Patent application title: Stimulation of the synthesis and of the activity of an isoform of lysyl oxidase-like LOXL for stimulating the formation of elastic fibers.
Eric Perrier (Les Cotes D'Arey, FR)
Valérie Cenizo (Vaulx-Milieu, FR)
Valérie Cenizo (Vaulx-Milieu, FR)
Charbel Bouez (Lyon, FR)
Pascal Sommer (Saint Genis Laval, FR)
Odile Damour (Saint Genis Laval, FR)
Claudine Gleyzal (Lyon, FR)
Valérie Andre (Ampuis, FR)
Corinne Reymermier (Charly, FR)
Corinne Reymermier (Charly, FR)
BASF Beauty Care Solutions France S.A.S.
IPC8 Class: AA61K3600FI
Class name: Drug, bio-affecting and body treating compositions plant material or plant extract of undetermined constitution as active ingredient (e.g., herbal remedy, herbal extract, powder, oil, etc.) containing or obtained from cinnamomum (e.g., cinnamon, etc.)
Publication date: 2010-02-18
Patent application number: 20100040710
The invention relates to the stimulation of the synthesis and of the
activity of an isoform of lysyl oxidase, and more particularly of the
LOXL (lysyl oxidase-like) isoform.
The Invention relates notably to a method of identifying an active
principle which stimulates the formation of elastic fibers.
The aim of the invention is mainly to provide such a method of
identification so as to provide compositions which enable stimulating the
formation of elastic fibers.
1. A method of stimulating the formation of skin elastic fibers comprising
applying to the skin an effective amount of a substance which stimulates
the enzymatic activity or expression of said lysyl oxidase isoform LOXL
having the peptide sequence of Sequence ID No1 and which stimulates the
expression of the protein elastin.
2. The method of claim 1 wherein the substance comprises a region which fixes to at least one part of the sequence of nucleotides of the cDNA encoding the lysyl oxidase isoform LOXL promoter of Sequence ID No3 or which modulates the expression of a protein which fixes to at least one part of the sequence of nucleotides of the cDNA encoding the lysyl oxidase isoform LOXL promoter of Sequence ID No3.
3. The method of claim 1 wherein the substance is selected from the group consisting of an extract of dill, an extract of cardamon, an extract of currant, an extract of black radish, an extract of small holly, an extract of cinnamon, a lactic bacteria-based fermentation, an extract of oats, an extract of potato, a silk protein, Asea foetida gumethyl hexenoate, methyl butyrate, ethyl decadienoate, and combinations thereof.
4. The method of claim 3 wherein the effective amount of the substance is between 0.1% and 5% (v/v).
5. The method of claim 1 wherein the stimulation of the skin elastic fibers provides a cosmetic benefit to the skin.
6. The method of claim 5 wherein the cosmetic benefit is combating against the loosening of tissues, for densifying the extracellular matrix, for firming up the subcutaneous tissues, for reducing skin wrinkles, for exerting an anti-wrinkle effect, for improving the appearance of scars, or for combating against stretch marks.
The invention relates to the stimulation of the activity of an
isoform of lysyl oxidase, and more particularly of the LOXL (lysyl
STATE OF THE ART
The properties of resistance and of elasticity of the skin and of the mucous membranes are essentially defined by the collagen fibers and elastin fibers of the dermis. Elastin is a protein which makes up the elastic fibers by combining itself with other molecules such as fibrillins and MAGPs (Microflbrillar Associated Glycoproteins).
The elastic fibers are formed of elastin deposited on the microfibrils. Elastin is synthesized in the form of soluble tropoelastin which acquires its physico-chemical properties (insolubility, elasticity) after the intra- and inter-molecular cross-linking of it by a lysyl oxidase, and its deposit on the microfibrils. The elastic fibers are responsible for the elastic property of the organs which contain them (vessels, pulmonary parenchyma, elastic cartilages, skin . . . ). The elastic fibers are mainly constituted of elastin deposited on the microfibrils. The name <<elastin>> was reserved to the protein which forms the amorphous portion of the elastic fibers and which imparted their elasticity to them. Recently, components of these elastic fibers have been shown in the epidermis.The collagen fibrils are formed by the trimeric assembly of chains of collagen. These collagen fibers are also cross-linked by a lysyl oxidase.
Ageing of skins and of mucous membranes is associated with a modification of these fibrillar networks, notably that of the elastic fibers which degrade and which do not re-form correctly. Similarly, in scars, the network of elastic fibers does not form correctly. Five known isoforms exist in the family of lysyl oxidases (LOs): LOX, LOXL, LOXL2, LOXL3, LOXL4 (Csiszar, Lysyl oxidases: A novel multifunctional amine oxidase family, Nucleic Acid Research and Molecular Biology, 2001, vol 70, p 2-28). LOX is clearly Involved in the cross-linking of collagen fibers (Seve et al, Expression analysis of recombinant lysyl oxidase (LOX) in myofibroblast-like cells, Connective Tissue Research, 2002, 43: 613-619).
Functional elastic fibers (which are mainly-found In the skin, the vessels, the macula of the retina and the inter-vertebral discs) are formed during prenatal development and immediately after birth. In the skin, these elastic fibers are formed by the fibroblasts of the dermis, but certain compounds, which are necessary for the elaboration of the elastic fibers, have also been found in the cells of the epidermis. The collagen fibers are synthesized in the connective tissues throughout the whole life.
The rate of replacement of the elastic fibers is very low in adult life, although the overall level of elastin of the skin can increase (Ashcroft et al., Age-related changes in the temporal and spatial distributions of fibrillin and elastin mRNAs and proteins in acute cutaneous wounds of healthy humans, J. Pathol., 1997, 183: 80-89). In the newly-born, the microfibrils are not all completely covered with elastin, and become so towards puberty. At 40 years, inclusions are seen to appear on the fibers, more frequently in women, and then the fragmentation of the elastic fibers and their disappearance under the dermal-epidermal junction (DEJ). This fractioning and/or this disappearance under the DEJ manifests itself by a loss of elasticity of the skin and the formation of wrinkles. The synthesis of non-functional elastic fibers is observed during photo-ageing, but this increase is accompanied with an accelerated loss of the elastic fibers under the DEJ. (Watson et al., Fibrilln-rich microfibrils are reduced in photo-aged skin. Distribution at the dermal-epidermal junction, J. Invest. Dermatol., 1999, 112: 782-787).
Furthermore, the neosynthesis of elastic fibers is carried out little in the scars of adult persons, while paradoxically, this property be in part found again in aged persons (more than 70 years old) whose elastic fibers produced are very fragmented. Yet the main components which Intervene In the final composition of the elastic fibers are present (elastin, microflbrils) and the overall lysyl oxidase activity is maintained (Pasquali-Ronchetti and Baccarani-Contri, Elastic fiber during development and aging, Microscopy Res. Tech., 1997, 38: 428-435). This suggested to the inventors that one or more factors were missing which enable the formation of functional elastic fibers in the adult, but which exist during embryonic development and during the first age.
The prior art does not enable providing criteria which enable evaluating the impact of an active principle in dermato-cosmetology on a functional neo-elastogenesis. In this context. It is also very difficult to obtain objective criteria enabling the Impact of these actives to be judged. The screening methods of actual active principles bear upon the evaluation of the expression of the genes Involved in the formation of the elastic fibers, such as elastin or the fibrillins.
Furthermore, at the present time, animal experimentation is forbidden in cosmetics in Europe and human experimentation is ethically disputed. It is therefore unacceptable to the inventors to carry out a screening method, for cosmetic applications, which makes use of animals or human beings.
In three-dimensional models, such as Mimeskin® (Coletica, Lyons, France), keratinocytes Induce the synthesis of tropoelastin and the deposit of tropoelastin on the microfibrils (Duplan-Perrat et al., Keratinocytes influence the maturation and organization of the elastin network in a skin equivalent. J. Invest. Dermatol. 114: 365-70, 1999). In the Mimeskin® model, the extracellular matrix showed an ultra-structural organization similar to that of the skin, with the collagen being organized in rays and elastic fibers which are constituted of elastin deposited on the microfibrils. This model has also been used for testing the effectiveness of certain molecules, such as inhibitors of lysyl oxidases. This has enabled proving that the inhibition of the lysyl oxidases induced a disorganization of the collagen fibers and the elastin fibers, but also a deviation from the program of differentiation of the keratinocytes, with a reduction of the level of expression of the labels of terminal differentiation, such as filaggrin (Farjanel et al., French patent 01 10443, CNRS, Use of inhibitors of lysyl oxidases for cell culture and tissue engineering (<<Utilisation d'inhibiteurs des lysyl oxydases pour la culture cellulaire and le genie tissulaire>>). In that patent, no distinction is made between the different isoforms of LO.
However, those studies did not enable developing a method of identifying active principles which enable stimulating the formation of functional elastic fibers.
The prior art does not therefore enable providing active principles which enable stimulating the formation of functional elastic fibers.
Furthermore, the prior art does not enable a dynamic tracking of the zone of expression of the Isoform of the lysyl oxidase LOXL, notably due to the fact that the methods provided by the prior art are imprecise.
AIMS OF THE INVENTION
The aim of the Invention is mainly to solve the technical problems set forth above and notably the technical problem aiming to provide a method of identifying active principles which stimulate the formation of functional elastic fibers. By "functional elastic fibers" is meant the usual meaning in the art as described above and notably in the context of the invention, elastic fibers which have elastic properties coming from a tridimensional structure.
The invention also relates to the use of the L isoform of lysyl oxidase or of an active principle which stimulates the enzymatic activity or the expression of the L isoform of lysyl oxidase (LOXL), notably for stimulating the formation of functional elastic fibers.
The invention enables solving the technical problem consisting of providing a method of locating the expression of LOXL and of tracking this expression.
SUMMARY OF THE INVENTION
In this text, by the term "LOXL", or "hLOXL", the inventors mean the L isoform of the human lysyl oxidase protein LOXL.
In this text, by the term "LOX", or "hLOX", the inventors mean the Initial isoform of the human lysyl oxidase protein LOX.
By "stimulating the expression of the isoform of the lysyl oxidase LOXL", the inventors mean the stimulation of the expression of the gene encoding LOXL or of its promoter, and notably the stimulation of the synthesis of the messenger RNA encoding LOXL, but also the stimulation of the synthesis of LOXL from this messenger RNA.
By "stimulating the expression of elastin", the Inventors mean the stimulation of the expression of the gene encoding the elastin protein or of its promoter, and notably the stimulation of the synthesis of the messenger RNA encoding the elastin protein, but also the stimulation of the synthesis of the protein elastin or of Its precursor, tropoelastin, from this messenger RNA.
In this invention, the Inventors thus aim to stimulate mainly either the expression of LOXL as described, or the enzymatic activity of LOXL.
This stimulation must be effective enough to enable stimulating the formation of functional elastic fibers.
Active principles are considered as effective which enable obtaining an activation or an increase of about 1.5 times the expression and/or the activity of LOXL on a model, which comprises at least one cell type which presents an expression and/or an activity of LOXL, upon contact of these active principles, with respect to the level of expression and/or of activity of LOXL in a control model (without placing the active principles in contact).
It is such that the present invention relates, according to a first aspect, to the use of the <<like>> isoform of lysyl oxidase having the Sequence ID No1, also called LOXL, or of an homologous or essentially homologous form thereof, or of a substance which promotes the activity and/or the expression of LOXL, for the manufacture of a composition for stimulating the formation of elastic fibers.
By "an homologous or essentially homologous form thereof" it is meant an homologous form of the Isoform of lysyl oxidase LOXL which has the same or similar activity as LOXL as defined herein.
Advantageously, the expression of LOXL is either the expression of a sequence of nucleotides encoding LOXL or the expression of a sequence of peptides constituting a fraction of the protein LOXL, said sequence of peptides being preferably selected from the Sequence ID No1.
Advantageously, said composition is a cosmetic, neutraceutical, medical or pharmaceutical composition.
Advantageously, the composition further comprises a second substance which stimulates the expression of the protein elastin, notably for stimulating the formation of elastic fibers, said second substance preferably being the substance which promotes the activity and/or the expression of LOXL.
Advantageously, said active substance comprises a region which fixes to at least one part of the sequence of nucleotides of the promoter of the human LOXL gene (Pr) (SEQ ID No3) or of an homologous or essentially homologous form thereof, or modulates the expression of a protein which fixes to at least one part of the sequence of nucleotides of the promoter of the human LOXL gene (Pr) (SEQ ID No3) or of an homologous or essentially homologous form thereof. This sequence is given from the nucleotide -2630 before the ATG codon, and the nucleotides from -2172 to -1 have In particular been studied.
Advantageously, the active substance is selected from the group consisting of dill, currant, cardamon, black radish, box holly, cinnamon, lactic bacteria-based fermentations, oats, potato, silk, Asa foetida gum, ethyl hexenoate and Its derivatives, methyl butyrate and its derivatives, and ethyl decadienoate and its derivatives.
Advantageously, the use described above is carried out for inducing a neo-elastogenesis of the tissues, and notably for stimulating the elasticity of the tissues thus obtained, and for reducing skin wrinkles.
Advantageously, the use described above is carried out for combating against the loosening of the tissues, notably when the loosening of the tissues is observed during ageing or during solar exposures, for densifying the extracellular matrix, for firming up the subcutaneous tissues, for reducing skin wrinkles, for exerting an anti-wrinkles effect, for improving the quality of scar tissue and the appearance of scars, notably dystrophic and keloid scars, or for combating against stretch marks.
According to a second aspect, the Invention relates to a cosmetic composition which comprises an active substance as defined above, optionally In a mixture with a cosmetically acceptable excipient.
According to a third aspect, the Invention relates to a neutraceutical composition which comprises an active substance as defined above, optionally in a mixture with an excipient acceptable for food.
According to a fourth aspect, the invention relates to a pharmaceutical composition which comprises an active substance as defined above, optionally in a mixture with a pharmaceutically acceptable excipient.
For the cosmetic or pharmaceutical compositions, the excipient contains for example at least one compound selected from the group consisting of preservatives, emollients, emulsifiers, surfactants, moisturizers, thickeners, conditioners, matifying agents, stabilizers, antioxidants, texture agents, brightening agents, filmogenic agents, solubillsers, pigments, dyes, perfumes and solar filters. These excipients are preferably selected from the group consisting of amino acids and their derivatives, polyglycerols, esters, polymers and derivatives of cellulose, lanolin derivatives, phospholipids, lactoferrins, lactoperoxidases, sucrose-based stabilisers, E vitamins and its derivatives, natural and synthetic waxes, plant oils, triglycerides, insaponifiables, phytosterols, plant esters, silicones and its derivatives, protein hydrolysates, jojoba oil and its derivatives, lipo/hydrosoluble esters, betaines, aminoxides, plant extracts, esters of sucrose, titanium dioxides, glycines, and parabens, and more preferably from the group consisting of butylene glycol, steareth-2, steareth-21, glycol-15 stearyl ether, cetearyl alcohol, phenoxyethanol, methylparaben, ethylparaben, propylparaben, butylparaben, butylene glycol, natural tocopherols, glycerol, sodium dihydroxycetyl, isopropyl hydroxycetyl ether, glycol stearate, triisononaoine, octyl cocoate, polyacrylamide, isoparaffin, laureth-7, a carbomer, propylene glycol, glycerol, bisabolol, dimethicone, sodium hydroxide, PEG 30-dipolyhydroxysterate, capric/caprylic triglycerides, cetearyl octanoate, dibutyl adipate, grape seed oil, jojoba oil, magnesium sulphate, EDTA, cyclomethicone, xanthan gum, citric acid, sodium lauryl sulphate, mineral waxes and oils, isostearyl isostearate, propylene glycol dipelargonate, propylene glycol isostearate, PEG 8 Beeswax, hydrogenated palm tree heart oil glycerides, hydrogenated palm oil glycerides, lanolin oil, sesame oil, cetyl lactate, lanolin alcohol, castor oil, titanium dioxide, lactose, sucrose, low density polyethylene, and an isotonic saline solution.
Advantageously, the compositions cited above are formulated in a form selected from the group consisting of a solution, which is aqueous or oily, an aqueous cream or gel or an oily gel, notably in a pot or in a tube, notably a shower gel, a shampoo; a milk; an emulsion, a microemulsion or a nanoemulsion, notably an oil-in-water or water-in-oil or multiple or silicone-containing microemulsion or nanoemulsion; a lotion, notably in a glass bottle, a plastic bottle or in a measure bottle or in an aerosol; an ampoule; a liquid soap; a dermatological bar; an ointment; a foam; an anhydrous product, preferably a liquid, pasty or solid anhydrous product, e.g. in the form of a stick, notably in the form of a lipstick.
Advantageously, the compositions which are sufficiently liquid can be administered, notably via the parenteral, ocular, pulmonary, oral or nasal route.
Advantageously, the pasty or dry compositions (pastes, powders, tablets, capsules, granules, suppositories . . . ), can be introduced into the body notably via the oral, sublingual, nasal or rectal route.
Advantageously, when the formulation of the composition allows it, the administration route is cutaneous or transmucosal, notably by application of the composition on the skin or on a mucous membrane.
Advantageously, from the various formulations and routes of administration, the person skilled in the art will select the one which is adequate for the effectiveness sought after.
According to a fifth aspect, the Invention relates to a method of cosmetic care characterized in that it comprises the use of a composition described above.
Advantageously, the cosmetic care is selected from the group consisting of combating against the loosening of the tissues, notably when the loosening of the tissues is observed during ageing or during solar exposures, densifying the extracellular matrix, firming up the subcutaneous tissues, reducing skin wrinkles, anti-wrinkles effects, improving the quality of scar tissue and the appearance of scars, notably dystrophic and keloid scars, and combating against stretch marks.
According to a sixth aspect, the invention relates to a screening method of a substance which promotes the activity of LOXL or of an homologous or essentially homologous form thereof, for stimulating the formation of elastic fibers, characterized in that it comprises: placing a potentially active substance in contact with LOXL at least one type of cells capable of expressing the isoform LOXL or of an homologous or essentially homologous form thereof, and a) analyzing the activity of LOXL or of an homologous or essentially homologous form thereof, notably with the aim of identifying whether said potentially active substance stimulates the activity of LOXL or of an homologous or essentially homologous form thereof, or b) analyzing the expression of LOXL or of an homologous or essentially homologous form thereof, notably with the aim of identifying whether said potentially active substance stimulates the expression of LOXL or of an homologous or essentially homologous form thereof. Within the context of analyzing the expression of LOXL or of an homologous or essentially homologous form thereof:
Advantageously, It is sought whether said potentially active substance stimulates: the expression of at least one sequence of nucleotides encoding the protein LOXL or of an homologous or essentially homologous form thereof, and/or the expression of a sequence of peptides essentially constituting a peptide fraction of the protein LOXL or of an homologous or essentially homologous form thereof.
Advantageously, the analysis of the expression of LOXL is carried out by qualitative and/or quantitative analysis of the expression of at least one part of a sequence of nucleotides encoding LOXL.
Advantageously, the sequence of nucleotides is the cDNA which is complementary to the mRNA encoding LOXL, the LOXL cDNA being defined by the sequence ID No2.
Advantageously, the analysis of the expression of LOXL makes use of a reverse transcription polymerase chain reaction (RT-PCR) which comprises the use of primers which hybridize with at least one part of the sequence of nucleotides of the complementary DNA encoding LOXL (SEQ ID No2), in order to amplify at least one part of the sequence of nucleotides encoding the LOXL.
Advantageously, the method also comprises a step locating the expression of LOXL which is carried out on a reconstructed skin model or on a a model based on biopsies by in situ hybridization, notably of at least one part of a sequence of nucleotides encoding LOXL for example by using at least one DNA probe which hybridizes with at least one part of the sequence of nucleotides of the complementary DNA encoding LOXL (SEQ ID No2); or by Immuno-detection notably by using at least one specific antibody of the LOXL.
The specific antibodies are itemized In Example 1.
Advantageously, the screening method comprises the comparison of the expression of LOXL with the expression of LOXL expressed in a control which does not comprise said potentially active substance.
Advantageously, the living cells comprise fibroblasts, originating notably from normal human skin, such as, for example, originating from the foreskin or from a skin of an adult subject.
Advantageously, the living cells comprise epithelial cells, for example keratinocytes, originating notably from normal human skin, such as, for example, originating from the foreskin or from a skin of an adult subject.
Advantageously, the living cells originate from at least one skin having a particular localization, for example from the face, from the abdomen, or from the breasts, and being able to be characterized as being <<aged>> or as being <<exposed>> to sun's radiation or not, or from a skin originating from a zone which has scars or stretch marks.
Advantageously, the screening method makes use of a reconstructed skin model, preferably at least one dermis model which comprises fibroblasts, or a model based on biopsies.
Advantageously, the screening method makes use of a reconstructed skin model or a model based on biopsies. The reconstructed skin model used is advantageously the Mimeskino reconstructed skin model but may also be a model of connective matrix, of epidermis or of epithelium, or of reconstructed skin or mucous membrane:
1) The three-dimensional connective matrix (dermis or chorion) culture model, comprises a support which is sown with stromal cells in order to form reconstructed dermis or reconstructed chorions. This support is preferably selected from: an inert support selected from the group consisting of a semi-permeable synthetic membrane, in particular a semi-permeable nitrocellulose membrane, a semi-permeable nylon membrane, a Teflon membrane or a Teflon sponge, a semi-permeable membrane of polycarbonate or polyethylene, polypropylene, or of polyethylene terephthalate (PET), a semi-permeable Anopore inorganic membrane, of cellulose acetate or cellulose ester (HATF) membrane, a semi-permeable Biopore-CM membrane, a semi-permeable polyester membrane, a membrane or a film of polyglycolic acid. In this group, the dermal models Skin2® model ZK1100 and Dermagraft® and Transcyte® (Advanced Tissue Sciences) for example, are found; a cell culture-treated plastic (formation of a dermal leaf: Michel M. et al. In In Vitro Cell. Dev Biol.-Animal (1999) 35: 318-326); a gel or a membrane based on hyaluronic acid (Hyalograft® 3D--Fidia Advanced Biopolymers) and/or on collagen and/or on fibronectin and/or on fibrin; in this group, dermal model Vitrix® (Organogenesis) for example is found; a porous matrix, which is surfaced or non-surfaced, made from collagen being able to contain one or more glycosaminoglycans and/or eventually chitosan (EP 0 296 078 A1 of the CNRS, WO 01/911821 and WO 01/92322 of Coletica).2) The three-dimensional Epidermis or Epithelium Culture Model
Comprises a support which is sown or not beforehand with stromal cells, particularly fibroblasts, and then with epithellal cells and in particular keratinocytes, so as to obtain reconstructed epithelia or epidermis. This support is preferably selected from an inert support selected from the group consisting of a semi-permeable synthetic membrane, in particular a semi-permeable nitrocellulose membrane, a semi-permeable nylon membrane, a Teflon membrane or a Teflon sponge, a semi-permeable membrane of polycarbonate or polyethylene, polypropylene, or of polyethylene terephthalate (PET), a semi-permeable Anopore inorganic membrane, of cellulose acetate or cellulose ester (HATF) membrane, a semi-permeable Biopore-CM membrane, a semi-permeable polyester membrane; in this group, the reconstructed models Epiderm and Epithelia (Skinethic®) are found, as well as the models EpiDerm®, EpiAirway®, EpiOccular® (Mattek Corporation); a film or a membrane based on hyaluronic acid and/or on collagen and/or on fibronectin and/or on fibrin. In this group, the models: Episkin® (L'Oreal) and Laserskin® (Fidia advanced Biopolymers), in particular, can be cited.
3) The Three-dimensional Reconstructed Skin or Mucous Membrane Culture Model
Comprises a matrix support (dermal or of chorion) which is sown with epithelial cells so as to obtain a reconstructed mucous membrane or with keratinocytes so as to obtain a reconstructed skin. This support is preferably selected from: an inert support selected from the group consisting of a semi-permeable synthetic membrane, in particular a semi-permeable nitrocellulose membrane, a semi-permeable nylon membrane, a Teflon membrane or a Teflon sponge, a semi-permeable membrane of polycarbonate or polyethylene, polypropylene, or of polyethylene terephthalate (PET), a semi-permeable Anopore Inorganic membrane, of cellulose acetate or cellulose ester (HATF) membrane, a semi-permeable Biopore-CM membrane, a semi-permeable polyester membrane, said inert support containing stromal cells, in particular fibroblasts, a gel based on collagen and/or hyaluronic acid and/or fibronectin, and/or on fibrin comprising stromal cells, in particular fibroblasts, a porous matrix, which is surfaced or non-surfaced, made from collagen being able to contain one or more glycosaminoglycans and/or eventually chitosan, these porous matrices integrating stromal cells, in particular fibroblasts, a human or animal de-epidermisised dermis or dead dermis.In this group, the models Apligraf® (Organogenesis), ATS-2000 (CellSystems® Biotechnologie Vertrieb), as well as Skin2® (ZK1200-1300-2000--Advanced Tissue Science), in particular, can be cited.
Furthermore, models exist which are dedicated to tissue therapy and which can also be the subject of such studies, The models Epidex® (Modex Therapeutiques), Epibase® (Laboratoire Genevrier), Epicell® (Genzyme), Autoderm® and Transderm® (Innogenetics), can be cited.
Advantageously, the screening method makes use of a reconstructed skin model, preferably at least one epidermis model which comprises keratinocytes.
Advantageously, the method comprises a step of analyzing the expression of a sequence at least of the protein elastin and/or tropoelastin, or of a sequence of nucleotides encoding the protein elastin, notably for detecting an eventual stimulation of the expression of the protein elastin when said active substance is in contact with said living cells.
Advantageously, the method comprises a step of immuno-detecting the expression of the protein LOXL, notably with the aim of performing the traceability of neo-elastogenesis, notably in the epithelial tissues and/or in the connective tissues, said tissues originating from at least one reconstructed skin model or from a model based on biopsies.
Advantageously, said active substance is selected from the group consisting of dill, currant, cardamon, black radish, box holly, cinnamon, lactic bacteria-based fermentations, oats, potato, silk, Asa foetida gum, ethyl hexenoate and its derivatives, methyl butyrate and Its derivatives, and ethyl decadlenoate and Its derivatives.
According to a seventh aspect, the Invention relates to a method of locating the expression of LOXL or of an homologous or essentially homologous form thereof in tissues with the aim of performing the traceability of neo-elastogenesis, notably in connective tissues, said tissues originating from at least one reconstructed skin model or from biopsies, characterized in that the method comprises a step of immuno-detecting the protein LOXL or of an homologous or essentially homologous form thereof or of in situ hybridizing at least one part of a sequence of nucleotides encoding LOXL or of an homologous or essentially homologous form thereof.
The invention also relates to a method of locating the expression of LOXL in tissues with the aim of performing the traceability of neo-elastogenesis, notably in epithelial tissues and/or in connective tissues, characterized in that the method comprises a step of immuno-detecting the protein LOXL or of in situ hybridizing the gene encoding LOXL.
The invention also relates to the use of an active principle which modifies the enzymatic activity or the expression of the protein LOXL for stimulating the formation of elastic fibers.
The invention also relates to a method of treatment of a deficiency associated with the enzymatic activity of the isoform of the protein lysyl oxidase LOXL which comprises administering, to a subject, a therapeutically effective amount of a composition which comprises the protein lysyl oxidase LOXL, or of an homologous or essentially homologous form thereof, or a compound which stimulates the enzymatic activity or the expression of the protein lysyl oxidase LOXL.
Advantageously, this method of treatment enables performing a treatment selected from combating against the loosening of the tissues, notably when the loosening of the tissues is observed during ageing or during solar exposures, densifying the extracellular matrix, firming up the subcutaneous tissues, reducing skin wrinkles, anti-wrinkles effects, improving the quality of scar tissue and the appearance of scars, notably of dystrophic scars, in particular dystrophic scars, and keloid scars, and combating against stretch marks.
DETAILED DESCRIPTION OF THE INVENTION
The inventors have unexpectedly demonstrated that the activity of LOXL was a main missing link in elastogenesis in the adult and that it was possible to reactivate the synthesis of this isoform of lysyl oxidase so as to obtain a stimulating effect on elastogenesis.
The inventors have in fact demonstrated that this Isoform of the family of lysyl oxidases (LO) is associated with elastogenesis in a reconstructed skin model producing elastic fibers. In seeking whether this isoform was present or absent in the skin of various ages and during skin alterations, the inventors noticed the simultaneous presence or absence of this isoform and of elastogenesis, and this enables indicating that the activity of this Isoform of LO corresponds to a missing link the synthesis of which it is necessary to modulate in order to orchestrate a functional elastogenesis.
The inventors have thus developed a method which enables visualizing increased expressions of this isoform of LO (LOXL), and then have sought active principles, notably amongst plant extracts or chemical molecules, which in particular stimulate the expression of the mRNAs encoding LOXL. The actives selected were then incorporated in cosmetic, dermo-pharmaceutical and pharmaceutical compositions in particular, for applications in the combat against loosening of the tissues during ageing, as well as in the improvement of the quality of scar tissue and the appearance of scars and stretch marks.
The inventors have developed specific antibodies of the mature LOXL forms (vide Examples 1 and 2), and have demonstrated in this way that the absence of this lysyl oxidase Isoform is correlated to the problems of the synthesis of functional elastic fibers, notably during ageing of the skin tissues, whether they be natural or induced by solar radiation.
The Isoforms LOXL2, LOXL3 and LOXL4 are not or are little expressed in the dermis and are not involved in elastogenesis (vide Example 4). The LOX isoform is present in the dermis and can be associated with the microfibrils, but LOX is involved in the formation of the functional collagen fibers and is not missing in adult skins. The absence of LOX is therefore not correlated with the loss of elasticity of the elastic fibers during ageing (vide Example 3).
This demonstration of the role of LOXL in elastogenesis was crucial for the implementation of the present invention (vide Example 5).
The association between LOXL and the elastic fibers or the microfibrils was clearly demonstrated by electron microscopy during the implementation of the present invention. LOXL associated with the microfibrils constitutes the framework on which the elastin is deposited.
LOXL is the enzyme which is responsible for the maturation of the elastin by cross-linking and thus enables the formation of functional elastic fibers.
Within the context of the present invention, the inventors have implemented a method of locating the expression of LOXL.
Notably, this method of location comprises the Immuno-detection of LOXL. It is also possible by this method to demonstrate the expression of the protein elastin. It has been demonstrated by the inventors' studies that LOXL is detected in association with the dense deposits or on the microfibrils, but not with the collagen fibers. Elastin was detected in the same dense deposits and In the microfibrils. This detection is made on reconstructed skin models, and notably on reconstructed skin models 30 days after the application of the keratinocytes (vide Example 5).
The association of LOXL with the microfibrils and with the elastic fibers was also confirmed on the skin of the foreskin, notably by transmission electron microscopy after Immuno-detection.
LOXL is expressed in the dermis of the skin of the foreskin taken from young patients (a few months), which have still a large synthesis of elastin. LOXL is not however expressed in the dermis of the skin, of the neck, of the breast, of the abdomen or of the face of adult persons. This absence of detection of LOXL in the dermis of the skin of the neck, of the breast, of the abdomen, or of the face, is confirmed in the adult whatever the age. A high expression was also observed of LOXL in the epidermis of human skin, with however a late extinction of the expression of this enzyme when the human skins originate from subjects aged about 80 years old and more (vide Example 6).
With regard to scars, LOXL was not observed in the dermis of these zones, neither three months after the scar, nor five years after the formation of the scar.
In this context, it is to be noted that the elastin which was present at three months is no longer present on this scar tissue zone five years after the formation of the scar.
The inventors have thus demonstrated the role of LOXL in the formation of elastic fibers, notably by using reconstructed skin models or dermis of the foreskin of young patients.
The inventors have also demonstrated the deficit of expression of LOXL in the scar tissue zones, as well as in the dermis of human skin of varying age, thus during ageing.
Amongst the Isoforms of the lysyl oxidases, LOXL is one of the isoforms which enables the cross-linking of the elastic fibers. However, only this isoform, LOXL, is missing in the adult for the cross-linking of the elastic fibers enabling functional fibers to be obtained.
The inventors have, from these unexpected discoveries, carried out a screening method of an active principle which stimulates the formation of functional elastic fibers with the view to identifying active principles for making cosmetic or pharmaceutical compositions.
The present invention further relates to the activation of the promoter of the human gene encoding LOXL (vide Example 7).
Various zones of activities of this promoter have been demonstrated. The sequence of this promoter is given in the annex and is designated in the following text by PrhLOXL.
On this promoter, the region corresponds to the nucleotides -712/-391 (according to the numbering defined from +1 of translation of the hLOXL gene) which possesses an up-regulating activity on the reporter gene luciferase, which is expressed for example after transitional transfection in fibroblasts of the skin of the human foreskin.
The Inventors have been able to define putative sites of regulation by nuclear factors. These factors have been correlated to cytokines or other factors known to act upon the transcription of certain genes.
Various regions of PrhLOXL have been able to be identified as being activating or inhibiting zones.
Notably, the regions -2172/-2002; -1438/-968; -712/-391; have been located as being activating regions; and the regions: -2002/-1438; -968/-712 have been identified as being inhibiting regions. The -80/-1 region is not active and is situated down from +1 of transcription. In this numbering, the putative transcription +1 is situated in position -342 with respect to the site of initiation of the translation. In this way, several sites of these regions have been shown which are susceptible to regulating the hLOXL gene. These sites are notably two putative sites of response to retinoic acid, two putative sites of response to TGF-β (Transforming Growth Factor 1), a putative site of response to EGF (Epidermal Growth Factor), three putative sites of response to oestrogens and two putative sites of response to glucocorticoids (GRE).
This Implies that the active principles which stimulate the neo-synthesis and/or the activity of hLOXL and thus which stimulate the formation of elastic fibers which act upon the promoter of the hLOXL gene, and notably in these zones, either directly or indirectly, in modulating the expression of a protein fixing onto these sites. It will therefore be possible for a substance to be considered as active when it will comprise a region which is capable either of associating with at least one part of the sequence of nucleotides of PrhLOXL, and in particular of associating with the putative sites defined above, or of modulating the expression of a protein which is capable of doing it.
The whole of the inventors' studies has enabled developing a method of Identifying an active principle which stimulates the formation of elastic fibers.
Within the context of the present invention, the Inventors have implemented an in situ hybridization which thus enables locating and verifying the presence of the expression of the messenger RNAs which encode LOXL in particular. This in situ hybridization is notably carried out by double strand DNA probes which are labeled with digoxigenin on sections of reconstructed skin models obtained 30 days after the addition of keratinocytes, and included in paraffin. This in situ hybridization was also carried out in order to verify the expression of the messenger RNAs of tropoelastin and of collagen α1(I) (vide Example 8).
The expression of the LOXL mRNAs is positive in the deep dermis and throughout the whole epidermis. The expression of the tropoelastin mRNA is located near to the dermal fibroblasts and in the epidermis. The expression of the collagen Iα1 mRNA is located in the dermis but not in the epidermis. This enables, within the context of the present invention, locating and verifying the presence of the expression of the LOXL mRNAs, notably in a reconstructed skin model, for example after application of an active principle which stimulates the formation of functional elastic fibers.
Within the context of the present invention, the hLOXL gene was activated by the addition of keratinocytes in a reconstructed skin model, and notably in the reconstructed skin model Mimeskine, (Coletica, Lyons, France). The induction of the synthesis of the LOXL mRNAs is concomitant notably with that of tropoelastin, and notably appears about 6 days after the addition of the keratinocytes on the dermis equivalent.
The present invention has also enabled demonstrating the decrease in the level of expression of notably the hLOXL gene, as well as of the human elastin gene, in fibroblasts originating from aged donors.
For this, the Inventors used 5 strains of fibroblasts from the foreskin (FF) (originating from young Infants) and 6 strains of adult fibroblasts (AF, of which 3 subjects of 20 years old on average, and 3 subjects of 60 years old on average) originating from plastic surgery on the abdomen. The expression of the gene encoding the protein LOX was also tested (vide Example 9).
The expression of these 3 genes of interest, as well as the one of actin, was analyzed by real time RT-PCR. The invention is not limited to this type of analysis. This technique enables precisely quantifying the expression of a gene in comparing it to that of actin which is considered to be constant. The regulation of the level of expression of this gene can therefore be quantified.
The results presented in FIG. 12 first of all show that the synthesis of LOXL mRNA drops spectacularly and statistically significantly in the fibroblasts of adults, with a drop of near to 70% with respect to the fibroblasts of the foreskin, whereas the elastin mRNA does not vary significantly with age. This piece of data is in agreement with the literature on elastin, since if the elastic tissue deteriorates and is not replaced, it does not seem to be due to an inhibition of the activity of the elastin gene.
The synthesis of LOX mRNA decreases on average by 40% in the AFs with respect to the FFs, but since this enzyme is not always expressed in normal human skin, this variation is not very indicative of the phenomena in vivo.
It is advantageous to also stimulate the expression of elastin so as to stimulate the formation of elastic fibers even more.
The present invention provides a method enabling identifying the expression of LOXL, notably in fibroblasts.
The present invention has aimed to implement these various techniques in a way as to identify active principles stimulating the formation of elastic fibers.
In general, the methods of the present invention Implement seeking the expression of the protein LOXL, and notably seeking the expression of the messenger RNAs encoding LOXL (vide Example 10).
The invention also relates to the active principles which stimulate the formation of elastic fibers (vide Examples 11 and 12).
The invention also relates to the use of the enzyme LOXL, or of a derivative form, or of the active principles as described above, for making cosmetic or pharmaceutical compositions (vide Examples 13 to 18). The stimulation of LOXL can be carried out at gene level, of the messenger RNA, or of the protein directly. This activation enables the formation of elastic fibers, notably by virtue of the cross-linking of the elastin by the enzyme LOXL.
Other aims, features and advantages of the invention will appear clearly to the person skilled in the art upon reading the explanatory description which makes reference to the following Examples.
The Examples make up an integral part of the present invention and any feature appearing novel with respect to any prior state of the art from the description taken in its entirety, including the Examples, makes up an integral part of the invention in its function and in its generality.
Thus, every Example is of general scope.
Furthermore, in the Examples, all the percentages are given by weight, unless indicated otherwise, the temperature is expressed in degrees Celsius unless indicated otherwise, and the pressure is atmospheric pressure, unless indicated otherwise.
The invention has first of all covered the development of novel specific antibodies of LOX and LOXL but which are capable of detecting their mature forms. The antibodies were developed against the mature regions of LOX and LOXL. The antigenic regions were selected in order to present the minimum of similarity with the corresponding regions on the other isoforms of the lysyl oxidases (LOs). The antibodies obtained against the regions of the peptides LOX.sup.V228-S280 (were called anti-LOXmat and similarly for the antibodies obtained against the region of the peptides LOXL.sup.R231-G368 called anti-LOXLpro.
In FIG. 1: description of the sequences of the LO defined for giving the specific antibodies: This figure represents the steps which have led to the selection of the antigenic regions in order to develop the anti-LOX and anti-LOXL antibodies.
FIG. 1(A): Schematic representation of hLOX (human LOX protein) and hLOXL (human LOXL protein).
The sequences of hLOX and hLOXL are indicated with open boxes, dotted in the C-terminal regions, in order to highlight the regions of high similarity. The position of the cleavage of the pre-region and of the site of cleavage by procollagen-C-protelnase (PCP), on the A22 and D169 residues of hLOX respectively, was indicated. The position of the cleavage of the pre-region of LOXL, before the Q26 residue, of the N-terminal maturation site of the 56 kDa precursor (before Q135), and the position of the cleavage sites by PCP of the LOXL precursor of 56 kDa (before the D 338 residues), are indicated. The corresponding LOXL proteins Q26-S574, D135-S574 and D338-S574 would display a deduced molecular mass of approximately 63 kDa, 54.6 kDa, and 26.7 kDa, respectively. The location of the recombinant peptides used for obtaining the anti-LOX antibodies were indicated: the G128-L212 peptide for the anti-LOXpro, the V228-S280 peptide for the anti-LOXmat, and the D305-N373 peptide for the anti-LOXcat. The location of the recombinant peptides used for developing the anti-LOXL antibodies were indicated: the R231-G368 peptide for the anti-LOXLpro and S355-D415 for the anti-LOXLmat.
FIG. 1(B): The percentage of similarity between the antigenic regions of LOX and LOXL with their equivalents on the LO isoforms was indicated in this Table.
In the Table of FIG. 1(B), hLOXL represents the human LOXL protein, bLOXL represents the bovine LOXL protein, mLOXL represents the mouse LOXL protein, hLOXL represent the human LOX protein, bLOX represents the bovine LOX protein, hLOXL2 represents the human LOXL2 protein, hLOXL3 represents the human LOXL3 protein, hLOXL4 represents the human LOXL4 protein.
The length column (aa) contains the value of the number of amino acids contained in the corresponding regions.
In order to obtain the antibodies, the chimeric genes were constructed by inserting the defined sequence of hLOXL or hLOX in phase with the gene of glutathion-5-transferase (GST), in the BamHI-XhoI sites of the expression plasmid pGEX-4T-3 (Amersham Biosciences).
The fusion gene GST-LOXL.sup.S355-D415 was constructed by introducing the cDNA sequence of HLOXL (cDNA hLOXL), produced by PCR with the sense primer 5'-TTGGATCCAGCGTAGGCAGCGTGTAC-3' (SEQ ID No17), and antisense primer 5'-AAACTCGAGCATCGTAGTCGGTGGC-3' (SEQ ID No18). The fusion gene GST-LOX.sup.G121-L212 was constructed by introducing hLOX cDNA amplified with sense primer 5'-TCGGATCCGGCTACTCGACATCTAGAGCC-3' (SEQ ID No18) and antisense primer 5'-GTCCTCGAGACCGTACTGGAAGTAGCC-3' (SEQ ID No19), respectively.
The fusion gene GST-LOX.sup.V228-S279 was constructed by introducing the hLOX sequence amplified with sense primer 5'-TTGGATCCGTGCAGAAGATGTCCATGTAC-3' (SEQ ID NO 20) and antisense primer 5'-TTTCGAGGCTGGGTAAGAAATCTGATG-3' (SEQ ID No21), respectively.
The fusion gene GST-LOX.sup.D306-N373 was constructed by introducing hLOX cDNA amplified with sense primer 5'-CACTATGGATCCCTTGATGCCAACACCC-3' (SEQ ID No22) and antisense primer 5'-CACGACCTTTAGGATATCGTITCCAGG-3' (SEQ ID No23), respectively.
For the whole of these amplifications by PCR, the Taq polymerase High Fidelity (Roche Diagnostic, Meyman, France) was used.
The fusion proteins GST-LOX and GST-LOXL, as well as the rabbit polyclonal antibodies were obtained and purified as described above for the fusion proteins originating from the expression of the fusion genes GST-LOXL.sup.S355-D415 and GST LOX.sup.G128-L212 (Decitre et al., Lab Invest, 78: 143-151, 1998; Borel et al., J. Biol. Chem., 276: 48944-49, 2001).
For the adsorption experiments, the antibodies were incubated for 3 hours at 20° C. with the fusion proteins, themselves adsorbed on a nitrocellulose membrane, Hybond-ECL membrane (Amersham Blosciences) before the immuno-detection.
These pieces of work have first of all enabled demonstrating the mature forms of LOX and LOXL, by virtue of the immunochemical and biochemical characterization of the mature proteins (vide Example 2, FIG. 2). The antibodies developed are distinguished from those used in the prior art for LOXL, which no not enable a recognition of the mature form of LOXL (Decitre et al., Lab Invest 78: 143-151, 1998; Borel et al, J. Biol. Chem., 276: 48944-49, 2001). The invention has been to use the anti-LOXLmat antibody and anti-LOXmat antibody, and this enabled demonstrating a protein of 31 kDa, which is recognized by the anti-LOXLmat but not by the anti-LOXmat, and which corresponds to the mature form of LOXL. This part of the invention demonstrates a real progress over the prior art, notably with reference to the patent of Csiszar et al., which describes all the proteins originating from the genes of the LO family without defining the features of them (WO 01/83702 A2 patent application: Novel members of the lysyl oxidase family of amine oxidases related applications).
Immuno-detection of LOX and LOXL of Muscle Cells by Virtue of the Novel Antibodies Anti LOX and Anti-LOXL
On FIG. 2:
FIG. 2 represents photographs of electrophoreses which were carried out as indicated below. These electrophoreses demonstrate the characterization of the mature proteins of LOX and LOXL, of smooth muscle cells (SMC) by virtue of the antibodies anti-LOX and anti-LOXL, Identified In Example 1.
The proteins of the cell strain (L) and of the cell culture medium (M) of a cell line of rat smooth muscle (developed by Jean-Marie Daniel Lamaziere, Bordeaux) were extracted and detected by western blotting by using the antibodies anti-LOXLmat, anti-LOXmat, anti-LOXLpro and anti-LOXpro. The cells were cultivated at 37° C. in an atmosphere of 5% CO2 in DMEM medium (Sigma) containing 10% foetal calf serum, 2 mM glutamine and 50 μg/ml gentamycin.
The cell strain proteins, which are washed twice with PBS buffer, were extracted for 2 hours at 4° C. with slow agitation in the lysis buffer (16 mM phosphate buffer pH 8, 0.5% NP40, protease Inhibitors (Complete Mini, Roche Diagnostics), and urea 6 M). The lysates were diluted with two volumes of 16 mM phosphate buffer pH 8, with protease inhibitors (Complete Mini, Roche Diagnostics, Meylan, France), and centrifuged for 5 minutes at 15,000 g. The soluble proteins were precipitated by adding 10% trichloroacetic acid (TCA) before the electrophoresis.
The proteins of the culture media of cells cultivated for 48 hours without serum, are recovered, precipitated by adding 10% TCA or 50% saturated ammonium sulphate.
For the immuno-detection, the proteins are separated by 10% SDS-polyacrylamide gel electrophoresis. The proteins were transferred onto a polyvinylidene fluoride (PVDF) membrane (Immobilon PSQ, Millipore) and were immuno-detected as described above (Borel et al, 2001).
The developed antibodies thus enable characterizing and locating the mature and immature forms of LOX and LOXL in the biological tissues.
Demonstration of the Role of LOX and LOXL in Elastocienesis
The inventors have demonstrated that the LOX and LOXL proteins can be associated with the formation of connective tissue in the dermis of reconstructed skin models by Immuno-histochemistry (FIG. 3). This demonstration was obtained without any ambiguity by virtue of the use of anti-LOX and anti-LOXL antibody couples, directed against the pro-enzymatic regions and mature regions of the two enzymes (LOX and LOXL).
On FIG. 3 representation is made of the immuno-histological detection of LOXL and LOX in the reconstructed skin (RS) and normal human skin.
The Immuno-detection of LOXL (A, C, E, G) on the reconstructed skin at days 16 (A), 35 (C), and 45 (E), by using anti-LOXL.sup.R231-G368 (A, C, E) or anti-LOXL.sup.R231-G368 adsorbed with the corresponding peptide GST-LOXL.sup.R21-G368 before the immuno-detection (G). The immuno-detection of LOX (B, D, F, H) at days 16 (B), 35 (D), and 45 (F), by using anti-LOX.sup.V228-S279 (B, D, F) or anti-LOX.sup.V228-S279 adsorbed with the corresponding peptide GST-LOX.sup.V228-S279 before the Immuno-detection (H). The Immuno-detection of LOXL (I) and of LOX (J) in the skin of human foreskin Is carried out by using anti-LOXL.sup.R231-G368 (I) and antl-LOX.sup.V228-S279 (J). The position of the dermal-epidermal junction is indicated with an open arrow, that of the dermal substrate with an arrow, and the location of the keratinocytes at day 16 is indicated with an arrow head.
The reconstructed skin (Mimeskin®, Coletica, Lyons, France) was prepared in Bouin's fixative (LOX, LOXL, elastin) or In a 10% formol solution (for the elastin), and then included in paraffin. 6 μm thick sections were ridded of paraffin and were whitened in glycine-HCl (100 mmol/l). The anti-LOX and anti-LOXL antibodies are described above.
The antibodies were used at the following dilution: 1:500 (anti-LOXL.sup.R231-G368), 1:100 (anti-LOX.sup.V228-S279, anti-LOXL.sup.S355-D416). The immune complexes were detected with a rabbit (goat) anti IgG conjugated with peroxidase (DAKO, Trappes, France), by using diaminobenzidine as substrate (DAKO).
LOXL is thus an excellent candidate for participating in elastogenesis in a reconstructed skin model notably such as Mimeskin®.
Demonstration of the Role of LOXL2, LOXL3, and LOXL4 in Elastogenesis
The invention also covers the development of two novel anti-LOXL2 antibodies, one of these antibodies theoretically also recognizing LOXL3 and LOXL4. This has enabled defining whether these enzymes are expressed with elastin in the dermis of a reconstructed skin model. The analysis by immunohistochemistry by using the two anti-LOXL2 antibodies does in fact show that this antigen, as well as the two antigenically linked proteins LOXL3 and LOXL4, are not or are little expressed in the dermis, and that therefore, they do not participate in the elastogenesis.
On FIG. 4: the immuno-detection is represented on the sections of reconstructed skin (16, 35 and 45 days) and of human foreskin skin with the antibody anti LOXL2517-581 (left column) and the antibody anti-LOXL2664-720 theoretically recognizing LOXL2, LOXL3, and LOXL4 (right column).
The anti-LOXL2 antibodies were obtained against fusion peptides GST-LOXL2, as described above (Decitre et al, Lab. Invest., 78, 143-151, 1998). The fusion gene GST-LOXL2517-581 was constructed by introducing the sequence 1543 to 1747 of the human LOXL2 gene (hLOXL2) in the plasmid, as described above.
This segment was generated by PCR with the sense primer 5-GAGCTGGGATCCGCGCACTGCC-3' and antisense primer 5'-GGCTGAGTCGACGAGGCAGTTCTCC-3'.
The fusion gene GST-LOXL2664-720 was constructed by introducing the corresponding hLOXL2 sequence, by virtue of the sense primer 5'-CACAGGATCCGAAGGAGACATCCAGAAG-3' and antisense primer 5'-CAGAGCTCCTGCATTTCATGATG-3'.
The fusion proteins and the anti-rabbit antibodies generated against these proteins were prepared as described above. The antibody against the 517-580 peptide was called anti-LOXL2, since this region is specific of LOXL2.
The antibody against the 664-734 peptide was called anti-LOXL-R (for <<relative to>>), since this region of LOXL2 possesses a high similarity with LOXL3 and LOXL4 (about 74.6% and 60.5%, respectively).
The reconstructed skins (Mimeskin®, Coletica, Lyons, France) at 16 days (RS-D16), 35 days (RS-D35) and 45 days (RS-D45), and the skin of the human foreskin are analyzed as above by immuno-histochemistry with the anti-LOXL2-R and anti-LOXL2 antibodies. Anti-LOXL2 shows an expression of LOXL2 in the epidermis and not in the dermis, while the antibody directed against the common C-terminal region of LOXL2, LOXL3, and LOXL4 confirms the expression of these enzymes in the epidermis and shows a low expression in the dermis but in an zone which does not correspond to the sites of elastogenesis.
LOXL2, LOXL3 and LOXL4 are therefore not involved in elastogenesis.
Demonstration of the Role of LOXL in Elastogenesis
The association between LOXL and LOX on the one hand, and the elastic fibers or the microfibrils on the other, was clearly demonstrated in transmission electron microscopy by the present invention.
LOX and LOXL associated with the microfibrils constitute the framework on which the elastin is deposited, whereas only LOX is associated with the formation of the collagen fibers (vide FIG. 5).
On FIG. 5: The immuno-detection of LOXL, of LOX and of elastin is represented by transmission electron microscopy in the dermal part of the reconstructed skin 30 days after the application of the keratinocytes, and of the normal human skin.
The tissues were fixed for 3 hours at 4° C. with 4% paraformaldehyde in PBS buffer containing 0.1% glutaraldehyde, and were then washed in phosphate buffer containing 0.4M of sucrose cacodylate and 0.2M lysine, dehydrated in solutions of ethanol, and included in LR White (Euromedex, France). The detection was carried out with primary antibodies diluted to 1:50 in Tris-HCl buffer at pH 8.2, to which 1% bovine serum albumin (BSA) is added. The immune complexes are detected with an rabbit anti-IgG antibody conjugated with colloidal gold particles of 10 and 20 nm (Biocell, Tebu, France) diluted to 1:40. The samples were contrasted with 3% aqueous uranyl acetate and lead citrate, and were then examined under a JEOL 1200 EX transmission electron microscope. The immuno-detection was carried out on the reconstructed skin (A-D) and on the skin of human foreskin (F-I).
On the reconstructed skin, it was carried out with the antibodies: anti-LOXL (A, B), anti-LOX (C), anti-elastin (Elm) (D), human anti-elastin antibodies being commercially available (Slgma, USA) and diluted to 1:50, and a negative control without primary antibody in the dermis (control) (E). A double labelling (F-I) was made on the skin of the human foreskin.
References A-D: Immuno-detection of LOXL, LOX and elastin by electron microscope in the dermal part of the reconstructed skins at 45 days.Reference E: Positive control with anti-elastin and anti-collagen I antibodies in the dermis of reconstructed skins at 45 days, i.e. 30 days after the addition of keratinocytes.References F and I: Double immuno-detection of LOXL, LOX, elastin and collagen by electron microscopy in in the dermal part of human foreskin.References G-H: Double-labelling in the dermal part of the human foreskin with the rabbit anti-LOXL antibody (the rabbit anti-IgG is conjugated with 10 nm gold particles) and the murine anti-elastin antibody (the mouse anti-IgG is conjugated with 20 nm gold particles).Keys in the Figure: m: microfibrils, c: collagen fibers, e: amorphous elastin. Bar of the scale: 500 nm.
LOXL (A-B) is detected in association with the dense deposits or on the microfibrils, but not with the collagen fibers which appear in white on these sections. The labelling of LOX (C) is low, although a few gold particles could be found with the dense deposits, the microfibrils and the collagen. The anti-elastin antibodies detected the same dense deposits and the microfibrils (D). The association of LOXL and LOX to the microfibrils and to the elastic fibers was confirmed in the skin of the human foreskin, by electron microscopy after immuno-detection (G-H). As In the observations on the reconstructed skin models, LOXL is not associated with the collagen fibers, opposite to the LOX which is very present with the collagen fibers and little present on the microfibrils. The LOXL antigens were detected in association with the microfibrils and around the elastic fibers of the skin of human foreskin. LOXL is not associated with the amorphous elastin which extends around the microfibrils, but is mainly observed on their periphery, and not with the collagen fibers.
LOXL is associated with the elastic fibers in the reconstructed skin models and in the skin of the human foreskin.
Demonstration of the Relationship between the Expression of LOXL and Elastogenesis
LOXL and LOX are expressed in the dermis of the skin of the foreskin taken from young patients (a few months) still having a high elastin synthesis. LOXL is not however expressed In the dermis of adult skin, of the neck, the breast, the abdomen or the face, whereas LOX is always expressed in the dermis (FIG. 6).
On FIG. 6: the immuno-detection of LOX and LOXL in the human skin is represented.
The antibodies anti-LOX (A, C, E, G) and anti-LOXL (B, D, F, H) were used for detecting the expression of LOX and LOXL in samples of skin of the foreskin (A, B), of the neck (C, D), of the breast (E, F) and of the abdomen (G, H) originating from the tissue bank of the Edouard Herriot Hospital, Lyons, France. The tissues were fixed with Bouin's reagent, included in paraffin, and treated for the immuno-detection as was carried out for the immuno-detections which are described above.
The absence of detection of LOXL in the dermis of the skin of the neck, of the breast, of the abdomen or of the face, is confirmed in the infant and the adult (FIG. 7: abdomen).
On FIG. 7: The immuno-detection of LOX and LOXL in skins of the human abdomen taken at various ages is represented.
The antibodies anti-LOX (A, C, E, G) and anti-LOXL (B, D, F, H) were used for detecting the expression of LOX and LOXL in samples of skin of the abdomen taken at 1.5 years old (A, B), 35 years old (C, D), 60 years old (E, F) and 91 years old (G, H) originating from the tissue bank of the Edouard Herriot Hospital. The tissues were fixed with Bouin's reagent, included in paraffin, and treated for the immuno-detection such as described for the preceding immuno-detections.
During the same pieces of work, a high expression was observed of LOX and LOXL in the epidermis of human skin, with a very late extinction of the expression of these 2 enzymes (91 years old) (FIG. 7).
In the scars, neither LOXL nor LOX were able to be observed in the scar tissue zones 3 months after the scar and 5 years after the scar. It is to be noted that the elastin was Immuno-detected at 3 months and disappeared at 5 years in this scar (FIG. 8).
On FIG. 8: The immuno-detection of LOX and LOXL in scar tissue skins at varying periods after the healing is represented.
The antibodies anti-LOX (A, D, G), anti-elastin (B, E, H) and anti-LOXL (C, F, I) were used for detecting the expression of LOX, of elastin and of LOXL in samples of skin of the neck of a patient of 17 years old, around the scar (<<normal>> zone, A-C), 3 months (D-F) or 5 years old (G-H) after a healing. The tissues were fixed with Bouin's reagent, included in paraffin, and treated for the immuno-detection such as was described for the preceding immuno-detections, The labeling of elastin requires a demasking with the aid of 0.2% hyaluronidase (Sigma).
By virtue of these Examples, the Invention demonstrates that there exists: (i) an undeniable implication of LOXL in the formation of elastic fibers in reconstructed skin models and in the dermis of the foreskins of young patients, and (ii) a veritable deficit of expression of LOXL in the dermis of the human skin at varying ages and in the scars. LOXL is therefore indeed the only lysyl oxidase isoform which is capable on the one hand of enabling the cross-linking of the functional elastic fibers and, on the other, of being missing in the situation wherein a cross-linking of the elastic fibers is necessary in order to produce functional fibers. LOX, which could also be associated with the formation of elastic fibers, is not missing in the skins of the adult.
Study of the Pre-transcriptional Regulation of the LOXL Gene
The invention has enabled demonstrating further that the human LOXL gene (hLOXL) can be activated at its promoter level (FIG. 9). The Sequence ID No. 3 describes the nucleotides going from -2730 to -1 of the sequence of this promoter. Several zones of activity of the hLOXL promoter were demonstrated. Notably, the region corresponding to the nucleotides -712/-391 (according to the numbering defined from +1 of translation of the LOXL gene) possesses an up-regulating activity on the reporter gene luciferase, expressed after transitional transfection in fibroblasts of skin of the human foreskin (FIG. 9).
It is therefore a pre-transcriptional up-regulation of the hLOXL gene, which indicates that it is possible to activate the synthesis of this gene, and therefore of the corresponding hLOXL protein, since the inventors had demonstrated beforehand that the variations of the expression of hLOXL can be traced concomitantly at the level of the genes and/or the proteins. This had also been demonstrated for LOX (Decitre et al., Lab. Invest., 78, 143-151, 1998).
A study of the nuclear sequence PrLOXL with the aid of the software Transfac® on the Internet has enabled us to define putative sites of regulation by nuclear factors. These factors were correlated with cytokines or other effectors known for acting on the transcription of certain genes via these transcription factors. The most interesting sites are presented in FIG. 10. This scheme recapitulates this analysis and indicates 2 putative sites of response to retinoic acid, 2 to TGF-β, 1 to EGF, 3 to cestrogens and 2 to glucocorticoids. We have thus been able to define several sites which could regulate the transcription of the LOXL gene, since they were studied in the regulating zones. These are the putative elements of response to retinoic acid, to TGF-β, to EGF and to glucocorticoids. The zones which correspond to these putative sites of regulation by retinoic acid and oestrogens seem to have a real activating effect on the transcription, since the activity of the promoter drops by about 50% and 60% respectively without these elements, The site of regulation by the TGF-β seems to lower the promoting activity.
The tools thus described can be used for the screening of active principles having an agonist or antagonist action upon the promoter of hLOXL, and more particularly upon the putative sites of recognition.
It is so that is advantageous to seek active principles comprising a region which hybridizes with at least one part of the sequence of nucleotides of the promoter of hLOXL, or which induces effector proteins having this property.
Functional Analysis of the Promotor of the hLOXL Gene
The promoter of the human LOXL gene (PrhLOXL) was defined by virtue of the sequences from the data bases. Since the site of initiation of the transcription was unknown, the inventors numbered it with respect to the +1 of translation. However, the EST (Expressed Sequence Tags) cDNA search, which corresponds to this region, did not give any sequence further up from position -342, which enables supposing that the initiation of the transcription of the hLOXL gene is done in this region (without TATA box). Specific primers were shown on this sequence In position -2172 and +189 (exon 1). They enabled the amplification and the isolation of the PrhLOXL from human genomic DNA originating from skin fibroblasts, it was cloned and sequenced, its sequence proving to be conform to that predicted. Then, the promoter, known as <<entire>>, going from -2172 to -1, was able to be sub-cloned in the pGL3-basic vector (Promega, Charbonnieres, France) for the study of it in eukaryotic cells. It was placed up from the reporter gene, the luciferase gene. Thus, the production of luciferase by the transfected cells is under the control of the PrhLOXL and therefore proportional to its activity. The cells are transfected at the same time with the promoter, intensely and reproducibly expressing β-galactosidase (β-Gal), enabling the results to be normalized. For a same condition, the luciferase and β-Gal enzyme activities are measured.
The PrhLOXL was progressively reduced (deletion 5') so as to determine the role of the regions taken out. The aim was to study the regulation of the PrhLOXL in the fibroblasts of human skin, the transfection of these cells was developed. In contrast to the cell lines which transfect easily, normal fibroblasts transfect very poorly. Superfect® (Qiagen, Courtaboeuf, France) was selected, since it enables the transfection of about 40% of fibroblasts in culture.
The constructions made, as well as their activity in the foreskin fibroblasts, are presented in FIG. 9. The inventors located three large activating regions of the transcription and two inhibiting regions. For example, the region -712→-391 is very activating since the activity of the promoter drops considerably when it is taken out (construction -391→-1). This study has enabled specifying the zones to be studied for stimulating the transcription of the PrhLOXL.
Notably, FIG. 9 represents the luciferase/β-galactosidase activity as a function of the sequence of the promoter PrhLOXL. This Figure enables facilitating the understanding of the definition of the activating and inhibiting regions of the promoter within a human fibroblast cell of the skin of the foreskin.
The successive reductions of the PrhLOXL in 5' have enabled generating 7 constructions, bearing shorter and shorter sequences of the promoter up from the reporter gene luciferase pLL-2172, pLL-2002, pLL-1438, pLL-712, -pLL-391, -pLL-81. The constructions were transfected in fibroblasts of the foreskin of human skin, and the luciferase activity was measured. In parallel, the cells are also transfected with a plasmid bearing the β-galactosldase gene, under the control of the promoter SV40, so as to serve as a transfection effectiveness control. The final values correspond to the luciferase activity (indicating the activity of the sequences of the hLOXL promoter) compared to the β-galactosidase activity (showing the effectiveness of transfection). The evolution of the activities enable defining several regulation zones on the promoter, including 3 activating zones, shown by the signs +(-2172→-2002-1438→-968; -712→-391) and 2 inhibiting zones, shown by the signs -(-2002→-1438; -9684→-712). The -81→-1 promoter is not active and is situated down from the +1 of transcription. The putative +1 of transcription is situated in position -342 with respect to the site of Initiation of the translation.
FIG. 10 represents a schematic view of the promoter of the hLOXL gene, and notably identifies the putative sites of regulation of the transcription of the hLOXL gene. The zones Indicated by a "+" sign correspond to a zone of activation of the expression of the gene, those represented by a "-" correspond to a zone of inhibition of this expression.
Demonstration of the Activation of LOXL by the Introduction of Keratinocytes in a Reconstructed Skin Model
The detection of the expression of the gene encoding LOXL is demonstrated by the in situ hybridization of the messenger RNA of LOXL with double strand DNA probes which are labeled with digoxigenin, on sections included in paraffin.
In FIG. 11, which represents skin model sections (Mimeskin®) at day 35:
(A) the expression of LOXL is positive in the deep dermis and throughout all the epidermis.(B) The expression of LOX is positive in all the dermis and in the suprabasal layers of the epidermis.(C) The expression of tropoelastin (TE) is found in association with the dermal flbroblasts and in the epidermis.(D) The expression of the gene COL1A1 (collagen α1(I)) is detected in the dermis but not in the epidermis.(E) Control without probe.
The position of the DEJ is indicated by an open arrow, the position of the porous dermal substrate is indicated with arrows, and the positive cells are indicated with arrow heads.
The double strand DNA probes are produced by PCR. The following primers were used, respectively:
For the gene of the Ialpha1 collagen, sense 5'-GTGGAGAGTACTGGATTG-3' (SEQ ID No14) and antisense 5'-TCGTGCAGCCATCGACAG-3' (SEQ ID No15), for tropoelastin, sense 5'-GTATATACCCAGGTGGCGTG-3' (SEQ ID No10) and antisense 5'-CGAACTTTGCTGCTGCTTTAG-3' (SEQ ID No11); for hLOX, sense 5'-GGTGGCCGACCCCTACTACATCC-3' (SEQ ID No12) and antisense 5'-GCAAATCGCCTCTGGTAGCCATAGTC-3' (SEQ ID No13); for hLOXL, sense 5'-GACATAACCGACGTGCAGCC-3' (SEQ ID N08) and antisense 5'-ATCCACGTTCGCTCCCTGAG-3' (SEQ ID No9).
The DNAs are amplified with Taq Polymerase (Promega, Charbonnieres, France) and Dig-11-dUTP (Roche Diagnostic, Meylan, France) as labeller nucleotide, and they are then purified after electrophoresis on agarose gel by using the QIAquick extraction kit (Qiagen, Courtaboeuf, France). The In situ hybridization was carried out on sections included in paraffin. The samples are ridded of paraffin and treated with proteinase K (Roche) at 2 μg/ml for 15 minutes at 20° C. The endogenous peroxidases are inhibited as indicated in the TSA.sup.+ amplification kit (NEN, Boston, USA). A pre-hybridization is carried out for 2 hours at 37° C. In 20 mM phosphate buffer at pH 7.4, with 50% delonised formamide, 2×SSC (sodium salt citrate), 5 mM EDTA, 2.5×Denhardt's solution, 200 μg/ml denatured herring DNA, 1 mg/ml salmon sperm DNA; and 10 mg/ml tRNA. The hybridization is carried out for 16 hours at 37° C. In 20 mM phosphate buffer, with 50% delonised formamide, 2×SSC, 5 mM EDTA, 2.5×Denhardt's solution, 200 μg/ml denatured herring sperm DNA, and 10% dextran sulphate, with or without the prior-denatured probe for 5 minutes in a boiling water bath. After the hybridization, the sections are washed at 20° C. (or 37° C. for the collagen) in 2×SSC/50% formamide, 1×SSC/50% formamide, 1×SSC, and 0.5×SSC. After dehydration, the hybrids labelled with the digoxigenin are detected with an anti-DIG antibody conjugated with horseradish peroxidase (Roche). The final detection of the complexes is carried out by using the TSA.sup.+ amplification kit (NEN). The positive signals correspond to the activity of the alkaline phosphatase linked to the amplification procedure of the TSA kit, after 2 hours of activity at ambient temperature, and defined by the precipitation of the tetrazolium salts formed (by using the Nitro Blue Tetrazolium/bromochlorylindolophosphate (NBT/BCIP) substrates).
The invention demonstrates that the LOXL and LOX genes can be activated by the addition of keratinocytes in a reconstructed skin model (Mimeskin®, Coletica, Lyons, France), as the tracking of the expression of the mRNAs by In situ hybridization demonstrates (FIG. 11).
The induction of the synthesis of LOXL is concomitant with that of tropoelastin (6 days after the addition of keratinocytes on the equivalent dermis).
The LOX gene is also activated after the addition of the keratinocytes, at the same time as the collagen Iα1 gene (Col1A1).
Demonstration of a Drop in the Level of the Expression of the LOX Gene in Adult Fibroblasts
The Inventors used five strains of fibroblasts from the foreskin (FF) (originating from young infants) and 6 strains of adult fibroblasts (AF, 3 of 20 years old on average, and 3 of 60 years old on average) originating from plastic surgery on the abdomen. The expression of the three genes of interest, as well as of actin, was analyzed by real time RT-PCR (quantitative reverse transcriptase polymerase chain reaction, FIG. 12). This technique enables precisely quantifying the expression of a gene in comparing it to that of the actin (considered as constant). The regulation of the level of expression of this gene can therefore be quantified.
The results presented in FIG. 12 first of all show that the synthesis of LOXL mRNA drops spectacularly and statistically significantly in the fibroblasts of adults, and this as from the age of 20 years, with a drop of near to 70% with respect to the fibroblasts of the foreskin, whereas the elastin mRNA does not vary significantly with age. This piece of data is in accordance with the literature on elastin: if the elastic tissue deteriorates and is not replaced, it does not seem to be due to an inhibition of the activity of the elastin gene.
The synthesis of LOX mRNA decreases on average by 40% in the AFs with respect to the FFs, but with the individual variability, this deviation is not very significant.
The total RNAs are purified with the <<SV 96Total RNA Isolation System>> kit (Promega, Charbonnieres, France). The purified RNAs are eluted In 100 μl of RNase-free water (Promega, Charbonnieres, France), determined and distributed into plates (96-well, 10 μl total RNA at 5 ng/μl by PCR). The primers selected for the implementation of this work are the following and are the subject of Table I:
TABLE-US-00001 TABLE I Position Melting Size on the tempera- (nucleo- Human human ture Gene Name tides) sequence gene (MT) ELN 1 Ela 20 GTA TAT ACC Sense: 62° C. CAG GTG GCG +443 TG 2 Ela 21 CGA ACT TTG Anti- 62° C. CTG CTG CTT sense: TAG +799 LOX Ox 64 21 ACG TAC GTG Sense: 60° C. CAG AAG ATG +676 TCC Ox 65 21 GGC TGG GTA Anti- 59° C. AGA AAT CTG sense: ATG +841 LOXL 30 L1 19 GAC TTC GGC Sense: 60° C. AAC CTC AAC +1480 C 30 L2 20 TGT TGC AGA Anti- 60° C. AAC GTA GCG sense: AC +1701 ACTIN Actin 20 GTG GGG CGC U sense 72° C. CCC AGG CAC CA Actin 24 CTC CTT AAT D 57° C. GTC ACG CAC anti- GAT TTC sense
The technique of real time RT-PCR is carried out with the <<Quanti Tect SYBR Green RT-PCR>> kit (Qlagen, France) on wells containing mRNA, in an OPTICON thermocycler, which carries out amplification cycles. The retrotranscription (RT) is performed for 30 minutes at 50° C., followed by 15 minutes at 95° C. in order to inhibit the reverse transcriptase, to activate the polymerase and to denature the complementary DNA (cDNA) obtained. 50-chain polymerization cycles (PCR) are carried out (15 seconds at 94° C., 30 seconds at 60° C., 30 seconds at 72° C.). At every cycle end, the fluorescence, which is proportional to the number of fragments amplified, is read. The level of expression is defined by the ratio of expression of each gene with respect to actin.
As the preceding pieces of work demonstrate the implication of LOXL in elastogenesis and Its disappearance in adults' skins, the following point of the invention has borne upon the levels of expression of the genes LOXL, of LOX and of elastin in fibroblasts of varying ages.
The Examples above demonstrate that the synthesis of the products of the genes LOXL and LOX can be activated at gene level. The activation of the synthesis of the mRNAs of LOXL and of tropoelastin is concomitant in reconstructed skin. The activation of the genes of LOXL and of tropoelastin enables the formation of the elastic fibers. A screening of active principles enables leading to the identification of molecules which can simultaneously re-induce the expression of the genes of elastin and of LOXL, so as to stimulate the elastogenesis.
In conclusion, the invention enables demonstrating the direct relationship of LOXL with the elastic fibers, the importance of LOXL for forming elastic fibers In reconstructed skin, and the absence of LOXL from tissues wherein the synthesis of functional elastic fibers does not take place (adult tissues, scars). The invention relates notably to a method of screening for detecting novel molecules which are capable of concomitantly inducing the synthesis of LOXL and of elastin with the view to re-inducing the expression of functional elastic fibers in reconstructed skin, skin biopsies, and human skin.
Analysis of the Expression of the Messenger RNAs of LOXL and/or of elastin, e.g. by Qualitative RT-PCR with or without the Placing in Contact of Active Principles the Activity of which is to be Tested
The active principles were tested at 1% (v/v) on fibroblasts of normal human skin (originating from the foreskin of the infant or originating from the adult). The culture was carried out, e.g. in a monolayer on 24-well culture plates, in a defined medium without serum (Fibroblast Basal Medium). The cells were sown, e.g. at 40,000 per cm2. At the confluence, the cells are placed in contact with the actives advantageously for 24 hours. In parallel, a non-treated control (medium alone) and three positive controls (TGF-β at 1 ng/ml, IL-1α at 50 μg/ml and Phytokine® (Coletica, Lyons, France) at 2% (v/v)) are advantageously carried out, e.g. on the same culture plate.
The TGF-β at 1 ng/ml and the IL-1α at 50 pg/ml were tested beforehand and the stimulation of the synthesis of elastin mRNA induced by these two cytokines at these concentrations was verified by an analysis of the mRNAs, e.g. by quantitative RT-PCR (×10 for TGF-β and x6 for IL-1 alpha). After the time of placing the actives in contact with the cells, e.g. 24 hours, the media are removed and the cells are preserved e.g. by dry freezing at -80° C. after a rinsing in phosphate buffer pH 7.4. The total RNAs are extracted e.g. with the aid of an extraction kit of 96 wells on silica columns and were determined on a 96-well spectrophotometer at 260 nm (purity indicator: protein determination at 280 nm). The RNAs are diluted e.g. to 5 ng/μl. The qualitative RT-PCR in 1 step is carried out e.g. on 50 ng of initial RNA on a 96-well plate, on the genes of actin, elastin, of LOX and of LOXL. The specific primers of each gene are used e.g. at 0.5 μM:
TABLE-US-00002 sense elastin gene: (SEQ ID No 24) 1Ela 5'-GTA TAT ACC CAG GTG GCG TG-3'; antisense elastin gene: (SEQ ID No 25) 2Ela 5'-CGA ACT TTG CTG CTG CTT TAG-3'; Sense LOXL gene: (SEQ ID No 26) 30L1 5'-GAC TTC GGC AAC CTC AAG C-3'; Antisense LOXL gene: (SEQ ID No 27) 31L1 5'-TGT TGC AGA AAC GTA GCG AC-3'; sense LOX gene: (SEQ ID No 28) Ox64 5'-ACG TAC GTG CAG AAG ATG TCC-3'; antisense LOX gene: (SEQ ID No 29) Ox65 5'-GGC TGG GTA AGA AAT CTG ATG-3'; sense Actin gene: (SEQ ID No 30) Actin U 5'-GTGGGGCGCCCCAGGCACCA-3'; antisense Actine gene: (SEQ ID No 31) Actin D 5'-CTCCTTAATGTCACGCACGATTTTC-3'.
The amplification parameters were advantageously the following 48° C., 30 min; 94° C., 2 min; (94° C., 30 seconds; 60° C., 30 seconds; 68° C., 30 seconds) 28 cycles for actin, 30 cycles for LOXL, 32 cycles for LOX, or 34 cycles for elastin; 68° C., 10 min; 14° C., infinity. After amplification, the products are for example mixed at the rate of 3 μl of actin amplification products +5 μl of elastin gene amplification products +5 μl of LOX gene amplification products +5 μl of LOXL gene amplification products. A loading buffer is added (2 μl) and the total volume (20 μl) is deposited on a pre-poured agarose gel (Invitrogen, France) e.g. at 2%. The inventors visualized the levels of expression by means known to the person skilled in the art and e.g.: the bands of the samples were visualized under UV in a black chamber after migration (15 minutes) and were photographed digitally. The photographs of the gels were analyzed by image analysis and quantification of the intensity of the bands (Phoretix1D, France). The level of expression of the genes of elastin, of LOX and of LOXL were expressed in percentage variation with respect to those obtained for the negative control (without treatment).
Interpretations of The results:
<<Young>> Cells and <<Mature>> Cells
It is noted that the cells of the foreskin express quantities of mRNA encoding elastin at a level which is Identical to that observed on average in the adult, while they are very much greater than the case of the mRNA encoding LOXL, as well as in the case of the mRNA encoding LOX. It is therefore possible to reverse this decrease of the expression of LOXL and eventually of LOX in the aged cells, and a screening of active principles in this sense was performed.
Screening of Active Principles:
The amounts of cDNA of each test are compared to the amount of actin cDNA and then to the negative controls (without actives). A preliminary analysis enabled considering the tests presenting an increase of elastin (Eln) mRNA of about 1.3 times, of about twice LOXL, to be significant. Of more than 900 molecules or active extracts tested, 13 actives meet these criteria at the concentrations tested and under the defined conditions. These actives are the following and are the subject of Table II
TABLE-US-00003 TABLE II Eln LOXL LOX Control Control Control multiplied multiplied multiplied Name by: by: by: Dill (fruit) 2.28 2.03 3.08 currant 4.11 2 4.55 Cardamon 2.08 2 5.57 Black radish 2.88 2.13 2.92 Box holly 1.58 2.4 2.53 Cinnamon 1.56 2.09 5.08 Lactic ferments 2.37 2.04 9.69 Potato 2.4 1.88 3.55 Silk protein 2 3.05 3.25 Oats 2.37 2.04 9.69 Asa foetida gum 1.35 2 3.19 ethyl hexenoate 1.5 2.33 3.09 methyl butyrate 1.43 3.24 5.08 ethyl decadienoate 2.04 2.32 3.64
The plant extracts were obtained in allowing the plants to soak at 2-5% (w/w) in a water/(alcohol, glycol or polyol) mixture (such as ethanol, glycerol, butylene glycol and other glycols, xylitol etc. . . . ) 100/0 to 0/100. The extracts obtained were then filtered or distilled so as to recover the soluble fraction which is then filtered in sterile manner. The chemical molecules originate from Sigma (Saint-Louis, USA) and are used diluted or dispersed at 1% in an alcohol or a glycol.
Conclusion: 13 actives from the bank of 960 actives are capable, under the conditions considered, of significantly activating the level of synthesis of mRNA of the genes encoding LOXL, LOX and elastin, in the fibroblasts of the abdomen of the mature age adult (donor of 63 years old in this case).
Study of Effectiveness of a Cosmetic or Dermopharmaceutical Active by e.g. Real Time RT-PCR
The actives selected after the first step of screening were tested at various concentrations of between 0.1% and 5%, (v/v) on fibroblasts of normal (adult) human skin. The culture was carried out e.g. in monolayer in 24-well plates, in a defined medium without serum (Fibroblast Basal Medium). The cells are sown e.g. at 40,000 per cm2. After the time of placing the actives in contact with the cells (24 hours), the media were removed and the cells were preserved e.g. by dry freezing at -80° C. after a rinsing with phosphate buffer at pH 7.4. At the end of experimentation, the content of mRNA of elastin, of LOXL and of actin is evaluated by an mRNA analysis technique, e.g. by real time RT-PCR. For this, the couples of primers enabling the amplification of specific fragments of these genes are those described above (Example 10).
After extraction e.g. with the aid of an extraction kit in 96-wells on silica columns and determination on a 96-well spectrophotometer at 260 nm, the RNAs are diluted e.g. to 5 ng/μl. The RT-PCR reactions (Reverse Polymerase Transcription Chain Reactions) were carried out by quantitative real time RT-PCR with the aid of the "Opticon" system (MJ Research). Advantageously, the reaction mixture (50 μl) introduced into the wells was the following, for each sample: 10 μl of RNA at a concentration of 5 ng/μl, The specific primers of the various labels sought after, Reaction mixture (Qiagen-25 μl 2×QuantiTect SYBR Green RT-PCR master mix containing 5 mM MgCl2+0.5 μl QuantITect RT mix), the label SYBR Green I inserting in the DNA double strands during the elongation step.The RT-PCR Conditions were Advantageously the Following:Reverse Transcription: 30 minutes at 50° C., then 15 minutes at 95° C.,PCR reactions: [15 seconds at 94° C., 30 seconds at 60° C. and 30 seconds at 72° C.], 50 cycles.
The absence of contamination and the purity of the amplified products were verified e.g. via the fusion curves of the amplified PCR products. The products presenting a double peak or an abnormal fusion temperature were eliminated.
Analysis and Method of Calculation:
The incorporation of fluorescence in the amplified DNA was evaluated continuously during the PCR cycles. This system enabled obtaining curves of fluorescence measurement as a function of the number of PCR cycles and thus enabled evaluating a relative amount of amplified DNA.
In order to take account of the cell population present, all the results were compared to the <<actin>> signal, which was used as housekeeping gene. According to the experimentation, the threshold of measurement of the C(T) (=Cycle Threshold) was fixed for T between 0.05 and 0.01, and then an arbitrary measurement unit is calculated for each gene according to the formula:
C (T)gene <<x>> signifying the number of cycles necessary to attain the cycle threshold of 0.01-0.05 of the gene <<x>>.
The values of the genes of interest were compared to the <<actin>> signal by calculation of the ratio:
These ratios were compared between the treated and non-treated samples, "x" being the LOXL gene, or the elastin gene.
Results: amongst the actives selected, the results obtained, as an example for two of them, are presented in Table III
TABLE-US-00004 TABLE III LOXL LOX El Control Control Control multiplied multiplied multiplied Name by: by: by: methyl butyrate at 0.01% 0.89 1.04 0.92 methyl butyrate at 0.1% 1.56 1.76* 0.96 methyl butyrate at 1% 2.10* 2.17* 1.25 methyl butyrate at 5% 0.91 0.91 1.70* Silk protein at 0.01% 1.18 0.96 1.00 Silk protein at 0.1% 1.46* 0.96 1.12 Silk protein at 1% 2.39* 1.16 1.24 Silk protein at 5% 2.05 1.37 1.37 *statistically significant results p < 0.05 (One Way Anova test)
Conclusion: the actives selected enable activating the level of synthesis of mRNA of the genes encoding LOXL, LOX and elastin, in the fibroblasts of the abdomen of mature age adult (donor of 63 years old in this case). The study made enables determining the optimal concentrations of use for each active selected.
For Examples 1 to 11: the person skilled in the art will know how to draw the adequate teaching from these Examples in order to make variants of the compositions (formulations) described.
Use of the Products of the Invention in Cosmetic or Pharmaceutical Formulations of Oil-in-water Emulsion Type
TABLE-US-00005  A water qsp 100 Butylene Glycol 2 Glycerol 3 Sodium Dihydroxycetyl 2 Phosphate, Isopropyl Hydroxycetyl Ether B Glycol Stearate SE 14 Triisononaoin 5 Octyl Cocoate 6 C Butylene Glycol, 2 Methylparaben, Ethylparaben, Propylparaben, pH adjusted to 5.5 D Products of the invention 0.01-10%
TABLE-US-00006  A Water qsp 100 Butylene Glycol 2 Glycerol 3 Polyacrylamide, Isoparaffin, 2.8 Laureth-7 B Butylene Glycol, Methylparaben, 2 Ethylparaben, Propylparaben; Phenoxyethanol, Methylparaben, 2 Propylparaben, Butylparaben, Ethylparaben Butylene Glycol 0.5 D Products of the invention 0.01-10%
TABLE-US-00007  A Carbomer 0.50 Propylene Glycol 3 Glycerol 5 Water qsp 100 B Octyl Cocoate 5 Bisabolol 0.30 Dimethicone 0.30 C Sodium Hydroxide 1.60 D Phenoxyethanol, Methylparaben, 0.50 Propylparaben, Butylparaben, Ethylparaben E Perfume 0.30 F Products of the invention 0.01-10%
Use of the Products of the Invention in a Water-in-oil Type Formulation
TABLE-US-00008  A PEG 30 - 3 dipolyhydroxystearate Capric Triglycerides 3 Cetearyl Octanoate 4 Dibutyl Adipate 3 Grape Seed Oil 1.5 Jojoba Oil 1.5 Phenoxyethanol, Methylparaben, 0.5 Propylparaben, Butylparaben, Ethylparaben B Glycerol 3 Butylene Glycol 3 Magnesium Sulphate 0.5 EDTA 0.05 water qsp 100 C Cyclomethicone 1 Dimethicone 1 D Perfume 0.3 E Products of the invention 0.01-10%
Use of the Products of the Invention in a Formulation of Shampoo or Shower Gel Type
TABLE-US-00009  A Xantham Gum 0.8 Water qsp 100 B Butylene Glycol, 0.5 Methylparaben, Ethylparaben, Propylparaben Phenoxyethanol, Methylparaben, 0.5 Propylparaben, Butylparaben, Ethylparaben C Citric acid 0.8 D Sodium Laureth Sulphate 40.0 E Product of the invention 0.01-10%
Use of the Products of the Invention in a Formulation of Lipstick and Other Anhydrous Product Type
TABLE-US-00010  A Mineral Wax 17.0 Isostearyl Isostearate 31.5 Propylene Glycol Dipelargonate 2.6 Propylene Glycol Isostearate 1.7 PEG 8 Beeswax 3.0 Hydrogenated Palm Kernel Oil 3.4 Glycerides, Hydrogenated Palm Glycerides Lanolin Oil 3.4 Sesame Oil 1.7 Cetyl Lactate 1.7 Mineral Oil, Lanolin Alcohol 3.0 B Castor Oil qsp 100 Titanium Dioxide 3.9 CI 15850:1 0.616 CI 45410:1 0.256 CI 19140:1 0.048 CI 77491 2.048 C Products of the invention 0.01-5%
Use of the Products of the Invention in a Formulation of Aqueous Gels (Eyeliners, Summers, etc.)
TABLE-US-00011  A water qsp 100 Carbomer 0.5 Butylene Glycol 15 Phenoxyethanol, Methylparaben, 0.5 Propylparaben, Butylparaben, Ethylparaben B Products of the invention 0.01-10%
Preparation of Pharmaceutical Formulations Containing LOXL
Formulation 17a: Preparation of Tablets
TABLE-US-00012  In g, per tablet A Excipients Lactose 0.359 Sucrose 0.240 B Extract of LOXL* 0.001-0.1 *The extract of LOXL is obtained for example according to the method of extraction described in Example 2, followed by a drying step.
Formulation 17b: Preparation of an Ointment
TABLE-US-00013  A Excipients Low density polyethylene 5.5 Liquid paraffin qsp 100 B Extract of LOXL* 0.001-0.1 *The extract of LOXL is obtained for example according to the method of extraction described in Example 2, optionally followed by a drying step.
Formulation 17c: Preparation of an Injectable Formula
TABLE-US-00014  A Excipient Saline isotonic solution 5 ml B Extract of LOXL* 0.001-0.1 g *The extract of LOXL is obtained for example according to the method of extraction described in Example 2, followed by a drying step.
Phase A and Phase B are packaged in separate ampoules and are mixed before use.
Evaluation of the Cosmetic Acceptance of a Preparation Containing the Subject of the Invention
Toxicology tests were carried out on the compounds obtained according to Examples 10 and 11 incorporated at 10% in a 0.5% xanthan gum, by an ocular evaluation in the rabbit, by the study of the absence of abnormal toxicity by single oral administration in the rat and by the study of the sensitizing power in the guinea pig.
Evaluation of the Primary Irritation of the Skin in the Rabbit:
The preparations described above were applied without dilution at the dose of 0.5 ml on the skin of 3 rabbits according to the method recommended by the OECD in relation to the study of <<the acute irritant/corrosive effect on the skin>>.
The products are classed according to the criteria defined in the Decision of Jan. 2, 1982 published in the Official Journal of the French Republic (the "JORF") of 21/02/82.
The results of these tests have enabled concluding that the preparation containing the compound obtained according to Example 11 was classed as non-irritant for the skin.
Evaluation of the Ocular Irritation in the Rabbit:
The preparations described above were instilled pure and in one batch at the rate of 0.1 ml in the eye of three rabbits according to the method recommended by the directive of the OECD NO. 405 of Feb. 24, 1987, in relation to the study of "the acute irritant/corrosive effect on the eyes".
The results of this test enable concluding that the preparations can be considered as non-irritant for the eyes, in the sense of the Directive 91/326 EEC, used pure or without dilution.
Test on the absence of Abnormal Toxicity by Single Oral Administration in the Rat:
The preparations described were administered In one batch orally at the dose of 5 g/Kg of body weight, to 5 male rats and 5 female rats of a protocol Inspired from the Directive of the OECD No. 401 of Feb. 24, 1987 and adapted to cosmetic products.
The LD0 and LD50 are found to be greater than 5,000 mg/Kg. The preparations tested are therefore not classed amongst the preparations which are dangerous by ingestion.
Evaluation of the Skin Sensitization Potential in the Guinea Pig:
The preparations described are subjected to the maximization test described by Magnusson and Kligmann, a protocol which is in agreement with the directive line No. 406 of the OECD.
The preparations are classed as non-sensitizing by contact with the skin.
Precisions on the sequences which are described:
Sequence ID No1: is the peptide sequence of the human protein LOXL.
Sequence ID No2: is the sequence of nucleotides of the cDNA encoding the human protein LOXL described in sequence ID No1.
Sequence ID No3: is the sequence of nucleotides of the cDNA encoding the promoter of the human gene encoding the protein LOXL described in sequence ID No1.
Sequence ID No4: is the peptide sequence of the human protein tropoelastin.
Sequence ID No5: is the sequence of nucleotides of the cDNA encoding the human protein tropoelastin described in sequence ID No4.
Sequence ID No6: Is the peptide sequence of the human protein LOX.
Sequence ID No7: is the sequence of nucleotides of the cDNA encoding the human protein LOX described in sequence ID No6.
For the double strand DNA probes
Sequence ID No8: is a sense primer of the DNA encoding the human protein LOXL described in sequence ID No1
Sequence ID No9: is an antisense primer of the DNA encoding the human protein LOXL described in sequence ID No1.
Sequence ID No10: is a sense primer of the DNA encoding the human protein tropoelastin described in sequence ID No4.
Sequence ID No11: is an antisense primer of the DNA encoding the human protein tropoelastin described in sequence ID No4.
Sequence ID No12: is a sense primer of the DNA encoding the human protein LOX described in sequence ID No6.
Sequence ID No13: is an antisense primer of the DNA encoding the human protein LOX described in sequence ID No6.
Sequence ID No14: is a sense primer of the DNA encoding the human protein collagen I α1L.
Sequence ID No15: is an antisense primer of the DNA encoding the human protein collagen I α1L.
For the fusion genes GST:
Sequence ID No16 is a sense primer of the DNA of the fusion gene GST S355-D415.
Sequence ID No17: is an antisense primer of the DNA of the fusion gene GST S355-D415.
Sequence ID No18: is a sense primer of the DNA of the fusion gene GST G128-L212.
Sequence ID No19 is an antisense primer of the DNA of the fusion gene GST G128-L212.
Sequence ID No20: is a sense primer of the DNA of the fusion gene GST V228-S279.
Sequence ID No21: is an antisense primer of the DNA of the fusion gene GST V228-S279.
Sequence ID No22: is a sense primer of the DNA of the fusion gene GST D306-N373.
Sequence ID No23: is an antisense primer of the DNA of the fusion gene GST D306-N373.
For the PCR primers
Sequence ID No24: is a sense primer for the RT-PCR of the mRNA encoding the human protein tropoelastin described in sequence ID No4.
Sequence ID No25: is an antisense primer for the RT-PCR of the mRNA encoding the human protein tropoelastin described in sequence ID No4.
Sequence ID No26: is a sense primer for the RT-PCR of the mRNA encoding the human protein LOXL described in sequence ID No1.
Sequence ID No27: is an antisense primer of the sequence of the mRNA encoding the human protein LOXL described in sequence ID No1.
Sequence ID No28: is a sense primer for the RT-PCR of the mRNA encoding the human protein LOX described in sequence ID No6.
Sequence ID No29: is an antisense primer for the RT-PCR of the mRNA encoding the human protein LOX described in sequence ID No6.
Sequence ID No30: is a sense primer for the RT-PCR of the mRNA encoding the human protein actin.
Sequence ID No31: is an antisense primer for the RT-PCR of the mRNA encoding the human protein actin.
311574PRTHomo sapiens 1Met Ala Leu Ala Arg Gly Ser Arg Gln Leu Gly Ala Leu Val Trp Gly1 5 10 15Ala Cys Leu Cys Val Leu Val His Gly Gln Gln Ala Gln Pro Gly Gln 20 25 30Gly Ser Asp Pro Ala Arg Trp Arg Gln Leu Ile Gln Trp Glu Asn Asn 35 40 45Gly Gln Val Tyr Ser Leu Leu Asn Ser Gly Ser Glu Tyr Val Pro Ala 50 55 60Gly Pro Gln Arg Ser Glu Ser Ser Ser Arg Val Leu Leu Ala Gly Ala65 70 75 80Pro Gln Ala Gln Gln Arg Arg Ser His Gly Ser Pro Arg Arg Arg Gln 85 90 95Ala Pro Ser Leu Pro Leu Pro Gly Arg Val Gly Ser Asp Thr Val Arg 100 105 110Gly Gln Ala Arg His Pro Phe Gly Phe Gly Gln Val Pro Asp Asn Trp 115 120 125Arg Glu Val Ala Val Gly Asp Ser Thr Gly Met Ala Leu Ala Arg Thr 130 135 140Ser Val Ser Gln Gln Arg His Gly Gly Ser Ala Ser Ser Val Ser Ala145 150 155 160Ser Ala Phe Ala Ser Thr Tyr Arg Gln Gln Pro Ser Tyr Pro Gln Gln 165 170 175Phe Pro Tyr Pro Gln Ala Pro Phe Val Ser Gln Tyr Glu Asn Tyr Asp 180 185 190Pro Ala Ser Arg Thr Tyr Asp Gln Gly Phe Val Tyr Tyr Arg Pro Ala 195 200 205Gly Gly Gly Val Gly Ala Gly Ala Ala Ala Val Ala Ser Ala Gly Val 210 215 220Ile Tyr Pro Tyr Gln Pro Arg Ala Arg Tyr Glu Glu Tyr Gly Gly Gly225 230 235 240Glu Glu Leu Pro Glu Tyr Pro Pro Gln Gly Phe Tyr Pro Ala Pro Glu 245 250 255Arg Pro Tyr Val Pro Pro Pro Pro Pro Pro Pro Asp Gly Leu Asp Arg 260 265 270Arg Tyr Ser His Ser Leu Tyr Ser Glu Gly Thr Pro Gly Phe Glu Gln 275 280 285Ala Tyr Pro Asp Pro Gly Pro Glu Ala Ala Gln Ala His Gly Gly Asp 290 295 300Pro Arg Leu Gly Trp Tyr Pro Pro Tyr Ala Asn Pro Pro Pro Glu Ala305 310 315 320Tyr Gly Pro Pro Arg Ala Leu Glu Pro Pro Tyr Leu Pro Val Arg Ser 325 330 335Ser Asp Thr Pro Pro Pro Gly Gly Glu Arg Asn Gly Ala Gln Gln Gly 340 345 350Arg Leu Ser Val Gly Ser Val Tyr Arg Pro Asn Gln Asn Gly Arg Gly 355 360 365Leu Pro Asp Leu Val Pro Asp Pro Asn Tyr Val Gln Ala Ser Thr Tyr 370 375 380Val Gln Arg Ala His Leu Tyr Ser Leu Arg Cys Ala Ala Glu Glu Lys385 390 395 400Cys Leu Ala Ser Thr Ala Tyr Ala Pro Glu Ala Thr Asp Tyr Asp Val 405 410 415Arg Val Leu Leu Arg Phe Pro Gln Arg Val Lys Asn Gln Gly Thr Ala 420 425 430Asp Phe Leu Pro Asn Arg Pro Arg His Thr Trp Glu Trp His Ser Cys 435 440 445His Gln His Tyr His Ser Met Asp Glu Phe Ser His Tyr Asp Leu Leu 450 455 460Asp Ala Ala Thr Gly Lys Lys Val Ala Glu Gly His Lys Ala Ser Phe465 470 475 480Cys Leu Glu Asp Ser Thr Cys Asp Phe Gly Asn Leu Lys Arg Tyr Ala 485 490 495Cys Thr Ser His Thr Gln Gly Leu Ser Pro Gly Cys Tyr Asp Thr Tyr 500 505 510Asn Ala Asp Ile Asp Cys Gln Trp Ile Asp Ile Thr Asp Val Gln Pro 515 520 525Gly Asn Tyr Ile Leu Lys Val His Val Asn Pro Lys Tyr Ile Val Leu 530 535 540Glu Ser Asp Phe Thr Asn Asn Val Val Arg Cys Asn Ile His Tyr Thr545 550 555 560Gly Arg Tyr Val Ser Ala Thr Asn Cys Lys Ile Val Gln Ser 565 57021725DNAHomo sapiens 2atggctctgg cccgaggcag ccggcagctg ggggccctgg tgtggggcgc ctgcctgtgc 60gtgctggtgc acgggcagca ggcgcagccc gggcagggct cggaccccgc ccgctggcgg 120cagctgatcc agtgggagaa caacgggcag gtgtacagct tgctcaactc gggctcagag 180tacgtgccgg ccggacctca gcgctccgag agtagctccc gggtgctgct ggccggcgcg 240ccccaggccc agcagcggcg cagccacggg agcccccggc gtcggcaggc gccgtccctg 300cccctgccgg ggcgcgtggg ctcggacacc gtgcgcggcc aggcgcggca cccattcggc 360tttggccagg tgcccgacaa ctggcgcgag gtggccgtcg gggacagcac gggcatggcc 420ctggcccgca cctccgtctc ccagcaacgg cacgggggct ccgcctcctc ggtctcggct 480tcggccttcg ccagcaccta ccgccagcag ccctcctacc cgcagcagtt cccctacccg 540caggcgccct tcgtcagcca gtacgagaac tacgaccccg cgtcgcggac ctacgaccag 600ggtttcgtgt actaccggcc cgcgggcggc ggcgtgggcg cgggggcggc ggccgtggcc 660tcggcggggg tcatctaccc ctaccagccc cgggcgcgct acgaggagta cggcggcggc 720gaagagctgc ccgagtaccc gcctcagggc ttctacccgg cccccgagag gccctacgtg 780ccgccgccgc cgccgccccc cgacggcctg gaccgccgct actcgcacag tctgtacagc 840gagggcaccc ccggcttcga gcaggcctac cctgaccccg gtcccgaggc ggcgcaggcc 900catggcggag acccacgcct gggctggtac ccgccctacg ccaacccgcc gcccgaggcg 960tacgggccgc cgcgcgcgct ggagccgccc tacctgccgg tgcgcagctc cgacacgccc 1020ccgccgggtg gggagcggaa cggcgcgcag cagggccgcc tcagcgtagg cagcgtgtac 1080cggcccaacc agaacggccg cggtctccct gacttggtcc cagaccccaa ctatgtgcaa 1140gcatccactt atgtgcagag agcccacctg tactccctgc gctgtgctgc ggaggagaag 1200tgtctggcca gcacagccta tgcccctgag gccaccgact acgatgtgcg ggtgctactg 1260cgcttccccc agcgcgtgaa gaaccagggc acagcagact tcctccccaa ccggccacgg 1320cacacctggg agtggcacag ctgccaccag cattaccaca gcatggacga gttcagccac 1380tacgacctac tggatgcagc cacaggcaag aaggtggccg agggccacaa ggccagtttc 1440tgcctggagg acagcacctg tgacttcggc aacctcaagc gctatgcatg cacctctcat 1500acccagggcc tgagcccagg ctgctatgac acctacaatg cggacatcga ctgccagtgg 1560atcgacataa ccgacgtgca gcctgggaac tacatcctca aggtgcacgt gaacccaaag 1620tatattgttt tggagtctga cttcaccaac aacgtggtga gatgcaacat tcactacaca 1680ggtcgctacg tttctgcaac aaactgcaaa attgtccaat cctga 172532731DNAHomo sapiens 3gtggaagtgg gggccacccc caagagataa ccccctcctt tctctcacaa gtcaagagaa 60agaagaaaat agtggccaca tgtctggctc tgtgctgggg acttttcata atcctctcat 120tccatcctct caaatatgcc acaagaaagg tatgattacc cccaggtcac agacgggact 180ctgaagctct gagagcttaa aaagcttccc cggggagtgg ctgggccagg ccaggtccct 240agctcaagtc atggtgtgga cccccaggtc tccatctcag cagggatggc tggcaggagc 300gcagtcctgg ccaggggagt ctgtgcagag gcccaggcta tgttcagagc agagtttatt 360caaatagagc ctaacaggaa acttggctcc tctgactcac tctgattcga ccttattaag 420aaaaaaagag agaggagcag gagccagcat cgggtaaggt ttcacgccaa gagctggctc 480tgaggcccgc tgagtggaat ggcatgtgcc ctgatccccc cacatccaaa gccttgaggc 540agcccctgcc ctgctgtccg agtcaaggcg aggggtcctg ccttcacgtt agggcaactg 600agttcccttc ctcacagcca tcctctgtcc tccctccact cctcttccct tccctccctg 660cctaggggta ccctgaggcc tgtttccatt tctccccctc ctctgctgca gcagctgccc 720atctggctgg cgggagggcc ctacaggacc ccagggattc caagcagctg aggccaacac 780tgcagggggc aagcaggagg gagggaaggc ttaaccctcc aggtcccggc cctcagtaag 840ccctgcctca gcatcttgct tggtgtcagt cacaccagtg gctctttgga ggaatcttgt 900ctggagtctg agatggaaac cccatgctgg ggagctgagg tgtcagcgtt gacaagttgc 960tggccaggag gtattggaag ccccctacct ctgggctgga ttctgggcat ttaaagggta 1020gagacatgct ggagtccaag cctcagtcct gaagaacagt gcaatgggtg aacaaatgct 1080gcctggcaag gatggggttg ggaggtctat agttcccaaa gaatgtccac tctagttgcc 1140tcctctctag gtgggctcca ggcatttcca aaataaagaa tatacaggag ccaaattaag 1200gacaggtcct tcataccttg tttataagcc caagagggca atttctgccc ttcggttccc 1260aggaacacgg ttggagaagg cagcccaggg gaggccagaa gagcagtatt tggagtgtga 1320tttcttggat gccaaagcgt tcaaacttct gggaccccct cctatccctg ccattcccag 1380aaggaacaga gaatctccca aagcactctc aggagccatc tggcctaatc ttccattggt 1440aagctgtggc acctggatca gatagagtaa gggactaagc cacacagcaa caggaccagg 1500ccaggtctcc ggaaccccct tctgttgttc aatgcttact ggcttctctg cctcacagtg 1560acccctgtcc catatcaaag acagccccca gtttcatttt atccatgtgt acactcaagt 1620tattcccagg ctatgcagag ccaagagatg taggacagaa accatacgtg atgtctggga 1680agttgatctc tcccaggatc tcacaagtgc ttttcagctc agggatacac cccactcttc 1740agactgggaa agtaagcccc tgaggtgtgc cacgagggaa aagctgcagc tccagtgcct 1800ctcttcgcag gcccaagagc tgcggtgcac ctgggacctg gaattagaga gtggtccctg 1860ttcagcatct ccccgaggag gcccaccaac aaagagggtg tgtctttttt ttttttttct 1920tcctattgag ggtgtgggat aatggtggaa ggaacatgca aagagggtgt gtcttaatta 1980gcactggctt tagggaacaa ggaaaaggga gaacccggga gtacgggaag gaggctgggg 2040cagacaggag tcagaggccc attccagccc aaacgagaag ccagtgagca aggtggagac 2100cagggatgct gtgaccaaag cagagaggaa tgggcggggt ggtgctgaca ccccagcccc 2160gttctgcctg ccagagcccc acttaccagg cccgagtccc cagaggtccc ctcctactcc 2220ctgctcgatt cccttcctca gaggcaggtc tgtggcttgg ctgggaactc cagggactga 2280gggagcactg cagctgtggg accggcgcat agctaaaagc cggcgggcca tagggccccg 2340cggaggaggc cccagcaggc ggaccaggag gccgaagcct cccgacgctc ccagcctgtt 2400gcttattcat tcagagtggg aaagcgccag ccgagcggcc agccagtgcg gggctggcca 2460tgtaaggccc acaggcggtc ctgcccgccc ggtgccctgc ggagagcctc gtgcagccct 2520gggcaccgcc cctgccctgc cctgacccct tggccttgaa atgctgtcat cggaggagcc 2580gtcccgctcg ggacaaggcc agcatggaca aagctagagc tggggcaagc aaggagcctt 2640cctgtcctcg aggccgtggg aagagaagca cgcccagggc cactcctgag agcctctctg 2700tccaccaggc ctctgcagag gggtcaccat g 27314788PRTHomo sapiens 4Met Ala Gly Leu Thr Ala Ala Ala Pro Arg Pro Gly Val Leu Leu Leu1 5 10 15Leu Leu Ser Ile Leu His Pro Ser Arg Pro Gly Gly Val Pro Gly Ala 20 25 30Ile Pro Gly Gly Val Pro Gly Gly Val Phe Tyr Pro Gly Ala Gly Leu 35 40 45Gly Ala Leu Gly Gly Gly Ala Leu Gly Pro Gly Gly Lys Pro Leu Lys 50 55 60Pro Val Pro Gly Gly Leu Ala Gly Ala Gly Leu Gly Ala Gly Leu Gly65 70 75 80Ala Phe Pro Ala Val Thr Phe Pro Gly Ala Leu Val Pro Gly Gly Val 85 90 95Ala Asp Ala Ala Ala Ala Tyr Lys Ala Ala Lys Ala Gly Ala Gly Leu 100 105 110Gly Gly Val Pro Gly Val Gly Gly Leu Gly Val Ser Ala Gly Ala Val 115 120 125Val Pro Gln Pro Gly Ala Gly Val Lys Pro Gly Lys Val Pro Gly Val 130 135 140Gly Leu Pro Gly Val Tyr Pro Gly Gly Val Leu Pro Gly Ala Arg Phe145 150 155 160Pro Gly Val Gly Val Leu Pro Gly Val Pro Thr Gly Ala Gly Val Lys 165 170 175Pro Lys Ala Pro Gly Val Gly Gly Ala Phe Ala Gly Ile Pro Gly Val 180 185 190Gly Pro Phe Gly Gly Pro Gln Pro Gly Val Pro Leu Gly Tyr Pro Ile 195 200 205Lys Ala Pro Lys Leu Pro Gly Gly Tyr Gly Leu Pro Tyr Thr Thr Gly 210 215 220Lys Leu Pro Tyr Gly Tyr Gly Pro Gly Gly Val Ala Gly Ala Ala Gly225 230 235 240Lys Ala Gly Tyr Pro Thr Gly Thr Gly Val Gly Pro Gln Ala Ala Ala 245 250 255Ala Ala Ala Ala Lys Ala Ala Ala Lys Phe Gly Ala Gly Ala Ala Gly 260 265 270Val Leu Pro Gly Val Gly Gly Ala Gly Val Pro Gly Val Pro Gly Ala 275 280 285Ile Pro Gly Ile Gly Gly Ile Ala Gly Val Gly Thr Pro Ala Ala Ala 290 295 300Ala Ala Ala Ala Ala Ala Ala Lys Ala Ala Lys Tyr Gly Ala Ala Ala305 310 315 320Gly Leu Val Pro Gly Gly Pro Gly Phe Gly Pro Gly Val Val Gly Val 325 330 335Pro Gly Ala Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly Ile Pro 340 345 350Val Val Pro Gly Ala Gly Ile Pro Gly Ala Ala Val Pro Gly Val Val 355 360 365Ser Pro Glu Ala Ala Ala Lys Ala Ala Ala Lys Ala Ala Lys Tyr Gly 370 375 380Ala Arg Pro Gly Val Gly Val Gly Gly Ile Pro Thr Tyr Gly Val Gly385 390 395 400Ala Gly Gly Phe Pro Gly Phe Gly Val Gly Val Gly Gly Ile Pro Gly 405 410 415Val Ala Gly Val Pro Ser Val Gly Gly Val Pro Gly Val Gly Gly Val 420 425 430Pro Gly Val Gly Ile Ser Pro Glu Ala Gln Ala Ala Ala Ala Ala Lys 435 440 445Ala Ala Lys Tyr Gly Ala Ala Gly Ala Gly Val Leu Gly Gly Leu Val 450 455 460Pro Gly Pro Gln Ala Ala Val Pro Gly Val Pro Gly Thr Gly Gly Val465 470 475 480Pro Gly Val Gly Thr Pro Ala Ala Ala Ala Ala Lys Ala Ala Ala Lys 485 490 495Ala Ala Gln Phe Gly Leu Val Pro Gly Val Gly Val Ala Pro Gly Val 500 505 510Gly Val Ala Pro Gly Val Gly Val Ala Pro Gly Val Gly Leu Ala Pro 515 520 525Gly Val Gly Val Ala Pro Gly Val Gly Val Ala Pro Gly Val Gly Val 530 535 540Ala Pro Gly Ile Gly Pro Gly Gly Val Ala Ala Ala Ala Lys Ser Ala545 550 555 560Ala Lys Val Ala Ala Lys Ala Gln Leu Arg Ala Ala Ala Gly Leu Gly 565 570 575Ala Gly Ile Pro Gly Leu Gly Val Gly Val Gly Val Pro Gly Leu Gly 580 585 590Val Gly Ala Gly Val Pro Gly Leu Gly Val Gly Ala Gly Val Pro Gly 595 600 605Phe Gly Ala Val Pro Gly Ala Asp Glu Gly Val Arg Arg Ser Leu Ser 610 615 620Pro Glu Leu Arg Glu Gly Asp Pro Ser Ser Ser Gln His Leu Pro Ser625 630 635 640Thr Pro Ser Ser Pro Arg Val Pro Gly Ala Leu Ala Ala Ala Lys Ala 645 650 655Ala Lys Tyr Gly Ala Ala Val Pro Gly Val Leu Gly Gly Leu Gly Ala 660 665 670Leu Gly Gly Val Gly Ile Pro Gly Gly Val Val Gly Ala Gly Pro Ala 675 680 685Ala Ala Ala Ala Ala Ala Lys Ala Ala Ala Lys Ala Ala Gln Phe Gly 690 695 700Leu Val Gly Ala Ala Gly Leu Gly Gly Leu Gly Val Gly Gly Leu Gly705 710 715 720Val Pro Gly Val Gly Gly Leu Gly Gly Ile Pro Pro Ala Ala Ala Ala 725 730 735Lys Ala Ala Lys Tyr Gly Ala Ala Gly Leu Gly Gly Val Leu Gly Gly 740 745 750Ala Gly Gln Phe Pro Leu Gly Gly Val Ala Ala Arg Pro Gly Phe Gly 755 760 765Leu Ser Pro Ile Phe Pro Gly Gly Ala Cys Leu Gly Lys Ala Cys Gly 770 775 780Arg Lys Arg Lys78552367DNAHomo sapiens 5atggcgggtc tgacggcggc ggccccgcgg cccggagtcc tcctgctcct gctgtccatc 60ctccacccct ctcggcctgg aggggtccct ggggccattc ctggtggagt tcctggagga 120gtcttttatc caggggctgg tctcggagcc cttggaggag gagcgctggg gcctggaggc 180aaacctctta agccagttcc cggagggctt gcgggtgctg gccttggggc agggctcggc 240gccttccccg cagttacctt tccgggggct ctggtgcctg gtggagtggc tgacgctgct 300gcagcctata aagctgctaa ggctggcgct gggcttggtg gtgtcccagg agttggtggc 360ttaggagtgt ctgcaggtgc ggtggttcct cagcctggag ccggagtgaa gcctgggaaa 420gtgccgggtg tggggctgcc aggtgtatac ccaggtggcg tgctcccagg agctcggttc 480cccggtgtgg gggtgctccc tggagttccc actggagcag gagttaagcc caaggctcca 540ggtgtaggtg gagcttttgc tggaatccca ggagttggac cctttggggg accgcaacct 600ggagtcccac tggggtatcc catcaaggcc cccaagctgc ctggtggcta tggactgccc 660tacaccacag ggaaactgcc ctatggctat gggcccggag gagtggctgg tgcagcgggc 720aaggctggtt acccaacagg gacaggggtt ggcccccagg cagcagcagc agcggcagct 780aaagcagcag caaagttcgg tgctggagca gccggagtcc tccctggtgt tggaggggct 840ggtgttcctg gcgtgcctgg ggcaattcct ggaattggag gcatcgcagg cgttgggact 900ccagctgcag ctgcagctgc agcagcagcc gctaaggcag ccaagtatgg agctgctgca 960ggcttagtgc ctggtgggcc aggctttggc ccgggagtag ttggtgtccc aggagctggc 1020gttccaggtg ttggtgtccc aggagctggg attccagttg tcccaggtgc tgggatccca 1080ggtgctgcgg ttccaggggt tgtgtcacca gaagcagctg ctaaggcagc tgcaaaggca 1140gccaaatacg gggccaggcc cggagtcgga gttggaggca ttcctactta cggggttgga 1200gctgggggct ttcccggctt tggtgtcgga gtcggaggta tccctggagt cgcaggtgtc 1260cctagtgtcg gaggtgttcc cggagtcgga ggtgtcccgg gagttggcat ttcccccgaa 1320gctcaggcag cagctgccgc caaggctgcc aagtacggtg ctgcaggagc aggagtgctg 1380ggtgggctag tgccaggtcc ccaggcggca gtcccaggtg tgccgggcac gggaggagtg 1440ccaggagtgg ggaccccagc agctgcagct gctaaagcag ccgccaaagc cgcccagttt 1500gggttagttc ctggtgtcgg ggtggctcct ggagttggcg tggctcctgg tgtcggtgtg 1560gctcctggag ttggcttggc tcctggagtt ggcgtggctc ctggagttgg tgtggctcct 1620ggcgttggcg tggctcccgg cattggccct ggtggagttg cagctgcagc aaaatccgct 1680gccaaggtgg ctgccaaagc ccagctccga gctgcagctg ggcttggtgc tggcatccct 1740ggacttggag ttggtgtcgg cgtccctgga cttggagttg gtgctggtgt tcctggactt 1800ggagttggtg ctggtgttcc tggcttcggg gcagtacctg gagccgatga gggagttagg 1860cggagcctgt cccctgagct cagggaagga gatccctcct cctctcagca cctccccagc 1920accccctcat cacccagggt acctggagcc ctggctgccg ctaaagcagc caaatatgga 1980gcagcagtgc ctggggtcct tggagggctc ggggctctcg gtggagtagg catcccaggc 2040ggtgtggtgg gagccggacc cgccgccgcc gctgccgcag ccaaagctgc tgccaaagcc 2100gcccagtttg gcctagtggg agccgctggg ctcggaggac tcggagtcgg agggcttgga 2160gttccaggtg ttgggggcct tggaggtata cctccagctg
cagccgctaa agcagctaaa 2220tacggtgctg ctggccttgg aggtgtccta gggggtgccg ggcagttccc acttggagga 2280gtggcagcaa gacctggctt cggattgtct cccattttcc caggtggggc ctgcctgggg 2340aaagcttgtg gccggaagag aaaatga 23676417PRTHomo sapiens 6Met Arg Phe Ala Trp Thr Val Leu Leu Leu Gly Pro Leu Gln Leu Cys1 5 10 15Ala Leu Val His Cys Ala Pro Pro Ala Ala Gly Gln Gln Gln Pro Pro 20 25 30Arg Glu Pro Pro Ala Ala Pro Gly Ala Trp Arg Gln Gln Ile Gln Trp 35 40 45Glu Asn Asn Gly Gln Val Phe Ser Leu Leu Ser Leu Gly Ser Gln Tyr 50 55 60Gln Pro Gln Arg Arg Arg Asp Pro Gly Ala Ala Val Pro Gly Ala Ala65 70 75 80Asn Ala Ser Ala Gln Gln Pro Arg Thr Pro Ile Leu Leu Ile Arg Asp 85 90 95Asn Arg Thr Ala Ala Ala Arg Thr Arg Thr Ala Gly Ser Ser Gly Val 100 105 110Thr Ala Gly Arg Pro Arg Pro Thr Ala Arg His Trp Phe Gln Ala Gly 115 120 125Tyr Ser Thr Ser Arg Ala Arg Glu Arg Gly Ala Ser Arg Ala Glu Asn 130 135 140Gln Thr Ala Pro Gly Glu Val Pro Ala Leu Ser Asn Leu Arg Pro Pro145 150 155 160Ser Arg Val Asp Gly Met Val Gly Asp Asp Pro Tyr Asn Pro Tyr Lys 165 170 175Tyr Ser Asp Asp Asn Pro Tyr Tyr Asn Tyr Tyr Asp Thr Tyr Glu Arg 180 185 190Pro Arg Pro Gly Gly Arg Tyr Arg Pro Gly Tyr Gly Thr Gly Tyr Phe 195 200 205Gln Tyr Gly Leu Pro Asp Leu Val Ala Asp Pro Tyr Tyr Ile Gln Ala 210 215 220Ser Thr Tyr Val Gln Lys Met Ser Met Tyr Asn Leu Arg Cys Ala Ala225 230 235 240Glu Glu Asn Cys Leu Ala Ser Thr Ala Tyr Arg Ala Asp Val Arg Asp 245 250 255Tyr Asp His Arg Val Leu Leu Arg Phe Pro Gln Arg Val Lys Asn Gln 260 265 270Gly Thr Ser Asp Phe Leu Pro Ser Arg Pro Arg Tyr Ser Trp Glu Trp 275 280 285His Ser Cys His Gln His Tyr His Ser Met Asp Glu Phe Ser His Tyr 290 295 300Asp Leu Leu Asp Ala Asn Thr Gln Arg Arg Val Ala Glu Gly His Lys305 310 315 320Ala Ser Phe Cys Leu Glu Asp Thr Ser Cys Asp Tyr Gly Tyr His Arg 325 330 335Arg Phe Ala Cys Thr Ala His Thr Gln Gly Leu Ser Pro Gly Cys Tyr 340 345 350Asp Thr Tyr Gly Ala Asp Ile Asp Cys Gln Trp Ile Asp Ile Thr Asp 355 360 365Val Lys Pro Gly Asn Tyr Ile Leu Lys Val Ser Val Asn Pro Ser Tyr 370 375 380Leu Val Pro Glu Ser Asp Tyr Thr Asn Asn Val Val Arg Cys Asp Ile385 390 395 400Arg Tyr Thr Gly His His Ala Tyr Ala Ser Gly Cys Thr Ile Ser Pro 405 410 415Tyr71254DNAHomo sapiens 7atgcgcttcg cctggaccgt gctcctgctc gggcctttgc agctctgcgc gctagtgcac 60tgcgcccctc ccgccgccgg ccaacagcag cccccgcgcg agccgccggc ggctccgggc 120gcctggcgcc agcagatcca atgggagaac aacgggcagg tgttcagctt gctgagcctg 180ggctcacagt accagcctca gcgccgccgg gacccgggcg ccgccgtccc tggtgcagcc 240aacgcctccg cccagcagcc ccgcactccg atcctgctga tccgcgacaa ccgcaccgcc 300gcggcgcgaa cgcggacggc cggctcatct ggagtcaccg ctggccgccc caggcccacc 360gcccgtcact ggttccaagc tggctactcg acatctagag cccgcgaacg tggcgcctcg 420cgcgcggaga accagacagc gccgggagaa gttcctgcgc tcagtaacct gcggccgccc 480agccgcgtgg acggcatggt gggcgacgac ccttacaacc cctacaagta ctctgacgac 540aacccttatt acaactacta cgatacttat gaaaggccca gacctggggg caggtaccgg 600cccggatacg gcactggcta cttccagtac ggtctcccag acctggtggc cgacccctac 660tacatccagg cgtccacgta cgtgcagaag atgtccatgt acaacctgag atgcgcggcg 720gaggaaaact gtctggccag tacagcatac agggcagatg tcagagatta tgatcacagg 780gtgctgctca gatttcccca aagagtgaaa aaccaaggga catcagattt cttacccagc 840cgaccaagat attcctggga atggcacagt tgtcatcaac attaccacag tatggatgag 900tttagccact atgacctgct tgatgccaac acccagagga gagtggctga aggccacaaa 960gcaagtttct gtcttgaaga cacatcctgt gactatggct accacaggcg atttgcatgt 1020actgcacaca cacagggatt gagtcctggc tgttatgata cctatggtgc agacatagac 1080tgccagtgga ttgatattac agatgtaaaa cctggaaact atatcctaaa ggtcagtgta 1140aaccccagct acctggttcc tgaatctgac tataccaaca atgttgtgcg ctgtgacatt 1200cgctacacag gacatcatgc gtatgcctca ggctgcacaa tttcaccgta ttag 1254820DNAHomo sapiensamorce 8gacataaccg acgtgcagcc 20920DNAHome sapiensamorce 9atccacgttc gctccctgag 201020DNAHomo sapiensamorce 10gtatataccc aggtggcgtg 201121DNAHome sapiensamorce 11cgaactttgc tgctgcttta g 211223DNAHomo sapiensamorce 12ggtggccgac ccctactaca tcc 231326DNAHome sapiensamorce 13gcaaatcgcc tctggtagcc atagtc 261418DNAHomo sapiensamorce 14gtggagagta ctggattg 181518DNAHomo sapiensamorce 15tcgtgcagcc atcgacag 181626DNAHomo sapiensamorce 16ttggatccag cgtaggcagc gtgtac 261725DNAHomo sapiensamorce 17aaactcgagc atcgtagtcg gtggc 251829DNAHomo sapiensamorce 18tcggatccgg ctactcgaca tctagagcc 291927DNAHomo sapiensamorce 19gtcctcgaga ccgtactgga agtagcc 272029DNAHomo sapiensamorce 20ttggatccgt gcagaagatg tccatgtac 292129DNAHomo sapiensamorce 21tttctcgagg ctgggtaaga aatctgatg 292228DNAHomo sapiensamorce 22cactatggat cccttgatgc caacaccc 282327DNAHomo sapiensamorce 23cacgaccttt aggatatcgt ttccagg 272420DNAHomo sapiensamorce PCR 24gtatataccc aggtggcgtg 202521DNAHomo sapiensamorce PCR 25cgaactttgc tgctgcttta g 212619DNAHomo sapiensamorce PCR 26gacttcggca acctcaagc 192720DNAHomo sapiensamorce PCR 27tgttgcagaa acgtagcgac 202821DNAHomo sapiensamorce PCR 28acgtacgtgc agaagatgtc c 212921DNAHomo sapiensamorce PCR 29ggctgggtaa gaaatctgat g 213020DNAHomo sapiensamorce PCR 30gtggggcgcc ccaggcacca 203124DNAHomo sapiensamorce PCR 31ctccttaatg tcacgcacga tttc 24
Patent applications by Charbel Bouez, Lyon FR
Patent applications by Claudine Gleyzal, Lyon FR
Patent applications by Corinne Reymermier, Charly FR
Patent applications by Eric Perrier, Les Cotes D'Arey FR
Patent applications by Odile Damour, Saint Genis Laval FR
Patent applications by Pascal Sommer, Saint Genis Laval FR
Patent applications by Valérie Andre, Ampuis FR
Patent applications by Valérie Cenizo, Vaulx-Milieu FR
Patent applications by BASF Beauty Care Solutions France S.A.S.
Patent applications in class Containing or obtained from Cinnamomum (e.g., cinnamon, etc.)
Patent applications in all subclasses Containing or obtained from Cinnamomum (e.g., cinnamon, etc.)