AU3750499A - Composition and method for treatment of aging skin - Google Patents

Description

WO99/55302 PCTIUS99/08497 COMPOSITION AND METHOD FOR TREATMENT OF AGING SKIN Field of the Invention The invention relates to cosmetic and pharmaceutical compositions. In particular, the invention relates to cosmetic and pharmaceutical compositions which enhance skin cell energy and rejuvenate aging skin cells. Background of the Invention As anyone into their middle years recognizes, the skin undergoes a number of mostly undesirable changes as it ages. These nature of these changes has now been fairly well documented on gross, tissue, and cellular levels. It is all too readily apparent to the naked eye that the skin of older individuals is drier, has more lines and wrinkles, and is generally more slack or loose than it was in youth. On the tissue level, many of these gross changes can be attributed to alterations in the production of collagen, which is greatly reduced in quantity, as well as being wasted and degraded in appearance. Interestingly, age-related changes can also be detected at the cellular and molecular level. As fibroblasts age, their cellular functions begin to decline. In particular, it has been observed that aging human fibroblasts in vitro are less responsive to stimuli which induce migration of cells in culture, and also replicate less frequently. Muggleton-Harris and Aroian, Somat. Cell Genet. 8: 41-50, 1982. It has been suggested that these changes are somehow related to age-related deficiencies in the normal cycle of ATP utilization and restoration. However, studies on the loss of replication have been said to indicate that it is not related to deficiencies in the ATP-restoring pathways (Goldstein et al. J. Cell Phys. 112: 419-424, 1982). Muggleton-Harris and 1 WO 99/55302 PCT/US99/08497 DeFuria(In Vitro Cell. Devel. Biol. 21: 271-276, 1985) in treating cells with metabolic poisons, showed that there is an age-dependent change in cells' ATP content and turnover rate, leading to the suggestion that aging cells lose some of their capacity to utilize ATP. Whatever the reason, however, the end result is an apparently inevitable reduced production in collagen, resulting in the typical skin atrophy seen in aged skin, and this loss of cellular function and decline in cell appearance appear to inextricably intertwined. It would be desirable to be able to alter this inexorable descent into cellular old age, in the hopes of prolonging the "youthful" energy exhibited by younger fibroblast cells. However, to date, there has been no remedy for this aspect of the aging of skin cells. The present invention now provides a means for enhancing the ATP levels of aged fibroblasts. Summary of the Invention The invention provides a method for increasing ATP levels in aging cells which comprises applying to the skin an effective amount of ADP, AMP, or oxaloacetic acid, or a combination thereof. The method is particularly useful in increasing energy in skin cells, particularly fibroblasts. The invention also provides cosmetic and pharmaceutical compositions for topical application to the skin comprising effective amounts of ADP, AMP, or oxaloacetate, or a combination thereof in a cosmetically or pharmaceutically acceptable carrier.. As used herein, the terms "ADP", "AMP" and "oxaloacetic acid" shall include safe and effective derivatives thereof which retain qualitatively the same activity. Detailed Description of the Invention 2 WO 99/55302 PCT/US99/08497 The present invention is based on an observation that the extrinsic provision of ADP, AMP or oxaloacetic acid(oxaloacetate) to skin cells results in an increase of cell energy present in the cells so treated. In particular, when ADP, AMP or oxaloacetic acid is provided to normal fibroblasts in culture, ATP levels can be increased substantially. Thus, use of ADP and/or AMP and/or oxaloacetic acid as active agents in the treatment and prevention of skin aging is indicated. The compositions of the invention comprise effective amounts of ADP, AMP or a combination thereof, in combination with a cosmetically or pharmaceutically acceptable carrier. By "effective amount" is meant that amount of the active agent which can increase the amount of ATP in a treated cell at least about 10%, preferably at least about 20%, more preferably at least about 30%, and most preferably at least about 40%, relative to untreated cells. In a typical composition, the concentration of the active agent will be from about 0.001-10%, preferably about 0.01-5%, by weight of the total composition; the concentration may be varied depending upon the intended frequency of use of the composition, lower concentrations being employed with more frequent applications. For topical application, the active components) can be formulated with a variety of cosmetically and/or pharmaceutically acceptable carriers. The term "pharmaceutically or cosmetically acceptable carrier" refers to a vehicle, for either pharmaceutical or cosmetic use, which vehicle delivers the active components to the intended target and which will not cause harm to humans or other recipient organisms. As used herein, "pharmaceutical" or "cosmetic" will be understood to encompass both human and animal pharmaceuticals or cosmetics. Useful carriers 3 WO99/55302 PCT/US99/08497 include, for example, water, acetone, ethanol, ethylene glycol, propylene glycol, butane-1,3-diol, isopropyl myristate, isopropyl palmitate, or mineral oil. Methodology and components for formulation of cosmetic and pharmaceutical compositions are well known, and can be found, for example, in Remington's Pharmaceutical Sciences, Eighteenth Edition, A.R. Gennaro, Ed., Mack Publishing Co. Easton Pennsylvania, 1990. The carrier may be in any form appropriate to the mode of delivery, for example, solutions, colloidal dispersions, emulsions(oil-in-water or water-in oil), suspensions, creams, lotions, gels, foams, mousses, sprays and the like. The active agents of the invention can be used alone to increase the energy level of skin cells generally, so as to delay or reverse the onset of the cellular symptoms of aging. By "aging", as used in the present specification and claims, is meant both photoaging, i.e., the premature aging which occurs as the result of excessive exposure to UV rays, and chronological aging, i.e., the naturally occurring, normal aging of the skin which occurs over time, even without prolonged sun exposure. The compositions of the invention are particularly suited for enhancing the energy levels of fibroblasts and keratinocytes in both normal and aging skin. Increase in energy levels of fibroblasts can be expected to delay or reverse the decrease in collagen and elastin production that characterizes aged fibroblasts. Similarly, the increase in energy levels of keratinocytes can be expected to result in a delay in the thinning of the epidermis observed in aging skin. The use of the active agents of the invention is not limited to skin cells, however, but may also be expected to aid in energy increase in, for example, aging muscle or other connective tissue cells. 4 WO 99/55302 PCT/US99/08497 Treatment of aging skin, or prevention of aging in normal, non-aged skin is preferably achieved by regular application of the composition over a period of time. A preferred method of obtaining the benefits of the composition is via chronic topical application of a safe and effective amount of a composition containing the mixture, to prevent or delay development of skin damage which may result from photo- or chronoaging, or to prevent worsening of or to reverse already established damage. It is suggested as an example that topical application of the composition, in an amount of from about 0.1 Pg/cm 2 to 2 mg/cm 2 of exposed skin, be performed from about once per week to about 4 or 5 times daily, preferably from about 3 times a week to about 3 times daily, most preferably about once or twice per day. By "chronic" application, it is meant herein that the period of topical application may be over the lifetime of the user, preferably for a period of at least about one month, more preferably from about three months to about twenty years, more preferably from about six months to about ten years, more preferably still from about one year to about five years, thereby resulting in the treatment or prevention of the external signs of photoaging and chronological aging. However, the compositions of the invention may also be used on a less frequent basis, for example, once or twice a month, as a sort of "spa treatment" with a higher level of active component provided on these occasions. The external signs of aging that the composition may alleviate include, but are not limited to, fine and deep lines and wrinkles, skin atrophy, reduction in skin thickness, changes in skin pigmentation, and reduction in hair growth. In their function as general anti-aging agents, the actives of the invention can also be combined with other 5 WO99/55302 PCT/US99/08497 anti-aging or skin-enhancing agents. For this purpose, the actives of the invention can be combined, for example, with one or more of the following products: alpha- or beta hydroxy acids, such as lactic acid, glycolic acid, citric acid, alpha-hydroxyoctanoic acid, alpha-hydroxydecanoic acid, alpha-hydroxylauric acid, tartaric acid, glucouronic acid, galactouronic acid, alpha-hydroxybutyric acid, alpha hydroxyisobutyric acid, malic acid, mandelic acid, pyruvic acid, and tartronic acid, and salicylic acid; retinoids, such as retinol, retinyl acetate, retinyl palmitate, retinyl butyrate, retinyl oleate, retinyl linoleate, and retinoic acid; and DHEA and derivatives thereof. The formulation, in addition to the carrier and active agents, also can comprise other components which may be chosen depending on the carrier and/or the intended use of the formulation. Additional components include, but are not limited to, water soluble colorants (such as FD&C Blue #1); oil soluble colorants (such as D&C Green #6); water soluble sunscreens (such as Eusolex 232); oil soluble sunscreens (such as octyl methoxycinnamate); particulate sunscreens (such as zinc oxide); antioxidants (such as BHT); chelating agents (such as disodium EDTA); emulsion stabilizers (such as carbomer); preservatives (such as methyl paraben); fragrances (such as pinene); flavoring agents (such as sorbitol); humectants (such as glycerine); waterproofing agents (such as PVP/Eicosene Copolymer); water soluble film-formers (such as hydroxypropyl methylcellulose); oil-soluble film formers (such as Hydrogenated C-9 Resin); cationic polymers (such as Polyguaternium 10); anionic polymers (such as xanthan gum); vitamins (such as tocopherol); and the like. As will be apparent, the compositions can be therapeutic products, ADP and/or AMP and/or oxaloacetic acid being the sole actives, 6 WO99/55302 PCT/US99/08497 or in combination with other actives. However, the compositions can also be a makeup products, for example, a lipstick, foundation, concealer, bronzer, blush, eyeshadow and the like. The invention is further illustrated by the following non-limiting examples. EXAMPLES Example I. This example illustrates the increase in cell energy levels produced by treatment of cells with ADP or AMP. Normal human dermal fibroblasts are grown to confluence in flasks, and then divided into three different treatment sets. Testing is done at the point at which the cells have undergone 32 population doublings. The treatments are with ADP(Sigma), AMP(Yamasa Shoyu Co., Inc.), and creatine monophosphate(Sigma), each at five different concentrations ranging from 0.01-1.0 mM. The growth medium serves as a control. The cells are treated for a period of 2 hours, and then trypsinized, washed and resuspended to 6X10 6 cells/ml. The cells are treated with a releasing agent, sodium lauryl sulfate, which causes the ATP contained within the cells to be released. The releasate is then added to a solution containing luciferin and luciferase. Luciferase utilizes the energy provided by free ATP to convert luciferin to oxyluciferin. The light released by this reaction is read on a spectrophotometer; the amount produced is proportional to the amount of ATP available. The ATP levels are calculated from an ATP standard curve, and then normalized on a per cell basis. The results are shown in Table 1. 7 WO 99/55302 PCT/US99/08497 TABLE 1 Sample ATP(pg/cell) % increase Media control 14.7 ADP 0.01mM 14.0 -5 0.05mM 17.7 20 0.10mM 18.7 27 0.50mM 23.0 56 1.00mM 20.7 41 AMP 0.01mM 14.7 0 0.05mM 19.0 29 0.10mM 19.0 29 0.50mM 20.0 36 1.00mM 19.3 31 Creatine 0.01mM 16.7 14 0.05mM 13.3 -9 0.10mM 14.7 0 0.50mM 12.7 -14 1.00mM 15.0 2 The results shown above indicate that ADP increases ATP levels in fibroblasts in a dose dependent manner. AMP is also found to increase the ATP levels of treated cells, but not to the same extent as ADP, and not in a dose dependent manner. Creatine monophosphate, however, does not significantly increase ATP levels at any dose tested. The maximal increase achieved by ADP is +56% at 0.5mM, whereas the maximal increase achieved by AMP is +36% at 0.5mM. 8 WO99/55302 PCTIUS99/08497 Example 2 This example illustrates the increase in cell energy levels produced by treatment of cells with oxaloacetic acid. Normal human dermal fibroblasts are grown to subconfluence in flasks, treated with oxaloacetic acid for two hours with three samples, having concentrations of 0.05 mM, 0.50mM and 1.0 mM. The cells are then trypsinized, washed and resuspended to 6X10 6 cells/ml. The treated cells have undergone four passages. The cells are further treated and analyzed as described in the previous example, with the amount of ATP calculated at the various dosages. The results are shown in Table 2. TABLE 2 Sample ATP(pg/cell) % increase Media control 1.67 OxAc 0.05mM 1.92 15 0.50mM 2.58 55 1.0mM 2.17 30 These data show that oxaloacetic acid at all concentrations increased ATP levels in treated cells, with a maximal increase of 55% observed at the 0.1mM dose. 9

Claims (18)

1. A cosmetic or pharmaceutical composition for topical application to the skin comprising an effective amount of ADP, AMP or oxaloacetic acid, or a combination thereof, with a cosmetically or pharmaceutically acceptable carrier.

2. The composition of claim 1 comprising from about 0.001 to about 10% of ADP, AMP, oxaloacetic acid or a combination thereof.

3. The composition of claim 2 comprising from about 0.1 to about 5% of ADP, AMP, oxaloacetic acid or a combination thereof.

4. The composition of claim 1 comprising ADP.

5. The composition of claim 1 comprising AMP.

6. The composition of claim 1 comprising oxaloacetic acid.

7. A method for increasing the energy level in cells which comprises applying to the skin an effective amount of the composition of claim 1.

8. The method of claim 7 in which the cells are skin cells.

9. The method of claim 8 in which the cells are fibroblasts.

10. A method for increasing the energy level in skin cells which comprises applying to the skin an effective amount of the composition of claim 2. 10 WO 99/55302 PCT/US99/08497

11. A method for increasing the energy level in skin cells which comprises applying to the skin an effective amount of the composition of claim 3.

12. A method for increasing the energy level in skin cells which comprises applying to the skin an effective amount of the composition of claim 4.

13. A method for increasing the energy level in skin cells which comprises applying to the skin an effective amount of the composition of claim 5.

14. A method for increasing the energy level in skin cells which comprises applying to the skin an effective amount of the composition of claim 6.

15. A method of treating or preventing symptoms of aging in the skin which comprises applying to the skin an effective amount of the composition of claim 1.

16. The method of claim 15 in which the symptom is skin atrophy.

17. The method of claim 15 in which the symptom is loss of skin thickness.

18. The method of claim 15 in which the symptoms are lines and wrinkles. 11