Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
1999-04-09
2003-09-16
Low, Christopher S. F. (Department: 1653)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C514S002600, C514S012200, C530S350000, C424S401000
Reexamination Certificate
active
06620790
ABSTRACT:
This application is a national stage filing under 35 U.S.C. §371 of International Patent Application No. PCT/FR97/01164, filed Jun. 30, 1997 and designating the United States, which International Application was published by the International Bureau on Jan. 8, 1998 in French, not in English, as WO 98/00095.
1. Field of the Invention
The invention relates to an isolated arachidonic heteromer. This heteromer can be used in particular to stimulate and/or induce hair growth and/or check hair loss or to accelerate the healing process or to test substances that affect the activity of fatty acids or further as a diagnostic tool.
2. Description of the Related Art
Fatty acids, such as arachidonic acid and its derivatives, play an important role in particular in the synthesis of cell or nuclear membranes, in the inflammation mechanism as precursors to mediators and as skin barriers, as well as in the control of skin cell differentiation and proliferation.
Furthermore, it is written that fatty acids affect the expression of many genes through nuclear receptors, called PPARs (peroxisome proliferator-activated nuclear receptors).
Due to their hydrophobic and brittle characteristics, fatty acids are disintegrated, stabilized and carried in the biological environment by specific cytosolic carrier proteins of low molecular weight (15 kDa) called FABP (fatty acid-binding proteins). The skin it the most active tissue as far as the synthesis of lipids is concerned. But, certain lipids called essential fatty acids, such as arachidonic acid and linoleic acid, cannot be synthesized by these cells and must therefore be acquired from humors, The capture and release mechanism of these fatty acids by the cells, and more specifically by the keratinocytes, is not known, However, it seems that this mechanism takes place by means of carrier proteins, even though to date the identification of these proteins remains unsuccessful. Recently, among the keratinocytes, a specific protein (called E-FABP) has been described as binding to epidermal fatty acids with a strong affinity for fatty acids containing 18 carbon atoms, such as stearic acid, oleic, linoleic and linolenic acid, but with a weak or nonexistent affinity for arachidonic acid and its derivatives (Siegenthaler, G. et al. (1994) Biochem. J. 302, 363-371). Therefore, this protein could play an important role in the transportation of certain fatty acids, but not in that of the arachidonic acid and its derivatives.
Furthermore, it is known that inflammatory illnesses, such as rheumatoid arthritis, cystic fibrosis, or skin disorders with an inflammatory component tied to the release of arachidonic acid or its derivatives, such a psoriasis, eczema, atopic dermatitis, or yet the healing of skin lesions, present a deregulation or a modification of the metabolism of fatty acids and more specifically of the arachidonic acid.
In the cosmetic field, we may think that a deregulation or modification of the metabolism of fatty acids is what causes dry skin or, on the contrary, oily skin, or yet a fragilization of the skin.
From this point of view, we sought to understand the capture, release and transportation mechanism of fatty acids by the skin cells, such as keratinocytes.
SUMMARY OF THE INVENTION
In this way, surprisingly so, we were able to identify and isolate, in particular from human keratinocytes, a heteromer that binds to fatty acids, and more specifically to arachidonic acid and/or oleic acid.
DETAILED DESCRIPTION OF THE INVENTION
Thus, the object of this invention is an isolated heteromer that binds arachidonic acid. More specifically, it also binds oleic acid.
This heteromer also binds another essential fatty acid, linoleic acid.
Because of this property, we can see the advantage of this heteromer in particular in the understanding of the capture, release or transportation mechanism of the arachidonic acid or its derivatives and/or in the identification of substances that are susceptible of modifying this transportation mechanism and therefore the biological activity resulting from the arachidonic acid. By derivatives of the arachidonic acid, we mean all biological derivatives of the arachidonic acid such as in particular, the hydroxyacids, the thromboxanes, the leukotrienes and the prostaglandins.
Therefore, another object of this invention is the use of this heteromer as a diagnostic tool, more specifically for inflammatory diseases or skin disorders with an inflammatory component linked to the release of the arachidonic acid or its derivatives (see above) In the same way, the heteromer allows for the evaluation of the biological activity of substances that are likely to be active in the treatment of inflammatory diseases or skin disorders with an inflammatory component linked to the release of the arachidonic acid or its derivatives (see above).
Indeed, thanks to the purification procedure of the heteromer that will be described later, we are aware that a skin with psoriasis contains approximately ten times more of this heteromer than a normal skin (whose purified quantity reaches approximately 4 pmol/mg of protein), The quantity of the heteromer found, in particular by the process of purification described later, is therefore one way of diagnosing a skin disorder, such as psoriasis.
From this discovery, we can also consider measuring the evolution of the quantity of isolated heteromer using the keratinocytes of a human being who is being treated with a substance that is likely to treat his inflammatory illness or skin disorder.
Preferably, this isolated heteromer shows a dissociation constant Kd that is less than or equal to 300 nM in relation to the arachidonic acid, and preferably less than or equal to 200 nM.
These dissociation constants are obtained by carrying out measurements using the carbon-dextran technique. This technique is as follows: aliquot portions of 0.5 &mgr;g of heteromer in solution in 100 &mgr;l of Tris buffer (50 mM Tris/HCl, 25 mM NaCl, 2.5 mM EDTA, 1 mM DTT at pH 7.5) containing 0.5% of gelatin and 1% of DMSO are incubated for 1 hour at 37° C. in the presence of increasing quantities of radiolabelled ligand, i.e, 0-10 &mgr;M for the oleic acid and the arachidonic acid. Then, each aliquot portion is treated with 50 &mgr;l a mixture of carbon-dextran T40® (5% and 0.05% respectively) at 0° C. for 10 minutes, then centrifuged at 10000 g and the radioactivity of the supernatant is measured. A saturation curve is established, and the Kd is calculated using the Scatchard plot.
In other respects, this heteromer shows a dissociation constant in relation to the oleic acid that is less than that of the E-FABP in relation to the same ligand, the E-FABP being the protein described in the Siegenthaler, G. et al. (1994) Biochem. J. 302, 363-371 publication.
More specifically, the heteromer consistent with the invention is a protein, and this protein has a molecular weight of approximately 34 kDa±10% (analysis in non denaturing conditions (filtration over gel)).
In general, the molecular weights have been determined by column filtration chromatography [(Superose 12) coupled with an HPLC]. The column, balanced with a 50 mM Tris/HCl buffer containing 0.2M of NaCl is graduated with protein standards of molecular weight ranging from 6.5 kDa to 150 kDa, then, an aliquot portion of heteromer is passed through the column to determine its molecular weight. The elution profile of the proteins is analyzed by the measurement of optical density (OD) at 280 nm.
This heteromer may also bind in quantity up to 1.5 times more arachidonic acid than oleic acid.
Indeed, when a determined quantity of heteromer in solution is incubated in the presence of 600 nM of radiolabelled arachidonic acid, then analyzed through a filtration column over gel [(Superose 12) balanced with a Tris/HCl buffer containing 0.2M NaCl and connected to a HPLC], the radioactive peak that co-elutes elutes at 34 kDa and corresponds to the radiolabelled arachidonic acid-heteromer complex is 1.5 times greater than if the experiment were performed in the same condi
Burns Doane Swecker & Mathis L.L.P.
Low Christopher S. F.
Lukton David
Societe L'Oreal S.A.
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