turned off and sodium acetate (154 g, 1.9 mol) was added while stirring. The temperature rose to 35° C. and stirring was continued for 30 minutes.
  • the reactor was evacuated to 40 mbar and the jacket temperature was set to 50° C. Over the period of 5 hours a mixture of acetic anhydride and acetic acid was removed by distillation (1343 g). The remaining slightly pasty but still stirrable reactor content was cooled to room temperature and water (5500 ml) was added (very carefully at first), maintaining the temperature of the mixture below 50° C. at all times. Gradually a fine, white suspension formed. The solids were isolated by filtration and washed with water (10 l).
  • EXAMPLE 2 METHOD FOR ISOLATING SODIUM ACETYL HYALURONATE BY SPRAY-DRYING TECHNIQUE
  • Spray drying of an aqueous 11% sodium acetyl hyaluronate solution was carried out using a GEA VERSATILE-SD′ 6.3 spray dryer, equipped with a rotary atomizer, using the following parameters:
  • the optimal concentration that can be used without inducing toxic effects on each cell type was determined as 0.1 mg/ml for fibroblasts and 0.5 mg/ml for melanocytes. These concentrations were used during the biological investigations.
  • a transcriptomic in vitro analysis was performed on two cell types present in the skin including melanocytes and fibroblasts. It was studied on 128 genes distributed on two transcriptomic plates. A first plate was specific for dermis function with many genes involved in extracellular matrix, matrix remodelling, oxidation defences, stress responses, wound healing and other biological functions. A second plate was specific for skin pigmentation functions with the most important genes involved in melanin synthesis, melanosomes formation and maturation and other functions linked to melanocytes.
  • the transcriptomic analysis was performed by Rt-qPCR (Real Time-quantification Polymerisation Chain Reaction) using TaqMan assays in triplicate for each cell types. Briefly after 24 h of cell stimulation at 0.1 mg/ml for fibroblast and 0.5 mg/ml for melanocytes, total RNA was extracted by the Trizol method. Total RNA was quantified by spectrometer and concentrations were adjusted to 400 ng/ ⁇ l. The quality of RNA was verified by migration on agarose gel. Total RNA was retro-transcripted to cDNA using a cDNA Verso kit following the supplier's recommendations. The retro-transcription was validated by classical PCR targeting housekeeping gene: RPL32 gene. cDNA was then used to perform RT-qPCR where 10 ng of cDNA was deposited per well using a TaqMan assay.
  • Results obtained are represented in the table 2 below.
  • An induction of more than 1.3 fold (positive or negative) in comparison with the untreated condition is considered to be indicative of a biological modification.
  • EXAMPLE 5 IMPACT OF SODIUM ACETYL HYALURONATE ON FIBROBLASTS (DERMIS)
  • MMP-1 and MMP-3 are two matrix metalloproteinases involved in matrix degradation, such as type I collagen, which is the most important collagen present in the dermis.
  • MMPs During the aging, the production of MMPs increases leading to intradermal matrix degradation that promotes the skin collapse and wrinkle formation. They represent the first visible signs of skin aging.
  • UV ultraviolet
  • oxidative stress in the skin that is responsible for a significant increasing of MMPs release, which in turn promotes the premature aging of the skin by severe matrix degradation.
  • fibroblasts were pre-conditioned with sodium acetyl hyaluronate (0.1 mg/ml) in basal medium for 2 h before the induction of oxidative stress. After the oxidative stress, fibroblasts were incubated 48 h in basal medium at 37° C. permitting MMPs to accumulate in the culture medium. The quantity of MMP-1 and MMP-3 released from supernatants was then estimated by the multiplex method using Luminex technology (principle of ELISA on beads).
  • Fibroblasts from abdominal biopsy of a Caucasian female (age 26) were seeded in 12-well plates at 200′000 cells/well in 1 ml of complete medium (ZenBio, ref. DF-1). 24 h after plating, the complete medium was replaced by basal medium without serum (Zen-Bio, ref. DF-2). After 24 h of culture in basal medium, the cells were pre-incubated with the test products at 0.1 mg/ml for 2 h at 37° C. and were then exposed to oxidative stress (H 2 O 2 200 ⁇ M) for 2 h at 37° C. The culture medium was replaced by basal medium and cells were incubated for another 48 h at 37° C. Supernatant was collected and stored at ⁇ 80° C. until analysis.
  • MMP-1 quantification was performed using the RayBio® Human MMP-1 ELISA kit, an in vitro enzyme-linked immunosorbent assay for the quantitative measurement of human MMP-1 pro and active forms in serum, plasma, and cell culture supernatants.
  • This assay employs an antibody specific for human MMP-1 coated on a 96-well plate.
  • Standards and samples are pipetted into the wells and MMP-1 present in a sample is bound to the wells by the immobilized antibody.
  • the wells are washed and biotinylated antihuman MMP-1 antibody is added. After washing away unbound biotinylated antibody, HRP conjugated streptavidin is pipetted to the wells.
  • TMB (3,3′,5′5′ tetramethylbenzidine) substrate solution is added to the wells and color develops in proportion to the amount of MMP-1 bound.
  • the Stop Solution changes the color from blue to yellow, and the intensity of the color is measured at 450 nm.
  • MMP-1 was measured after a 1/100 dilution of samples, in triplicate. Standard curve was done in duplicate. From MMP-1 standard concentrations of 18′000, 6′000, 2′000, 666.7, 222.2, 74.07 and 24.69 pg/ml, a four-parameter logistic regression was calculated.
  • a pre-incubation with the sodium acetylated hyaluronate of the present invention (a) induced a significant reduction of stress-mediated MMP-1 production, while the both other products showed an inverse effect with +63% and +12% for monoacetate hyaluronate (b) and acetylated hyaluronic acid from Sisheido (c), respectively.
  • sodium acetylated hyaluronate of the present invention presents an efficient activity on the inhibition MMP-1 release in basal and in presence of oxidative stress (aging like).
  • MMPs are released under two forms including inactive pro-form (pro-MMP) and active form with the pro-peptide cleaved (MMP). Only the active MMPs are able to induce collagen degradation. DQ-type I collagen that is a type I collagen fluorescent linked to a quencher was used. When it is degraded it promotes the quencher release and fluorescence emission. Finally, the emitted fluorescence which is proportional to collagen degradation was measured. This experiment was performed exactly under the same experimental conditions that were used to quantify the secretion of MMP-1 and MMP-3. Briefly, supernatant was incubated with DQ-collagen for 4 h at room temperature and the fluorescence was measured by Microplate Reader (TECAN) with excitation wavelength 495 nm and emission wavelength 515 nm.
  • TECAN Microplate Reader
  • sodium acetyl hyaluronate decreased the DQ-collagen degradation like observed by the decreasing of fluorescence level (table 5).
  • sodium acetyl hyaluronate limits the release of active forms of MMP-1 and MMP-3 responsible of DQ-collagen degradation.
  • sodium acetyl hyaluronate possesses an anti-aging activity through an impact on matrix remodelling which is involved in the visible signs of skin aging. Indeed, it limits this by a down-expression of MMP-1 and MMP-3 that control the matrix degradation such as type I collagen.
  • sodium hyaluronate was not able to promote the same results confirming that the bioactivity is linked to the acetylated modification (table 5).
  • the medium was then replaced by culture medium with or without sodium acetyl hyaluronate or unmodified sodium hyaluronate at concentrations of 0.1, 0.25, 0.5, and 1 mg/ml, respectively, or reference (L-tyrosine 1 mM). Cells were incubated for 10 days with two renewals of the treatment after 3 and 7 days of incubation.
  • melanocytes were incubated in 24-wells plates for 24 h of culture. The medium was then replaced by culture medium containing L-tyrosine (1 mM) supplemented by sodium acetyl hyaluronate or unmodified sodium hyaluronate at concentrations of 0.1, 0.25, 0.5, and 1 mg/ml, respectively, or reference (Lipoic Acid 5 ⁇ g/ml). Cells were incubated for 10 days with two renewals of the treatment after 3 and 7 days of incubation.

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