HK1190921B - Modulation of dynein in skin - Google Patents

Description

Regulation of dynein in skin

Related applications

This application claims priority to U.S. Provisional Patent Application Serial No. 61/428,272, filed December 30, 2010, the contents of which are hereby incorporated by reference in their entirety.

Field of the Invention

The present invention relates generally to methods for improving the aesthetic appearance and health of human skin and also to methods for identifying compounds useful for treating skin. In particular, the present invention relates to compounds that modulate dynein levels in skin.

Background Art

Dyneins are a family of proteins that move along microtubules in a minus-end direction. Dyneins convert the chemical energy from ATP hydrolysis into the mechanical energy required for stepwise movement along microtubules. Axial dyneins are found only in cells with cilia and flagella, where they cause microtubules to slide within the axoneme of those structures. Cytoplasmic dyneins, on the other hand, are thought to be present in all animal cells. They have been identified and characterized more recently than axial dyneins. See Paschal B.M., Shpetner H.S., Vallee R.B., “MAP1C is a microtubule-activated ATPase which translocates microtubules in vitro and has dynein-like properties,” J. Cell Biol., 1987 Sep; 105(3): 1273-82; and Shpetner H.S., Paschal B.M., and Vallee R.B., “Characterization of the microtubule-activated ATPase of brain cytoplasmic dynein (MAP1C),” J. Cell Biol., 1988 Sep; 107(3): 1001-9, the disclosures of which are incorporated herein by reference.

Cytoplasmic dynein is a cytoskeletal molecular motor that is responsible for mitosis, cell polarization, intracellular transport of vesicles, endosomes, and lysosomes, as well as other movements within the cell. Cytoplasmic dynein has a molecular weight of approximately 1.5 megadaltons (MDa) and has twelve or more polypeptide subunits, each comprising two identical heavy chains of approximately 530 kDa/chain, two intermediate chains of approximately 74 kDa, four intermediate chains of approximately 53 to 59 kDa, and several light chains of approximately 10 to approximately 14 kDa. Each heavy chain contains four ATP binding sites (including an ATP-hydrolysis site) and a microtubule binding site. The intermediate chain is believed to bind dynein to its cargo.

Because dyneins generate the movement of many materials in cells, they are critical for numerous cellular and developmental functions, including organelle movement, localization of developmental determinants, and potentially long-range signaling in neurons. Dyneins have also been shown to regulate centrosome reorientation, cytoskeletal remodeling, leading edge formation, and cell migration during wound healing in monolayer cell cultures. They also mediate the perinuclear accumulation of phagocytosed melanosomes in human keratinocytes, which leads to the formation of supranuclear melanin.

Defects in dynein function have been implicated in the neuronal degeneration seen in motor neuron diseases, which are caused by impaired axonal transport in neurons. Disruption of the gene encoding the cytoplasmic dynein chain has been associated with the respiratory disease, primary ciliary dyskinesia (Kartagner syndrome), and may be implicated in motor neuron diseases such as amyotrophic lateral sclerosis. To date, the role of dynein in maintaining healthy skin and slowing skin aging has not been recognized.

It is an object of the present invention to provide compositions and methods for treating, ameliorating, inhibiting and/or preventing dermatological signs of aging. Another object of the present invention is to provide methods for treating, ameliorating, inhibiting and/or preventing dermatological signs of aging by modulating dynein levels in skin cells. A further object of the present invention is to provide methods for identifying compounds useful for treating, ameliorating, inhibiting and/or preventing dermatological signs of aging based on their ability to modulate dynein levels in skin cells.

The above discussion is presented only to provide a better understanding of the nature of the problems faced in the prior art and should not be construed in any way as an admission of the prior art.

SUMMARY OF THE INVENTION

In accordance with the foregoing objectives and other objectives, the present invention provides modulators of dynein, preferably cytoplasmic dynein, to improve one or more dermatological signs of aging. Depending on the desired effect, the modulators can upregulate or downregulate the level of dynein in the skin. It is believed that upregulators of dynein produced in skin fibroblasts and keratinocytes will lead to increased protein production in the cells, including the fibroblast-specific proteins collagen, elastin, and fibrillin, as well as the keratinocyte protein keratin. Given the importance of proteins produced by fibroblasts to overall skin strength and health, upregulating dynein will have a beneficial effect in reducing the appearance of aging on the skin.

The present invention also includes modulators that downregulate the levels of dynein in the skin. Because dynein also mediates the perinuclear accumulation of phagocytosed melanosomes in human keratinocytes (which leads to the formation of supranuclear melanin), downregulators of dynein (particularly in keratinocytes and/or melanocytes) would be useful for treating age spots and other forms of hyperpigmentation in the skin. It is also contemplated that downregulators of dynein would be effective in treating proliferative disorders of the skin, including psoriasis.

In one aspect of the present invention, methods are provided for screening candidate substances to identify active ingredients that can be used to improve the aesthetic appearance of skin. The methods comprise determining the ability of a candidate substance to modulate dynein expression in skin fibroblasts or keratinocytes. The methods generally involve incubating human skin fibroblasts or keratinocytes with a candidate substance and subsequently measuring the level of mRNA encoding the cytoplasmic dynein heavy chain subunit DYNC1H1 by quantitative techniques, such as quantitative polymerase chain reaction (qPCR).

Thus, methods for improving the aesthetic appearance of human skin are provided, comprising topically applying to an area of skin in need thereof an effective amount of a substance that modulates dynein levels in human skin fibroblasts and keratinocytes. The dynein modulators are referred to herein as "active substances" and can be up-regulators or down-regulators, and are typically formulated in a cosmetically acceptable carrier and topically applied to human skin (e.g., the skin of the face, neck, hands, chest, legs, etc.) for a period of time sufficient to enhance its health or aesthetic appearance.

In one aspect of the present invention, a method for improving the aesthetic appearance of human skin is provided, comprising topically applying to an area of skin in need thereof an effective amount of an active agent that modulates (e.g., upregulates or downregulates) the cellular level of cytoplasmic dynein, wherein the ability of the active agent to modulate cytoplasmic dynein is determined by measuring the expression level of cytoplasmic dynein in cells contacted with the active agent. The cells used in the assay can be any cell that expresses dynein, but are preferably mammalian cells, more preferably human or mouse cells. The cells can be skin cells, such as fibroblasts or keratinocytes. The assay can measure the level of any fragment or marker of dynein, including the intact complex, but typically measures the expression level of the cytoplasmic dynein heavy chain subunit DYNC1H1, which is expressed by the human gene DYNC1H1 or the mouse gene Dync1h1. The expression level of the human gene DYNC1H1 or the mouse gene Dync1h1 can be determined, for example, by measuring the expression level of the corresponding mRNA using any suitable technique, such as quantitative polymerase chain reaction (qPCR).

Provided are methods for treating wrinkles and/or fine lines, the methods comprising topically applying to an area of skin in need thereof an effective amount of an active agent that upregulates dynein, wherein the ability of the active agent to modulate cytoplasmic dynein is determined by measuring the level of expression of cytoplasmic dynein in cells contacted with the active agent. The assay most typically comprises incubating human skin fibroblasts with a candidate agent and subsequently measuring the level of mRNA encoding the cytoplasmic dynein heavy chain subunit DYNC1H1, wherein the candidate agent is selected for use as the active agent based in part on its ability to upregulate dynein expression.

Also provided are methods for treating hyperpigmentation, the methods comprising topically applying to an area of skin in need thereof an effective amount of an active agent that downregulates dynein, wherein the ability of the active agent to modulate cytoplasmic dynein is determined by measuring the level of expression of cytoplasmic dynein in cells contacted with the active agent. The measuring step in this practice generally comprises incubating human skin keratinocytes with a candidate agent and then measuring the level of mRNA encoding the cytoplasmic dynein heavy chain subunit DYNC1H1, wherein the candidate agent is selected for use as the active agent based in part on its ability to downregulate dynein expression.

Other aspects, features and advantages of the present invention will be better understood after reading the detailed description of the invention.

Detailed Description of the Invention

Unless otherwise provided, all terms used herein are intended to have their ordinary meanings. "Cosmetically acceptable" means that the particular ingredient is generally regarded as safe and non-toxic at the levels employed. As used herein, the term "prevent" includes delaying the onset or progression of particular signs of skin aging. The term "thin skin" includes skin that becomes thinner with chronological aging as well as premature thinning of skin, which can be caused by, for example, photoaging. The term "individual in need" refers to a person, male or female, who can benefit from improved skin appearance or health. The term "skin" includes, but is not limited to, the skin of the lips, face, hands, arms, neck and chest. As used herein, the term "consisting essentially of..." is intended to limit the invention to the specified materials or steps and those materials or steps that do not materially affect the basic and novel characteristics of the claimed invention, as understood from a reading of this specification.

As used herein, the term dynein refers to cytoplasmic dynein and ciliary axis dynein, although preferred regulators according to the present invention will regulate the level of cytoplasmic dynein. Cytoplasmic dynein includes cytoplasmic dynein 1 complex, and cytoplasmic dynein 2 complex, although preferred regulators according to the present invention will regulate the level of cytoplasmic dynein 1 complex. Dynein can be from any animal, but is typically human or mouse dynein, and preferably human dynein. The nomenclature used for the cytoplasmic dynein described herein is Pfister et al., "Cytoplasmic dynein nomenclature" J. Cell Biol., 2005 November 7; 171 (3): 411–413, which is incorporated herein by reference and summarized in Table 1.

Table 1

The term "regulator" encompasses any substance, including but not limited to organic molecules, biomolecules (such as peptides, proteins, antibodies, nucleic acid oligomers, etc.), and combinations of substances, such as plant extracts. The regulator regulates the cellular level of dynamin, which means that the cellular level of dynamin is increased or decreased by the active agent. The term "regulate" may refer to upregulating, inducing, stimulating, enhancing, and/or alleviating inhibition, as well as inhibiting, reducing and/or downregulating or suppressing. The regulator can be, but is not limited to, an inhibitor or antagonist, which is, for example, a compound that binds, partially or completely blocks stimulation, reduces, prevents, delays activation, inactivates, desensitizes, or downregulates the expression level of a gene or dynamin or peptide. The regulator can also be, but is not limited to, an activator or agonist, which is, for example, a compound that binds, stimulates, increases, opens, activates, promotes, enhances activation, sensitizes, or upregulates the expression level of a gene or dynamin or peptide. The mechanism of regulating protein levels is unimportant.

As used herein, the term "expression level" refers to the amount of a gene and/or protein expressed in a cell. As used herein, a "gene" includes a polynucleotide containing at least one open reading frame capable of encoding a specific polypeptide. As used herein, the term "polynucleotide" is synonymous with "oligonucleotide" and includes a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof, including but not limited to mRNA, DNA, cDNA, primers, probes, and the like.

The discovery of the association between dynamin expression levels and dermatological aging has produced a screening method for identifying potential dynamin regulators. In one embodiment, a determination is provided for determining dynamin expression levels after treating, incubating or otherwise contacting cells with a candidate substance. The term "candidate substance" refers to any substance tested for its activity as a dynamin regulator, whether or not the substance is suspected of having such activity. The cell can be any cell expressing dynamin, preferably cytoplasmic dynamin. In one embodiment, the cell is a mammalian fibroblast. In another embodiment, the cell is a mammalian keratinocyte. Preferably, the cell is a human or mouse cell. After incubating the cell with the candidate substance for a sufficiently long time to provide a measurable change in expression level (this will typically be at least 1 hour, and more typically about 12 hours to about 72 hours), the cell is then lysed to release cellular components, such as dynamin and mRNA encoding dynamin. The amount of dynamin or any of its subunits can then be measured by any suitable technique for detecting and quantifying peptides and proteins and/or polynucleotides (e.g., mRNA).

A preferred method for measuring dynein expression levels involves quantifying mRNA expression. Suitable methods for determining mRNA expression include quantitative PCR (QPCR), real-time QPCR, reverse transcription PCR (RT-PCR), and quantitative reverse transcription PCR (QRT-PCR), as are well known in the art. As described in detail in U.S. Patent Nos. 7,101,663 and 7,662,561 (the disclosures of which are incorporated herein by reference), a quantitative reverse transcriptase polymerase chain reaction (QRT-PCR) for detecting mRNA may include the following steps: (a) incubating an RNA sample from a cell lysate with a reverse transcriptase and a high concentration of a target sequence-specific reverse transcriptase primer under conditions suitable for producing cDNA; (b) then adding suitable polymerase chain reaction (PCR) reagents to the reverse transcriptase reaction, including adding a high concentration of a PCR primer set specific for the cDNA and a thermostable DNA polymerase to the reverse transcriptase reaction; and (c) cycling the PCR reaction for a desired number of cycles and under suitable conditions to produce a PCR product ("amplicon") specific for the cDNA. After a fixed number of PCR cycles, the products of the QRT-PCR process can be compared, for example, by Southern blotting, to determine the relative amounts of RNA species relative to a given reporter gene. More generally, the progress of the PCR reaction is monitored by analyzing the relative rates of amplicon production for each PCR primer set, for example, by (1) nonspecific fluorescent dyes that intercalate into any double-stranded DNA, and/or (2) sequence-specific DNA probes consisting of oligonucleotides labeled with a fluorescent reporter (which allows detection only after hybridization of the probe to its complementary DNA target).

The mRNA can be any mRNA related to dynein, including mRNAs encoding the subunits identified in Table 1. In one embodiment, the mRNA encodes the heavy chain subunit DYNC1H1, which is synonymously known in the literature as "cytoplasmic dynein 1 heavy chain" or "conventional cytoplasmic dynein heavy chain", etc. In a preferred embodiment, the mRNA encodes human DYNC1H1.

The expression level of mRNA can be compared with the control that is not processed with candidate's substance to determine the relative degree of regulation.In certain embodiments, candidate's substance will raise mRNA expression by at least about 10%, more appropriately at least about 20%, at least about 30%, at least about 40%, or at least about 50%.Preferably, described candidate's substance will raise mRNA expression by at least about 60%, at least about 70%, at least about 80%, at least about 90% or at least about 100%.In other embodiments, described candidate's substance will lower mRNA expression by at least about 10%, more appropriately at least about 20%, at least about 30%, at least about 40% or at least about 50%.Can select candidate's substance that meets these conditions to be used for further evaluation.

The dynein modulators identified by the aforementioned screening protocols can be used as active agents in cosmetic preparations and can be formulated, along with other cosmetically acceptable components (e.g., carriers), into compositions for topical application to the skin. The compositions are topically applied to the skin in an effective amount sufficient to achieve a measurable improvement in skin health or to reduce one or more dermatological signs of aging. The signs of skin aging include, but are not limited to, the following:

(a) treating, reducing and/or preventing fine lines or wrinkles,

(b) reduce the size of skin pores,

(c) improving skin thickness, plumpness, and/or firmness;

(d) improving skin flexibility and/or softness;

(e) improving skin tone, radiance, and/or clarity;

(f) improving procollagen and/or collagen production;

(g) Improved elastin maintenance and remodeling;

(h) improving skin texture and/or promoting retexturization;

(i) improving skin barrier repair and/or function;

(j) improving the appearance of skin contours;

(k) Restoring skin radiance and/or brightness;

(l) replenishing key nutrients and/or ingredients in the skin;

(m) reduced by aging and/or menopause;

(n) Improve skin moisturizing properties;

(o) increase skin elasticity and/or resilience;

(p) Treat, reduce and/or prevent skin sagging and/or

(q) Reduce pigment spots.

In practice, the compositions of the present invention (including an agent that modulates dynein expression levels), alone or in a cosmetically acceptable carrier, are applied to skin in need of treatment. That is, skin that suffers from a deficiency or loss of any of the aforementioned properties, or that would otherwise benefit from an improvement in any of the aforementioned skin properties. The skin is typically treated once or twice daily. Treatment may continue for one week, two weeks, four weeks, eight weeks, six months, or longer.

In one embodiment, the active agent is topically applied (in a cosmetically acceptable carrier) to skin afflicted with fine lines and/or wrinkles to prevent, treat and/or improve the appearance of fine lines and/or wrinkles in the skin. In this case, the composition is applied to skin in need of treatment, which refers to skin that already has wrinkles and/or fine lines or skin that is at risk of developing fine lines and/or wrinkles. Preferably, the composition is applied directly to the fine lines and/or wrinkles on the skin of the face, neck, chest and/or hands. Preferred agents according to this embodiment are upregulators of dynamin expression. In preferred embodiments, upregulators of dynamin expression cause enhanced production of collagen, elastin and/or fibrillin in skin fibroblasts.

In one embodiment, the present invention relates to a method for improving the aesthetic appearance of skin by increasing the production of collagen, elastin and/or fibrillin in the skin, the method comprising topically applying to an area of skin in need thereof an effective amount of an agent that upregulates dynein expression, wherein the compound used is identified by an assay that determines the ability of a substance to modulate dynein expression levels, including the assays described herein. Preferably, the assay measures dynein mRNA expression levels in skin fibroblasts.

In one embodiment, the dynein regulator comprises a natural plant material, such as a plant extract. Among the natural plant materials and plant extracts that upregulate dynamin, mention may be made of extracts from the following species: Orthosiphon grandiflorus, Clinacanthus nutans, Cistanchetubulosa, Erythrina indica, Operculina turpethum, Gynandropsis gynandra, Erthrina Flabelliformis, Helichrysum Odoratissimum, Desmanthus illinoensi, Ozothamnus Obcordatus, Tagetes erecta Linn, Caesalpinia sappan Linn, Medemia nobilis, Calatropis gigantean, Loropetalum chinense, Heliotropium indicum, Dianella ensifolia, Breynia fruticosa), Dendranthema indicum, Sambucus chinensis, Scoparis dulcis, Acacia melanoxylon, Clerodendrum floribundum, Commersonia bartramia, Dodonaea viscose, Duboisa myoporoides, Ehretia acuminate, Ficus coronata, Goodenia ovata, Hymenosporum flavum, Lavatera plebeian, Melaleuca quinquernervia, Omolanthes populifolius, and combinations thereof.

Other substances that upregulate dynein include the following:

Compound 1

Compound 2

Compound 3

Compound 4

Compound 5

Also suitable for upregulating dynein is chalmicrin, which is 10-methyl-trans-monocyclic farnesol linked to D-mannitol as an allyl ether isolated from Chalara microspores; i.e., 1-O-[(E)-3-methyl-5-(2,5,6,6-tetramethyl-1-cyclohexen-1-yl)-2-pentenyl]-D-mannitol.

In one embodiment, the present invention relates to a method for reducing the appearance of age spots and other manifestations of hyperpigmentation in the skin, the method comprising topically applying to an area of skin in need thereof an effective amount of an agent that downregulates dynein expression, wherein the compound used is identified by an assay that determines the ability of a substance to modulate dynein expression levels, including the assays described herein. Preferably, the assay measures the expression level of dynein mRNA in skin keratinocytes. Downregulation of dynein may also be effective in treating proliferative disorders of the skin, including psoriasis.

Among the natural plant materials and plant extracts that downregulate dynamin are Amorphophallus campanulatus, Gomphrena globosa Linn., Fibraretinum resica Pierre (Fructus Fibrosum), Vernonia cinerea Linn. Less, Cayratia japonica, Pteris semipinnata, and combinations thereof.

Other substances that downregulate dynein include 5-chlorosalicylic acid, 3,6-nonadienol, cis-6-nonenol, and the following:

Compound 6

Compound 7

Compound 8

In an embodiment, a method for improving the aesthetic appearance of human skin and/or improving the appearance of aged and/or photodamaged skin comprises topically applying an effective amount of a composition comprising a natural plant extract selected from the group consisting of Mammea siamensis (flower extract), Medemia nobilis (seed extract), Raphia farinifera (seed extract), Erythrina chinensis (leaf extract), Clerodendron fragrans (whole plant extract), Ceratonia ovata (whole plant extract, without roots), or a combination thereof and a cosmetically acceptable carrier.

The plant material can be in any form, including but not limited to the whole plant, dried plant, ground plant, or parts thereof (including but not limited to seeds, needles, leaves, roots, bark, cones, stems, rhizomes, callus cells, protoplasts, flowers, and meristems), or components and/or ingredients found or isolated from the natural plant material and/or plant parts, or any combination thereof. In one embodiment, the natural plant material is in the form of an extract derived from the whole plant or from a selected part of the plant (e.g., leaves of the plant). It will be understood that "natural plant material" also includes raw materials, components, ingredients, or extracts derived from natural plant materials. In another embodiment, plant extracts as used herein also include "synthetic" extracts, i.e., various combinations of known plant components and/or ingredients that have been combined to substantially mimic the composition and/or activity of plant extracts from natural sources. Such synthetic extracts are included in the term "plant extract." Synthetic extracts will have two or more, three or more, or four or more active ingredients shared with the plant. Most preferably, the synthetic extract will have substantially the same number of active ingredients as the natural extract. The correspondence in the numerical appearance of active ingredients between a synthetic extract and a plant or natural extract can also be described in terms of "percent commonality." Preferably, the synthetic extract has about 50 percent or more commonality in chemical composition with the plant or natural extract. In other words, the synthetic extract has about 50 percent or more of the active ingredients found in the plant or natural extract. More preferably, the synthetic extract has about 70 percent or more commonality in chemical composition with the plant or natural extract. Optimally, the synthetic extract has about 90 percent or more commonality in chemical composition with the plant or natural extract.

For use in the compositions of the present disclosure, the plant extracts or components and/or active ingredients are preferably derived directly from the plant. The components may be in pure, semi-purified, or unpurified form. In one embodiment, the components are in the form of extracts obtained by extraction with aqueous or organic solvents. Non-limiting examples of organic solvents include acetic acid, ethyl acetate, lower alcohols (such as methanol, ethanol, isopropanol, butanol), dichloromethane, hexane, toluene, xylene, petroleum ether, and combinations thereof. The solvent may be polar or non-polar, protic or aprotic, water-soluble or immiscible with water. The pH may be acidic, neutral, or alkaline. Methods well known in the art may be used for aqueous or organic solvent extraction. An extraction time of about 1-8 hours at a temperature between about 30°C and about 90°C is generally suitable. The collected extract is then finely filtered to remove debris and may be used directly, or concentrated (e.g., by distilling the solvent or by other conventional processing), and the extract may also be provided in powder form.

Cosmetic compositions according to the present invention can be formulated in various forms for topical application and will contain from about 0.00001% to about 90% by weight of one or more dynein-modulating actives, and preferably will contain the actives in an amount from about 0.001% to about 25% by weight, more preferably from about 0.001% to about 1% by weight.

The composition may include a cosmetically acceptable carrier. Such carriers may take any form known in the art suitable for application to the skin and may include, but are not limited to, water, vegetable oils, mineral oils, esters such as octalpalmitate, isopropyl myristate, and isopropyl palmitate, ethers such as dicaprylyl ether and dimethyl isosorbide, alcohols such as ethanol and isopropyl alcohol, fatty alcohols such as cetyl alcohol, cetearyl alcohol, stearyl alcohol, and biphenylmethanol, isoparaffins such as isooctane, isododecane, and isohexadecane, silicone oils such as cyclomethicone, hydrocarbon oils such as mineral oil, petrolatum, isoeicosane, and polyisobutene, polyols such as propylene glycol, glycerin, butylene glycol, pentylene glycol, and hexylene glycol, liposomes, waxes, or any combination or mixture of the foregoing.

The carrier may comprise an aqueous phase, an oil phase, an alcohol, a silicone phase or mixtures thereof and may be in the form of an emulsion. Non-limiting examples of suitable emulsions include water-in-oil emulsions, oil-in-water emulsions, silicone-in-water emulsions, water-in-silicone emulsions, glycerol-in-oil emulsions, wax-in-water emulsions, water-in-oil-in-water triple emulsions, and the like. The emulsion may include an emulsifier, such as a nonionic, anionic or zwitterionic surfactant, or a gelling agent.

The topical composition will typically have a pH in the range of about 2 to about 8, preferably a pH in the range of 3 to 7. In some embodiments, the composition will have a pH in the range of 3.5 to 5.5. Suitable pH adjusters (e.g., citric acid and triethanolamine) may be added to bring the pH within the desired range.

In one embodiment of the present invention, the composition may include other skin active substances, including but not limited to botanicals, keratolytics, desquamation agents, keratinocyte proliferation enhancers, collagenase inhibitors, elastase inhibitors, depigmenting agents, anti-inflammatory agents, steroids, anti-acne agents, antioxidants, and advanced glycation endproduct (AGE) inhibitors.

The composition may contain other active ingredients having anti-aging benefits, and it is expected that synergistic improvements may be obtained with such combinations. Exemplary anti-aging components include, but are not limited to, botanicals (e.g., Butea mays extract); phytol; thiodipropionic acid (TDPA) and its esters; retinoids (e.g., 9-cis retinoic acid, 13-cis retinoic acid, all-trans retinoic acid and its derivatives, phytanic acid, retinol (vitamin A) and its esters, such as retinyl palmitate, retinyl acetate and retinyl propionate, and their salts and others); hydroxy acids (including α-hydroxy acids and β-hydroxy acids), salicylic acid and alkyl salicylic acids; exfoliants (e.g., glycolic acid, 3,6,9-trioxaundecanedioic acid, etc.), estrogen synthase stimulating compounds (e.g., caffeine and derivatives); compounds capable of inhibiting 5α-reductase activity (e.g., linolenic acid, linoleic acid, finasteride, and mixtures thereof); and barrier function enhancers (e.g., ceramides, glycerides, cholesterol and its esters, α-hydroxy and ω-hydroxy fatty acids and their esters, etc.). Exemplary retinoids include, without limitation, retinoic acid (eg, all-trans or 13-cis) and its derivatives, retinol (vitamin A) and its esters, such as retinyl palmitate, retinyl acetate, and retinyl propionate, and salts thereof.

In another embodiment, the topical compositions of the present invention may further include one or more of the following: skin penetration enhancers, lubricants such as isopropyl myristate, petrolatum, silicones (e.g., methicone, dimethicone), oils, mineral oils, and fatty acid esters; humectants such as glycerin or caprylyl glycol, skin plumpers such as palmitoyl oligopeptide, collagen, or collagen and/or glycosaminoglycan (GAG) enhancers, sunscreens such as avobenzone, exfoliants, and antioxidants.

Suitable exfoliants include, for example, α-hydroxy acids, β-hydroxy acids, oxacids, oxadiazine acids, and their derivatives such as their esters, anhydrides, and salts. Suitable hydroxy acids include, for example, glycolic acid, lactic acid, malic acid, tartaric acid, citric acid, 2-hydroxyalkanoic acid, mandelic acid, salicylic acid, and their derivatives. A preferred exfoliant is glycolic acid. When present, the exfoliant may comprise from about 0.1 wt % to about 80 wt % of the composition.

Examples of antioxidants that can be used in the present composition include compounds having a phenolic hydroxyl function, such as ascorbic acid and its derivatives/esters, β-carotene, catechin, curcumin, ferulic acid derivatives (such as ethyl ferulate, sodium ferulate), gallic acid derivatives (such as propyl gallate), lycopene, reducing acid, rosmarinic acid, tannic acid, tetrahydrocurcumin, tocopherol and its derivatives, uric acid, or any mixture thereof. Other suitable antioxidants are those having one or more sulfhydryl functions (-SH) (in reduced or unreduced form), such as glutathione, lipoic acid, thioglycolic acid, and other sulfhydryl compounds. The antioxidant can be inorganic, such as bisulfite, metabisulfite, sulfite, or other sulfur-containing inorganic salts and acids. The composition of the present invention may contain an antioxidant, preferably in an amount of about 0.001 wt % to about 10 wt %, and more preferably in an amount of about 0.01 wt % to about 5 wt %, based on the total weight of the composition.

Other conventional additives include vitamins such as tocopherol and ascorbic acid; vitamin derivatives such as ascorbyl monopalmitate; thickeners such as hydroxyalkylcellulose; gelling agents; structuring agents; metal chelators such as EDTA; pigments; colorants and pH adjusters. The composition may optionally contain other components known to those skilled in the art, including but not limited to film formers, humectants, minerals, viscosity and/or rheology modifiers, anti-acne agents, insect repellents, skin cooling compounds, skin protectants, lubricants, fragrances, preservatives, stabilizers, and mixtures thereof. In addition to the foregoing, the cosmetic compositions of the present invention may contain any other compound useful for treating skin conditions.

The composition can be formulated into various product forms, such as emulsions, lotions, creams, serums, sprays, aerosols, cakes, ointments, serums, gels, pastes, patches, pens, wipes, masks, sticks, foams, elixirs, concentrates, etc., especially for topical administration. Preferably, the composition is formulated into an emulsion, lotion, cream, ointment, serum or gel.

The present invention provides methods for treating aging skin by topically applying to the affected area a composition comprising an active agent that modulates dynein, preferably in a cosmetically acceptable carrier, for a period of time sufficient to reduce, ameliorate, reverse or prevent dermatological signs of aging.

Generally, the improvement in condition and/or aesthetic appearance is selected from the group consisting of: reducing dermatological signs of chronological aging, photoaging, hormonal aging and/or actinic aging; preventing and/or reducing the appearance of lines and/or wrinkles; reducing the perceptibility of facial lines and wrinkles, facial wrinkles on the cheeks, forehead, vertical wrinkles between the eyes, parallel wrinkles above the eyes, and around the mouth, marionette lines, particularly deep wrinkles or folds; improving the appearance of infraorbital and/or periorbital lines; reducing the appearance of crow's feet; rejuvenating and/or revitalizing the skin, particularly aging skin; reducing skin fragility; preventing and/or reversing the loss of glycosaminoglycans and/or collagen; ameliorating the effects of estrogen imbalance; preventing skin atrophy; preventing, reducing and/or treating hyperpigmentation or hypopigmentation; minimizing skin discoloration; improving skin tone, radiance, clarity and and/or firmness; prevent, reduce and/or improve skin sagging; improve skin firmness, plumpness, flexibility and/or softness; improve procollagen and/or collagen production; improve skin texture and/or promote retexturing; improve skin barrier repair and/or function; improve the appearance of skin contours; restore skin radiance and/or brightness; minimize dermatological signs of fatigue and/or stress; protect against environmental stressors; replenish skin components that are reduced by aging and/or menopause; improve communication between skin cells; increase cell proliferation and/or growth; increase skin cell metabolism that is reduced by aging and/or menopause; delay cellular aging; improve skin moisturization; enhance skin thickness; slow down or stop skin thinning; increase skin elasticity and/or resilience; enhance exfoliation; improve microcirculation; reduce and/or prevent the formation of cellulite; and any combination thereof.

In one embodiment, the composition will comprise from about 0.00001% to about 20%, more typically from about 0.001% to about 10%, including from about 0.01% to about 1% dynein modulator by weight. Preferably, the modulator can be an upregulator of dynein in fibroblasts. Also preferably, the modulator can be an upregulator of dynein in keratinocytes. The modulator can be a downregulator of dynein in keratinocytes. Less preferably (although still within the scope of the invention), the modulator can be a downregulator of dynein in fibroblasts. In one embodiment, the modulator is an upregulator of dynein in both fibroblasts and keratinocytes. Combinations of modulators are also contemplated. In one embodiment, the modulator is a combination of two or more substances that are upregulators of dynein in fibroblasts and/or keratinocytes.

The composition will typically be applied to the skin once, twice, or three times daily, as needed to achieve the desired results. Treatment regimens may include daily application for at least one week, at least two weeks, at least four weeks, at least eight weeks, or at least twelve weeks or more. Chronic treatment regimens are also contemplated. The effect of the composition on the formation or appearance of fine lines and wrinkles can be evaluated qualitatively (e.g., by visual inspection) or quantitatively (e.g., by microscopic or computer-assisted measurement of wrinkle morphology (e.g., the number, depth, length, area, volume, and/or width of wrinkles per unit skin area)). In one embodiment, the composition of the present invention is applied to the skin in an amount of about 0.001 to about 100 mg/ ^cm2 , more typically about 0.01 to about 20 mg/ ^cm2 , and preferably about 0.1 to about 10 mg/ ^cm2 .

It is also contemplated that the compositions of the present invention can be used to treat thin skin by topically applying the composition to the thin skin of an individual in need thereof. "Thin skin" is intended to include skin that has thinned due to chronological aging, menopause, or photodamage, as well as skin that has thinned prematurely. In certain embodiments, the treatment is for thin skin in men, while other embodiments treat thin skin in women (pre-menopausal or post-menopausal), as it is believed that skin thinning with age differs in men and women (particularly in women at different stages of life).

The methods of the present invention can be employed prophylactically to forestall aging, including in individuals who do not yet exhibit signs of skin aging (most commonly in individuals under the age of 25). The methods can also be used to reverse or treat signs of aging once they have manifested, as is common in individuals over the age of 25, or to slow the progression of dermatological aging in such individuals.

Example

The following examples describe certain aspects of the present invention to illustrate the present invention but should not be construed as limiting the present invention as the examples merely provide specific methods that can be used to understand and practice the present invention and its various aspects.

Example 1

Age-related expression of dynein in skin fibroblasts

Normal skin fibroblasts from newborns (n=3) and female donors aged 31-39 years (n=3) were plated in 10 cm culture dishes in DMEM (10% FBS, 4.5 g/L glucose) and cultured to sub-confluence. Cells were washed with cold PBS and RNA was isolated using an RNAqueous kit according to the manufacturer's instructions. RNA integrity and concentration were confirmed using an RNA nanochip and an Agilent Bioanalyzer 2100. Gene expression analysis was performed using isolated RNA and an ABI7900HT RT-PCR instrument. The dynamin probe used was Homo sapiens, cytoplasmic, heavy chain DYNC1H1, with ABI encoding DYNC1H1-Hs00300243_m1. An internal control was included and dynamin gene expression was normalized relative to GAPDH. Compared to newborn cells, a significant (~14%) reduction in dynamin expression was found in cells from donors aged 30+ years. Intrinsic (normalized) gene expression levels are provided in Table 1 below.

In another experiment, human biopsies were analyzed for the expression of DYNC1H1 after exposure to UV radiation. Exploratory biopsy skin biopsies from the dorsal forearm (light exposed) and lower upper arm (light protected) of healthy female Caucasian donors from young (age 18-25 years, n=15) and old (age 45-65 years, n=15) were collected and formalin fixed and paraffin embedded. Five micron sections were cut and placed on slides. IHC was performed using a rabbit polyclonal antibody against dynamin and observed using vector blue conjugated with goat anti-rabbit secondary antibodies. In the younger age group, 62% of the subjects showed increased dynamin expression, while only 33% of the older subjects showed an increase. These data show that older skin is much less effective in producing dynamin when needed. The increase in gene expression levels during UV exposure is also provided in Table 2 below.

Table 2

The data in Table 2 show that cytoplasmic dynein expression decreases with age, as does the skin's ability to upregulate dynein expression in response to stressors, such as UV exposure. Modulating dynein is therefore believed to be a reasonable approach to combat age- and environmental-related skin aging.

Example 2

Dynein regulation assay

Normal human dermal fibroblasts or keratinocytes were cultured in 96-well tissue culture-treated plates containing the appropriate culture medium. Stock solutions of the active substances were prepared in an appropriate vehicle (water/ethanol/propylene glycol). Cells were treated with the test substances or the corresponding vehicle control diluted in growth medium for 24 hours in a humidified 37°C incubator containing 10% _CO₂ . Following incubation, the growth medium was removed from each plate, 100 μL of lysis buffer was added to the wells, and the cells were placed in a 37°C incubator containing 10% _CO₂ for 30 minutes. At the end of the incubation period, cells were harvested in a freezing plate and placed in a -80°C freezer until analysis. Changes in dynein mRNA following treatment were analyzed using the Panomics Quantigene multiplex assay using branched DNA technology, according to the manufacturer's instructions (Affymetrix, CA). The percentage increase (upregulation) or decrease (downregulation) in mRNA for the cytoplasmic dynein heavy chain, DYNC1H1, was calculated by comparing the test results with the control. The percentage increases or decreases were converted to scale scores as shown in Table 3 below.

Table 3

Example 3

Upregulation of dynein

Various plant extracts were tested for their ability to upregulate dynein according to the method of Example 2. The results are provided in Table 4 below. The concentration of each extract is provided based on the dry weight of the given plant extract, which refers to the weight of the extract after removal of the volatile extraction solvent. The cells tested were either keratinocytes (K) or fibroblasts (F).

Table 4

Large-flowered Ginseng 0.0001 K +++ Alligator Mouth Flower 0.01 K ++++ Cistanche tubulosa 0.01 K ++++ Erythrina 0.1 K +++ Erythrina 0.01 F +

Erythrina 0.001 F + box fruit vine 0.1 K ++++ White cauliflower 0.1 K +++ Erthrina Flabelliformis 0.01 F ++++ Erthrina Flabelliformis 0.001 K ++++ Immortelle 0.01 K ++++ Illinois Albizia 0.01 F ++++ Ozothamnus Obcordatus 0.01 K +++ marigold 0.01 K ++ marigold 0.001 K +++ Sumu 0.01 K ++++ Medemia nobilis 0.01 K ++++ Ox-horned melon 0.001 F ++++ Ox-horned melon 0.0001 F ++++ Jimu 0.1 K +++ Big Tail Shake 0.1 K +++ Big Tail Shake 0.01 K + Yamasuga 0.1 K +++ Black-Faced God 0.1 K +++ Shennong Fragrant Chrysanthemum 0.1 K +++ Shennong Fragrant Chrysanthemum 0.01 K + comfrey 0.1 K ++++ comfrey 0.01 K + Wild Licorice 0.1 K ++++ Wild Licorice 0.01 K ++ Acacia nigra 0.001 K +++ Clerodendrum floribundum 0.01 K ++++ Clerodendrum floribundum 0.001 K + Mountain hemp tree 0.001 K +++

Che Sangzi 0.01 F ++++ Che Sangzi 0.001 K + Che Sangzi 0.0001 K +++ Australian eggplant 0.01 F ++++ Australian eggplant 0.01 K ++++ Australian eggplant 0.001 F ++ Australian eggplant 0.001 K ++++ Thick-shelled tree 0.01 K ++ Thick-shelled tree 0.001 F ++++ Thick-shelled tree 0.001 K + Fairy Fruit 0.001 F + Fairy Fruit 0.001 K ++ Fairy Fruit 0.0001 F +++ Fairy Fruit 0.0001 K + Goodenia ovata 0.01 F ++ Goodenia ovata 0.001 K ++++ Goodenia ovata 0.0001 K +++ Yellow Pittosporum flowers 0.01 F + Yellow Pittosporum flowers 0.001 F ++++ Yellow Pittosporum flowers 0.001 K ++++ common sunflower 0.01 F + common sunflower 0.01 K ++++ Melaleuca alternifolia 0.001 K ++++ Melaleuca alternifolia 0.0001 K +++ Omolanthes populifolius 0.0001 K +++ Melicope hayesii 0.01 K ++++ Medemia nobilis 0.01 K ++++

Compounds 1-5 and chalmicrin were also tested for their ability to upregulate dynein according to the method of Example 2. The results are reported in Table 5.

Table 5

0.00005 F ++ 0.0005 K ++ 0.00005 F ++ 0.00005 F ++ 0.00001 F ++ Chalmicrin 0.0001 K ++++

Example 4

Downregulation of dynein

Various compounds and plant extracts were tested for their ability to downregulate dynein according to the method of Example 2. The results are provided in Table 6 below, where the concentrations of the plant extracts are provided based on the dry weight of the given plant extract. Statistical significance was achieved at a level of p < 0.05 or better in each case.

Table 6

Salicylic acid derivatives provided the greatest downregulation of mRNA for the cytoplasmic dynein heavy chain DYNC1H1, producing a 45.03% reduction in keratinocytes (p=0.02), followed by Tliliacora Triandra, which gave a 36.7% reduction in fibroblasts (p=0.01).

Example 5

In vivo upregulation of dynein

The ability of the plant extract to upregulate dynein in vivo was tested. 33 healthy female Caucasian subjects aged 30-65 years with skin type II or III were treated with the raw material on the dorsal forearm for 3 weeks (3 consecutive rounds of 5x24 hour patches under semi-occlusive conditions). The test article and vehicle were applied to five sites randomly assigned on each forearm. The applied dose was 2 mg/cm ² . After treatment, a 2 mm punch biopsy was obtained from each treatment site and fixed in 4% paraformaldehyde and processed for IHC studies. IHC was performed using a rabbit polyclonal antibody against dynein and observed using vector blue conjugated to a goat anti-rabbit secondary antibody, as described in Example 1. The results shown in Table 7 below are expressed as the number of patients (N) who showed upregulation, no change, or downregulation of dynein levels.

Table 7

As shown in Table 7, nearly all of the plant extracts tested upregulated dynein in vivo when applied topically to the skin. While P. truncatula did not show significant upregulation or downregulation in this study, based on its in vitro efficacy, it is believed that the results would be improved by using penetration enhancers to improve delivery to the skin and/or increasing the dose or duration of treatment.

All references cited herein (including patent applications and publications) are hereby incorporated by reference in their entirety and for all purposes to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Many modifications and variations of the present invention may be made without departing from its spirit and scope, as will be apparent to those skilled in the art. The specific embodiments described herein are provided by way of example only, and the invention is to be limited only by the terms of the appended claims and the full scope of equivalents to which such claims are entitled.

Claims (1)

1. Use of an active agent that upregulates dynein in the preparation of a formulation for treating wrinkles and/or fine lines, said treatment comprising applying said formulation topically to the skin of the face daily for at least one week at a dose of 0.001 mg/cm² to 100 mg/cm², wherein said active agent is an extract of the plant Medemia nobilis.