Topical compositions associating sodium hyaluronate fragments and retinoid

European).

15 / P23705PLOO

EP 1 689 356 B1

Description

The present invention relates to compositions for external use based on hyaluronan, their use in cosmetology and in dermatology.

Hyaluronan (HA) is the main component of the extracellular matrix. It is especially present in connective tissues called "soft" as opposed to other glucosaminoglucans, such as chondroityabark acid, present in "hard" tissues such as cartilage. Thus, it is found in large quantities mainly in the skin.

HA is a non-sulfate linear glucosaminoglucan composed of repeating units of D-glucuronic acid and N-acetyl-D-glucosamine (R. Tammi, UM Agren, AL Tuhkanen, M. Tammi, Hyaluronan metabolism in skin, Progress in Histochemistry & Cytochemistry, 29 ( 2): 1-81, 1994).

In normal skin, HA is synthesized essentially by dermis fibroblasts and epidermal keranocytes (R. Tammi, already cited). Due to its negative charge residues, HA plays the role of a water pump, which maintains skin elasticity. HA has a major role in controlling the diffusion of nutrients, hormones, vitamins and inorganic salts from connective tissue, and in cleansing from metabolic waste that can cause inflammatory reactions. With age, the amount of HA and the degree of its polymerization decrease, resulting in a decrease in the amount of water retained in the connective tissue. The skin then undergoes the aging process, which leads to an increase in fibrosis and a decrease in the content of elastic fibers.

In normal human skin, HA exists in the form of a polymer with a high molecular weight (600,000-1,000,000 Da). Physiological degradation of HA in the skin occurs as a result of (i) internalization by keratinocytes via CD44 and (ii) intracellular fragmentation by hyaluronidase into intermediate sized fragments (60,000-300,000 Da).

Fragmented Ha is released by keratinocytes, passes through the basement membrane and is released directly into lymphatic vessels (R. Tammi et al. Already cited).

Accumulation of low molecular weight HA forms has been shown in animals under inflammation. During inflammation, chemotactic platelet factors, such as fibrin, stimulate the inflow and activation of fibroblasts, which degrade HA by secreting hyaluronidase, resulting in high concentrations of tissues with small HA fragments. The creation of these small HA fragments also occurs through various mechanisms, such as depolymerization by reactive oxygen species released by granulocytes, or in skin exposed by ultraviolet rays, or the synthesis of fragments with low molecular weight again. Several studies have suggested that HA with high and low molecular weight may have different biological effects on cells and tissues (CM.McKee, MB.Penno, M.Cowman, MD.Burdick, RM.Strieter, C.Bao, PW.Noble, Hyaluronan (HA) fragments induce chemokine gene expression in alveolar macrophages. The role of HA size and CD44., Journal of Clinical Investigation, 98 (10): 2403-13, 1996; CC.Termeer,

J. Hennies, U. Voith, T. Ahrens, JM Weiss, P.Prehm, JC.Simon, Oligosaccharides of hyaluronan are potent activators of dendritic cells, Journal of Immunology, 165 (4): 1863-70,

2000; KA.Fitzgerald, AG.Bowie, BS.Skeffington, LA.O'Neill, Ras, Protein kinase C zeta and I kappa B kinases 1 and 2 are downstream effectors of CD44 during the activation of NFkappa B by hyaluronic acid fragments in T- 24 carcinoma cells, Journal of Immunology, 164 (4): 2053-63, 2000).

It has been shown that intermediate molecular weight (50,000-250,000 Da) HA applied to the skin of mice and humans passes through the layers of the epidermis and dermis. Degradation products appear in the serum 2 hours after applying HA to the skin. The molecular weight of HA recovered from serum is slightly lower than the molecular weight of HA applied to the skin, thus showing that the passage of HA through the skin is not limited to fragments of smaller dimensions (100-10,000 Da) (TJ Brown, D. Alcorn, JR. Fraser, Absorption of hyaluronan applied to the surface of intact skin, Journal of Investigative Dermatology, 113 (5): 740-6, 1999).

Vitamin A (retinol) and its natural and synthetic derivatives, together designated as retinoids, create a great variety of substances that have a significant effect especially in embryogenesis, reproduction, vision mechanism, growth regulation and the phenomenon of cell differentiation (R. Blomhoff et al., 1991, Physiol. Rev., 71: 951-990; MBSporn et al.

(1994) in the Retinoids, 2nd ed. Raven Press, NY).

Natural retinoids are defined by the following general formula (I) as defined in (IUPAC-IUB Joint Commission on Biochemical Nomenclature in Eur. J. Biochem. (1982), 129,

1-5) :

(I) in which

R = CH₂OH corresponds to retinol (ROL)

R = CHO corresponds to retinal (RAL)

R = COOH corresponds to retinoic acid (RA).

The biological effect of retinoids is through their interaction with nuclear receptors such as RAR (retinoic acid receptors) and RXR (retinoic acid X receptor). The known RAR receptor ligand is the trans isomer of vitamin A (trans-retinoic acid).

In many cell types, including endothelial cells,

ROL is metabolized into retinyl esters.

The importance of retinoids in dermatology goes back to the first observation in animals with skin anomalies caused by vitamin A deficiency (Wolbach and Howe, J.exp.Med., 43: 753).

Today, many retinoids have been synthesized. Dermatological indications such as actinic keratosis, acne and, generally, external or oral treatment of keratinization disorders such as psoriasis and Darier's disease remain the main areas of use.

In addition, a number of combinations or combinations with retinoids are known:

- combinations of molecules that act on cell differentiation and immunomodulation, e.g. a combination of retinoids and chemotherapy in skin lymphomas (Thestrup-Petersen et al., Br. J. Dermatol., 118: 811-818),

- a combination of retinoids and phototherapy, e.g. in combination with psoralens (Saurat et al., Dermatology, 177: 218-224),

- a combination of molecules that affect retinoid metabolism. This is e.g. the case of interaction between azoles and vitamin D derivatives that inhibit retinoid metabolism and allow for an increase in intracellular indicators of the active hormone (Kato et al., Biochem.J. (1992) 286: 755760, Jugert et al., Skin Pharmacol., (1998).

It is also known that irritation is one of the main secondary effects of retinoids during external use. This effect may prevent strict adherence to treatment.

Retinoic aldehyde (RAL), a member of the retinoid family, is a natural metabolite of vitamin A currently used for external use in humans. It has been shown that RAL has biological activity in the skin, causing epidermal hyperplasia, as well as a significant increase in CD44 and HA expression in the epidermis and intercellular mice of C57BL / 6 and SKH1 hairless (SKH1 hairless) mice. These effects have also been observed in connection with the external use of retinoic acid (RA) and retinol (ROL). However, CD44 and HA expression increased more strongly in RAL treated mice than in RA or ROL treated mice.

CD44 is a transmembrane polymorphic glycoprotein that has several isoforms formed by alternative assembly and post-translational modifications. In a recent study, we showed that the two main functions of CD44 in mouse skin are (i) regulation of keratinocyte proliferation in response to extracellular stimuli, and (ii) maintenance of HA local homeostasis (G. Kaya, I. Rodriguez, JL. Jorcano, P. Vassalli,

I. Stamenkovic, Selective suppression of CD44 in keratinocytes of mice bearing an antisense CD44 transgene driven by a tissue-specific promoter disrupts hyaluronate metabolism in skin and impairs keratinocyte proliferation, Genes & Development, 11 (8): 996-1007, 1997). We also observed a reduction in CD44 expression in the epidermis in patients suffering from scleroderma atrophy, which is potentially responsible for the deposition of HA in the dermis and epidermal atrophy in this disease (G. Kaya, E. Augers, K. Stamenkovic, JH.Saurat, Decrease in epidermal CD44 expression as a potential mechanism for abnormal hyaluronate accumulation in superficial dermis in lichen sclerosus and atrophicus,

Journal of Investigate Dermatology, 115 (6): 1054 = 8, 2000).

CD44 is involved in cell-cell and cell-matrix interactions. Recent research shows that the conjugated form formed by CD44 and HA fragments (CD44-HAF) is an inducer of HA mitosis and neosynthesis (TC.Laurent, UB.Laurent, JR.Fraser, The structure and function of hyaluronan: An overview, Immunol.Cell Biol., 79 (2): 1-7, 1996). Therefore, CD44 seems to be the mediator in HA and RAL on the epidermis and dermis.

There are currently pharmaceutical and cosmetic preparations containing high molecular weight inorganic HA salts, especially Healon, Hyalgan, Provisc, Vitrax and those cited in the Martindale The Complete Drug list, 32nd edition, 1999, The Pharmaceutical Press Editor.

However, HA is difficult to pass through the skin, given its high molecular weight.

This is why, in the international application WO 02/76470 A1, the authors propose a composition combining N-acetylglucosamine with a retinoid. This combination allows synergistic enhancement of hyaluronic acid synthesis by epidermal cells in vitro. No result obtained after application of the preparation in vivo is reported.

Japanese application 11279042 describes compositions based on hyaluronic acid fragments with sulfate groups, said fragments having a molecular weight of between 1,000 and 50,000 Da, and sulfate groups occupy 10 to 90% of the total R1, R substituents₂, R3 and R4 in the formula. These low molecular weight fragments are very active in maintaining skin elasticity and avoiding keratinization. On the contrary, hyaluronic acid fragments without sulfate groups turn out to be inactive in the test.

International application WO 01/03657 A1 describes a composition that avoids premature aging of the skin in good condition, containing retinol and hyaluronan with a molecular weight comprised between 11,000,000 Da.

International application WO 2005/039532 A1 describes compositions that can be used externally, in the form of microemulsions and containing a retinoid such as 13-cisretinic acid as an active agent in combination with a hyaluronic acid derivative with a molecular weight comprised between 400 and 1,000,000 Da, and more particularly between 400 and 200,000 Da.

Application EP 0 197 718 describes the use of hyaluronic fragments with a molecular weight comprised between 50,000 and 750,000 Da in combination with another dermatologically active substance.

Well, the inventors have unexpectedly shown that HA without sulfate groups, hydrolyzed into fragments with molecular weight comprised between 50,000 and 750,000 Da has biological activity on the skin that is enhanced when these fragments are attached to the retinal.

Therefore, the present invention relates to compositions intended for external use characterized in that they contain as active ingredient one or several fragments of low molecular weight hyaluronan comprised between 50,000 and 750,000 Da and in addition contain retinal.

In a preferred embodiment of the invention, the molecular weight of hyaluronan fragments is between 50,000 and 250,000 Da or between 250,000 and 750,000 Da.

The combination of hyaluronan fragments with a molecular weight comprised between 50,000 and 750,000 Da with retinal has a synergistic effect on the synthesis of hyaluronic acid by keratinocytes.

Hyaluronan means any salt, especially sodium hyaluronate.

The external compositions of the invention may also contain dyes, silicone oils, retinoids or coloring pigments, antiseptics, vegetable oils, antioxidants, mineral salts, thickeners, pH modifiers, ultraviolet absorbents, vitamins or any other dermatology and pharmaceutically acceptable excipient.

The compositions of the invention may be used in cosmetology and in dermatology to prevent or improve the condition of wrinkled skin, dry skin ... and allow the skin to remain firm and moist.

The compositions of the invention can be used in preparations for external use in the field of dermatology or cosmetology, for the prevention or treatment of dermatoses associated with atrophy of the skin tissue, for which it will be necessary to improve the hydration state of the skin, reduce skin atrophy, e.g. in secondary effects of corticoid treatment, reduction of wrinkles, combating skin aging, whether induced or not caused by light, resumption of epidermal and skin cells, skin firming, increasing its elasticity.

The invention is illustrated by the examples and figures that follow.

Figure 1 shows the amount of HA measured in the epidermis of untreated animals or animals treated twice with formulation 5, formulation 3 or formulation 7 obtained according to the operating procedure described in examples 1 and 2.

Fig. 2 shows the dermal HA assay in animals not treated or treated with formulation 5, 3 or 7, such as those prepared in examples 1 and 2.

COMPARATIVE EXAMPLE 1: Action of externally applied hyaluronan fragments

1. MATERIALS AND METHODS

1.1.Preparaty

Three types of hyaluronan (HAF) fragments were evaluated:

• HAF with very high molecular weight (1,000,000,000,000 Da) • HAF with low molecular weight (50,000-750,000 Da)

- HAF (250,000 - 750,000 Da)

- HAF (50,000-250,000 Da) • HAF with very low molecular weight (1000-20,000 Da).

These fragments were included in typical cosmetic preparations, of which two examples of compositions are given in Table 1 below:

<td>Ingredients</td><td>Quantities%</td><td>Quantities%</td>

<td>PEG 600</td><td> 5</td><td> -</td>

<td>PEG 400</td><td> -</td><td> 1</td>

<td>sorbitol</td><td> -</td><td> 2</td>

<td>Glycerine</td><td> -</td><td> 10</td>

<td>BHT</td><td> 0,02</td><td> -</td>

<td>Sodium lauryl sulfate</td><td> -</td><td> 0,25</td>

<td>carbopol</td><td> 1</td><td> -</td>

<td>Vitamin E Acetate</td><td> 0,5</td><td> -</td>

<td>spermaceti</td><td> -</td><td> 6</td>

<td>Cetyl stearyl alcohol</td><td> -</td><td> 3</td>

<td>Thick vaseline oil</td><td> 5</td><td> -</td>

<td>Cremophor RH 40</td><td> 2</td><td> -</td>

<td>Sorbic acid</td><td> 0,05</td><td> -</td>

<td>nipagine</td><td> 0,15</td><td> -</td>

<td>Fenonip</td><td></td><td> 1</td>

<td>triethanolamine</td><td> 2,48</td><td> -</td>

<td>Water</td><td>qsad 100</td><td>qsad 100</td>

1.2. Measurement of HAF activity

SKH1 mice without hair (SKH1 hairless) are used in an amount of 3 animals per group.

Various preparations are used externally.

The thickness of the epidermis is measured through a scaled eyepiece (Zeiss); magnification 40 times, an average of 5 fields per mouse is drawn.

Dermal cellularity is assessed by calculating the number of skin cells, at 40 times magnification, drawing an average of 5 fields per mouse.

The number of epidermal and dermal proliferative cells is shown by immuno-labeling of Ki 67 with anti-Ki-67 antibody (rat anti-mouse, Dako).

The presence of hyaluronan in the skin is measured, after applying various preparations in the amount of two applications per day, according to the ELISA technique (Corgenix) and hyaluronan appears on histological cross-sections by colloidal iron staining.

2. RESULTS

2.1. Effects on epidermal thickness, dermal cellularity and proliferative cell number

The results are collected in Tables 2 and 3.

Formulations 2 and 3 containing low molecular weight HAF according to the invention and applied externally, significantly increasing the thickness of the epidermis and dermal cellularity in SKH1 mice without hair (SKH1 hairless}, compared to formulations 1 and 4 containing high and very low HAF, respectively molecular weight (Table 2). Preparation 3 also significantly increases the number of epidermal and skin proliferative cells in SKH1 mice without hair (SKH1 hairless), compared to preparations 1 and 4 (Table 3).

External application of formulations 2 and 3 causes increased cellularity in surface and deep skin. The increased cells are mainly fibroblasts, which show the phenotype of activation with well developed intracytoplasmic reticulum.

2.2. Effect on HA index in the skin

The results are summarized in table 4.

The presence of HA in the skin was detected for formulations 2 and 3 (formulation 3&gt; formulation 2)

The HA indicator cannot be detected after applying Formulations 1 and 4.

So the inventors have shown that the external application of low molecular weight HAF has two main effects:

1. Great epidermal growth occurs by increasing cell proliferation;

2. Focal accumulation of HA in surface skin with a large increase in fibroblasts;

effects that are not observed for HAF with high or very low molecular weight.

EXAMPLE 2: Operation of a low molecular weight HAF retinal combination

1. MATERIALS AND METHODS

The fragments of Table 5 are included in typical cosmetic preparations such as those previously provided in the examples.

2. RESULTS

They were collected in tables 6 and 7 and in figures 1 and 2. External application of formulations 6 and 7 increases the number of foci of dense epidermis.

The effect of applying RAL alone (formulation 5) is comparable to the effect obtained for formulation 8, combining HAF with a small molecular size with RAL, which proves that there is no synergy between RAL and fragments with very low molecular weight.

The synergistic effect of the HAF-RAL combination is particularly evident at the dermal cellularity level of preparations 6 and 7 (see Table 5).

External application of formulations 6 and 7 also increases the color of HA dermis (formulation 7&gt; formulation 6) (see Table 7).

The synergistic effect of the HAF-RAL combination is also demonstrated by ELISA determination of the presence of HA in the dermis and epidermis of mice that have been treated with the various preparations mentioned above.

The results show a significant increase in HA production both in the dermis (Fig. 2) and in the epidermis (Fig. 1) after treatment with the HAF-RAL combination (formulation 7) compared with treatment by RAL alone (formulation 5) or HAF alone (formulation

3) .