HK1170412B - Injectable composition combining a filling agent and a fibroblast growth medium - Google Patents

Description

Injectable composition combining a bulking agent and a fibroblast growth medium

Technical Field

The present invention has been accomplished on the basis of the development of an injectable solution for treating wrinkles.

The present invention proposes to combine classical bulking agents (e.g. hyaluronic acid) with fibroblast growth media of well-defined composition that activate the dermis.

Prior Art

Skin is a tissue that is constantly renewed and includes a variety of cells and specialized structures. In long-term contact with the environment, the skin forms a protective barrier for the body. In addition, it involves many physiological processes that enable the body to maintain a constant, fixed temperature. Moreover, the skin plays an important role in the immune system, which protects the body from diseases.

Structurally, the skin consists of three layers:

-an outer layer, a skin,

-an inner layer, subcutaneous tissue,

and an intermediate layer, commonly known as dermis.

The natural human epidermis is composed of three types of cells: the majority of keratinocytes and melanocytes, and Langerhans cells (cells de Langerhans). The epidermis, which is the outer layer, acts as a barrier to foreign matter.

The epidermis itself has 5 distinct layers; from the deepest layer to the outermost layer:

a basal layer or a stratum germinativum (stratum germinativum),

-the Malpighian layer (coeche de Malpighi) or the spinous layer (stratum spinosum),

-a granular or granular layer (stratum granulosum),

a transparent layer (stratum lucidum), and

the stratum corneum or stratum corneum (stratum corneum).

The dermis provides the epidermis with solid support and nutrients. Essentially, the dermis is composed of fibroblasts and an extracellular matrix (ECM) composed mainly of collagen, elastin and a substance called the "substrate". All these components are synthesized by fibroblasts. Leukocytes, erythrocytes and even tissue macrophages are also found in the dermis. In addition, it is also traversed by blood vessels and nerve fibers.

The subcutaneous tissue or hypodermis (hypoderm) is a layer of fat and connective tissue that covers nerves and large blood vessels.

During aging, various characteristic markers appear on the skin, indicating changes in its structure and function. The main hallmark of skin aging is the appearance of fine lines and/or wrinkles, which increase with age. These wrinkles may be deeper or intermediate or superficial, especially in the nasolabial folds, periorbital areas, the labial contour, the area between the forehead and the eyebrows (lion lines) (rides du lion). These wrinkles and fine lines are seen as depressions or folds in the skin surface.

Deep wrinkles are believed to be due to dermal-subdermal changes, while superficial wrinkles may be explained by dermal and possibly epidermal changes.

Wrinkles are often caused by loss of skin elasticity (especially tissue softening) and the creation of fine lines of varying thickness. When the dermis loses elasticity, it is weakened and begins to form deeper wrinkles. As wrinkles develop, collagen fibers, which are responsible for skin elasticity and structure, lose their properties with the overproduction of metalloproteases. This abnormal amount of enzyme degrades the collagen matrix, resulting in the development of deep wrinkles. Thus, the dermis becomes thinner over time, particularly its collagen layer.

Other factors (e.g., free radicals, exposure to sunlight, pollution, smoking, alcohol intake, or ozone) can damage the skin by the same phenomenon of activating metalloproteases and collagen breakdown.

In recent years, the management of unwanted skin changes, particularly those associated with aging, has progressed rapidly and has made tremendous progress.

Various treatments have been proposed, in particular the injection of natural or synthetic substances, to repair skin changes.

In particular, we should mention the use of inactivated botulinum toxinLocal injection and the use of laser techniques, or both techniques may be used together.

An alternative to these solutions is to inject a filling product (so-called dermal filler) into the dermis. Such filling may be performed with non-resorbable products, such as polyacrylamide gel or Polymethylmethacrylate (PMMA) particles. However, these compounds can lead to inflammatory or hypersensitivity intolerance reactions.

For these reasons, it has been considered to use resorbable products, such as proteins or lipids. At present, the preferred solution is to use substances existing in the human body in a natural state, such as collagen or hyaluronic acid, which is the basis of most of the products on the market at present.

However, these resorbable products have the disadvantage of decomposing rather rapidly in the organism, which reduces their efficacy and requires repeated injections at regular intervals.

An example of a naturally occurring resorbable product is hyaluronic acid, which is one preferred compound.

It should be mentioned that Hyaluronic Acid (HA) is a natural component of the dermis in which it plays an important role in maintaining hydration and skin elasticity. However, its quantity and quality decrease with age, leading to dry and thin skin, which then wrinkles. Hyaluronic acid is one of the most widely used drugs, since it is also very soluble in water and forms a highly viscous solution in water.

Currently, hyaluronic acid used in pharmaceutical or medical devices intended for the treatment of wrinkles is available in the form of sodium or potassium hyaluronate gels. However, these sodium or potassium hyaluronate gels are quite rapidly bioresorbable (typically for 4-6 months), which means that repeated injections must be made at regular, close intervals.

In order to prolong the duration of action of hyaluronic acid, a stable form of hyaluronic acid has been developed. In particular, these hyaluronic acids are chemically cross-linked HA gels. This crosslinking, by intramolecular or intermolecular bridging, is believed to extend the duration of the product in the dermis. As an alternative, encapsulation of hyaluronic acid is also contemplated (WO 2008/147817).

Furthermore, recent advances in filled products have focused on combining multiple active ingredients for this application.

Hyaluronic acid has been envisaged as a mechanical filler in combination with other substances active herein for transdermal application. For example, document WO2008/139122 combines HA with an inhibitor of hyaluronic acid degradation in vivo to ensure that a certain amount of injected HA molecules is preserved.

Despite the different alternatives appearing on the market, there is still a need to develop a technical solution that ensures effective skin repair for a long time and is as painless as possible to the skin.

Disclosure of Invention

In view of this situation, the applicant has adopted a completely new approach.

Although the prior art advocates combining a filler (e.g. hyaluronic acid) with an agent protecting said filler, the present invention aims to act at two different levels in order to reestablish a good skin appearance, in particular thereby serving an anti-ageing effect.

The present invention relates to injectable dermatological, cosmetic or therapeutic formulations combining known "mechanical" fillers with fibroblast growth media.

In practice, this means both physically filling the irregularities or folds that form the wrinkles and stimulating the growth of fibroblasts in the dermis. Fibroblasts can also grow in the folds and additionally synthesize substances that aid in skin regeneration, such as elastin and collagen. Furthermore, the growth medium is likely to stabilize and protect the filler of the present invention.

Thus, the first component of the combination of the invention is a mechanical filler, the main function of which is to build up volume within the wrinkles.

In this context, mention may in particular be made of dextran sulphate, elastin, collagen and hyaluronic acid. Synthetic fillers (e.g., silicone or polyurethane gels) are also contemplated by the present invention.

In seeking a technical solution which is as compatible as possible with the skin, it is preferable to use the natural polymers present in the skin. This interferes as little as possible with the dermal composition and reduces the risk of allergic or inflammatory reactions. The advantage is greater when the natural polymer is hyaluronic acid.

Hyaluronic acid is known to exist in different forms: salts, derivatives (e.g. esters or amides), linear or chemically cross-linked forms. All of these forms can be included in the present invention. Although cross-linking extends the life of the hyaluronic acid molecule in an organism, it affects its physical/chemical properties, its biological properties and its potential immunogenicity.

In the search for technical solutions that are as neutral as possible for the skin (that is to say biomimetic solutions), non-crosslinked hyaluronic acid and its physiologically acceptable salts are preferred, since this molecule is a natural component of the dermis. Physiologically acceptable salts of hyaluronic acid mean in particular the sodium and potassium salts and mixtures thereof.

The filler (preferably hyaluronic acid) is present in an amount of 0.07-3%, more preferably 0.8-2.5% by weight of the total composition.

It should be noted that the degree of crosslinking and the molecular weight of the hyaluronic acid chosen may depend on the application aimed at, in particular on the depth of the wrinkles to be treated.

The second essential component of the composition of the present invention is a fibroblast growth medium.

For this invention, fibroblast growth medium is defined as a complete medium that not only maintains fibroblast survival but also stimulates its proliferation. The growth function assay can be used to determine whether a given medium is a fibroblast growth medium of the present invention. In particular, a suitable functional assay well known to those skilled in The art is colorimetric visualization of viable Cell density using WST-1 reagents and reading The results at 450nm (Berridge, M.V. et al (1996): The Biochemical and cellular basis of Cell Proliferation Assays Using Tetrazolium salts. Biochemical 4, 15-19.)

An example of a commercially available fibroblast growth medium is DMEM standard medium (Sigma) supplemented with 10 wt% of cell growth factor FCS (fetal calf serum).

In general, such a medium contains an extract of animal or cellular origin, which in fact does stimulate the growth of fibroblasts, but has the disadvantage of being of undefined composition or of containing exogenous factors of untraceable origin, such as FCS (bovine pituitary extract), the cell growth factors EGF (epidermal growth factor) or FGF (fibroblast growth factor), insulin or cholera toxin, hydrocortisone, piperazine, etc.

Advantageously, the fibroblast growth medium used in the present invention is free of cell growth factors or biological extracts of animal or cellular origin, particularly when these factors or extracts cannot be traced to an undefined origin and/or composition.

The expression "untracked source" or "untracked source" means that the source of the biological material is at issue and/or cannot be confirmed or verified by the treatment to which the biological material is subjected.

In practice, the culture medium is preferably free of biological extracts of animal or cellular origin, free of cellular compounds or growth factors or hormones.

In a preferred embodiment, the fibroblast growth medium, which is as compatible as possible with the natural components of the skin, is introduced into the dermis by injection. This is a medium containing components that are biological dermis (naturally contained in the skin), biomimetic, and/or biocompatible (biomimetic or neutral to the skin).

The medium will provide the fibroblasts with, inter alia, vitamins, trace elements, amino acids, inorganic salts, simple sugars (e.g. glucose, ribose, deoxyribose) and/or optimal nutrients in the form of complexes (e.g. "HA") as well as natural growth factors in the form of nucleic acid components (nucleotide bases and pentoses required for the formation of nucleotides and nucleosides). Advantageously, the physiological pH values are 6.5-7.9, 7.4-7.6 and the osmotic pressure is 280-450mOsm, preferably 300-350 mOsm.

It should be noted that HA may be either a component of the growth medium or a bulking agent. The difference is the form of HA (which must be hyaluronate in the medium) and its amount (which is much lower in the medium).

According to a particular embodiment, all the components of the culture medium are naturally present (dermal components) in the skin (continuits dermiques). However, to stimulate the growth of fibroblasts, such media can be enriched with substances that are not present in the skin but are of natural, traceable origin and well-defined composition. Substances that meet this definition are, for example, peptide mixtures extracted from milk, or MPCs (milk peptide complexes), obtained by continuous precipitation from milk followed by enzymatic hydrolysis to isolate certain proteins. The substance in the form of a dehydrated powder is advantageously added to the culture medium at 0.5-5mg/ml, preferably at 4-5 mg/ml.

According to another preferred embodiment, the fibroblast growth medium used in the composition according to the invention does not contain EDTA or its salts or lipoic acid as metalloproteinase inhibitor.

As an example, the applicant has developed a complex medium that satisfies this definition, combining about 60 precisely quantified components as follows:

the compositions of the invention may also comprise other ingredients or excipients commonly used for this application, in particular derivatives or purified fractions of HA. However, according to a specific embodiment, the injectable composition consists of only two components as described above.

As previously mentioned, the composition is intended for injection and thus resembles an injectable implant.

Thus, the implant of the invention is intended to be injected subcutaneously or intradermally into the superficial, intermediate or deep dermis, preferably the face.

According to an advantageous embodiment, the composition is in the form of a gel, since the use in injectable form is an object of the present invention. Notably, this restriction is preferably compatible with the fibroblast growth media described above, which can be formulated into a gel by introducing HA without the addition of exogenous excipients.

For even greater advantage, the composition is in the form of a single phase hydrogel, i.e., a hydrogel in a single homogeneous phase. The viscosity of the resulting composition can be easily adjusted, particularly by adjusting the composition and amount of the filler. In the case of hyaluronic acid, the concentration may be adjusted, which generally varies from 0.07% to 3% by weight of the composition, as well as its degree of crosslinking or its molecular weight.

The injectable composition of the present invention may also form part of a kit which may contain the composition and additionally include a syringe. For example, these syringes may be single dose syringes of 0.5-1.5 ml. In such a kit, the 2 necessary components of the composition may be present in one syringe as a mixture, or in 2 different syringes for immediate mixing.

In view of the intended treatment, the composition is preferably sterilized, preferably using cold sterilization to avoid denaturation of the components. This stage can be performed by filtering the fibroblast growth media with a 0.22 μm membrane and sterilizing the HA alone using procedures well known to those skilled in the art.

In view of their complementary modes of action, the two components of the composition of the invention may be applied simultaneously, separately or in a spread over time.

As mentioned above, the composition of the invention is intended to correct all skin irregularities and is particularly useful for treating, ameliorating and/or preventing skin aging. Thus, wrinkles, fine lines, skin depressions and scars, particularly facial or forehead areas expressing wrinkles, are addressed.

It can be used for cosmetic or therapeutic purposes. Both dermatology and orthopedics involve the composition of the invention.

In other words, the present invention relates to a cosmetic or therapeutic method comprising the injection of a composition as defined in the present application. It should be noted that the two essential components of the composition may be mixed instantaneously. Also, they do not require simultaneous injection.

Due to the specific composition of the invention, two complementary physiological effects are targeted and obtained: one is a mechanical filling effect on irregularities and the other is an effect of promoting cell renewal and thus the synthesis of newly formed components (in particular collagen and elastin) by fibroblasts. This results in the reconstruction of the extracellular matrix and the neogenesis of the dermis.

Therefore, mechanical filling was performed immediately after injection, and finally cells were regenerated. Thus, it is contemplated that the concentration of bulking agent may be reduced during this treatment. In fact, once fibroblast growth has begun, the mechanical filler becomes less necessary and, in fact, the amount of filler can be reduced.

Furthermore, in a preferred embodiment, where the composition consists essentially of components naturally found in the dermis, the composition is completely biocompatible with the skin. For this reason, the microenvironment of the skin is not disturbed, thereby reducing the risk of inflammatory or allergic reactions. Furthermore, the biomimetic and biocompatible media have been shown to allow for the stimulation of fibroblast growth in the presence of serum. Therefore, they are particularly suitable candidates for dermal injection, as the dermis is rich in blood vessels.

The invention will now be illustrated by the following non-limiting examples with the aid of the accompanying drawings.

Drawings

FIG. 1 shows comparative growth of human fibroblasts in cultures of fibroblast growth medium of the invention without growth factors and standard DMEM medium (Sigma).

Figure 2 shows the collagen concentration in the superficial and medial dermis of healthy skin, skin altered by UV radiation and then treated with fibroblast growth media of the present invention.

Fig. 3 is a tissue section showing collagen fibers staining in skin after healthy skin, altered by UV radiation, and then treated with fibroblast growth media of the present invention.

Figure 4 shows the amount of elastin in healthy skin, skin altered by UV radiation and then treated with fibroblast growth media of the present invention.

Fig. 5 is a tissue section showing staining of elastin fibers in healthy skin, after irradiation modification and then treatment with fibroblast growth media of the present invention.

Figure 6 shows GAG concentrations in the superficial and intermediate dermis of healthy skin, skin altered by UV radiation and then treated with fibroblast growth media of the present invention.

Figure 7 is a tissue section showing GAG staining in skin after healthy skin, altered by UV radiation, and then treated with fibroblast growth media of the present invention.

Examples of embodiments

Use of fibroblast growth medium in injectable compositions

a)The culture medium comprises the following components:

b)human fibroblast cultures

Scheme(s)

Human fibroblasts were seeded at low density in 96-well plates in DMEM standard medium supplemented with FCS (fetal calf serum) cell growth factor.

After 24 hours, they were cultured in the pure medium of the present invention or in DMEM standard medium without growth factors.

No medium change during the experiment.

Viable cell densities at T0 and then after 2, 4, 7 and 9 days were determined using a colorimetric method (WST-1 reagent).

Results

The medium of the invention alone maintained fibroblast growth for 9 days. From day 7, a slow cell growth was observed, which can be explained by the fact that the medium was not changed (fig. 1).

In DMEM medium without FCS, a decrease in cell viability was observed after 2 days and no cell growth was observed throughout the study (fig. 1).

In summary, it appears that the fibroblast growth media used in the present invention allows normal fibroblasts to survive and stimulate their growth in the absence of exogenous growth factors.

c)Promoting repair of dermal components

Scheme(s)

Skin fragments were taken from 8 donors, placed in the insert and placed in culture medium.

Low dose UVA radiation (decrease fibroblast metabolism, alter connective tissue macromolecules) given at D0 and D2.

Addition of the culture medium of the invention to the skin surface at D3 to D14 (impregnated paper).

As negative control skin without irradiation (healthy control) or without application of the culture medium of the invention (UV radiation only) was used.

Histological examination (staining):

collagen (sirius red) and elastic fibers (catechin) > the percentage of area occupied in the superficial and middle dermis was evaluated (computer-aided image analysis).

Glycosaminoglycan (GAG) (Hale staining) > semi-quantitative score (staining intensity).

Biochemical measurements (spectrophotometry):

total collagen: on the skin fragments after enzymatic digestion and homogenization.

Soluble elastin: in the culture supernatant.

GAG: on pieces of homogenised skin.

Results

The amount of collagen and elastin in the superficial and deep dermis was significantly reduced after UV irradiation (fig. 2 to 5).

The staining of collagen and elastin with a statistically significant increase (fibrous repair) after treatment with the medium of the invention (figures 2 to 5).

The medium of the invention stimulates the metabolism of fibroblasts, in which the concentration of elastin increases significantly and collagen has an increasing trend (figures 2 to 5).

A significant increase in total GAG amount in the dermis after administration of the media of the invention (fig. 6 and 7).

The model allows to quantify the connective tissue repair properties of the fibroblast growth medium on histological sections.

In conclusion, it is clear that the culture medium stimulates the repair and recovery of the essential components of the dermis (collagen fibers, elastin, GAG) when the tissue has changed.

2 preparation of injectable gel for treating wrinkles:

-fibroblast growth medium.

-adding up to 3% HA by weight of the total composition, wherein preferably: 0.8% for treating superficial wrinkles, 1.6% for treating intermediate deep wrinkles, and 2% for treating deep wrinkles.

-a gel formulation: HA was dissolved in fibroblast culture medium. The HA concentration determines the viscosity of the final formulation. For example, HA used is sodium hyaluronate with a molecular weight of 1.3-1.8 MDa. The injectable gels of the present invention do not contain any additives and all components of the formulation function as excipients and active ingredients.

-sterilization: the fibroblast growth medium was filtered through a 0.22 μm membrane and the HA was separately sterilized using procedures known to those skilled in the art.

-an injection protocol: one or more stages are designed according to the area to be treated and the depth of the wrinkles. To maintain the results, it may be necessary to repeat the procedure over a 6 month interval, the longer the filling wrinkle lasts, the younger the skin.

Claims (10)

1. A composition for subcutaneous or intradermal injection comprising:

-a filler which is non-crosslinked hyaluronic acid or a physiologically acceptable salt thereof; and

-a fibroblast growth medium consisting of:

2. the composition of claim 1, characterized in that it consists of said bulking agent and said fibroblast growth medium.

3. Composition according to claim 1 or 2, characterized in that it is in the form of a gel.

4. Composition according to claim 1 or 2, characterized in that the filler represents 0.07-3% by weight of the composition.

5. The composition of claim 1 or 2, characterized in that the fibroblast growth medium is enriched in milk peptide complexes.

6. A kit in the form of a syringe comprising the composition of any one of claims 1 to 5.

7. Use of a composition according to any one of claims 1 to 5 for the preparation of a kit for filling wrinkles, fine lines, skin depressions and/or scars.

8. Use of a composition according to any one of claims 1 to 5 for the preparation of a medicament intended for the prevention or treatment of skin ageing.

9. Use of a composition according to any one of claims 1 to 5 for the preparation of a cosmetic product for filling wrinkles and/or fine lines.

10. Use of a composition according to any one of claims 1 to 5 for the preparation of a combination for simultaneous, separate or sequential administration and for the prevention or treatment of wrinkles, fine lines, skin depressions and/or scars.