EP2459162A2

EP2459162A2 - Injectable composition combining a filling agent and a fibroblast growth medium

Info

Publication number: EP2459162A2
Application number: EP10742206A
Authority: EP
Inventors: Jean-Noël THOREL; Hugues Gatto
Original assignee: Individual
Current assignee: Individual
Priority date: 2009-07-27
Filing date: 2010-07-06
Publication date: 2012-06-06
Also published as: WO2011015744A3; WO2011015744A2; KR101779905B1; BR112012001497A2; KR20120089433A; BR112012001497B1; ES2648391T3; JP2013500315A; FR2948286A1; EP2727581B1; FR2948286B1; EP2727581A1; JP5735965B2; CN102497851A; US9339450B2; US20120121534A1

Abstract

The invention relates a composition that can be subcutaneously or intradermally injected, including: a filling agent; and a fibroblast growth medium.

Description

INJECTABLE COMPOSITION COMPRISING A FILLING AGENT AND A FIBROBLAST GROWTH MEDIUM

The present invention is part of the development of injectable solutions for the treatment of wrinkles.

It proposes to associate a conventional filler, such as hyaluronic acid, with a fibroblast growth medium of well-defined composition for a revitalization of the dermis.

Prior state of the art

The skin is a continuously renewed tissue comprising a plurality of cells and specialized structures. Because of its constant contact with the environment, the skin is a protective barrier for the body. In addition, it is involved in many physiological processes that allow the body to maintain a fixed and constant temperature. In addition, the skin plays an important role in the immune system that protects the body from disease. At the structural level, the skin has three layers:

an outer layer, called epidermis,

an inner layer, called subcutaneous tissue,

and an intermediate layer, commonly referred to as a dermis. The natural human epidermis is composed mainly of three types of cells, namely the very major keratinocytes, melanocytes and Langerhans cells. The epidermis, as an outer layer, acts as a barrier against external agents. The epidermis itself comprises 5 distinct layers, from the depth to the surface: the basal layer or stratum germinativum,

the layer of Malpighi or stratum spinosum,

the granular layer or stratum granulosum,

the clear layer or stratum lucidum,

- and the stratum corneum or stratum corneum. The dermis, on the other hand, provides the epidemic with a solid support and acts as a nurturing element vis-à-vis the epidermis. The dermis consists essentially of fibroblasts and an extracellular matrix (ECM), which is mainly composed of collagen, elastin and a substance called the "fundamental substance". All of these components are synthesized by the fibroblasts. Leukocytes, hematocytes or tissue macrophages are also found in the dermis. In addition, it is crossed by blood vessels and nerve fibers.

Finally, the subcutaneous tissue or hypodermis is a layer of adipose and connective tissue that covers the nerves and large blood vessels.

During the aging process, various characteristic signs appear on the skin, reflecting a change in the structure and cutaneous functions. The main signs of aging skin include the appearance of fine lines and / or wrinkles, increasing with age. These wrinkles can be deep, medium or superficial and particularly affect the nasolabial folds, the periorbital area, the contour of the lips, the forehead, the intersurillary zone (lion's wrinkles). These lines and wrinkles result in depression or furrows on the surface of the skin.

Deep wrinkles are due to dermo-hypodermic changes, while superficial wrinkles can be explained by dermal and possibly epidermal changes. Wrinkles are often due to the loss of elasticity of the skin, in particular a relaxation of the tissues, as well as the production of fine lines of different thicknesses. When the dermis loses its elasticity, it weakens and begins to form deeper wrinkles. During the production of wrinkles, the collagen fibers, responsible for the elasticity and the structure of the skin, lose their characteristics, with an overproduction of metalloproteinase type enzymes. This abnormal amount of enzymes degrades the collagen matrix and thus leads to the production of deep wrinkles. Thus, over the years, the dermis tends to become thinner, especially at the level of its collagen layer. Other factors, such as free radicals, sun exposure, pollution, smoking, alcohol consumption or ozone, can cause damage to the skin, through the same phenomenon of activation of metalloproteinases and decomposition of collagen.

In recent years, the treatment of unsightly skin changes, particularly related to aging, has seen a significant rise and made enormous progress. Thus, it has been proposed various treatments, including the injection of natural or synthetic substances, to remedy skin disorders.

It may be noted in particular the application, in the form of local injections, botulinum toxin deactivated (Botox ^® ) or the implementation of laser techniques, or their use in combination.

An alternative to these technical solutions is the injection into the dermis of dermal filler. This filling can be carried out using non-resorbable products, such as polyacrylamide gels or polymethyl methacrylate (PMMA) particles. However, these components may cause intolerance reactions of the type inflammation or hypersensitivity.

For these reasons, the use of resorbable products, such as proteins or fats, has been considered. To date, the preferred technical solution is the use of substances naturally present in the human body, such as collagen or hyaluronic acid, which are at the origin of the majority of the products currently available on the market.

These absorbable products however have the disadvantage of being degraded fairly quickly in the body, which reduces their effectiveness and requires repeated and regular injections.

As a particular example of a natural resorbable product, hyaluronic acid is a preferred compound.

It should be mentioned that hyaluronic acid (HA) is a natural constituent of the dermis, where it plays an important role in the hydration and elasticity of the skin. he however decreases in quantity and quality with age, resulting in drying and thinning of the skin that wrinkles. Since hyaluronic acid is also highly hydrous and forms solutions with high viscosity in water, it is one of the most widely used pharmaceutical products.

To date, hyaluronic acid used in pharmaceutical products or medical devices for the treatment of wrinkles is in the form of a sodium or potassium hyaluronate gel. However, these sodium or potassium hyaluronate gels have a fairly rapid bioresorbability (typically ranging between 4 and 6 months), which requires that injections be repeated at close and regular intervals.

In an attempt to increase the duration of action of hyaluronic acid, stabilized forms of HA have been developed. In particular, these are chemically crosslinked HA gels. This crosslinking, via intramolecular or intermolecular bridging, would increase the persistence of the product inside the dermis. Alternatively, it has been envisaged to encapsulate hyaluronic acid (WO2008 / 147817).

In addition, the latest developments in filling products have focused on the combination of different active ingredients in this application.

Thus, it has been envisaged to associate hyaluronic acid, acting by mechanical filler effect, with the cutaneous administration of other active substances in this context. By way of example, the document WO2008 / 139122 associates HA with a hyaluronic acid degradation inhibitor acting in vivo to ensure a certain preservation of the injected HA molecules.

It appears however that, despite the different alternatives available on the market, there is still the need to develop technical solutions to ensure effective skin repair, lasting over time and being as painless as possible for the skin. Description of the invention

In this context, the Claimant has entered a totally new approach. While the prior art advocated associating a filler, such as hyaluronic acid, with a protective agent thereof, the present invention proposes acting at two distinct levels to restore good skin appearance. , especially to fight against aging. Thus, the present invention relates to a composition (dermatological, cosmetic or therapeutic) injectable, combining both a so-called mechanical filler, known as such, and secondly, a fibroblast growth medium. In practice, it is therefore both to act physically to fill the asperities or furrows that constitute wrinkles and secondly to stimulate the growth of fibroblasts in the dermis. Fibroblasts can also grow inside furrows and in addition synthesize substances such as elastin and collagens that promote regeneration of the dermis. In addition, the growth medium is capable of stabilizing and protecting the filling agent in the presence.

The first component of the combination according to the invention is therefore a mechanical filler whose essential function is to create a volume inside the wrinkles.

In particular, mention may be made of dextran sulphate, elastin, collagen or hyaluronic acid. Synthetic fillers, such as silicones or polyurethane gels, are also covered by the present invention.

In the search for a technical solution that is as compatible as possible with the skin, the use of polymers naturally present in the skin is preferred. This makes it possible to disturb the composition of the dermis as little as possible and reduces the risk of allergic or inflammatory reactions. Even more advantageously, the natural polymer is hyaluronic acid. In a known manner, hyaluronic acid can be in various forms: salts, derivatives such as esters or amides, in linear form or crosslinked chemically. All these forms are conceivable in the context of the present invention. While crosslinking can increase the life of hyaluronic acid molecules in the body, these changes affect its physico-chemical characteristics, its biological properties and its potential immunogenicity.

In the search for a technical solution as neutral as possible for the skin, called "biomimetic", non-crosslinked hyaluronic acid, as well as its physio-logically acceptable salts, is therefore preferred insofar as this molecule is a natural constituent of dermis. The term physiologically acceptable salts of hyaluronic acid, especially the sodium and potassium salts, and their mixture.

Advantageously, the filler, advantageously hyaluronic acid, represents from 0.07 to 3% of the total weight of the composition, advantageously from 0.8 to 2.5%. It should be noted that the degree of crosslinking and the molecular weight of the hyaluronic acid chosen may depend on the intended application, in particular on the depth of the wrinkles to be treated.

The second necessary component of the composition according to the invention is therefore a fibroblast growth medium.

In the context of the present invention, a fibroblast growth medium is defined as a complete medium allowing not only maintenance of the fibroblasts alive, but also stimulating their multiplication. The implementation of a functional growth test makes it possible to determine whether a given medium corresponds to a fibroblast growth medium according to the invention. In particular, a suitable functional test well known to those skilled in the art is the test for the observation of the density of living cells by colorimetric method, using the WST-I reagent and a reading at 450 nm (Berridge, MV et al. (1996): The Biochemical and Cellular Basis of this Proliferation Assays That Use Tetrazolium Salts, Biochemica 4, 15-19.) A fibroblast growth medium is for example commercially available: it is the standard culture medium DMEM (Sigma) supplemented with cell growth factor SVF (fetal calf serum), at a level of 10% by weight.

In general, such media contain extracts of animal or cellular origin which have a stimulating effect on the growth of fibroblasts, but which have the drawback of not having a specific composition or of containing non-traceable exogenous elements. such as FCS, beef pituitary stem extracts, EGF ("epidermal growth factor") cell growth factors, FGF ("fibroblast growth factors"), insulin or cholera toxin, hydrocortisone, piperazine, ...

Advantageously, the fibroblast growth medium used in the context of the present invention does not contain cell growth factor or biological extract of animal or cellular origin, in particular if these are not traced. or traceable and / or not of definite composition.

The term "non-traced" or "non-traceable" means that the source of the biological material in question and / or the treatment to which it is subjected can not be established or controlled.

In practice, said medium is advantageously devoid of (free) biological extract of animal or cellular origin, cell growth factor or compound, or a hormone.

In a preferred embodiment, it is therefore a question of introducing, by injection into the dermis, a fibroblast growth medium as compatible as possible with the natural composition of the skin, namely a medium comprising biodermal constituents (contained in the invention). natural state in the skin), biomimetic and / or biocompatible (biological elements mimetic or neutral for the skin).

Such a medium makes it possible to provide the fibroblasts with optimized nutritional intake in the form of vitamins, trace elements, amino acids, mineral salts, simple sugars (such as glucose, ribose, deoxyribose) and / or complex (such as than HA), and natural growth factors in the form of nucleic acid constituents (nitrogenous and pentose bases required for the formation nucleotides, as well as nucleosides). It also advantageously has pH characteristics (between 6.5 and 7.9, advantageously between 7.4 and 7.6 and osmolarity (between 280 and 450 mOsm, advantageously between 300 and 350 mOsm) physiological.

It should be noted that HA can be both an ingredient of the growth medium and a filler. The difference lies both in the form of HA (necessarily a hyaluronate salt in the medium) and in its quantity (much lower amounts in the medium).

According to a particular embodiment, all the constituents of the medium are naturally present in the skin (dermal constituents). However, and to stimulate the growth of fibroblasts, such a medium can be enriched with a substance exogenous to the skin but of natural origin, traceable and well-defined composition. A substance corresponding to this definition is, for example, a mixture of peptides extracted from milk, or MPC complex ("MiIk Peptide Complex"), obtained by successive precipitations of the milk and then separation of certain proteins subjected to enzymatic hydrolysis. This substance, which is in the form of a dehydrated powder, is advantageously added to the medium in an amount of 0.5 to 5 mg / ml, and even more advantageously in the range of 4 to 5 mg / ml.

According to another preferred embodiment, the fibroblast growth medium used in the composition according to the invention is devoid of EDTA and its salts and / or lipoic acid as metalloproteinase inhibiting agents.

By way of example, a complex medium corresponding to such a definition has been developed by the Applicant and corresponds to the association of about sixty components in precisely defined quantities as follows:

A composition according to the invention may further contain other ingredients or excipients, commonly used in this application, including derivatives or purified fractions of the HA. However, according to a particular embodiment, the injectable composition comprises only the two components described above.

As already said, this composition is intended to be injected and is therefore comparable to an injectable implant.

The implant according to the present invention is therefore intended to be injected into the superficial, medium or deep dermis, subcutaneously or intradermally, advantageously at the level of the face.

According to an advantageous embodiment, the composition is in the form of a gel, because of the injectable application subject of the invention. Remarkably, this constraint is fully compatible with the fibroblast growth media described above, which is formable in gel form, by incorporating HA without the addition of exogenous excipients.

Even more advantageously, the composition is in the form of a monophasic hydrogel, namely a hydrogel in the form of a single homogeneous phase. The viscosity of the composition obtained can be easily adjusted, in particular by adjusting the composition and the amount of the filler. In the case of hyaluronic acid, one can play both on the concentration which typically varies between 0.07% and 3% by weight of the composition, but also in terms of its degree of crosslinking or its molecular weight.

The injectable composition according to the invention may also be the subject of a kit which further comprises syringes that may contain said composition. This is for example single-dose syringes of 0.5 to 1.5 ml. In such a kit, the 2 essential components of the composition may be presented as a mixture in one syringe, or in 2 separate syringes for extemporaneous mixing.

Given the therapeutic aim, such a composition is advantageously subjected to sterilization, sterilization taking place advantageously cold so as not to denature the components in the presence. This step may, for example, be carried out by filtration on a 0.22 μm membrane for the growth medium of the fibroblasts, and by separate sterilization according to a process known to those skilled in the art for HA.

Given their complementary mode of action, the two constituents of the composition according to the invention can be administered simultaneously, separately or spread over time.

As already stated, a composition according to the invention is intended to correct all cutaneous irregularities, and in particular to the treatment, improvement and / or prevention of skin aging. This includes wrinkles, fine lines, fibroblastic depressions and scars, especially in the areas of the face or forehead marked by expression lines.

It may as well be a cosmetic treatment that therapeutic. A composition according to the invention relates both to dermatology and to reconstructive surgery.

In other words, the present invention relates to a cosmetic or therapeutic treatment method of injecting the composition defined in the present application. Note that the mixture of the two essential constituents of this composition can be done extemporaneously. Likewise, their injection can take place in a non-simultaneous manner.

Thanks to the particular composition according to the present invention, two complementary physiological actions are therefore targeted and obtained: on the one hand a mechanical action of filling irregularities, but also the action of promoting cellular renewal and, in fact, the synthesis of constituents neo-synthesized by fibroblasts, including collagen and elastin. This results in a remodeling of the extracellular matrix and a revitalization of the dermis.

There is thus a phenomenon of immediate mechanical filling from the injection and a long-term effect of cellular regeneration. It can therefore be envisaged to reduce the concentrations of filler during treatment. Indeed, once the growth of the fibroblasts takes over, the mechanical filler becomes less necessary and in practice, the amounts of filler can be decreased. Furthermore, in a preferred embodiment, such a composition is completely biocompatible with the skin, insofar as it consists essentially of constituents naturally present in the dermis. In fact, it does not disturb the microenvironment of the skin, thus reducing the risk of inflammatory or allergic reactions. Moreover, it has been shown that such biomimetic and biocompatible media allow stimulated fibroblast growth in the presence of serum. They are therefore particularly suitable candidates in the context of a dermal injection, since the dermis is richly vascularized.

The invention will now be listed in a nonlimiting manner by the following examples, in support of the appended figures.

Legends of the figures

FIG. 1 illustrates the comparative growth of human fibroblasts in culture in a fibroblast growth medium according to the invention and the standard medium.

DMEM (sigma), without growth factor.

FIG. 2 represents the collagen levels in the superficial and middle dermis of healthy skin, altered by UV radiation and after treatment with a fibroblast growth medium according to the invention.

Figure 3 corresponds to histological sections showing the marking of collagen fibers in healthy skin, altered by irradiation and after treatment with a fibroblast growth medium according to the invention.

FIG. 4 illustrates the dosage of elastin in healthy skins, then modified by UV radiation and then after treatment with the fibroblast growth medium according to the invention.

Figure 5 corresponds to histological sections showing the labeling of elastin fibers in healthy skin, skin altered by irradiation and then treated with the fibroblast growth medium according to the invention.

FIG. 6 represents the GAG levels in the superficial and middle dermis of healthy skin, skin damaged by UV radiation and after treatment with the fibroblast growth medium according to the invention.

Figure 7 corresponds to histological sections showing the labeling of GAGS in healthy skin, skin altered by UV irradiation and after treatment with the fibroblast growth medium according to the invention. Examples of realization

1 / Use of a fibroblast growth medium in an injectable composition: a) Composition of the medium:

b) Culture of human fibroblasts

Protocol

- Human fibroblasts seeded at low density in 96-well plates under standard culture medium DMEM supplemented with cell growth factor SVF (Fetal calf serum);

After 24 h, culture in the medium according to the invention or in the standard DMEM medium without growth factor;

- Lack of environmental renewal during the experiment;

Density of the living cells evaluated at TO and then after 2, 4, 7 and 9 days, by colorimetric method (reagent WST-I).

Results

The culture medium according to the invention supports, alone, the growth of fibroblasts over a period of 9 days. From the 7th day, a slowing down of cell growth is observed, explained by the non-renewal of the medium (Fig. 1).

In DMEM without FCS, there was a decrease in cell viability after 2 days and no cell growth throughout the study (Fig. 1). In conclusion, it appears that the fibroblast growth medium used according to the invention allows survival and stimulates the growth of normal human fibroblasts in the absence of exogenous growth factors. c) Aid for the repair of dermal components

Protocol

- Skin fragments taken from 8 donors, deposited in inserts and placed in culture medium.

- Low dose UVA irradiation at OJ and J2 (reduction of fibroblast metabolism, alteration of macromolecules of the connective tissue).

Medium according to the invention added to the surface of the skin from D3 to D14 (imbibed paper).

Negative control without irradiation (healthy controls) or without application of the medium according to the invention (UV only)

- Histological analysis (marking):

Collagen (Sirius Red) and elastic fibers (Catechin)>% surface area occupied at the level of the superficial and average dermis (computer-assisted image analysis).

• Glycosaminoglycans (GAGs) (heme stain)> semiquantitative score (intensity of the staining).

- Biochemical determination (spectrocolorimetric):

Total collagen: on skin fragments after enzymatic digestion and grinding.

• Elastin soluble: in culture supernatants.

GAGs: on fragments of ground skin.

Results

Significant reduction in the amount of collagen and elastin in the superficial and deep dermis following UV irradiation (Figs 2 to 5).

Statistically significant increase in the labeling of collagen and elastin after treatment with the medium according to the invention (fiber repair) (Figs 2 to 5).

Stimulation of fibroblast metabolism by the medium according to the invention with a significant increase in elastin dosage and a tendency to increase the level of collagen (Figs 2 to 5). Significant and significant increase in the amount of total GAGs in the dermis following the application of the medium according to the invention (Figures 6 and 7).

This model made it possible to quantify, on histological sections, the connective tissue repair properties of the fibroblast growth medium used.

In conclusion, it appears that this medium stimulates the repair and restoration of essential components of the dermis (collagen fibers, elastin, GAGs) during tissue alterations.

2 / Preparation of an injectable salt for the treatment of wrinkles:

Growth medium of fibroblasts.

HA added up to 3% by weight of the total composition, with preferentially: 0.8% for the treatment of superficial wrinkles, 1.6% for the treatment of middle wrinkles and 2% for the treatment of deep wrinkles . Formulation of a gel: HA is dissolved in the fibroblast culture medium. The concentration of HA determines the viscosity of the final preparation. By way of example, the HA used is sodium hyaluronate, the molecular weight of which is between 1.3 and 1.8 MDa. The injectable gel according to the invention does not contain any additive, all of the components of the formula acting both as excipients and active ingredients.

Sterilization: by 0.22 μm membrane filtration for the fibroblast growth medium, and by separate sterilization according to a process known to those skilled in the art for HA.

Injection protocol: Depending on the area to be treated and the depth of the wrinkles one or more sessions are envisaged. To maintain the results, it may be necessary to perform biannual retouching, the filling of wrinkles for as long as the skin is younger.

Claims

1 / Injectable composition by subcutaneous or intradermal route comprising:

a dextran sulfate, elastin, collagen, hyaluronic acid, silicone or polyurethane gel fillers; and

a fibroblast growth medium free of any cell growth factor or any biological extract of animal or cellular origin, not marked and / or of undetermined composition. 2 / A composition according to claim 1, characterized in that it consists of the filler and fibroblast growth medium.

3 / Composition according to one of the preceding claims, characterized in that it is in the form of a gel.

4 / Composition according to one of the preceding claims, characterized in that the filler is hyaluronic acid or a salt thereof.

5 / Composition according to one of the preceding claims, characterized in that the filler, preferably hyaluronic acid, is from 0.07 to 3% by weight of the composition.

6 / A composition according to one of the preceding claims, characterized in that the fibroblast growth medium consists of dermal components, and advantageously enriched with a mixture of milk peptides (MPC).

Composition according to one of the preceding claims, characterized in that the fibroblast growth medium comprises:

nucleic acid constituents,

amino acids,

simple and complex sugars,

vitamins,

and an inorganic fraction containing trace elements and inorganic salts. 8 / Kit being in the form of syringes and containing a composition according to one of claims 1 to 7. 9 / composition according to one of claims 1 to 7 or kit according to claim 8 for its use in the filling of wrinkles, fine lines, skin depressions and / or scars. 10 / Use of a composition according to one of claims 1 to 7 for the preparation of a medicament for preventing or treating skin aging.

11 / Cosmetic process for filling wrinkles and / or fine lines, comprising injecting at least one composition according to one of Claims 1 to 7.

12 / composition according to one of claims 1 to 7 as a combination product for simultaneous use, separate or spread over time, for the prevention or treatment of wrinkles, fine lines, skin depressions and / or scars.