JP4954450B2 - Induction of lysyl oxidase isoform activity to address disease states due to failure, loss or disorder of elastic fiber formation - Google Patents

Description

The present invention relates to the induction of lysyl oxidase isoforms, more preferably the activity of LOX (lysyl oxidase) isoforms.

Properties of skin and mucous membranes such as resistance and elasticity are essentially determined by dermal collagen fibers and elastin fibers. Elastin is a protein that forms an elastic fiber that is also composed of other molecules such as fibrillin and MAGP (microfibrillar associated glycoprotein).

“Elastic fibers” are composed of elastin deposited on microfibrils. Elastin is synthesized as soluble tropoelastin, and tropoelastin exhibits its physicochemical properties (insoluble and elastic) when deposited on microfibrils by cross-linking between molecules and molecules with lysyl oxidase. The elastic fiber imparts contractility to an organ (tube, lung soft tissue, elastic cartilage, skin, etc.) containing the elastic fiber. Elastic fibers are mainly composed of elastin deposited on microfibrils. The name “elastin” is used for a protein that forms an amorphous part of an elastic fiber and gives elasticity to the elastic fiber.
Recently, it has been found that this component constituting the elastic fiber is present in the epidermis.

“Collagen fine fibers” are composed of three chains of collagen. This collagen fiber is also cross-linked by lysyl oxidase. The aging of the skin and mucous membrane is related to the denaturation of the fibrillar network, particularly the denaturation of elastic fibers that deteriorate and do not regenerate correctly. Similarly, the network of scar elastic fibers is not accurate. Five isoforms of LOX, LOXL, LOXL2, LOXL3 and LOXL4 are known in the lysyl oxidase (LO) family (Non-patent Document 1). It has been found that LOX is involved in the crosslinking of collagen fibers (Non-patent Document 2). LOX is also involved in the maintenance of cell homeostasis and is considered to be a regulator necessary for the maintenance of cell homeostasis by controlling NFκB (Non-patent Document 3).

Collagen synthesis is concomitantly deregulated in the case of pathological, disordered and / or nonfunctional elastic fiber formation, such as fibrosis and photoelastic fibrosis, and in the case of certain cancer pathologies. Unlike collagen and LOX expression, which involves densification of the matrix as a defense mechanism against cell invasion, the synthesis of collagen that does not express LOX involves pathological changes in the extracellular matrix, as in cancer ( Non-patent document 4).

Elastin synthesis is deregulated in the case of pathological, disordered and / or non-functional elastic fiber formation, such as fibrosis and photoelastic fibrosis, and in the case of certain cancer pathologies. This disordered elastin synthesis leads to the formation of derived peptides that are generally considered to be favorable for cellular tumor potential (Non-Patent Document 5).

In the prior art, there is no standard that can evaluate the influence of an active ingredient in skin / cosmetics on the new formation of functional elastic fibers. In this situation, it is also difficult to define a desired standard that can evaluate the effect of the active ingredient to be investigated. The actual screening method for active ingredients affects the evaluation of the expression of genes involved in the formation of elastic fibers such as elastin and fibrillin.

Currently, animal experiments are prohibited in cosmetics in Europe, and human experiments are ethically challenged. For this reason, the present inventors cannot carry out screening using animals or humans for cosmetic applications.

In three-dimensional models such as Mimeskin® (Coletica, Lyon, France), keratinocytes induce the synthesis of tropoelastin and the deposition of tropoelastin on microfibrils (Non-Patent Document 6). In the Mimeskin® model, the extracellular matrix had radial collagen and elastic fibers made of elastin deposited on microfibrils, and exhibited an ultrastructure similar to the ultrastructure of the skin. This model has also been used to test the effectiveness of certain molecules such as lysyl oxidase inhibitors. Using this model, inhibition of lysyl oxidase induces collagen fiber and elastin fiber rupture, but the expression level of terminal differentiation markers (filaggrin, etc.) is also reduced, so deviation from the keratinocyte differentiation program Has also been demonstrated to be induced (Patent Document 1). In this patent document, no distinction is made between different LO isoforms.

In these studies, pathological, disordered and / or non-functional elastic fiber formation, such as in the case of fibrosis, photoelastic fibrosis, skin streaks, and / or dystrophic scars and cancer pathologies In some cases, a screening method for active ingredients that induces the expression of LOX and down-regulates the formation of elastic fibers could not be developed.

Thus, in the prior art, in the case of pathological, disordered and / or non-functional elastic fiber formation, such as in the case of fibrosis, photoelastic fibrosis, cutaneous striatum, and / or dystrophic scar, It is not possible to provide an active ingredient that induces the expression of and down-regulates the formation of elastic fibers.

Furthermore, in the conventional technology, the expression method of the lysyl oxidase LOX isoform cannot be accurately specified because the provided method is inaccurate and not sufficiently specific for various isoforms of lysyl oxidase. .

French patent 01 10443 Csiszar, Lysyl oxidases: A novel multifunctional amine oxidase family, Nucleic Acid Research and Molecular Biology, 2001, vol 70, p2-28 Seve et al., Expression analysis of recombinant lysyl oxidase (LOX) in myoblast-like cells, Connective Tissue Research, 2002, 43: 613-619. Jaye S, Pianetti S, Kagan HM, Sonenshein GE. Lysyl oxidase inhibits ras-mediated transformation by presenting of NF-kappaB. Mol Cell Biol 23: 2251-63, 2003 Decitre et al. Lab. Invest. 78, 143-151, 1998 Brassart B, Fuchs P, Huet E, Alix AJ, Wallach J, Tamburro AM, Delacoux F, Haye B, Emerald H, Hornebeck W, Debell L. Conformational dependency of collagenase (matrix metalloproteinase-1) up-regulation by elastin peptides in cultured fibroblasts. J. et al. Biol. Chem. 276 (7): 5222-7, 2001 Duplan-Perrat et al., Keratinocytes influencing the maturation and organization of the elastin network in a skin equivalent. J. et al. Invest. Dermatol. 114: 365-70, 1999

The purpose of the present invention is mainly to treat the above mentioned technical problems, in particular pathologic, disordered and / or nonfunctional, such as in the case of fibrosis, photoelastic fibrosis, skin streaks and / or dystrophic scars. To solve the technical problem for the purpose of providing a screening method for an active ingredient that induces the expression of LOX or its homologues or essential homologues to control the formation of elastic fibers in the case of forming elastic fibers That is. The term “functional elastic fiber” is used in the usual sense in the above-described conventional technology, but particularly in the present invention, it means an elastic fiber having contractility derived from a three-dimensional structure.

The present invention provides elastic fiber formation in the case of pathological, disordered and / or non-functional elastic fiber formation, particularly as in the case of fibrosis, photoelastic fibrosis, skin striatum, and / or dystrophic scar. It also relates to the use of an isoform of lysyl oxidase LOX and an active ingredient that inhibits the expression of the protein elastin to control. Thus, the resulting effect is synergistic.

The present invention provides elastic fiber formation in the case of pathological, disordered and / or non-functional elastic fiber formation, particularly as in the case of fibrosis, photoelastic fibrosis, skin striatum, and / or dystrophic scar. Use of an active ingredient to induce enzymatic activity or expression of an isoform of lysyl oxidase LOX to control, wherein the active ingredient inhibits the expression of protein elastin or the expression of protein elastin It also relates to a use that is in combination with a second active ingredient that inhibits. Thus, the resulting effect is synergistic.

The present invention can also solve the technical problem consisting of providing an effective method for identifying the location of LOX expression and tracking this expression.

In the present description, we mean the isoform of human lysyl oxidase protein LOX having the sequence of SEQ ID NO: 1 by the term “LOX” or “hLOX”.
In the present description, the inventors mean the human tropoelastin protein or the elastin protein having the sequence of SEQ ID NO: 3 by the term “elastin”.
In this description, the inventors mean various collagens present in human skin tissue by the term “collagen”.

In the present description, by the term “LOXL” or “hLOXL” we mean the isoform-like (L) human lysyl oxidase protein LOXL having the sequence of SEQ ID NO: 6.
In the present description, the present inventors not only induced the expression of LOX-encoding gene, particularly the synthesis of LOX-encoding messenger RNA by “inducing the expression of lysyl oxidase LOX isoform”. It also means the induction of the protein LOX synthesized from messenger RNA. The LOX cDNA has the sequence of SEQ ID NO: 2.

In the present description, the present inventors not only inhibit the expression of the gene encoding the protein elastin, particularly the inhibition of the synthesis of the messenger RNA encoding the protein elastin by “inhibiting the expression of the protein elastin”. Also means the inhibition of the synthesis of the protein elastin from or the precursor tropoelastin. The cDNA for the protein tropoelastin has the sequence SEQ ID NO: 4.

In the present description, the present inventors have not only induced the expression of the gene encoding collagen Ialpha1, particularly the induction of the synthesis of messenger RNA encoding collagen Ialpha1, by “inducing the expression of collagen I”. It also means the induction of the synthesis of collagen I or its precursor procollagen I from this messenger RNA.

This induction of LOX expression is associated with elasticity in the case of pathological, disordered and / or non-functional elastic fiber formation, such as in the case of fibrosis, photoelastic fibrosis, cutaneous striatum, and / or dystrophic scars. It must be implemented to be sufficiently effective to contribute to the control of fiber formation. In certain medical conditions, it is desirable to inhibit the expression of the protein elastin so as to obtain an effective effect of controlling the disordered elastic fiber formation seen especially in the case of photoelastic fibrosis or fibrosis.

That is, in the present invention, the present inventors induce expression of LOX or a homologue or essential homologue thereof, or enzymatic activity of LOX or a homologue or essential homologue thereof, and protein elastin Is intended to inhibit the expression of.
By "its homologue or essential homologue" is meant a homologue of the lysyl oxidase isoform LOXL having the same or similar activity as LOXL as defined herein.

As an active ingredient, when a model containing at least one cell type exhibiting LOX expression and / or activity is brought into contact with the active ingredient, the expression level of LOX in the control model (not brought into contact with the active ingredient) and / or Alternatively, it is effective to increase the expression and / or activity of LOX by about 2-fold compared to the amount of activity, and at the same time reduce the expression of elastin (Eln) mRNA by about 0.5-fold. Conceivable.

According to the first aspect of the present invention, the component that induces the activity and / or formation of LOX or a homologue or essential homologue thereof and controls the formation of elastic fibers in the case of abnormal or pathological elastic fiber formation. A method of screening for:
Contacting the potential active ingredient with at least one cell that is preferably alive and capable of expressing LOX or a homologue or essential homologue thereof;
The expression of LOX or its homologues or essential homologues, in particular for the purpose of confirming whether the potential active substance induces the expression of LOX or its homologues or essential homologues. It relates to a method characterized in that it consists of analyzing.

Preferably, the method comprises contacting the potential active ingredient with at least one cell capable of expressing the protein elastin, which may be the same as expressing LOX. Particularly, analyzing the expression of the protein elastin for the purpose of confirming whether the potential active ingredient induces a reduction or inhibition of elastin synthesis.

Preferably, the active ingredient is:
Whether to induce the expression of at least part of the nucleotide sequence encoding the protein LOX, and / or
Whether to induce the expression of a peptide sequence present in the structure of the protein LOX; and whether to inhibit the expression of at least part of the nucleotide sequence encoding the protein elastin and / or
Whether it inhibits the expression of peptide sequences present in the structure of the protein elastin.

Preferably, analysis of the expression of LOX and / or the protein elastin is performed by qualitative and / or quantitative analysis of the expression of the nucleotide sequence encoding LOX and / or tropoelastin.

Preferably, analysis of the expression of LOX and / or the protein elastin comprises
Use of a primer that hybridizes with at least a portion of the nucleotide sequence of complementary DNA encoding LOX (SEQ ID NO: 2) to amplify at least a portion of the nucleotide sequence encoding LOX, and / or
Reverse transcription polymerase comprising the use of a primer that hybridizes with at least a portion of the nucleotide sequence of complementary DNA encoding tropoelastin (SEQ ID NO: 5) for amplifying at least a portion of the nucleotide sequence encoding tropoelastin A chain reaction (RT-PCR) is used.

Preferably, the method is:
In particular by using at least one DNA probe that hybridizes with at least part of the nucleotide sequence encoding LOX, in particular by at least part of the nucleotide sequence encoding LOX, and / or eg By in situ hybridization with a DNA probe that hybridizes with at least part of the nucleotide sequence encoding poelaestin, in particular at least part of the nucleotide sequence encoding tropoelastin and / or
-At least one of the peptide sequences encoding in particular LOX, in particular by using at least one antibody capable of recognizing all or part of LOX and / or at least one antibody recognizing all or part of elastin By immunodetection,
The method further includes the step of identifying the position of LOX expression performed on the regenerated skin model or biopsy.

Preferably, the immunodetection or in situ hybridization step allows for the investigation of LOX and elastin traceability, particularly in epithelial and / or connective tissue, said tissue comprising at least one regeneration It is derived from a skin model or biopsy.
Suitably, the screening method compares LOX expression with expression of LOX expressed in a control without active ingredient and / or expression of protein elastin and expressed in control without active ingredient Includes comparison with expression of the protein elastin.
Preferably, the cells include fibroblasts, particularly fibroblasts derived from normal human skin, such as fibroblasts derived from foreskin or adult subject's skin.

Preferably, the cells include epithelial cells such as keratinocytes, in particular epithelial cells derived from normal human skin, such as foreskin or epithelial cells derived from the skin of an adult subject.
Preferably, the living cells originate from a particular site such as the face, abdomen or chest and are at least one skin that can be considered “photo-aged” or “regularly exposed” to sunlight, etc. It is derived from.

Suitably, the screening method utilizes a regenerated skin model, preferably at least one dermis model comprising fibroblasts, or a model based on biopsy.
Suitably, the screening method utilizes a regenerated skin model, preferably at least one epidermis model comprising keratinocytes.
The skin model used is preferably a Mimeskin® regenerated skin model, but may be a model of a binding matrix, epidermis or epithelium, or regenerated skin or mucous membrane.

(1) <Three-dimensional binding matrix (dermis or chorion) culture model>
Includes a carrier seeded with stromal cells to form regenerated dermis or regenerated chorion. This carrier is preferably
-Synthetic semipermeable membranes, specifically nitrocellulose semipermeable membranes, nylon semipermeable membranes, Teflon membranes or Teflon sponges, polycarbonate or polyethylene, polypropylene or polyethylene terephthalate (PET) semipermeable membranes, Anopore ^TM inorganic semipermeable membranes, An inert carrier selected from the group consisting of a cellulose acetate or cellulose ester (HATF) membrane, a Biopore-CM ^™ semipermeable membrane, a polyester semipermeable membrane or a polyglycolic acid membrane or thin film (in the above group, for example, the dermis Models include Skin ² ™ model ZK1100, Dermagraft® and Transcyt® (Advanced Tissue Sciences)
-Cell culture treated resin (Structure of a dermal leaf: Michel M. et al., In Vitro Cell. Dev Biol.-Animal (1999) 35: 318-326)
-Gels or membranes based on hyaluronic acid (Hyagraft (R) 3D-Fidia Advanced Biopolymers) and / or collagen and / or fibronectin and / or fibrin (in the above group, for example Vitrix (R), which is a dermis model) ( Organogenesis))
-Surfaced, made from collagen that can contain one or more glycosaminoglycans and / or finally chitosan (CNRS EP0296078 A1, Coletica WO01 / 911821 and WO01 / 92322) Alternatively, it is selected from a non-floating porous matrix.

(2) <Three-dimensional epidermis or epithelial culture model>
Comprises a carrier that is seeded with stromal cells, in particular fibroblasts and then with epithelial cells, in particular keratinocytes, or has not been seeded in order to obtain a regenerated epithelium or regenerated epidermis. This carrier is preferably
-Synthetic semipermeable membrane, specifically nitrocellulose semipermeable membrane, nylon semipermeable membrane, Teflon membrane or Teflon sponge, polycarbonate or polyethylene, polypropylene or polyethylene terephthalate (PET) semipermeable membrane, Anopore inorganic semipermeable membrane, acetic acid An inert carrier selected from the group consisting of a membrane of cellulose or cellulose ester (HATF), a Biopore-CM semipermeable membrane or a polyester semipermeable membrane (in the above group, a model of regenerated epidermis and epithelium (Skinetic®), And models such as EpiDerm (R), EpiAirway (R), and EpiOccular (R) (Mattek Corporation).
-Thin films or membranes based on hyaluronic acid and / or collagen and / or fibronectin and / or fibrin (in the above group, in particular models such as Episkin (R) (L'Oreal) and Laserskin (R) (Fidia advanced Biopolymers)) Can be mentioned)
Selected from.

(3) <Three-dimensional regenerated skin or mucosal culture model>
Comprises a matrix carrier (dermal or chorionic) seeded with epithelial cells to obtain regenerated mucosa or keratinocytes to obtain regenerated skin. This carrier is preferably
-Synthetic semipermeable membrane, specifically nitrocellulose semipermeable membrane, nylon semipermeable membrane, Teflon membrane or Teflon sponge, polycarbonate or polyethylene, polypropylene or polyethylene terephthalate (PET) semipermeable membrane, Anopore inorganic semipermeable membrane, acetic acid An inert carrier selected from the group consisting of cellulose or cellulose ester (HATF) membrane, Biopore-CM semipermeable membrane or polyester semipermeable membrane (the inactive carrier includes stromal cells, particularly fibroblasts)
-Gels based on collagen and / or hyaluronic acid and / or fibronectin and / or fibrin, including stromal cells, in particular fibroblasts-one or more glycosaminoglycans and / or finally chitosan A porous matrix that is made of collagen that is capable of containing, is floating or not floating (the porous matrix is an integral of stromal cells, especially fibroblasts)
-Selected from the peeled or dead dermis of humans or animals.

In the above group, models such as Aplygraf® (organogenesis), ATS-2000 (CellSystems® Biotechnology Vertrieb), particularly Skin ^2TM (ZK1200-1300-2000, Advanced Tissue Science) can be mentioned.

Preferably, the active ingredient is selected from the group consisting of plant or synthetic polyphenols, galega or cardamom extracts, saturated fatty acids, polyols such as xylitol, adragant gum, cereal extracts such as barley or oats. Is.

According to a second aspect, the present invention induces a reduction or inhibition of elastin synthesis to produce a composition intended to control elastic fiber formation, especially in the case of abnormal or pathological elastic fiber formation. To the use of a LOX having the sequence SEQ ID NO: 1 or a homologue or essential homologue thereof, or a first component that induces the activity and / or formation of LOX. This composition is preferably intended to induce enzymatic activity or expression of an isoform of lysyl oxidase LOX or a homologue or essential homologue thereof and inhibit the synthesis of the protein elastin.

Preferably, abnormal or pathological elastic fiber formation is particularly associated with fibrosis, photoelastic fibrosis, skin streaks, dystrophic scars, eczema, and / or cancer involving disordered and / or non-functional elastic tissue It is seen in the case of a stromal reaction to
Preferably, the first component and the second component are the same.

Suitably, said second component is one that induces a reduction or inhibition of synthesis of tropoelastin having a sequence of SEQ ID NO: 3 which is a precursor protein of elastin, or a homologue or essential homologue thereof.

Preferably, the second component is:
i) a region that binds to the nucleotide sequence of the human elastin gene promoter (Pr) (SEQ ID NO: 4) or a homologue thereof or an essential homologue thereof, or
ii) by regulating the expression of a protein that binds to the nucleotide sequence of the human elastin gene promoter (Pr) (SEQ ID NO: 4) or a homologue thereof or at least part of an essential homologue thereof,
It reduces or inhibits the synthesis of tropoelastin.

Preferably, the active ingredient is selected from the group consisting of plant or synthetic polyphenols, galega or cardamom extracts, saturated fatty acids, polyols such as xylitol, adragant gum, cereal extracts such as barley or oats. Is.

According to a third aspect, the present invention relates to a cosmetic composition characterized in that it contains the above-mentioned active ingredient, optionally mixed with a cosmetically acceptable excipient.
According to a fourth aspect, the present invention relates to a nutritional supplement composition comprising the above-mentioned active ingredient mixed with excipients acceptable for food as needed.
According to a fifth aspect, the present invention relates to a pharmaceutical composition characterized in that it contains the above-mentioned active ingredient, optionally mixed with a pharmaceutically acceptable excipient.

The active ingredient or combination of active ingredients, or the combination of the enzyme LOX or a derivative thereof and the active ingredient that inhibits the synthesis of the protein elastin is preferably mixed with excipients that are acceptable to the human body. . For example, excipients are preservatives, emollients, emulsifiers, surfactants, moisturizers, thickeners, modifiers, matting agents, stabilizers, antioxidants, texture modifiers, whitening agents, filming And at least one compound selected from the group consisting of a filmogenic agent, a solubilizer, a pigment, a dye, a fragrance, and a solar filter. The excipient is preferably an amino acid and derivatives thereof, polyglycerin, cellulose esters, polymers and derivatives, lanolin derivatives, phospholipids, lactoferrin, milk peroxidase, sucrose stabilizer, vitamin E and derivatives thereof, natural And synthetic waxes, vegetable oils, triglycerides, unsaponifiables, plant sterols, plant esters, silicones and derivatives thereof, protein hydrolysates, jojoba oil and derivatives thereof, fat-soluble / water-soluble esters, betaines, amine oxides, plant extracts , Sucrose ester, titanium dioxide, glycine, and paraben. More preferably, butylene glycol, steareth-2, steareth-21, glycol-15 stearyl ether, cetearyl alcohol, phenoxyethanol, methyl paraben, ethyl paraben, propyl paraben, butyl paraben, butylene glycol, natural tocopherol, glycerin, dihydroxycetyl sodium, Isopropyl hydroxycetyl ether, glycol stearate, triisononaoine, coconut oil fatty acid octyl, polyacrylamide, isoparaffin, laureth-7, carbomer, propylene glycol, glycerin, bisabolol, dimethicone, sodium hydroxide, fragrance, PEG30- Dipolyhydroxystearate, tri (capryl / capric) glyceryl, Cetearyl octoate, dibutyl adipate, grape seed oil, jojoba oil, magnesium sulfate, EDTA, cyclomethicone, xanthan gum, citric acid, sodium lauryl sulfate, mineral wax and mineral oil, isostearyl isostearate, propylene glycol dipelargonate (propylene glycol) dipelargonate), propylene glycol isostearate, PEG-8 beeswax, hydrogenated palm tree oil fatty acid glyceride, hydrogenated palm oil fatty acid glyceride (hydrogenated palm oil oil) Castor oil, titanium dioxide, coloring Selected from the group consisting of agents and pigments, lactose, sucrose, low density polyethylene and isotonic saline.

Advantageously, the composition is an aqueous or oily solution, aqueous cream, aqueous gel or oily gel, in particular body soap, shampoo; emulsion, emulsion, microemulsion or nanoemulsion, especially in bottles or tubes. , Especially oil-in-water or water-in-oil or multiphase or silicone-containing microemulsions or nanoemulsions; lotions, especially in glass bottles, plastic bottles or measuring bottles; or aerosol-like lotions; ampoules; liquid soaps; dermatology Formulated in a form selected from the group consisting of dermatologic bars; ointments; foams; anhydrous products, preferably liquid, pasty or solid anhydrous products such as sticks, in particular lipsticks.

Advantageously, if the composition is sufficiently liquid, it can be administered, especially subcutaneously, in the eyes, lungs, mouth or nose.

Advantageously, the pasty or solid composition (paste, powder, tablet, capsule, granule, suppository, etc.) can be introduced into the body, especially from the mouth, tongue, nose or rectum.

Advantageously, if possible in the formulation of the composition, the composition is administered through the skin or mucosa, in particular by application to the skin or mucosa.

Advantageously, one skilled in the art can select from a variety of formulations and routes of administration that are suitable for the desired effect.

According to a sixth aspect, the present invention provides an effective amount of at least one active ingredient as described above or a combination of one type, or a composition as described above, in particular photoelastic fibrosis, skin streaks and / or dystrophic scars. In this case, the present invention relates to a cosmetic care method characterized by being applied.

According to a seventh aspect, the present invention is derived from at least one regenerative skin model or a biopsy based model for the purpose of investigating the traceability of collagen fibril formation and / or elastic fibril formation. A method for identifying the expression position of LOX or homologues or essential homologues thereof and optionally the protein elastin in tissues, in particular epithelial and / or connective tissues,
It relates to a method characterized in that it comprises the step of immunodetection of the protein LOX and optionally the protein elastin, for example with a specific antibody.

The present invention also provides an isoform of lysyl oxidase LOX or an isoform thereof comprising an immunodetection or in situ hybridization step, particularly in epithelial and / or connective tissue, specifically for traceability studies of elastic fibrogenesis. It also relates to a method for specifying the expression position of a homologue or an essential homologue.

The present invention also provides a composition or combination type of active ingredients as described above that enhances the enzymatic activity of the protein lysyl oxidase LOX or a homologue or essential homologue thereof and inhibits the synthesis of the protein elastin. It also relates to the treatment of loss of enzyme activity of protein lysyl oxidase LOX isoforms or homologues or essentially homologues, comprising administering to a subject a therapeutically effective amount.

Preferably, the method of treatment is for fibrosis, photoelastic fibrosis, skin streak, dystrophic scar, eczema, and / or stromal response to cancer involving disordered and / or non-functional elastic tissue. It is used to control elastic fiber formation in the case of pathological, disordered and / or non-functional elastic fiber formation.

Detailed description of the invention The inventors surprisingly have the case of pathological, disordered and / or non-functional elastic fiber formation, such as in the case of photoelastic fibrosis, cutaneous striatum and / or dystrophic scars. It was proved that the lysyl oxidase isoform LOX was not sufficiently induced locally to control the elastic fiber formation. The inventors have surprisingly aging the skin, especially in the case of pathological, disordered and / or non-functional elastic fiber formation, such as in the case of photoelastic fibrosis, cutaneous striatum and / or dystrophic scars. Reduces the synthesis of elastin (which tends to be non-functionally overproduced and precipitated in the above cases), to protect against UV or in the case of eczema and other similar malfunctions In some cases, it was proved necessary to be able to reinduce the expression of LOX.

The inventors have now shown that this isoform of the lysyl oxidase (LO) family is involved in elastic fiber formation in regenerative skin models that produce elastic fibers. When we examined the presence of this isoform in the process of skin changes in the skin of subjects of different ages, we found that this isoform and elastic fiber formation were present or absent at the same time. Thereby reactivating the expression of this isoform of LO (LOX) together with inhibition of elastin synthesis, as in fibrosis, photoelastic fibrosis, skin striae and / or dystrophic scars, It is suggested that elastic fiber formation in the case of pathological, disordered and / or non-functional elastic fiber formation can be controlled.

In this way, we have found a way to visualize this increase in the expression of LOX isoforms, specifically inducing the expression of mRNA encoding LOX, especially from plant extracts or chemicals. We searched for active ingredients and active ingredients that can inhibit the expression of the protein elastin simultaneously or non-simultaneously. Cosmetic formulations and skin for use in reducing pathological, disordered and / or non-functional elastic fiber formation by combining the selected active ingredient or active ingredient that inhibits the expression of the protein elastin with the enzyme LOX Formulated in a medical preparation.

The inventors have developed specific antibodies against mature forms of LOX and LOXL (see Examples 1 and 2), whereby the lack of lysyl oxidase isoform LOX is mainly due to the pathological, disordered and / or Or proved to be the cause of collagen fiber formation problems in non-functional elastic fiber formation.

Isoforms LOXL2, LOXL3, and LOXL4 are not or hardly expressed in the dermis and are not involved in elastic fiber formation (see Example 4). LOX is an enzyme involved in collagen maturation (by cross-linking), which indicates that this enzyme may be involved in interstitial densification.

In the present invention, the present inventors have completed a method for specifying the position of LOX expression. In particular, this specific method involves immunodetection of LOX. The expression of the protein elastin can also be demonstrated by this method. Our study demonstrated that LOX is involved in microfibrils and the formation of collagen fibers (FIG. 5). Elastin was detected in the same dense deposit and in microfibrils. This detection is performed on the regenerated skin model, and in particular in the regenerated skin model, it is observed 30 days after the deposition of keratinocytes. The binding of LOX to microfibrils and collagen fibers was also confirmed in the foreskin skin, especially by electron microscopy after immunodetection. LOX is expressed in the dermis of foreskin skin taken from young patients (2-3 months) who are still synthesizing elastin in large quantities. LOX is always expressed in the dermis of adults, especially in the dermis of the neck, chest, abdomen or facial skin. Although many LOXs are also expressed in the epidermis of human skin, it was observed that the expression of this enzyme gradually disappears when human skin is derived from an elderly subject (91 years old) (implementation) (See Example 6).

With respect to scarring, LOX was not observed in these areas, neither after 3 months nor after 5 years. In this regard, it should be noted that elastin, which is expressed at 3 months, is no longer present in this scar tissue 5 years after scar formation.

In this way, the inventors have clarified the effect of LOX in the formation of collagen fibers and elastic fibers, especially using regenerated skin models or the dermis of the foreskin of young patients.
The inventors also revealed a loss of LOX expression in the scar tissue area.

Our research as a whole enables the development of screening methods for active ingredients that control elastic fiber formation in the case of pathological, disordered and / or non-functional elastic fiber formation, such as photoelastic fibrosis I made it. This control is manifested in particular by the cross-linking of collagen and elastin.

In the present invention, the present inventors have performed an in situ hybridization method, and in particular could identify and confirm the presence of expression of messenger RNA encoding LOX. This in situ hybridization is performed in particular on a cross section of a 35 day regenerated skin model with paraffin added using a digoxigenin labeled double stranded DNA probe. This in situ hybridization was also performed to demonstrate the expression of tropoelastin and collagen Iα1 messenger RNA (see Example 7).

LOX mRNA expression is observed throughout the dermis. In the epidermis, cells in the basal layer express little or no LOX, whereas cells in the spinous and granular layers express it strongly. Collagen Iα1 mRNA expression is specified in the dermis but not in the epidermis.

The purpose of this is therefore after application of the active ingredient which ultimately controls elastic fiber formation in the case of pathological, disordered and / or non-functional elastic fiber formation, for example fibrosis or photoelastic fibrosis To the induction of LOX mRNA and ultimately inhibition of elastin mRNA in a regenerative skin model.

In the present invention, the hLOX gene was activated by adding keratinocytes to a regenerative skin model, in particular the regenerative skin model Mimeskin® (Coletica, Lyon, France). Induction of LOX mRNA synthesis is observed simultaneously with that of collagen Iα1 (COL1 α1) mRNA, and in particular, appears about 6 days after the addition of keratinocytes in the equivalent of the dermis.

The expression of the target gene encoding LOX, elastin, and LOXL and the gene encoding actin were examined by real-time RT-PCR. According to this method, the expression of a gene can be accurately quantified as compared with the expression of actin which is considered to be constant. This makes it possible to quantify the control of the expression level of these genes, particularly the expression level of the gene encoding LOX and the gene encoding elastin.

LOX mRNA synthesis is reduced by an average of 40% in fibroblasts derived from aged donors compared to fibroblasts derived from foreskin (see Example 8). The present invention provides a method that can quantify LOX expression, particularly in fibroblasts. The present invention uses these various techniques to identify active ingredients that can control elastic fiber formation in the case of pathological, disordered and / or non-functional elastic fiber formation, such as fibrosis or photoelastic fibrosis. For the purpose.

In general, the method of the present invention searches for the expression of the proteins LOX and elastin, in particular messenger RNA encoding LOX and its expression encoding elastin (see Example 9).

The invention also induces the expression of LOX and inhibits the expression of the protein elastin to form an elastic fiber in the case of pathological, disordered and / or non-functional elastic fiber formation, such as fibrosis or photoelastic fibrosis It also relates to active ingredients that control (see Examples 9-11). The invention also relates to the use of these active ingredients for preparing cosmetic or pharmaceutical compositions (see Examples 12-18).

Variations in LOX and elastin expression can be observed directly in genes, messenger RNAs or their proteins. These fluctuations can control elastic fiber formation in the case of pathological, disordered and / or non-functional elastic fiber formation, such as fibrosis or photoelastic fibrosis, in particular by collagen cross-linking with the enzyme LOX To.

Other objects, features and advantages of the present invention will become apparent to those skilled in the art upon reading the description with reference to the following examples.

The embodiments constitute an indispensable part of the present invention, and all the features that are considered to be novel from the entire description including the embodiments, compared with the state of the art, are in the function and generality of the present invention. It constitutes an indispensable part.
That is, each embodiment covers a general range.

Also, in the examples, all percentages are expressed on a weight basis unless otherwise indicated, temperatures are expressed in degrees Celsius unless otherwise indicated, and pressures are atmospheric unless otherwise indicated.

“Production of mature antibodies of LOX and LOXL”
The present invention first made novel antibodies specific to mature forms of LOX and LOXL. This antibody was raised against the mature region of LOX and LOXL. The antigenic region was selected to minimize similarity to the corresponding region on other lysyl oxidase (LO) isoforms. The antibody obtained against the region of peptide LOX ^V228-S280 was referred to as anti-LOXmat antibody, and similarly the antibody obtained against the region of peptide LOXL ^R231-G368 was referred to as anti-LOXLpro antibody.

FIG. 1: Description of the sequence of LO determined to make a specific antibody: This figure represents the step of selecting antigenic regions to make anti-LOX and anti-LOXL antibodies.
FIG. 1 (A): Schematic of hLOX (human LOX protein) and hLOXL (human LOXL protein).

The hLOX and hLOXL sequences are shown as outlined rectangles, and the C-terminal region is dotted to highlight regions of high similarity. The position of the cleavage site in the front region and the cleavage site by procollagen C proteinase (PCP) are shown on the A22 and D169 residues of hLOX, respectively. For LOXL, the position of the cleavage region of the previous region at the N-terminal maturation site of the 56 kDa precursor (before Q26 residue) and the cleavage site by PCP of the 56 kDa LOXL precursor were determined in front of D135 residue and D338. Each is shown before the residue. The molecular weights of the corresponding LOXL proteins Q ²⁶ -S ⁵⁷⁴ , D ¹³⁵ -S ⁵⁷⁴ and D ³³⁸ -S ⁵⁷⁴ are estimated to be about 63 kDa, 54.6 kDa and 26.7 kDa, respectively. The position of the recombinant peptide used to make the anti-LOX antibody is shown as peptide G128-L212 for the anti-LOXpro antibody, peptide V228-S280 for the anti-LOXmat antibody, and peptide D305-N373 for the anti-LOXcat antibody. The location of the recombinant peptide used to make the anti-LOXL antibody is shown as peptide R231-G368 for the anti-LOXLpro antibody and peptide S355-D415 for the anti-LOXLmat antibody.

FIG. 1 (B): This table shows the similarity between the LOX and LOXL antigenic regions and the corresponding regions on the LO isoform.
In the table of FIG. 1B, hLOXL is human LOXL protein, bLOXL is bovine LOXL protein, mLOXL is mouse LOXL protein, hLOX is human LOX protein, bLOX is bovine LOX protein, hLOXL2 is human LOXL2 protein, hLOXL3 is human LOXL3 The protein, hLOXL4, represents the human LOXL4 protein.
In the length (aa) column, the number of amino acids contained in the corresponding region is described.

In order to produce an antibody, the determined hLOXL or hLOX sequence is inserted into the BamHI-XhoI site of the expression plasmid pGEX-4T-3 (Amersham Biosciences) together with the gene for glutathione S-transferase (GST), and the chimeric gene Built.

The cDNA sequence of HLOXL (hLOXL cDNA) prepared by PCR using the sense primer 5'-TTGGATCCAGCGTAGGCAGCGTGTAC-3 '(SEQ ID NO: 16) and the antisense primer 5'-AAACTCGAGCATCGTAGCGGGGC-3' (SEQ ID NO: 17) is introduced. Thus, the fusion gene GST-LOXL ^S355-D415 was constructed. By introducing hLOX cDNA amplified using the sense primer 5′-TCGGATCCGCCTACTGACATCATGAGCCC-3 ′ (SEQ ID NO: 18) and the antisense primer 5′-GTCCTCGAGACCGTACTGGGAAGTAGCC-3 ′ (SEQ ID NO: 19), respectively, the fusion gene GST-LOX ^G128-L212 was constructed. By introducing the hLOX sequence amplified using the sense primer 5′-TTGGATCCGTGCCAGAAGATGCTCGTTAC-3 ′ (SEQ ID NO: 20) and the antisense primer 5′-TTTCTCGAGGCTGGTAAGAAATCTGATG-3 ′ (SEQ ID NO: 21), respectively, the fusion gene GST- LOX ^V228-S279 was constructed. By introducing hLOX cDNA amplified using the sense primer 5′-CACTATGATCCCTGATGCCACAACCACC-3 ′ (SEQ ID NO: 22) and the antisense primer 5′-CACGACCTTTAGGATATCGTTCCAGG-3 ′ (SEQ ID NO: 23), respectively, the fusion gene GST-LOX ^D306-N373 was constructed. Taq polymerase High Fidelity (Roche Diagnostics, Meyman, France) was used for all these PCR amplifications.

The fusion proteins GST-LOX and GST-LOXL and rabbit polyclonal antibodies were obtained and the fusion protein derived from the expression of the fusion genes GST-LOXL ^S355-D415 and GST-LOX ^G128-L212 was purified by the method described above (Borel) J. Biol. Chem, 276: 48944-49, 2001; Decitre et al., Lab. Invest., 78: 143-151, 1998). The adsorption experiment was performed by incubating the antibody at 20 ° C. for 3 hours together with a fusion protein adsorbed on a nitrocellulose membrane such as Hybond-ECL membrane (Amersham Bioscience) prior to immunodetection.

Performing the above experiments, it was possible to first elucidate mature forms of LOX and LOXL based on the immunochemical and biochemical properties of the mature protein (see Example 2 (FIG. 2)). ). The resulting antibody is distinct from the antibody that has not been used to recognize the mature form of LOXL that was previously used against LOXL (Decitre et al., Lab Invest, 78: 143-151, 1998). In the present invention, an anti-LOXLmat antibody and an anti-LOXmat antibody were used, which revealed a 31 kDa protein corresponding to the mature form of LOXL that is recognized by the anti-LOXLmat antibody but not the anti-LOXLmat antibody. I was able to. From this, it can be seen that the present invention has made substantial progress over the prior art, particularly the Csiszar et al. Patent, which describes all proteins derived from the LO family of genes but does not define their characteristics. I understand (WO01 / 83702A2: an application related to a novel enzyme belonging to the lysyl oxidase family in the amine oxidase family).

“Immunodetection of LOX and LOXL in myocytes with novel antibodies, anti-LOX and anti-LOXL antibodies”
FIG. 2 is a photograph of electrophoresis performed by the method described below. This electrophoresis reveals the properties of LOX and LOXL mature proteins in smooth muscle cells (SMC) by using the anti-LOX and anti-LOXL antibodies revealed in Example 1.

Extract protein from cell line (L) and cell culture medium (M) of rat smooth muscle cell line (developed by Jean-Marie Daniel Lamazeire (Bordeaux)), anti-LOXLmat antibody, anti-LOXmat antibody, anti-LOXLpro antibody and anti-LOXLpro antibody Was detected by Western blotting. Cells were cultured in DMEM medium (Sigma) containing 10% fetal calf serum, glutamine 2 mM and gentamicin 50 μg / ml at 37 ° C. in a 5% CO ₂ atmosphere.

The cell line protein was washed twice with PBS buffer and slowly stirred at 4 ° C. over 2 hours while dissolving the lysis buffer (16 mM phosphate buffer (pH 8), 0.5% NP40, protease inhibitor (complete mini, Roche Diagnostics) and urea 6M). The lysate was diluted with 2 volumes of 16 mM phosphate buffer (pH 8) containing protease inhibitors (Complete Mini, Roche Diagnostics, Meylan, France) and centrifuged at 15000 g for 20 minutes. . Soluble protein was precipitated by adding 10% trichloroacetic acid (TCA) and subjected to electrophoresis. The protein of the culture was recovered by adding 10% TCA or 50% saturated ammonium sulfate from the culture medium of the cells cultured for 48 hours without adding serum and precipitated.

Proteins were separated by 10% SDS polyacrylamide gel electrophoresis for immunodetection. The proteins were transferred onto polyvinylidene fluoride (PVDF) membrane (Immobilon ^{P SQ,} Millipore) were immunized detected as described above (Borel et al., J.Biol.Chem, 276: 48944-49, 2001 years).

Thus, by using the antibody produced above, mature and immature forms of LOX and LOXL in living tissue can be characterized and confirmed.

"Demonstration of LOX action in elastic fiber formation"
The inventors have revealed by immunohistochemical techniques that LOX and LOXL proteins may be involved in the formation of connective tissue in the dermis in a regenerated skin model (FIG. 3). This was clarified by using anti-LOX and anti-LOXL antibodies against the enzyme precursor region and mature region of the two enzymes (LOX and LOXL).

FIG. 3 shows the results of immunohistological detection of LOXL and LOX in regenerated skin (RS) and normal human skin.
^{-Immunodetection} of LOXL (A, C, E, G) in regenerated skin after 16 days (A), 35 days (C) and 45 days (E), anti-LOXL ^R231-G368 antibody (A, C, E) Or what was performed using the corresponding peptide GST-LOXL ^R231-G368 and the adsorbed anti-LOXL ^R231-G368 antibody (G) before ^{immunodetection} .
Immunodetection of LOX (B, D, F, H) in regenerated skin after 16 days (B), 35 days (D) and 45 days (F), anti-LOX ^V228-S279 antibody (B, D, F) Or what was performed using the corresponding peptide GST-LOX ^V228-S279 and the adsorbed anti-LOX ^V228-S279 antibody (H) before ^{immunodetection} .
^{-Immunodetection} of LOXL (I) and LOX (J) in human foreskin skin using anti-LOXL ^R231-G368 antibody (I) and anti-LOX ^V228-S279 antibody (J). The boundary between the dermis and epidermis is indicated by a white arrow, the substrate of the dermis is indicated by an arrow, and the position of the corneocytes after 16 days is indicated by the tip of the arrow.

Regenerated skin (Mimeskin®, Coletica, Lyon, France) was prepared in bouin fixative (LOX, LOXL, elastin) or 10% formalin solution (for elastin) and then embedded in paraffin. Paraffin was removed from a 6 μm thick section and bleached in a hydrochloric acid solution of glycine (100 mmol / L). Anti-LOX and anti-LOXL antibodies are as described above.

The antibodies were used diluted as follows: 500-fold (anti-LOXL ^R231-G368 antibody), 100-fold (anti-LOX ^V228-S279 antibody, anti-LOXL ^S355-D416 antibody). The immune complex was detected with a peroxidase-labeled anti-IgG rabbit (goat) antibody (Dako, Trap, France) using diaminobenzidine (Dako) as a substrate.

From the above, LOX and LOXL may be involved in the formation of elastic fibers in regenerative skin models (particularly Mimeskin®).

“Demonstration of the effects of LOXL2, LOXL3 and LOXL4 in the formation of elastic fibers”
The present invention also created two new anti-LOXL2 antibodies, one of which also theoretically recognizes LOXL3 and LOXL4. Using this, it was possible to determine whether these enzymes are expressed with elastin in the dermis in a regenerated skin model. As a result of immunohistochemical examination using the above-mentioned two types of anti-LOXL2 antibodies, these antigens and two antigenic related proteins, LOXL3 and LOXL4, were not expressed at all or hardly in the dermis, and therefore elastic. It was actually found that it was not involved in fiber formation.

Figure 4: in reconstructed skin (16 and 35, and 45 days later) and human foreskin skin section, anti ^{LOXL2 517-581} antibody (left column), and, theoretically LOXL2, LOXL3 and recognizes LOXL4 anti ^{LOXL2 664-} The result of the immunodetection performed using ⁷²⁰ antibody (right column) is shown. Anti-LOXL2 antibody was obtained as described above for the fusion peptide GST-LOXL2 (Decitre et al., Lab. Invest., 78, 143-151, 1998). The fusion gene GST- ^{LOXL2 517-581} was constructed as described above with the human LOXL2 gene (hLOXL2) sequence 1543-1747 introduced into the plasmid.

This fragment was prepared by PCR using the sense primer 5-GAGCTGGGATCCGCGCACTGCCC-3 '(SEQ ID NO: 32) and the antisense primer 5'-GGCTGAGGTCGACGAGGCAGTTCTCC-3' (SEQ ID NO: 33).

By introducing the corresponding hLOXL2 sequence using the sense primer 5′-CACAGGATCCGAAGGAGACATCATGAAG-3 ′ (SEQ ID NO: 34) and the antisense primer 5′-TTTCTGAGCTCCTGCATTTCATGATG-3 ′ (SEQ ID NO: 35), the fusion gene GST- ^{LOXL2 664-720} was constructed.

The fusion protein and anti-rabbit antibody prepared against the above protein were prepared as described above. The antibody against the 517-580 peptide was called anti-LOXL2 antibody because this region is unique to LOXL2.

Antibodies against the 664-734 peptide were identified as anti-LOXL-R (“relevant”) because this region of LOXL2 is highly similar to LOXL3 and LOXL4 (approximately 74.6% and 60.5%, respectively). ) Antibody.

Regenerated skin after 16 days (RS-D16), 35 days (RS-D35) and 45 days (RS-D45) (Mimeskin®, Coletica, Lyon, France) and human foreskin skin As described above, the anti-LOXL2-R antibody and the anti-LOXL2 antibody were examined by immunohistochemical technique. It was found by anti-LOXL2 antibody that LOXL2 was expressed in the epidermis but not in the dermis. On the other hand, antibodies against the C-terminal region common to LOXL2, LOXL3 and LOXL4 can confirm that these enzymes are expressed in the epidermis, and these enzymes are slightly expressed in the dermis. It was found that it was not expressed in the part corresponding to the part forming the fiber.

LOXL2, LOXL3 and LOXL4 are not involved in the formation of elastic fibers.

"Demonstration of LOX action in elastic fiber formation"
The binding of LOXL and LOX to elastic fibers or microfibrils was clearly demonstrated by the present invention using a transmission electron microscope.

LOXL and LOX bound to microfibrils constitute a scaffold on which elastin is deposited, but only LOX is associated with collagen fiber formation (see FIG. 5).

FIG. 5 shows the results of immunodetection of LOXL, LOX and elastin performed using transmission electron microscopy on regenerated skin after 45 days and on the dermis of normal human skin.

Tissue was fixed with 4% paraformaldehyde dissolved in PBS buffer (containing 0.1% glutaraldehyde) for 3 hours at 4 ° C., and phosphate buffer (0.4 M sucrose-cacodylic acid and 0.2 M lysine). And then dehydrated in an ethanol solution and embedded in LR White (Euromedex, France). Detection was carried out using a primary antibody diluted 1/50 with Tris-HCl buffer (pH 8.2) and 1% bovine serum albumin (BSA). Immunocomplexes were detected using colloidal gold particle (10-20 nm) labeled anti-IgG rabbit antibody (Biocell, Tebu, France) diluted 40-fold. The sample was stained with 3% uranyl acetate and lead citrate and examined with a JEOL 1200EX transmission electron microscope. This immunodetection was performed on regenerated skin (AD) and human foreskin skin (FI).

In the regenerated skin, the following antibodies were used: anti-LOXL antibody (A, B), anti-LOX antibody (C), anti-elastin antibody (Elm) (D), and primary antibody in the dermis as a negative control. (E) which does not contain (Control). Double labeling was performed on human foreskin skin (FI).

“AD”: immunodetection of LOXL, LOX and elastin using electron microscopy in the dermis of regenerated skin after 45 days.
“E”: Positive control using anti-elastin antibody and anti-collagen I antibody in the dermis of regenerated skin 45 days later, ie 30 days after addition of corneocytes.
“F-I”: Double immunodetection of LOXL, LOX, elastin and collagen using electron microscopy in the dermis part of human foreskin.
“GH”: human foreskin using anti-LOXL rabbit antibody (anti-IgG rabbit antibody labeled with 10 nm gold particles) and anti-elastin mouse antibody (anti-IgG mouse antibody labeled with 20 nm gold particles) Double label in the dermis of
Symbols in the figure: m: microfibril, c: collagen fiber, e: amorphous elastin. Scale bar: 500 nm.

LOXL (A, B) was detected in association with dense deposits or on microfibrils but not with collagen fibers appearing in white on the cross section. For LOX (C) labeling, gold particles were observed slightly associated with dense deposits, microfibrils and collagen, but the labeling was thin. Similarly, dense deposits and microfibrils were detected by anti-elastin antibody (D). Similar to the observations in the regenerated skin model, LOXL is not associated with collagen fibers and is the opposite of LOX (which is abundant with collagen fibers and rarely present on microfibrils). The antigen LOXL was bound to microfibrils and was detected around elastic fibers in human foreskin skin.
LOX labeling was not observed with elastic fibers, but was associated with collagen fibers and skin microfibrils (I).

“Demonstration of the relationship between LOX expression and elastic fiber formation”
LOXL and LOX are expressed in the dermis of foreskin skin taken from a young patient (a few months old) who still has high elastin synthesis. LOXL is not expressed in the dermis of the skin of the neck, chest, abdomen or face, whereas LOX is always expressed in the dermis (FIG. 6).

FIG. 6 shows the results of immunodetection of LOX and LOXL at various positions in human skin.
Foreskin (A, B) provided by tissue bank of Ed Earl Hospital (Lyon, France) using anti-LOX antibody (A, C, E, G) and anti-LOXL antibody (B, D, F, H), LOX and LOXL expression was detected in skin samples from the neck (C, D), breast (E, F) and abdomen (G, H). These tissues were fixed with a Buan reagent, embedded in paraffin, and immunodetection was performed in the same manner as described above.

FIG. 7 shows the results of immunodetection of LOX and LOXL in abdominal skin collected from people of different ages.
1.5 years old (A, B), 35 years old, provided by the tissue bank of Ed Earlier Hospital using anti-LOX antibodies (A, C, E, G) and anti-LOXL antibodies (B, D, F, H) Expression of LOX and LOXL was detected in samples of abdominal skin taken from (C, D), 60 years old (E, F) and 91 years old (G, H). These tissues were fixed with Buan reagent, embedded in paraffin, and immunodetection was performed as described above.

In this experiment, it was observed that LOX and LOXL were highly expressed in human skin epidermis, and the expression of these two enzymes lasted very long (91 years) (FIG. 7).

FIG. 8: LOX and LOXL show the results of immunodetection in the skin of scar tissue with different elapsed time after treatment.
Using anti-LOX antibodies (A, D, G), anti-elastin antibodies (B, E, H) and anti-LOXL antibodies (C, F, I), around the scar on the neck skin of a 17-year-old patient (“normal” Part, A to C), the expression of LOX, elastin and LOXL in samples after 3 months (DF) and 5 years (G, H) after treatment was detected. These tissues were fixed with Buan reagent, embedded in paraffin, and immunodetection was performed as described above.

In scars, neither LOXL nor LOX could be observed in the scar tissue area 3 months after scar formation and 5 years after scar formation. Note that in these scars, elastin was immunodetected at 3 months and disappeared at 5 years (FIG. 8).

With this embodiment of the invention, in scars (i) LOX is definitely involved in tissue formation (but not in various age reconstructions), but (ii) LOX expression is lost. It is proved to be missing. This example demonstrates that LOX is present at the site of elastic fiber formation but is defective in reconstructed scar tissue regions that have lost the ability to form functional elastic fibers.

“Demonstration of LOX activation by introduction of corneocytes in regenerative skin model”
Expression of the gene encoding LOX was detected by performing in situ hybridization between LOX messenger RNA and a digoxigenin-labeled double-stranded DNA probe in a paraffin-embedded section.
In FIG. 9 showing the skin model cross section (Mimeskin (R)) after 35 days,
(A) LOXL expression is positive across the deep dermis and the entire epidermis.
(B) LOX expression is observed throughout the dermis. In the epidermis, cells in the basal layer do not express LOX, whereas cells in the spinous and granular layers are strongly expressed.
(C) Expression of tropoelastin (TE) is found in dermal fibroblasts and epidermis.
(D) Expression of the gene COL1A1 (collagen Iα1) is detected in the dermis and not in the epidermis.
(E) Control without probe.
DEJ is indicated by an open arrow, porous dermal matrix is indicated by an arrow, and positive cells are indicated by an arrow tip.

Double stranded DNA probes were made by PCR. The following primers were used respectively.
Sense 5'-GTGGAGAGTACACTGGATTG-3 '(SEQ ID NO: 14) and antisense 5'-TCGTGCAGCCATCGACAG-3' (SEQ ID NO: 15) are included in the gene for Iα1 collagen, and sense 5'-GTATATACCCAGTGGGCGTG-3 '(in the tropoelastin). SEQ ID NO: 10) and antisense 5′-CGAACTTTGCTGGCTGCTTTTAG-3 ′ (SEQ ID NO: 11), hLOX is sense 5′-GGTGGGCCGACCCCTACTACCATCC-3 ′ (SEQ ID NO: 12) and antisense 5′-GCAAATCGCCCTCTGGTAGCCATAGTC-3 ′ (SEQ ID NO: 11) 13), hLOXL includes sense 5′-GACATACACCGACGTGAGCC-3 ′ (SEQ ID NO: 8) and antisense 5′-ATCCACGTTC CTCCCTGAG-3 'was used (SEQ ID NO: 9).

DNA was amplified using Taq polymerase (Promega, Charbonnier, France) and Dig-11-dUTP (Roche Diagnostics, Meylan, France) as the labeled nucleotide. Agarose gel electrophoresis was then performed and the DNA was purified using a QIAquick extraction kit (Qiagen, Courtaboeuf, France). Thereafter, in situ hybridization was performed on the section embedded in paraffin. Paraffin was removed from the sample and treated with 2 μg / ml proteinase K (Roche) at 20 ° C. for 15 minutes. Endogenous peroxidase was inhibited as directed by the TSA ⁺ amplification kit (NEN, Boston, USA). Prehybridization was carried out at 37 ° C. for 2 hours with 20 mM phosphate buffer (pH 7.4) (50% deionized formamide, 2XSSC (sodium citrate), EDTA (5 mM), 2.5 × Denhart solution, 200 μg / ml denatured herring. DNA, 1 mg / ml salmon semen DNA, and tRNA (10 mg / ml)). Hybridization in 20 mM phosphate buffer (containing 50% deionized formamide, 2X SSC, EDTA (5 mM), 2.5X Denhart solution, 200 μg / ml denatured herring semen DNA, 10% dextran sulfate) for 16 hours at 37 ° C. I went there. As the modified probe, those treated for 5 minutes in a boiling water bath and those not treated were used. After hybridization, sections were washed in 2XSSC / 50% formamide, 1XSSC / 50% formamide, 1XSSC, and 0.5XSSC at 20 ° C (37 ° C for collagen). After drying, digoxigenin labeled hybrids were detected using horseradish peroxidase labeled anti-DIG antibody (Roche). This complex was finally detected using a TSA ⁺ amplification kit (NEN). A positive signal corresponds to the activity of alkaline phosphatase. This activity is defined as the activity after 2 hours at room temperature based on the amplification procedure of the TSA kit, and can be seen by precipitation of a tetrazolium salt (using nitroblue tetrazolium / bromochloroindolyl phosphate (NBT / BCIP) as a substrate).

In the present invention, as a result of tracing mRNA expression by in situ hybridization, LOXL gene and LOX gene were activated by adding keratinocytes to a regenerated skin model (Mimeskin®, Coleta, Lyon, France). It can be seen that there is a possibility that. (FIG. 9).

The LOX gene is also activated simultaneously with the collagen Iα1 gene (Col1α1) after the addition of keratinocytes.

“Demonstration of reduced expression of LOX gene in fibroblasts of elderly adults”
We have 5 strains of fibroblasts derived from foreskin (FF) (from infants) and 6 strains of adult fibroblasts (AF) collected during abdominal plastic surgery (of which 3 The average age is 20 years old, and the average age of the remaining 3 people is 60 years old). The expression of the above three genes of interest and actin were examined by real-time RT-PCR (quantitative reverse transcription polymerase chain reaction, FIG. 10). According to this method, the expression of a gene can be accurately quantified in comparison with the expression of actin (which is assumed to be constant). Therefore, control of the amount of this gene can be quantified.

FIG. 10: FIG. 10 represents changes in gene expression levels of elastin, LOX and LOXL with aging. These results were obtained by analyzing the expression levels of LOX, LOXL and elastin in foreskin and adult fibroblasts by real-time RT-PCR. This result represents the average expression level of each gene in the FF5 and AF6 strains. The genes examined were hLOXL, hLOX and human elastin genes. RT-PCR values are compared to actin amplification. It should be noted that if the expression of the elastin gene does not appear to be inhibited by aging, that of the LOX gene is reduced by 40% compared to 20 years old. This data is consistent with the literature on elastin: if elastic tissue deteriorates and does not replace it, it is likely not due to inhibition of the activity of the elastin gene.

Total RNA was purified using the “SV96 Total RNA Isolation System” kit (Promega, Charbonnier, France). The purified RNA was dissolved in 100 μl of RNase-free water (Promega, Charbonnier, France) and measured, and then dispensed onto a plate (96 wells, 10 μl each (total RNA by PCR: 5 ng / μl)). The primers used in this experiment are as shown in Table I below.

Real-time RT-PCR was performed on wells containing mRNA in a thermal cycler manufactured by OPTICON using the “Quant Tect SYBR Green RT-PCR” kit (Qiagen, France) for amplification cycles. After carrying out a reverse transcription (RT) reaction at 50 ° C. for 30 minutes and then maintaining at 95 ° C. for 15 minutes, the reverse transcriptase is inhibited, the polymerase is activated, and the resulting complementary DNA (cDNA) is denatured. It was. Thereafter, 50 cycles of chain polymerization (PCR) were carried out (94 ° C. for 15 seconds, 60 ° C. for 30 seconds, 72 ° C. for 30 seconds). At the end of each cycle, the amount of fluorescence proportional to the number of amplified fragments was read. The expression level was expressed as the ratio of the expression level of each gene to actin.

The above examples show that LOXL gene and product synthesis by LOX gene may be activated at the gene level. Activation of the gene encoding LOX allows the formation of cross-linked collagen fibers. In order to induce the synthesis of cross-linked collagen by screening active ingredients and at the same time avoid the formation of an amorphous precipitate of elastin and to re-induce the expression of standard tissues in pathological tissues, It becomes possible to identify compounds that can induce increased LOX expression and decreased elastin expression simultaneously or non-simultaneously.

“Analysis of LOX and / or elastin messenger RNA expression (eg, by qualitative RT-PCR performed with or without contact with an active ingredient to be tested for activity)”
The active ingredient was tested at a concentration of 1% (v / v) against fibroblasts from normal human skin (from healthy “old” adults). The culture was carried out, for example, in a well-known serum-free medium (Fibroblast Basal Medium, Promocell, Germany) in a single layer in a 24-well culture plate. For example, 40,000 cells were seeded per cm ² . When confluent, the cells were contacted with the active ingredient, preferably for 24 hours. Advantageously, in parallel, one untreated control (medium only) and three positive controls (1 ng / ml TGF-β, 50 pg / ml IL-1α, and 2% (v / v) Phytokine. (R) (Coretica, France) was also tested, for example, in the same culture plate.

Pre-tested for 1 ng / ml of TGF-β and 50 pg / ml of IL-1α, it was shown that stimulation of elastin mRNA synthesis was induced by two cytokines at this concentration, mRNA analysis, eg quantitative RT-PCR (10 times for TGF-β, 6 times for IL-1α). After contacting the active ingredient with the cells (for example, 24 hours later), the medium was removed, and the cells were washed, for example, in phosphate buffer (pH 7.4) and lyophilized at −80 ° C. and stored. Total RNA was extracted (eg, using a 96-well extraction kit with a silica column) and measured with a 96-well spectrophotometer at 260 nm (purity indicator: protein measurement at 280 nm). The RNA is diluted, for example to 5 ng / μl. One-step qualitative RT-PCR was performed on 96-well plates using, for example, 50 ng of initial RNA for the actin gene, elastin gene and LOX gene. Specific primers for each gene were used at 0.5 μM, for example.

-Sense elastin gene: 1Ela 5'-GTA TAT ACC CAG GTG GCG TG-3 '(SEQ ID NO: 26);
-Antisense elastin gene: 2Ela 5'-CGA ACT TTG CTG CTG CTG CTT TAG-3 '(SEQ ID NO: 27);
-Sense LOX gene: Ox64 5'-ACG TAC GTG CAG AAG ATG TCC-3 '(SEQ ID NO: 24);
-Antisense LOX gene: Ox65 5'-GGC TGG GTA AGA AAT CTG ATG-3 '(SEQ ID NO: 25);
-Sense actin gene: actin U 5'-GTGGGGCGCCCCAGGCACCA-3 (SEQ ID NO: 30);
-Antisense actin gene: D 5'-CTCCTTAATGTCACGCACGATTTC-3 '(SEQ ID NO: 31)

Advantageously, the amplification parameters were as follows: 48 ° C., 30 minutes: 94 ° C., 2 minutes: (94 ° C., 30 seconds; 60 ° C., 30 seconds; 68 ° C., 30 seconds) 28 cycles for actin, 32 cycles for LOX, and 34 cycles for elastin: 68 ℃ 10 minutes: 14 ℃, infinite. After amplification, the products were mixed at a ratio of, for example, (actin amplification product 3 μl + elastin gene amplification product 5 μl + LOX gene amplification product 5 μl). Loading buffer (2 μl) was added and the entire volume (20 μl) was loaded on a pre-filled agarose gel (eg 2%). The present inventors visualized the expression level by a method known to those skilled in the art (for example, the band of the sample after electrophoresis was visualized under ultraviolet light in a dark room (15 minutes) and digitally photographed). Gel photographs were image analyzed and the intensity of the bands was quantified (Phoretrix 1D Quantifier, Non Linear Dynamics, USA). The expression levels of the elastin gene and the LOX gene were expressed as a percentage of the value in the negative control (no treatment).

<Explanation of results>
It can be seen that aged cells express an amount of elastin-encoding mRNA that is about the same as that measured in young cells, whereas the mRNA encoding LOX is very strongly reduced (−40%). Therefore, since it was possible to reversely increase the decrease in LOX expression in the aged cells, the active ingredient was screened.

"Screening of active ingredients"
The amount of cDNA in each test was compared with the amount of actin cDNA, followed by comparison with a negative control (no active ingredient). Preliminary experiments indicate that it is important to increase LOX mRNA by about 2-fold and elastin (Eln) mRNA by about 0.5-fold. Of over 900 molecules or active extracts tested, 8 active ingredients met this criterion at the concentrations tested and under the conditions specified. These active ingredients are as shown in Table II below.

Plants in water / (alcohol, glycol or polyol) mixtures (glycols such as ethanol, glycerin and butylene glycol, xylitol, etc.) at a concentration of 2 to 5% (w / w) at a concentration of 100/0 to 0/100 A plant extract was obtained by soaking. The obtained extract was filtered or distilled to recover the soluble fraction, and further filtered under aseptic conditions. The chemical substance was provided by Sigma (St. Louis, USA), and was used after diluting or dispersing to a concentration of 1% with alcohol or glycol.

<Conclusion>
Eight out of 960 active ingredients were able to significantly activate the amount of mRNA synthesis of LOX-encoding gene and reduce elastin in the abdominal fibroblasts of “old” adults under the test conditions .

“Research on the effectiveness of cosmetic or dermatological active ingredients (eg by real-time RT-PCR)”
The active ingredients selected in the first stage of the screening were tested against normal human (adult) skin fibroblasts at varying concentrations ranging from 0.001% to 5% (v / v). The culture was performed, for example, in a well-known serum-free medium (Fibroblast Basal Medium, Promocell, Germany) in a single layer in a 24-well plate. For example, 40,000 cells were seeded per cm ² . After contacting the active ingredient with the cells (after 24 hours), the medium was removed, and the cells were washed, for example, in phosphate buffer (pH 7.4) and lyophilized at -80 ° C and stored. After the test, the amounts of elastin, LOX and actin mRNA were evaluated by mRNA analysis, for example, real-time RT-PCR. In order to perform this evaluation, the above primer pairs were used (Example 9) which can amplify fragments peculiar to these genes.

RNA was extracted (eg, using a 96-well extraction kit with a silica column), measured with a 96-well spectrophotometer at 260 nm, and then diluted (eg, 5 ng / μl). The RT-PCR reaction (reverse transcription polymerase chain reaction) was performed by quantitative real-time RT-PCR using the “Opticon” system (MJ Research, USA). Advantageously, the reaction mixture (50 μl) added to the wells was as follows for each sample:
-10 μl of RNA at a concentration of 5 ng / μl,
Various desired labeled specific primers,
- The reaction mixture was inserted into (Qiagen, 2X "QuantiTect SYBR Green RT-PCR master mix" (MgCl ₂ 5 mM containing) 25 [mu] l + "QuantiTect RTmix" 0.5 [mu] l), DNA 2 stranded in the label SYBR Green I extension process.

“Advantageously, RT-PCR was performed under the following conditions”
Reverse transcription: 30 minutes at 50 ° C, then 15 minutes at 95 ° C.
PCR reaction: 50 cycles of (94 ° C., 15 seconds; 60 ° C., 30 seconds; 72 ° C., 30 seconds).
Confirmation of the absence of contamination and confirmation of the purity of the amplified product were performed by, for example, a melting curve of the amplified PCR product. Products showing double peaks or unusual melting temperatures were removed.

“Analysis and Calculation Methods”
The amount of fluorescence incorporated into the amplified DNA was measured continuously during the PCR cycle. Thereby, a curve of the fluorescence amount with respect to the number of PCR cycles was obtained, and the relative amount of the amplified DNA could be examined.

All of the above results were compared to the << actin >> signal used as a housekeeping gene to take into account the cell number. From the experiment, the measurement threshold value (= cycle threshold value) of C (T) was fixed between T = 0.05 and 0.01, and an arbitrary measurement unit was calculated by the following formula for each gene.
Sgene << x >> = 10 ⁷ X (1/2) ^{C (T) gene << x >>}
C (T) gene << x >> represents the number of cycles necessary for the fluorescence threshold of gene << x >> to be 0.01 to 0.05.
This value for the gene to be examined was compared to the << actin >> signal by the following ratio.
R = Sgene << x >> / S actin This ratio was compared between the treated and untreated samples. << x >> is a LOX gene or an elastin gene.

<Result>
The results for two of the selected active ingredients are shown in Table III as examples.

<Conclusion>
Cardamom extract significantly induces LOX at all concentrations and significantly inhibits elastin (Eln) at all concentrations. The buckwheat extract significantly induces LOX at 0.1% and 1% and significantly inhibits elastin (Eln) at 5%.

"Analysis of LOX and / or elastin messenger RNA expression (eg, by qualitative RT-PCR performed in or without contact with an active ingredient to be tested for activity in cells from scar tissue regions)"
The active ingredient was tested in fibroblasts of human scar tissue skin (surgical removal of hypertrophic scar after caesarean section) at early passage (less than 5 passages). The culture was carried out, for example, in a well-known serum-free medium (Fibroblast Basal Medium, Promocell, Germany) in a single layer in a 24-well culture plate. For example, 40,000 cells were seeded per cm ² . When confluent, the cells were contacted with the active ingredient, preferably for 24 hours. Advantageously, in parallel, one untreated control (medium only) and three positive controls (1 ng / ml TGF-β, 50 pg / ml IL-1α, and 2% (v / v) Phytokine. (R) (Coretica, France) was also tested, for example, in the same culture plate. Pre-tested for 1 ng / ml of TGF-β and 50 pg / ml of IL-1α, it was shown that stimulation of elastin mRNA synthesis was induced by two cytokines at this concentration, mRNA analysis, eg quantitative RT-PCR (10 times for TGF-β, 6 times for IL-1α). After contacting the active ingredient with the cells (for example, 24 hours later), the medium was removed, and the cells were washed, for example, in phosphate buffer (pH 7.4) and lyophilized at −80 ° C. and stored. Total RNA was extracted (eg, using a 96-well extraction kit with a silica column) and measured with a 96-well spectrophotometer at 260 nm (purity indicator: protein measurement at 280 nm). The RNA is diluted, for example to 5 ng / μl. One-step qualitative RT-PCR was performed in 96-well plates using, for example, 50 ng of initial RNA for the actin gene, collagen Iα1 gene and LOX gene. Specific primers for each gene were used at 0.5 μM, for example.

CALL1 sense 5′-CAG AGG GAA GCC GCA AGA-3 ′ (SEQ ID NO: 36) CALL1 antisense 5′-CTG GCC GCC ATA CTC GAA C-3 ′ (SEQ ID NO: 37) LOX sense 5′-ACG TAC GTG CAGAAAG ATG TCC-3 ′ (SEQ ID NO: 24) LOX Antisense 5′-GGC TGG GTA AGA AAT CTG ATG-3 ′ (SEQ ID NO: 25) Actin Sense 5′-GTG GGG CGC CCC AGG CAC CA-3 ′ (SEQ ID NO: 30) ) Actin antisense 5'-CTC CTT AAT GTC ACG CAC GAT TTC-3 '(SEQ ID NO: 31)

Advantageously, the amplification parameters were as follows: 50 ° C., 30 minutes: 94 ° C., 2 minutes: (94 ° C., 30 seconds; 60 ° C., 30 seconds; 68 ° C., 30 seconds) for 28 cycles for actin, 26 cycles for COLL1, 32 cycles for LOX: 72 ° C. 10 minutes: 14 ° C., infinite. After amplification, the products were mixed at a ratio of, for example, (actin amplification product 3 μl + collagen I gene amplification product 5 μl + LOX gene amplification product 5 μl). Loading buffer (2 μl) was added and the entire volume (20 μl) was loaded on a pre-filled agarose gel (eg 2%). The present inventors visualized the expression level by a method known to those skilled in the art (for example, the band of the sample after electrophoresis was visualized under ultraviolet light in a dark room (15 minutes) and digitally photographed). The photograph of the gel was image-analyzed and the density of the band was quantified. The expression levels of the collagen I gene and the LOX gene were expressed as a percentage of the value in the negative control (no treatment) and compared with the results obtained in the positive control.

The selected active ingredient increases LOX mRNA approximately 2-fold and normalizes or decreases COL1 mRNA expression. Of the over 900 active compounds or extracts examined, the soba extract meets these criteria particularly critically.

For Examples 12-16: Those skilled in the art will know suitable methods for making compositions in which the compositions (formulations) described herein are varied.

<Prescription 12a>
"Use of the product of the invention in the formulation of an oil-in-water emulsion type cosmetic or pharmaceutical"
A water qsp100
Butylene glycol 2
Glycerin 3
Sodium dihydroxycetyl phosphate, 2
Isopropyl hydroxycetyl ether B glycol stearate SE 14
Triisononaoin 5
(Triisononaoine)
Palm oil fatty acid octyl 6
C adjusted to pH 5.5 2
Butylene glycol,
Methylparaben,
Ethylparaben, propylparaben D Product of the invention 0.01-10%

<Prescription 12b>
A water qsp100
Butylene glycol 2
Glycerin 3
Polyacrylamide, 2.8
Isoparaffin, Laureth-7
B Butylene glycol, 2
Methylparaben,
Ethylparaben, propylparaben;
2
Phenoxyethanol,
Methylparaben,
Propylparaben,
Butyl paraben,
Ethylparaben 0.5
Butylene glycol D Product of the invention 0.01-10%

<Prescription 12c>
A Carbomer 0.50
Propylene glycol 3
Glycerin 5
Water qsp100
B Palm oil fatty acid octyl 5
Bisabolol 0.30
Dimethicone 0.30
C Sodium hydroxide 1.60
D Phenoxyethanol, 0.50
Methylparaben,
Propylparaben, butylparaben,
Ethylparaben E Fragrance 0.30
F Product of the invention 0.01-10%

"Use of products of the invention in water-in-oil formulations"
A PEG30-dipolyhydroxystearic acid 3
Capric acid triglyceride 3
Cetearyl octoate 4
Dibutyl adipate 3
Grape seed oil 1.5
Jojoba oil 1.5
Phenoxyethanol, 0.5
Methylparaben, propylparaben,
Butylparaben, ethylparaben B Glycerin 3
Butylene glycol 3
Magnesium sulfate 0.5
EDTA 0.05
Water qsp100
C Cyclomethicone 1
Dimethicone 1
D Fragrance 0.3
E Product of the invention 0.01-10%

"Use of the products of the invention in shampoo or body soap formulations"
A Xanthan gum 0.8
Water qsp100
B Butylene glycol, 0.5
Methylparaben,
Ethylparaben, propylparaben phenoxyethanol, 0.5
Methylparaben,
Propylparaben, butylparaben,
Ethylparaben C citric acid 0.8
D Sodium laureth sulfate 40.0
E Product of the invention 0.01-10%

"Use of products of the present invention in formulations of anhydrous products such as lipsticks"
A mineral wax 17.0
Isostearyl isostearate 31.5
Propylene glycol dipelargon 2.6
(Propylene glycol dipelargonate)
Propylene glycol isostearate 1.7
PEG-8 beeswax 3.0
Hydrogenated palm kernel fatty acid glycerides, 3.4
Hydrogenated palm oil fatty acid glycerides
Lanolin oil 3.4
Sesame oil 1.7
Cetyl lactate 1.7
Mineral oil, lanolin alcohol 3.0
B castor oil qsp100
Titanium dioxide 3.9
CI 15850: 1 0.616
CI 45410: 1 0.256
CI 19140: 1 0.048
CI 77491 2.048
Product of the present invention 0.01-5%

“Use of products of the present invention in the formulation of aqueous gels (eyeliners, slimming agents, etc.)”
A water qsp100
Carbomer 0.5
Butylene glycol 15
Phenoxyethanol, 0.5
Methylparaben,
Propylparaben, butylparaben,
Ethylparaben B Product of the invention 0.01-10%

“Preparation of prescriptions for pharmaceuticals containing LOXL”
<Prescription 17a> Preparation of tablets A excipient g unit, 1 tablet Lactose 0.359
Sucrose 0.240
B LOXL extract ^* 0.001-0.1
(LOXL extract ^* is obtained, for example, by extraction as described in Example 2 and then drying.)

<Prescription 17b> Preparation of ointment A Excipient Low density polyethylene 5.5
Liquid paraffin qsp100
B LOXL extract ^* 0.001-0.1
(The LOXL extract ^* is obtained, for example, by extracting as described in Example 2 and then drying if necessary.)

<Prescription 17c> Preparation of injection formulation A excipient isotonic saline 5 ml
B LOXL extract ^* 0.001-0.1g
(LOXL extract ^* is obtained, for example, by extraction as described in Example 2 and then drying.)
Wrap Phase A and Phase B separately in ampoules and mix before use.

"Evaluation of cosmetic acceptability of the preparation containing the product of the present invention"
The toxicity obtained by mixing the compounds obtained in Examples 9 to 11 with 0.5% xanthan gum at a rate of 10% was confirmed as an eye irritation test in rabbits and no abnormal toxicity when once administered orally to rats. It was examined by a test and a sensitization test in a guinea pig.

“Evaluation of primary irritation in rabbit skin”
The above formulations were administered in 0.5 ml aliquots to the skin of 3 rabbits according to the OECD recommended method for the study of “Acute Irritation / Corrosion to Skin”.

The products were classified according to the criteria defined in the February 1, 1982 resolution published in the official newspaper of the French Republic on February 21, 1982 ("JORF").

From the above test results, it can be seen that the preparation containing the substance obtained in Example 11 is classified as having no irritation to the skin.

"Eye irritation test in rabbits"
In the pure state of the above formulation according to the method officially recommended by the OECD Directive 405 on February 24, 1987 for the study of << acute eye irritation / corrosion >> It was dripped in 3 eyes.
From the results of this study, these formulations are considered non-irritating to the eye in the sense of EEC 91/326 when used in the pure state or undiluted.

"Confirmation test that there is no abnormal toxicity after single oral administration to rats"
The above formulation was orally administered in a single dose of 5 g / kg body weight to 5 male rats and 5 female rats in accordance with a protocol applied to cosmetics inspired by OECD Directive No. 401 of February 24, 1987 did.
LD0 and LD50 have been found to be 5000 mg / kg or higher. Therefore, the tested formulations are not classified as formulations that are dangerous for oral consumption.

"Potential skin sensitization test in guinea pigs"
The above-mentioned preparation was subjected to a Magnusson-Kligmann maximum test, which is a protocol according to the OECD No. 406 directive.
These formulations are classified as non-sensitizing on contact with the skin.

Description of described sequences SEQ ID NO: 1: Peptide sequence of human protein LOX.
SEQ ID NO: 2 is the nucleotide sequence of cDNA encoding the human protein LOX described in SEQ ID NO: 1.
SEQ ID NO: 3 is the peptide sequence of the human protein tropoelastin.
SEQ ID NO: 4: Nucleotide sequence of cDNA encoding promoter of human gene encoding human elastin protein (base pair from −2171 upstream from ATG).
SEQ ID NO: 5: Nucleotide sequence of cDNA encoding the human protein tropoelastin described in SEQ ID NO: 3.
SEQ ID NO: 6 is the peptide sequence of human protein LOXL.
SEQ ID NO: 7: nucleotide sequence of cDNA encoding human protein LOXL described in SEQ ID NO: 6.

Regarding the double-stranded DNA probe, it is a sense primer for DNA encoding the human protein LOXL described in SEQ ID NO: 8: SEQ ID NO: 6.
SEQ ID NO: 9: antisense primer of DNA encoding human protein LOXL described in SEQ ID NO: 6.
SEQ ID NO: 10: sense primer for DNA encoding the human protein tropoelastin described in SEQ ID NO: 3.
SEQ ID NO: 11: antisense primer of DNA encoding human protein tropoelastin described in SEQ ID NO: 3.
SEQ ID NO: 12: sense primer for DNA encoding human protein LOX set forth in SEQ ID NO: 1
SEQ ID NO: 13: antisense primer of DNA encoding human protein LOX described in SEQ ID NO: 1.
SEQ ID NO: 14: sense primer for DNA encoding human protein collagen Iα1L
SEQ ID NO: 15: antisense primer for DNA encoding human protein collagen Iα1L

Regarding GST fusion gene, it is a sense primer of DNA of SEQ ID NO: 16: fusion gene GST S355-D415.
Sequence number 17: It is an antisense primer of DNA of fusion gene GST S355-D415.
SEQ ID NO: 18: sense primer for DNA of fusion gene GST G128-L212
Sequence number 19: It is an antisense primer of DNA of fusion gene GST G128-L212.
SEQ ID NO: 20: sense primer for DNA of fusion gene GST V228-S279
SEQ ID NO: 21: antisense primer for DNA of fusion gene GST V228-S279
SEQ ID NO: 22: sense primer for DNA of fusion gene GST D306-N373
Sequence number 23: It is an antisense primer of DNA of fusion gene GST D306-N373.

The PCR primer is an RT-PCR sense primer for mRNA encoding the human protein LOX described in SEQ ID NO: 24: SEQ ID NO: 1.
SEQ ID NO: 25: RT-PCR antisense primer for mRNA encoding the human protein LOX described in SEQ ID NO: 1.
SEQ ID NO: 26: RT-PCR sense primer for mRNA encoding the human protein tropoelastin described in SEQ ID NO: 3.
SEQ ID NO: 27: RT-PCR antisense primer of mRNA sequence encoding human protein tropoelastin described in SEQ ID NO: 3.
SEQ ID NO: 28: RT-PCR sense primer for mRNA encoding human protein LOXL described in SEQ ID NO: 6.
SEQ ID NO: 29: antisense primer for mRNA sequence encoding human protein LOXL described in SEQ ID NO: 6.
SEQ ID NO: 30: RT-PCR sense primer for mRNA encoding human protein actin.
SEQ ID NO: 31: RT-PCR antisense primer for mRNA encoding human protein actin.

SEQ ID NO: 32: sense primer for DNA of fusion gene GST 517-581
Sequence number 33: It is an antisense primer of DNA of fusion gene GST 517-581.
SEQ ID NO: 34: sense primer for DNA of fusion gene GST 664-720
SEQ ID NO: 35: antisense primer for DNA of fusion gene GST 664-720
SEQ ID NO: 36: sense primer for DNA encoding collagen Iα1 gene
SEQ ID NO: 37: antisense primer for DNA encoding collagen Iα1 gene

A description of the sequence of LOs determined to produce specific antibodies. FIG. 1 (A): Schematic of hLOX (human LOX protein) and hLOXL (human LOXL protein). FIG. 1 (B): shows the similarity between LOX and LOXL antigenic regions and the corresponding region on the LO isoform. Photo of electrophoresis performed on smooth muscle cells (SMC) using anti-LOX antibody and anti-LOXL antibody. Immunohistological detection of LOXL and LOX in regenerated skin (RS) and normal human skin. In reconstructed skin (16 and 35, and 45 days later) and human foreskin skin section, anti ^{LOXL2 517-581} antibody (left column), and, theoretically LOXL2, recognize LOXL3 and LOXL4 anti ^{LOXL2 664-720} antibody ( Immunodetection performed using (right column). Immunodetection of LOXL, LOX and elastin performed using transmission electron microscopy in regenerated skin after 45 days and in the dermis of normal human skin. Immunodetection of LOX and LOXL in human skin. Immunodetection of LOX and LOXL in abdominal skin taken from people of different ages. Immunodetection of LOX and LOXL in the skin of scar tissue with different duration after treatment. Detection of the expression of the gene encoding LOXL by in situ hybridization performed on the skin model cross section after 35 days. Changes in gene expression levels of elastin, LOX and LOXL with aging.

Claims (24)

Abnormal or pathological elastic fiber formation seen in fibrosis, photoelastic fibrosis, skin streak, dystrophic scar, eczema, or stromal response to cancer involving disordered or non-functional elastic tissue In combination with a second component that induces a reduction or inhibition of elastin synthesis to produce a composition intended to control elastic fiber formation of
LOX having the sequence of SEQ ID NO: 1 or
Use of a first component to induce LOX activity or formation,
The first component and the second component are the same or different and are selected from the group consisting of an extract of garega, an extract of cardamom , xylitol, an adragant gum, an extract of barley and an extract of buckwheat. Is,
Use wherein the extract is obtained using one or more solvents selected from water, alcohols, glycols and polyols. Use according to claim 1, characterized in that the second component induces a reduction or inhibition of the synthesis of tropoelastin having the sequence SEQ ID NO: 3 which is a precursor protein of elastin. The second ingredient is:
i) includes a region that binds to at least a portion of the nucleotide sequence (SEQ ID NO: 4) of the human elastin gene promoter (Pr), or
ii) by regulating the expression of a protein that binds to at least a portion of the nucleotide sequence (SEQ ID NO: 4) of the human elastin gene promoter (Pr),
Use according to claim 1, characterized in that it reduces or inhibits the synthesis of tropoelastin. The first component and the second component are the same or different and are selected from the group consisting of an extract of cardamom and an extract of buckwheat,
Use according to claim 1, wherein the extract is obtained using one or more solvents selected from water, alcohols, glycols and polyols. Selected from the group consisting of an extract of garega, an extract of cardamom, xylitol, an adragant gum, an extract of barley and an extract of buckwheat, wherein the extract is selected from water, alcohol, glycol and polyol Inducing reduction or inhibition of elastin synthesis, characterized in that it contains one or more active ingredients that are one or more solvents, optionally mixed with cosmetically acceptable excipients , Cosmetic composition for inducing activity or formation . Selected from the group consisting of an extract of garega, an extract of cardamom, xylitol, an adragant gum, an extract of barley and an extract of buckwheat, wherein the extract is selected from water, alcohol, glycol and polyol LOX activity, which induces reduction or inhibition of elastin synthesis, characterized in that it contains one or more active ingredients that are one or more solvents, optionally mixed with pharmaceutically acceptable excipients Or a pharmaceutical composition for inducing formation . A method of screening for components that induce LOX activity or formation to control elastic fiber formation, comprising:
Contacting the potential active ingredient with at least one cell that is alive and capable of expressing LOX;
A method comprising analyzing the expression of LOX for the purpose of ascertaining whether the potential active substance induces the expression of LOX. Screen for ingredients that control elastic fiber formation in fibrosis, photoelastic fibrosis, cutaneous streak, dystrophic scar, eczema, or stromal response to cancer involving disordered or nonfunctional elastic tissue The screening method according to claim 7. The method comprises contacting a potential active ingredient with at least one cell capable of expressing the protein elastin, which may be the same as that expressing LOX, said potential 8. The screening method according to claim 7, comprising analyzing the expression of the protein elastin for the purpose of confirming whether or not the active ingredient induces reduction or inhibition of elastin synthesis. The active ingredients are:
Whether to induce the expression of at least part of a nucleotide sequence encoding the protein LOX, or
Whether to induce the expression of a peptide sequence present in the structure of the protein LOX; and whether to inhibit the expression of at least a part of the nucleotide sequence encoding the protein elastin, or
The screening method according to claim 7, wherein whether or not the expression of a peptide sequence present in the structure of the protein elastin is inhibited is examined. The screening method according to claim 7, wherein the analysis of the expression of LOX or protein elastin is performed by qualitative or quantitative analysis of the expression of a nucleotide sequence encoding LOX or tropoelastin. Analysis of LOX or protein elastin expression
Use of a primer that hybridizes with at least a portion of the nucleotide sequence of complementary DNA encoding LOX (SEQ ID NO: 2) to amplify at least a portion of the nucleotide sequence encoding LOX, or
Reverse transcription polymerase comprising the use of a primer that hybridizes with at least a portion of the nucleotide sequence of complementary DNA encoding tropoelastin (SEQ ID NO: 5) for amplifying at least a portion of the nucleotide sequence encoding tropoelastin The screening method according to claim 7, wherein a chain reaction (RT-PCR) is used. The method is:
-By in situ hybridization of at least part of the nucleotide sequence encoding LOX, or by in situ hybridization of at least part of the nucleotide sequence encoding tropoelastin, or
By immunodetection by using at least one antibody capable of recognizing at least part of the peptide sequence encoding LOX, or all or part of elastin,
The screening method according to claim 7, further comprising a step of specifying an expression position of LOX performed on a regenerated skin model or biopsy. The step of immunodetection or in situ hybridization allows investigation of the traceability of LOX and elastin in epithelial or connective tissue, said tissue being derived from at least one regenerative skin model or biopsy The screening method according to claim 13, wherein: The screening method can be used to compare the expression of LOX with the expression of LOX expressed in a control containing no active ingredient, or the expression of protein elastin and the expression of protein elastin expressed in a control containing no active ingredient. The screening method according to claim 7, comprising a comparison. The screening method according to claim 7, wherein the cells include fibroblasts. The screening method according to claim 7, wherein the cells include fibroblasts derived from foreskin or skin of an adult subject. The screening method according to claim 7, wherein the cells include epithelial cells. The screening method according to claim 7, wherein the cells include keratinocytes derived from foreskin or skin of an adult subject. The screening method according to claim 7, wherein the living cells are derived from the face, abdomen or chest and are “photo-aged” or “regularly exposed” to sunlight. The screening method according to claim 7, wherein the screening method uses a regenerated dermis model containing fibroblasts or a model based on biopsy. The screening method according to claim 7, wherein the screening method uses a regenerated epidermis model containing keratinocytes. Active ingredient, an extract of Garega, extract of cardamom, key xylitol, Adoraganto (adragant) rubber, it is those that are selected from the group consisting of barley extract and oat extract,
The screening method according to claim 7, wherein the extract is obtained using one or more solvents selected from water, alcohol, glycol, and polyol. Ray elastic fibrosis, or for treating striae Cosmetic composition according to claim 5.