Modulator from fractions of vegetable unsaponifiables

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Filagrinol is a clear liposoluble active ingredient, composed of fractions of vegetable unsaponifiables, modulating filaggrin production, and carrying out a specific epidermal moisturising action.

Chemically, it is a combination predominantly existing of polyions from vegetable oil unsaponifiables (wheat germ, olives and soybean) and the lipid fraction of entomophilous pollen. Filagrinol is a clear yellow-amber liquid (T=20°C), with characteristic odour, soluble in lipid systems. It does not contain any preservative. Its INCI Name is: Pollen Extract, Glycine Soja (Soybean) Oil Unsaponifiables, Olea Europaea (Olive) Oil Unsaponifiables, Triticum Vulgare (Wheat) Germ Oil Unsaponifiables. In particular, referring to pollen, the one used in Filagrinol is not anemophilous but exclusively entomophilous; entomophilous Acacia Farnesiana (Real Acacia) Pollen Extract. Therefore, it has no allergy risk.

Filaggrin

In mammalian epidermis, the cells are organised in specific layers that correspond to different developmental stages. The cells of the basal layer, on the membrane that divides dermis and epidermis, constitute the inner layer; they give origin to the prickle-cells, characterised by the presence of cytoplasmic filaments, and then to the granulose cells layer, which bear both filaments and keratohyalin granules. From this layer, completely keratinised cells of the stratum corneum originate, without nucleus, granules and other cellular organelles, with a thick membrane and keratin filaments packed and ’soaked’ in an amorphous proteic matrix of basic nature. It was shown in vitro that this basic protein of the stratum corneum is able to interact specifically with keratin filaments, in a ratio of one molecule for a triplet of keratin couples, giving origin to fibrous aggregates. For this reason, this protein was called Filaggrin (FILament AGGRegating proteIN). It is an epidermal protein (MW about 37,000 daltons) that has a key role in the keratinisation process. Filaggrin is at first synthesised in the epidermis cells in the form of its precursor, profilaggrin, in the granulose cells layer. This protein, mainly present in the keratohyalin granules, has a high molecular weight (ca. 300,000 daltons) and is composed of 10-12 tandem repetitions and it is a highly phosphorylated protein. It is highly enriched in histidine, up to 7% of its molecular weight. During the maturation process, profilaggrin is de-phosphorylated by a phosphatase and it is broken down into single proteins by a protease. Filaggrin is fundamental in maintaining integrity and good health of the stratum corneum and therefore of all epidermis, mainly due to its functions outlined below:

•  It promotes the organisation and aggregation of keratin protein chains of the stratum corneum. In fact, light keratin (MW from 46,000 to 58,000 daltons), which is usually found only in the basal elements, during the maturation from the epidermal basal layers to the superficial ones, polymerises and gives origin to heavy keratins (in the most external layers their MW ranges from 63,000 to 67,000 daltons). This process of keratinic maturation which allows the ‘packing’ of polypeptide chains (filaments) is allowed by means of the protein filaggrin. One molecule of this protein is linked to each triplet of keratinic couples to promote their organisation and aggregation.
•  As a consequence of its degradation, filaggrin generates a pool of hydrosoluble molecules which composes the natural moisturising factor (NMF). This is mainly composed by aminoacids (40%) and their metabolites (among these, the most abundant are urocanic acid and pyrrolidone carboxylic acid), urea (8%), lactic acid (12%), inorganic ions (K+, Ca2+, Cl–). These molecules play a role in the stratum corneum by maintaining the right level of hydration and thus flexibility, and in protecting it from UV rays.

Epidermal alterations and filaggrin

Changes in the amount and distribution of filaggrin may be associated with some keratinisation diseases and therefore also associated with modification of the structural integrity of the stratum corneum. For example, in patients affected by psoriasis, it was shown that the levels of urocanic acid in blisters is considerably lower, along with a lower amount of histidine. The decrease in urocanic acid levels may be caused by a minor formation starting from histidine, or to its degradation due to the enzyme, urocanase. However, patients affected by psoriasis do not show a decrease in filaggrin production, and urocanase is not present in blisters. Therefore it is reasonable to think that in the cutis of psoriasis-affected people, histidine is mainly converted in histamine due to histidine-decarboxylase, rather than being transformed in urocanic acid by histidase. Therefore Filagrinol, which activates the process ‘filaggrin-histidineurocanic acid’ by increasing the final amount of filaggrin, finally causes a considerable increase of levels of urocanic acid, essential for good moisturisation of epidermis, despite the altered conversion of histidine in histamine rather than urocanic acid in psoriasis-affected people. Therefore, Filagrinol constitutes a valuable and natural method of defence and prevention.

Applications in cosmetic products

The ingredient can be used for treatment of dehydrated, sensitive, dry and reddening skin; skin blemishes and other forms of reddening; treatment of skin ageing, sun damages; functional make-up preparations. Filagrinol should be added to the lipid phase. The recommended use levels are between 3% and 10%, for the formulation of O/W and W/O emulsions, lotions, masks, oils and ointments. Continuous use: 3%-5%; shock treatment: 6%-10%.

 Functional moisturisation

Since Filagrinol (now referred to as ‘the filaggrin modulator’) exerts its activity in the upper layers of the skin, it is recommended to be used in association with Hyaluramine-S, an active that carries out a deep and long lasting moisturising action. In fact, skin moisturisation can be performed in three ways:

•  By impermeabilisation, that is, by occluding the skin with a thin film which prevents water loss (passive moisturisation).
•  By applying hydrophilic substances which retain moisture within the epidermis (direct moisturisation).
•  By modulating the production of intra-epidermal moisture (active moisturisation).

 Impermeabilisation, as the name suggests, has the advantage of taking immediate effect, since it forces water to be retained in the most superficial layers of the skin. Its disadvantage is that the skin becomes dependent on a film. Furthermore, the epidermis is part of an organ – the skin – which is constantly being renewed, and suffocating is also a way of modifying its capacity to renew itself and thus of preventing the natural elimination not only of water but also of catabolitic waste. In other words, it can lead to a sort of paralysis of the skin. Passive hydration modifies skin’s fundamental ability to absorb and its important property to metabolise applied substances. Such inconveniences, reversible within a few hours, should not be underestimated when occlusive substances are used in cosmetic emulsions. Active hydration does not alter these functions. Hyaluramine, the oligomer of mucopolysaccharides, is a direct moisturiser since, by enriching the skin in mucopolysaccharides, it allows moisture to be stored through a direct link. The filaggrin modulator modulates the moisture content in the epidermis and therefore it is an active moisturiser. Therefore, moisturisation of epidermis is efficient only if:

•  Water supplied by dermis is sufficient to balance the skin Perspiratio insensibilis.
•  Water retention capacity of the upper layers of the skin system is effective and constant. This can be achieved through functional moisturisation (direct moisturisation + active moisturisation = Hyaluramine-S + Filagrinol).

Therefore, the correct management of the lipids used in the chosen cosmetic form is involved too. This management should take account of any modifications of their ratio during the whole process of differentiation and maturation of keratinocytes. The filaggrin modulator is also helpful against free radicals as it exerts a strong antilipoperoxidant activity.

Mechanism of activity

The filaggrin modulator accomplishes its action on specific metabolic steps of the epidermal keratinisation process. The evaluation of its efficacy has been carried out through a thorough series of histochemical, immunohistochemical and clinical studies. Following are some of the most significant of these.

ATPase (Adenosine TriPhosphatase) activity

The stratum granulosum is so called because of the keratohyalin granules in the cells. These granules contain medium to high molecular weight keratins, profilaggrin and filaggrin, together with other filamentous proteins. Profilaggrin, rich in histidine and highly phosphorylated, is transformed in filaggrin, the active form of the protein with a lower molecular weight, thanks to a maturation process genetically determined and carried out by specific enzymes. Profilaggrin proteins contain ATP molecules bound to the end of the protein filament and degradation is carried out by ATPase enzymes. Therefore ATPase activity is involved in the maturation process from profilaggrin (precursor) to filaggrin in epidermis. This degradation allows the cell to gain the necessary energy to pack and organise the keratin filaments in the stratum corneum. It has been demonstrated that the filaggrin modulator increases the ATPase activity, involved in the maturation process. Compared with controls, a significant enrichment in ATPase enzymes was shown at the level of the stratum granulosum (Figs. 1 and 2).

Histochemical determination of bound histidine

An increase in the ATPase activity led to the hypothesis of a parallel increase in profilaggrin and filaggrin molecules. These are proteins with a high content of histidine (profilaggrin is composed of 7% histidine). Therefore, the Pauly reagent (sulfanilic acid) was used in order to highlight the bound histidine inside a protein chain, determining a thin orange strip. Appropriate precautions have been taken in order to avoid the positive reaction of urocanic acid and tyrosine. The intensity of the colouration clearly marking the stratum granulosum is proportional to the presence of filaggrin: a proportional quantitative increase of the colour intensity was shown at the level of the stratum granulosum, due both to an increase of the granules in the single cells, and to an increase in the number of the cells themselves (Figs. 3 and 4).

Immunohistochemical determination of filaggrin

Filaggrin is a marker of the final process of keratinisation and it states the proper epithelial maturation. Using a specific anti-filaggrin antibody (Dale Method), the effect of the filaggrin modulator has been studied by checking the production of filaggrin after topical application. A red-brownish colouration highlights the epidermal cells that contain filaggrin, both in the stratum granulosum and in the first layers of the stratum corneum. It was possible to state that, besides a general healthy appearance of the epidermis and the dermis, filaggrin quantity was higher in the treated subjects compared to controls (Figs. 5 and 6). Clinical evaluation Since the filaggrin modulator was shown to be non-toxic, non-irritant and devoid of mutagenic effects, it was considered interesting to verify its moisturising properties by cutaneous application in humans. Treatment with an 8% filaggrin modulator O/W emulsion, applied on 30 volunteers (40-50 year old women – double blind) clearly showed that this product penetrates through the epidermis and determines a progressive improvement both of the quantitative parameters chosen to measure the hydration level of the skin, and of the global aspect of the skin to a subjective evaluation.

Protective action against lipid peroxidation caused by UV rays [Okawa]

One of the biochemical mechanisms that are actually able to explain the physiological pathogenesis of skin ageing is that of lipid peroxidation. This kind of chain reaction is usually triggered by free radicals. It is therefore reasonable to evaluate the capacity of a product to slow down the process of skin ageing by measuring its inhibiting properties against the lipoperoxidation process, artificially induced by UV radiation. Protective action of the filaggrin modulator was assessed by quantification of its inhibitory effect toward malondialdehyde production, a typical product of lipid peroxidation process. After treatment with an 8% filaggrin modulator O/W emulsion, a proportional decrease in nalondialdehyde production was shown:

•  35.4% – single topical application.
•  49.8% – after 10 days of repeated topical application.

The comparison between the single application and the continuous treatment clearly showed that the protective properties of the filaggrin modulator are cumulative, probably due to a progressive intake of molecules with antioxidant capacities, able to remove the reactive species generated by the lipid peroxidation process.

Safety profile

The filaggrin modulator is a safe product, characterised by the DCG trademark (Dermo Cosmetic Grade), which identifies the highly specialised products of Vevy Europe Fine Chemical division and certifies that each product is designed and developed for cutaneous application and tested according to rigorous toxicological, cryptotoxicological, enzyme-kinetic and dermatological activity protocols, and consequently, does not interfere with the biochemistry of the cutaneous apparatus. Products are also updated on the basis of scientific progress and compliant with national and international regulations. These premises lead to the development of projects and products, regardless of their source, that are characterised by:

•  Safety (absence of harmful effects).
•  Effectiveness (compliance with the promised and expected requirements).
•  Stability (persistence of the stated characteristics). Specifically, the filaggrin modulator has an excellent and rigorous toxicological profile.

Conclusion

The importance of a correct production of filaggrin is undeniable for maintaining a suitable level of epidermal moisturisation, as far as to ensure a physiological defence and a real barrier function of the skin as a whole. Filagrinol, being an active modulator of the filaggrinogenesis and biochemical base for a complete aggregation of fibrous keratin, is at the base of corneous hydration and elasticity, and leads to the formation of a highly efficient cornified barrier. Therefore, its use is particularly suitable in cosmetics for the treatment of sensitive, dry and couperose skin, skin blemishes and other forms of cutaneous reddening, senile, blotchy skin, sunscreen products (dehydrated actinic skin) to prevent solar keratosis, ageing skin care, and functional make-up preparations.