MXPA99007883A - Methods and compositions for preventing and treating chronological aging in human skin - Google Patents

Abstract

The deleterious effects of the passage of time on human skin (i.e., chronological aging of human skin) can be prevented and treated with the topical application of a retinoid, preferably retinol. We have found that some of the same pathways (namely the stress-activated pathways, SAPs) activated in photoaging of human skin (i.e., sun-induced premature skin aging) are similarly elevated in the skin of elderly people. We have also found that other pathways (namely the mitogen-activated ERK pathway) is depressed in the same skin. Treatment of chronologically-aged skin with a retinoid both inhibits degradation of dermal collagen and promotes procollagen synthesis. Biopsied sections from skin of elderly (80+ years old) show that a single treatment can increase epidermal thickness, improve the dermal collagen density, and promote the formation of rete pegs and dermal papillae (see Fig. 13), and can decrease the amount of c-Jun and increase the amounts of Types I and III procollagen (see Fig. 18). Such benefits are also helpful in preventing bruising, tearing, and ulceration of elderly skin.

Description

METHODS AND COMPOSITIONS FOR PREVENTING AND TREATING CHRONOLOGICAL AGING IN HUMAN SKIN DESCRIPTION OF THE INVENTION This application is based in part on co-pending provisional applications 60 / 040,594, filed on February 25, 1997, and 60 / 042,976, filed on 7 April 1997, the description of which are incorporated herein by reference. This invention relates to methods and compositions, especially those comprising retinoids, preferably applied topically, which are useful for improving the proliferation of keratinocytes and fibroblasts, decreasing the expression of the metalloproteinase (MMP) matrix, and improving the synthesis of collagen in aged skin, thereby providing as an effect the rejuvenation of aging skin. As for mammals, humans are essentially hairless; that is, most of the skin of the human body can be seen without hair interference. The skin in this way is exposed to any type of inclemency (natural and human) that the environment can create. Since at first it was thought that the sun causes erythema, people took measures to avoid their "harmful rays". A century ago, in England during the period of Queen Elizabeth, it was fashionable to avoid the sun at any cost. Even so, the skin of those people still wrinkled and displayed other signs of chronological aging. Human skin is a complex organ which extends throughout the body. There are different types of skin in different parts of the body; For example, the facial skin is different from that of the scalp, and even the skin of the palm of the hand is different from that of the torso of the hand. Although the type of skin can vary throughout the body in the person, the skin is usually composed of two main layers of tissue. The epidermis or cuticle, the outermost layer, is composed of superficial layers (from the outside inwards: stratum comeum, stra tum lucidem and stra tum granulosum) and deeper layers (stra tum spinosum and stratum basale). The dermis, cutis vera, or natural skin, is composed of a papillary layer on top and a reticular layer underneath. For a long time, a variety of substances have been applied to the skin to improve its appearance, generally affecting the outermost layer of the skin, or to treat some skin disease, generally affecting the natural skin. More recently, efforts have been made to rejuvenate the skin and claim the elasticity and softness losses from exposure to sunlight (UV radiation) and weather.

There is a difference between the physiology of chronologically aged skin or intrinsically aged skin (old skin) compared to that of photoaged skin (ie, skin that looks old due to damage by UV solar irradiation). Old skin typically maintains an appearance without flaw compared to the leathery, stained and often deeply wrinkled appearance of photoaged skin. The epidermis of old skin is typically thinner than normal, whereas that of old photoaged skin is typically thicker than normal (acantotic) and atrophies over time.

Photoaged skin usually has a large Grenz area (a broad band of eosinophilic material just below the epidermis, and the formation of collagen and structures that indicate the healing of lesions, which is absent in chronologically aged skin.) See also NA Fenske and CW Lober, "Structural and functional changes of normal aging skin ", J. Am. Acad. Derma tol., 15: 571-585 (1986) Kligman et al., in EP-A2-0 379,367 describes a method for the treatment or prevention of intrinsically aged skin with retinoids, Kligman et al., tested all trans-retinoic acids (such as Retin-A® cream) in albino hairless mice and in 5 older Caucasian women, only clinical observations were made in the women before and after the study, and only one biopsy was reported and this occurred six months after the treatment (ie, there is no description on this publication of a reference biopsy taken from the subject subjected to biopsy before treatment or an early period of treatment). * U.S. Patent Nos. 3,932,665 and 4,934,114 describe the use of retinol (Vitamin A aldehyde), for the treatment of acne and for the treatment of skin keratosis, respectively, see also United States Patent NO. . 3,060,229. Retinol and its derivatives have also been suggested as useful in the treatment of such conditions as wrinkles, pettiness, psoriasis, eczema, dandruff and the like (see EP-A2-0 391 033). There is also evidence indicating that tretinoin (all trans retinoic acids) improve the appearance of photoaged skin. Albert M. Kligman, "Current Status of Topical Tretinoin in the Treatment of Photoaged Skin," Drug & amp;; Aging, 2 (1): 7-13 (1992); and Chas. N. Ellis et al., "Tretinoin: Its Use in Repair of Photodamage", and A.S. Zelickson et al., "Topical Tretinoin in Photoaging: An Ultrastructural Study, "both in Journal of Cutaneous Aging & Cosmetic Dermatology, Vol. 1, No. 1, p. 33-40 and 41-47 (1988). Burger et al. , in the United States Patent ,665,367, describes compositions for topical applications on the skin containing naringenin and / or quercetin, and a retinoid. The compositions are described as being useful for treating many unrelated skin conditions, such as wrinkles, acne, skin lightening, and age spots. The action of its composition on human skin is described with respect to an enzyme (transglutaminase) important in the formation of the envelope of the cell and thus the epidermis. In contrast, the present invention is directed to changes in the dermis and in the proliferation of cells of beneficial dermal structures. The main invention is the discovery of a method to rejuvenate human skin. As used with respect to the description and claims of this invention, "rejuvenate" includes the steps of simultaneously preventing the degradation of collagen and stimulating the formation of new collagen in aged human skin. The invention is based on biopsies of human skin protected from the treated and untreated sun of older volunteers (80 or more years old) compared to protected skin biopsies of younger individuals. Compared to the skin of younger people, aged skin is thin, has few cells in the epidermis (keratinocytes) and the dermis (fibroblasts), has less density and more disorganized connective tissue, has higher levels of kinase activity cJun and matrix metalloproteinases (MMP), and has reduced levels of ERK activity, cyclin D2, and procollagen of Types I and III. In summary, it has been found as an invention that aged human skin can be rejuvenated by topical administration of one or more compounds in effective amounts to inhibit collagen degradation and to promote the synthesis of procollagen, the application preferably being performed in a regular basis. The preferred class of compounds that perform both functions are retinoids, especially retinol and all trans-retinoic acids. Aged human skin can benefit by improving the synthesis of procollagen. It has been found as another invention that procollagen levels can be increased in aged human skin by the preferably regular application on the skin of effective amounts of a retinoid, in preferred embodiments, the treatment also includes the inhibition of collagen degradation by the use of an MMP inhibitor. In addition to treating and / or preventing chronological aging of the skin, the discovery that effective amounts of a retinoid applied to the skin can increase the synthesis of procollagen provides another invention in the prevention (prophylaxis) of skin ulcers. By increasing the collagen content of the skin, the shape, strength and function of the skin are improved. The increase in procollagen synthesis, and thus an increase in collagen content of the skin, mitigate the loss of strength and support due to reduced collagen degraded, and thus the synthesis of improved procollagen is expected to decrease the occurrence and / or severity of skin ulcers. In conjunction with the above, it has been found as yet other inventions, that keratinocytes and fibroblasts, each beneficial to the integrity of the skin, are increased in number by the topical application of the retinoid, once again preferably applied on a base regular. The fibroblasts are tropic to the epidermis; under normal conditions they secrete a number of growth factors (eg, FGF, IGF, and KGF, among others) and produce procollagen that enters the dermal matrix and becomes structural collagen. Yet another invention is the decrease of cJUN activity and / or reducing the amount of c-Jun protein present in the skin, and increasing ERK activity, both in aged skin, by the topical application of an effective amount of the retinoid on the skin. aged skin In further embodiments, the prophylaxis and treatment of chronological aging of the dermis is each improved by applying it together with the active ingredient to at least one additional compound selected from: a sunscreen, at least one of UVA1, UVA2 and UVB; an antioxidant; an MMP inhibitor (metalloproteinase matrix); and mixtures thereof. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 represents the representation of certain pathways of ERK and SAP in an idealized skin cell. Figure 2 represents the density of fibroblasts (2A), the characteristics of dermal connective tissue (2B), in the epidermal thickness (2C) and the density of keratinocytes (2D) in populations of all ages that were studied and through biopsy. Figure 3 represents the relative growth potential (measured as the number of cell colonies per biopsy) of human keratinocytes (3A) and fibroblasts (3B) in populations of various ages studied. Figure 4 represents the relative activity of three MMPs (MMP-1 in 4A, MMP-9 in 4B, and MMP-2 in 4C) found in each of the populations of different ages studied. Figure 5 depicts the differences in expression of interstitial collagenase in vivo between young and aged skin. Figure 6 depicts the differences in expression of Type I procollagen in vivo between young skin and aged skin.

Figure 7 depicts the differences in ERK activity in vivo between young skin and aged skin. Figure 8 depicts the differences in the expression of phosphorylated (activated) ERK in vivo between young skin and aged skin. Figure 9 depicts differences in the expression of cyclin D2 in vivo between young skin and aged skin. Figure 10 depicts differences in cJUN kinase activity in vivo between young skin and aged skin. Figure 11 represents the change in fibroblast density (HA), the characteristics of dermal connective tissue (11B), epidermal thickness (11C), and keratinocyte density (11D) in aged skin (80 years or more), when applying a retinoid (1% retinol, one application, occluded, and examined seven days later). Figure 12 depicts the effect of retinoid (retinol) treatment on the growth of human keratinocyte fibroblast cells cultured ex vivo from the biopsy samples. Figure 13 shows cross-sections and cross-sections stained from the skin of a young individual (13A and 13B), an older individual (13C and 13D) and the same older individual (13E and 13F) after a single application of 1% retinol (occluded, for seven days, and then the biopsy was performed). Figure 14 describes the effect on the activities of three MMP enzymes (MMP 1, 2 and 9, analogous to Figure 4), after a single application of retinol to the aged skin as determined by the biopsy. Figure 15 depicts the induction of retinoic mediated acid from the synthesis of Type I collagen in cultured human skin fibroblasts. Figure 16 depicts the in vivo induction of procollagen to (III) mRNA of Type III caused by the administration of retinol to aged skin. Figure 17 depicts the in vivo induction of ERK activity caused by the administration of retinol to aged skin. Figure 18 is a photomicrograph showing in stained biopsies stained of aged skin that, seven days after a single application of 1% retinol, the c-Jun protein and the increase in the amounts of procollagen of the types I and III on the skin. Figure 1 represents certain degrading pathways that verify the functioning of an idealized skin cell based on discoveries. The particularly important causes of the chronological aging of human skin probably vary among a population of older humans, including such factors as diet, genetics, and the environment. In general, although it is believed that the chronological aging of the skin is due to the activation of trajectories activated by stress (SAP), and to a repression of trajectories activated by mitogens (ERK). Contrary to conventional knowledge, compared to inventions that are related to the photo-aging of human skin, it has been found that chronological aging and photo-aging of human skin have similar molecular pathophysiology. The ERK serves as a mediator of the actions of the growth factors necessary for healthy skin. Interference with ERK can lead to thinning of the chronologically aged skin due to the reduced number of cells in the epidermis and dermis. Almost on the contrary, SAPs activate factors (for example, c-Jun) that promote both the inhibition of procollagen synthesis and the degradation of mature collagen, and with this they lead to a reduced form, strength and function of the skin. It can be expected that the chronological aging of the skin includes some interference with the ERK and / or some activation of the SAP. It has been found that both cases occur in chronologically aged human skin. As shown in Figure 1, the idealized skin cell 101 has a cell membrane 103 through which various compounds pass or in which they interact with the receptors via the cell on the surface of the cell. An input group indicated by 105 activates the ERK path which activates ERK by phosphorylation. Activated ERK induces D2 107 cyclin formation in the nucleus of the cell, with the result that the growth of the cell is promoted. Another input group is indicated by 109, which activates the pathway activated by stress, which leads to the increase in cJUN kinase activity. Once activated (once again phosphorylation), c-Jun becomes a component of AP-1, which leads to the formation of MMP 111 and the export of MMPs from the cell, resulting in the degradation of collagen in the dermal matrix. Matrices of metalloproteinases (MMPs) include collagenases, gelatinases and other enzymes that occur naturally in human skin that degrade extracellular matrix molecules, such as collagen. While not wishing to be bound by a particular theory, it is believed that the increase in the activity of the kinase cJUN interferes with the synthesis of procollagen (precursor of soluble collagen), which is then exported (113, with reference to the Figure 1) of the cell within the matrix to become structural collagen (insoluble collagen). It is believed that activated cJUN inhibits one or more steps in the synthesis of procollagen, as shown in the figure.

The invention is generally directed to the topical administration, preferably on a regular basis, of an amount of retinoid, preferably retinol or retinoic acid, on the skin of an elderly person in effective amounts to induce the proliferation of at least one of keratinocytes and fibroblasts, to reduce the expression of MMP, to stimulate the synthesis of procollagen in the skin of the elderly back to normal levels, and / or to increase the level of activated ERK and to reduce the activity of the cJUN kinase and to reduce the level of c-Jun protein in aged skin. Analysis of Aging Skin It has been found that there is a decrease associated with age in the number of keratinocytes and fibroblasts as determined for the comparisons of the biopsy repetitions with 4 mm perforations obtained from the skin protected from the sun of 40 individuals , varying from 18 to more than 80 years. Keratinocytes are the main cells that comprise the epidermis. The cells of the epidermis arise from the differentiation of the basal keratinocytes, some of which differentiate through successive underlying layers to become the comet. (outermost layer of the skin). Figures 2A, 2B, 2C and 2D represent the morphometric data based on the entire study of the population that was conducted. The biopsies of our voluntary population showed an average decrease of 27% (keratinocytes) and 39% (fibroblasts) when the group of younger age (18-29 years) was compared with the older group (80 years or older) ) (p < 0.1 for both cell types), as shown in Figures 2A and 2D; It was also noted that the changes associated with age on the skin are seen as a decrease in fibroblasts above the age of 30 and another decrease above the age of 80 (Figure 2A). This population study also evidenced an increase in the number of features of undesirable dermal connective tissue above the age of 60 (Figure 2B) and a decrease in epidermal thickness above the age of 60 (Figure 2C). These data show that there is an increase associated with age in connective tissue disorganization and / or degeneration (a double increase of 2.25 in the older age group compared to the younger age group, p <0.05), as it was shown in Figure 2B. The disorganization and degeneration of the connective tissues were measured by a microscopic histological examination of the skin tissue taken from the biopsy obtained from these older subjects and compared with the skin histology taken from the biopsy of the younger individuals. These results were later confirmed by the discovery of the decreases associated with age in keratinocytes (54% decrease) and the growth of fibroblasts (50% decrease), determined as the number of cell colonies, in the culture of the tissue taken from the biopsy, compared between the same two age groups (p < 0.1 for both cell types), as shown in Figure 3. As shown in Figure 4, an increase related to the age in the relative activities of MMP-1, MMP-2 and MMP-9, when these groups of young and old individuals are compared (respectively, average increases of 40%, 82% and 53%, respectively p <0.01) , p < .001 and P < 0.01). these results were determined using repeat biopsies with 4-mm perforations of the 40 volunteer subjects. As shown in Figure 5, measurements of skin interstitial collagenase taken from the biopsy of 16 individuals in each of the two groups (young and old), revealed that the relative expression of the collagenase protein was present in the skin of older subjects in amounts close to double compared to that found in young subjects. Figure 6 represents the analysis to determine the presence of Type I procollagen in in vivo samples of the unexposed skin taken from each of the two groups of tested individuals (young and old). It has been found that about twice as much procollagen of Type I is expressed in the skin of young subjects not exposed (protected from the sun) than in the skin of subjects of unexposed age. Figure 7 shows the discoveries for the activity in uniform state of ERK in the skin biopsies of our volunteers, both of those of age (over 80 years) and young people (19-29 years), generally according to the methods described here. The histography in Figure 7 shows that the relative activity of ERK in the skin of aged volunteers is almost half of their activity level in the skin of young people. The reduced activity of ERK can result from reduced levels of ERK or reduced ERK activation in aged skin, or a combination of both. Therefore, fifteen volunteers were tested to determine the relative amounts of ERK present in the skin, both in the total form and in the activated form (phosphorylated), the activated form stimulates cell growth. As shown in Figure 8, the elderly subjects have essentially the same amount of total ERK in their skin as the younger half-century subjects, but they have significantly less ERK in their active phosphorylated form. Accordingly, it has been found that the reduced activity of ERK in the skin of the elderly is not due to a reduction in the total amount of ERK, but to the low concentration of the activated form.

In the trajectory of the ERK shown in Figure 1, ERK induces cyclin D2, which is required for cell growth. Again, it is expected that interference with this ERK determinant will lead to delayed cell repair and growth, and therefore the effects of aging on the skin will be promoted. Figure 9 represents the results analyzed in twelve young subjects and eleven elderly subjects for the expression of cyclin D2 in normally covered skin (protected from the sun). The histography of Figure 9 shows that the amount of cyclin D2 expressed in chronologically aged skin is significantly reduced compared to its expression in young skin. Fifteen subjects were tested in two age groups, young subjects (19-29) and subjects of age (over 80), to determine the degree of activation of stress-activated protein kinase ("SAPK") in each group; the activity of the SAPK is measured by the phosphorylation of the cJUN protein. Figure 10 shows that the in vivo samples of the unexposed skin of the young subjects had approximately 25% of the relative activity of the cJUN kinase compared to the unexposed skin of the elderly subjects. From these results, one can expect that high levels of c-Jun activated in the skin of elderly people lead to increased activity of MMP.

Treatment of Aging with Retinoid Skin Returning again to Figure 1, the activation of SAPs (stress-activated pathways 109) promotes the degradation of collagen in the skin through the production of MMPS, which includes collagenase and gelatinase 92 kDa SAPs can be activated or regulated by UV radiation (as described in co-pending application 08 / 588,771, registered on January 16, 1996, and provisional applications 60 / 048,520, filed on June 4, 1997, and 60 / 057,976 , filed on September 5, 1997, all relating to the photo-aging of human skin, the descriptions of which are incorporated herein by reference), the factors of tumor necrosis (eg, TNF-a), interlucin (eg, IL- la), and other stress. As described in the aforementioned application 771 and provisional applications 520 and 976, AP-1 induces MMPs, enzymes that degrade collagen. While it has been shown in the aforementioned photoenvexment patent applications that the UV-induced production of the collagenases may be interfered with, there are additional trajectories involved with chronological aging, and such trajectories determine the chronological aging of the skin. (for example, the ERK trajectory), do not necessarily intend to determine when a patient should be treated by photoaging of the skin (for example, using a compound that only inhibits the formation of MMPs will not necessarily determine the ERK path). To investigate the treatment of chronically aged skin, 17 subjects aged at least 80 years were provided with a topical treatment with 1% retinol or with the vehicle alone, the test area was occluded for seven days, and then biopsies were taken from the test area. The composition of the vehicle was a mixture of ethanol and polyethylene glycol in a volumetric ratio of 70:30. In comparison, the skin treated with the vehicle with the untreated skin of individuals of the same age range, there were no statistically significant differences in any of the parameters described with reference to Figure 2, that is, the number of fibroblasts and keratinocytes, the epidermal thickness and the characteristics of undesirable dermal connective tissue. When comparing the retinol-treated skin with the vehicle-treated skin of the same individuals, as shown in Figure 11, the numbers of keratinocytes (HA) and fibroblasts (11D) were increased per retinol-treated section of skin (273). % (p < 0.001) and 30% (p < 0.05), average increases, respectively). In addition, treatment with retinoids increased the epidermal thickness substantially (11C). Due to the short duration of treatment with retinoid (Rethol), there were small obvious changes in the number of features of harmful dermal connective tissue (11B). Figures 12A and 12B demonstrate the effects of retinol treatment for 7 days in vivo on an ex vivo growth of keratinocytes and fibroblasts extracted from sun-protected skin biopsies taken from our volunteer individuals over 80 years of age. That is, an old volunteer was treated with retinol (a 1% retinol application), occluded for seven days), biopsies were taken from the treated area, and the keratinocytes and fibroblasts from the biopsy were cultured ex vivo. This retinol treatment of the cells in vivo resulted in a substantial increase in the ex vivo growth of both cell types. In particular, the growth of the keratinocytes increased approximately 30% (12A) while the growth of the fibroblasts increased approximately 200% (12B), with p < 0.05 for both. Accordingly, it is expected that the topical application of a retinol on aged skin will increase the number of keratinocytes and / or the number of fibroblasts in the skin. The graphic results of the novel treatments are shown in Figure 13. Figures 13A and 13B are photomicrographs showing the histological appearance of sun-protected skin of a 22-year-old individual, the portion in Figure 13B is a view enlarged area within a box in Figure 13A. As shown within it, the skin is composed of the epidermis E underlying the dermis D. Part of the adhesion between the epidermis and the dermis is facilitated by a large interfacial area between the epidermis and the dermis. This interface is defined by the projections or ridges R that extend downwards from the epidermis into the dermis and by the dermal papilla P that extends upwards from the dermis into the epidermis. This projection and papilla create the double cross sectional views of Figure 13A and increase the interfacial surface area between the two layers of skin. The section at the bottom (Figure 13B) shows a more detailed view of the dermis that contains few C cells and is mostly collagen L. As seen in this section of a young individual, the collagen in the dermis is relatively dense and of a uniform structure. Figures 13C and 13D show the histology of the skin protected from the sun treated with the vehicle of an 86-year-old individual. As seen in Figure 13C, the epidermis is thinner in the aged skin and there are essentially no protrusions and essentially no dermal papilla. This detailed view in Figure 13D shows that the dermis of aged skin generally has fewer cells and has collagen that is less dense and denser evenly distributed than that found in younger individuals. The thinner epidermis and the decrease in interfacial surface area between the epidermis and the dermis tend to cause older people to have a higher incidence of bruising and ulcerative conditions such as Bateman's purpura. Figures 13E and 13F depict skin protected from the sun, treated with retinol from the same individual of which a biopsy was shown in Figures 13C and 13D after 7 days of being treated as described above (an application of retinol). The changes shown on the skin are really remarkable and unexpected. The thickened epidermis, the interfacial surface area increased as evidenced by the presence of new protrusions and dermal papilla and as shown in the detailed view the dermal collagen became denser and more regular in its appearance. Thus, the topical application of an effective amount of a retinoid acts to increase the thickness of the epidermis (eg, normalize with respect to young skin), to promote the formation of protrusions and dermal papilla, and to increase the amount , density and regularity of collagen in the dermis. These changes reversed the apparent histological changes seen in aged skin and helped prevent similar re-stunning, ripping, ulceration and trauma in aged skin that does not occur in young skin.

Biopsies of the aforementioned volunteers were also used to determine the effect with the same retinoid treatment on the relative activity of the same collagenase enzymes studied for the results shown in Figure 4. Figures 14A, 14B and 14C represent the levels of average activity of MMP (for collagenase MMP-1 and gelatinases MMP-9 and MMP-2, respectively), after treatment of elderly individuals with retinol. As shown in Figure 14, seven days after a single treatment with retinol, the activities of MMP-1 and MMP-9 both decreased (48% and 39% decrease, respectively with p <0.001 for both enzymes ). No significant changes were observed in the activity of MMP-2. Enzyme activity levels were significantly reduced for MMP 1 and 9, as compared to vehicle control treatment alone. (In Figures 14A-14C, the number of subjects for each figure was 10, "*" indicates p <0.5 versus values of 18-29 years of age, "**" indicates p <0.1, and " *** "indicates p <0.001). Using the above and analogous techniques, using samples from seven volunteers over 80 years old, and cultured fibroblasts from biopsy samples of their unexposed skin (protected from the sun), a retinoic acid concentration of 0.25 μg / ml was found. increased the relative expression of collagen to triple that of the untreated cells, and 0.5 and 1.0 μg / ml generally increased the biosynthesis of collagen in these cells grown to the fivefold from untreated cells as shown in Figure 15. Seven Individuals over 80 years of age were treated clinically with a 1% retinol cream once applied to the skin protected from the sun, covered with a patch, left undisturbed for seven days. Biopsies from these areas treated under the patch revealed that procollagen Type III mRNA, increased in the skin of these individuals approximately 2.5 times compared to the control areas (treated with vehicle) treated with the same method (one application and covers for seven days). These results are shown in Figure 16. Using the same technique previously described for treatment with retinoid (1% retinol applied to the skin protected from the sun, occluded and examined seven days later), they were treated and then three of the volunteers over 80 years of age to determine the effect of retinoid treatment on ERK activity. The results of the biopsies of these individuals showed that after the treatment, the ERK activity was more than doubled compared to a biopsy of the area treated with the vehicle of the same volunteers. These results are shown in Figure 17 (in which the control, the skin treated with the vehicle, was normalized to a value of one). Additional stained and sectioned biopsies of the age group (greater than 80) of the volunteers after retinoid treatment are depicted in Figure 18. As mentioned above, AP-1 induces the formation of MMP and is formed by the heterodimerization of proteins c-and c-Fos. Figure 18A shows that after one week of treatment with retinoid (retinol, "ROL"), the level of c-in the aged skin (c-being stained red) significantly decreased (almost absent) compared to a section taken from the biopsy of the skin treated with the vehicle. Thus, the invention appears to inhibit the formation of c-and thereby inhibit the formation of AP-1 and the resulting MMPs. Figures 18B and 18C show that procollagen levels Types I and III (red staining) are improved on skin treated with retinoid. (The seemingly lower density of procollagen in the dermis in Figure 18C is an artifact of the section made - clearly, there is a higher level of staining in the section treated with retinol than in the section treated with vehicle). Therefore, in one embodiment, the invention comprises a method for rejuvenating aged skin, by applying a non-toxic effective amount of a retinoid for an effective period of time. The effective period of time is usually daily, preferably a single application / administration of the composition each day. The treatment and preferred maintenance regimens utilize an effective amount of approximately 0.4% retinoid, although higher doses can be used where warranted. Retinol is a preferred retinoid. In another embodiment, the invention provides a method for inducing keratinocytes and / or proliferating fibroblasts in vivo by topically administering a nontoxic effective amount of retinoid (preferably retinol or all trans retinoic acids) for an effective period of time. Again, the treatment is preferably daily, once or twice, with the retinoid amount preferably from about 0.1% to about 1.0%. In yet another embodiment, the invention reduces and / or inhibits the expression of MMP-1 and / or MMP-9 in aged skin, by topical administration of an effective amount of a retinoid for an effective period of time.; once again the preferred retinoids are retinoids and all trans retinoic acids. Again, the treatment preferably is daily, once or twice, with the amount of retinoid preferably between about 0.1% to about 1.0%.

As mentioned above, the decrease in keratinocytes and fibroblasts, and the increase in the expression of MMP, can be seen as an age-related condition without resorting to grievances such as sun damage. This invention thus provides prophylaxis against a detrimental change in any of these age-related parameters, as well as providing a treatment to ameliorate these age-induced detrimental etiological changes. In another aspect, this invention is directed to improve, or stimulate, the synthesis of procollagen types I and III in the skin of the elderly. It has been found that there is a significant reduction in the synthesis of procollagen types I and III in the skin protected from the sun of many individuals of age (at least 50% of) and that this condition can be treated by topical application of the retinoid. Procollagen is a protein synthesized by fibroblast cells from the skin and then secreted into the extracellular environment, where it is converted to collagen by naturally occurring enzymes. The synthesis of reduced procollagen in the skin of an elderly person is manifested as the reduced presence of procollagen protein both in the upper (extracellular) dermis and in the fibroblasts through the dermis, and can be determined (for example) by immunohistochemistry .

Taken together, the investigation of the control trajectories shown in Figure 1 indicates that the chronological growth of the skin may be caused by the deactivation of the ERK and / or by the activation of the SAPs. In fact, it has been found that both cases occur in culture skins. The results presented in Figure 7-9 show that the skin protected from the sun of the elderly (whose skin has not usually been exposed to the sun on a chronic basis) has reduced amounts of active ERK and a reduced amount of cyclin D2, which leads to a reduction in cell growth. It has also been shown on the skin of older people that there is a reduction in the amount of procollagen synthesized from Types I and III (see Figure 6). If there is less cell growth in the dermis, then the epidermal cover is also likely to be compromised. The results of several ERK trajectory signaling components important for promoting growth in human skin indicate that chronological aging in human skin is characterized by a reduction in the activation of at least two components of the human skin. trajectory that promotes the growth of the cell. As the skin ages, the reduced activation of ERK and the reduced production of cyclin D2 lead to reduced cell growth which ultimately results in aging skin. The results shown in Figures 15-17 prove the in vivo effectiveness of retinoid treatment by increasing the level of ERK activity and procollagen production of Types I and III. On the other hand (the degradation of the dermis as opposed to the creation of a new dermis), the results represented in Figures 4, 5 and 10 show that the unexposed skin of the elderly has increased the activities of the cJUN kinase and MMP. The up-regulation of degrading MMP enzymes and the down-regulation of procollagen synthesis results in a deficiency of collagen, causing the aging of the skin and the impediment to repair aged skin. An increase in path activity that causes an increased rate of skin breakdown (such as through MMP-mediated degradation of the dermal matrix and inhibition of procollagen synthesis), concomitant with the decrease in activity of the skin. the trajectory that promotes cell growth (such as the decrease in ERK activity) both contribute to the chronological aging of human skin. Methods to prevent and rejuvenate chronologically aged skin, tested on non-exposed skin, protected from the sun, very often with occlusion of the treated place, are applicable to treat the chronological aging of the skin throughout the body, including the face and hands. Taken together with the teachings of the aforementioned patent and provisional applications aimed at photoaging, the daily application of a retinoid on the skin will lessen the effects of natural aging, as well as the effects of the sun's exacerbation on the aging of the skin. Thus, since it is not desired to be subject to a particular theory of function, it is believed that the results shown, for example, in Figures 13E and 13F are due, in part due to some combination of enhanced ERK activation, decreased amounts of c-Jun, and increased amounts of procollagen of Types I and / or III. Using the same technique described for the treatment with retinoid (1% retinol applied on the skin protected from the sun, occluded, and examined seven days later), three of the volunteers older than 80 years were tested. The results of the biopsies of these individuals show that after treatment, the activity of the ERK in vivo was more than doubled, as shown in Figure 13 (in which the skin treated with the control vehicle was normalized to a value of one) . Retinoids are a class of MMP inhibitors. The MMP inhibitors can act directly on the MMP and / or on the transcription factors AP-1 and NF-KB by which the MMPs are naturally produced. Aspirin and E5510 (described by Fujimori, T., et al., Jpn J Pharmacol (1991) 55 (1): 81-91) inhibits NF-? B activation. Retinoids such as those described in U.S. Patent No. 4,877,805 and dissociated retinoids that are specific for Al antagonism (such as those described by Fanjul et al., In Nature (1994) 372: 104-110), glucocorticoids, and Vitamin D3 target AP-1. Compounds for improving the therapeutic effect of vitamin D3 are described in copending application serial number 08 / 832,865 (J. Voorhees et al., "Method for Assessing 125 (OH) 2D3"), filed on April 4, 1997, the description of which is incorporated herein for reference. Other retinoids in addition to retinol, include natural and synthetic analogs of vitamin A (retinol), vitamin A aldehyde (retinal), vitamin A acid (retinoic acid (RA)), which include all trans, 9- cis and retinoic acids. 13-cis), etretinate and others as described in EP-A2-0 379367, and U.S. Patents 4,887,805 and 4,888,342 (the descriptions of which are incorporated herein by reference). It is expected that various retinoids and synthetic compounds having retinoid activity will be useful in this invention, to the extent that they exhibit retinoid activity in vivo, and such are described in several patents assigned in their phase to Allergan Inc., such as in the following United States Patents numbers: 5,514,825; 5,698,700; ,696,162; 5,688,957; 5,677,451; 5,677,323; 5,677,320; 5,675,033; 5,675,024; 5,672,710; 5,688,175; 5,663,367; 5,663,357; 5,663,347; 5,648,514; 5,648,503; 5,618,943; 5,618,931; 5,618,836; 5,605,915; 5,602,130. Still other described compounds having retinoid activity are described in other numbered US Patents: 5,648,563; 5,648,385; 5,618,839; 5,559,248; 5,616,712; 5,616,597; 5,602,135; 5,599,819; 5,556,996; 5,534,516; 5,516,904; 5,498,755; 5,470,999; 5,468,879; 5,455,265; 5,451,605; 5,343,173; 5,426,118, 5,414,007; 5,407,937; 5,399,586, 5,399,561; 5,391,753; and the like, the descriptions of all of the above and the following patents and literature references are incorporated herein by reference. MMPs are also inhibited by BB2284 (described by Gearing, AJH et al., Nature (1994) 370: 555-557), GI129471 (described by McGeehan GM, et al., Nature (1994) 370: 558-561), and TIMP (tissue inhibitors of metalloproteinases, which they inhibit vertebrate collagenases and other metalloproteinases, including gelatinase and stromelysin). Still other compounds useful for the present invention include hydroxamate and hydroxy urea derivatives, such as Galardin, Batimastat and Marimastat, and those described in EP-A1-0 558635 and EP-A1-0558648 (described herein as useful for inhibiting MMP in the treatment of, among other etiologies, skin ulcers, skin cancer and bullous epidermolysis). They have been reported by Goldsmith, L.A. (Physiology, Biochemistry, and Molecular Biology of the Skin, 2nd. De. (New York: Oxford Univ. Press, 1991), chapter 17) that retinoids cause an increase in steady state TIMP mRNA levels suggesting a control transcriptional; although, it has been found that this is not true based on the findings here on human skin in vivo. Still other MMP inhibitors that can be applied topically to apply the claimed invention include the tetracyclines and derivatives thereof, such as minocycline, roliteraciclines, chlortetracyclines, metacycline, oxytetracycline, doxycycline, demeclocycline and the various salts thereof. Due to possible allergic sensitivity reactions, topical administration of tetracyclines should be carefully monitored so that there are no such undesirable reactions. Other MMP inhibitors include genistein and quercetin (as described in U.S. Patent Nos. 5637703 5665367 and FR-A-2, 671, 724, the descriptions of which are incorporated herein by reference) and related compounds, as well as other antioxidants such as NAC (N-acetyl cysteine), green tea extract and others. The effective amount of the active ingredient applied on the skin is preferably in the range of about 0.001-5% by weight, more preferably about 0.01-2% by weight, and even more preferably 0.1-1% by weight of the total weight of the animal. composition. The compositions are formulated to preferably provide about 5 μg / cm2 of skin when applied. For example, a preferred composition for use in this invention is Retin-A® gel retinoic acid and cream (available from Ortho Pharmaceuticals for the treatment of acne vulgaris), in concentrations from 0.01% to 0.1%; the vehicle preferably includes, depending on the particular formulation, at least one of butylated hydroxytoluene, alcohol (denatured with t-butyl alcohol and brucine sulfate), stearic acid, isopropyl myristate, polyoxyl 40 stearate, stearyl alcohol, and the like , and compatible mixtures of these. Fifteen subjects were tested in two different age groups, young subjects (19-29 years of age) and subjects of age (over 80 years) to determine the degree of activation of the SAP kinase in each group. The activity of the SAP kinase is measured by the phosphorylation of c-Jun. Figure 10 shows that the in vivo samples of the unexposed skin of the young subjects had approximately 25% relative kinase activity relative to the unexposed skin of the elderly subjects. From these results, one can correspondingly expect high levels of AP-1 and MMP in the skin of the elderly subjects.

It has unexpectedly been found that chronically aged skin topical treatment of elderly subjects with a retinoid results in a restitution of intracellular procollagen protein levels similar to that seen in young individuals (such as those with ages of 40). and younger). In particular, it has been found that a single application of 1% retinol on chronologically aged skin, covered with an air permeable adhesive bandage, and examined seven days later, resulted in procollagen protein levels comparable to those found in the skin protected from the sun by significantly younger individuals (for example below 40 years). It would be more preferable that retinoids be applied to elderly people once or twice daily to maintain a therapeutic regimen, although in people it can be applied less frequently, but preferably on a regular basis (for example, a day yes, a day no , or twice a week). It may also be desirable for the skin to be occluded from the inclemency of the environment, particularly UV light sources, detergents and other potent chemicals, and the like. Accordingly, it would be beneficial to mention a UV sunscreen, an antioxidant and the like for the retinoid composition. The stimulation of procollagen production is an important factor in obtaining the integrity of chronologically aged skin. The aged skin is thin and fragile due in part, to the reduced content of collagen and the harmonization of reduced collagen fibers. It is expected that the stimulation of the synthesis of procollagen by retinoids and their subsequent conversion to collagen, reduce the fragility, increase in thickness, and improve the appearance of aged skin. Accordingly, this invention provides methods to increase procollagen concentrations, both intra and extracellularly, and also to improve the concentrations of collagens, all in the chronologically aged skin. Additionally, as shown here, MMP levels increase in aged skin compared to those found on the skin of older people. Improved procollagen production would be frustrated if the resulting collagen were degraded in the skin, and therefore the treatment of aged skin with both a retinoid and an MMP inhibitor is important to achieve the desired benefits of improved procollagen biosynthesis. In fact, it has been found that in a skin that does not have a reduced level of collagen, treatment with a retinoid does not elevate collagen levels above normal; therefore, the invention shows that the application of a retinoid can restore collagen levels to their desired baseline value. In this way, the inventive treatment with a retinoid increases both the production of fibroblasts and that of (pro) collagen and interferes with the activities of MMP that cause thinning of the epidermis. Although retinol is the preferred compound for topical administration, effective retinol derivatives that are expected to be useful for practicing this invention include retinal, retinoic acid (including all trans, 9-cis, and 13-cis isomers) and derivatives thereof (such as 7,8-didehydroretinoic acid), and others as described by Kligman et al., referenced above, the disclosure of which is incorporated herein by reference, including salts, reversible esters and esters of these cosmetically acceptable, conjugates of these and mixtures thereof. The compositions described herein formulated on a commercial basis may include various conventional colorants, fragrances, thickeners (such as xanthan gum), preservatives, humectants, emollients, emulsifiers, surfactants, dispersants, improved penetration, and the like may be added to provide additional benefits and improve the texture and / or appearance of the topical preparation. In the same way, the composition can be formulated as a cream, lotion, ointment, soap or body lotion, shampoo, or a mask. The above description and the following methods are intended to be illustrative of the invention and not limiting.

Various changes, modifications and additions may be obvious to those skilled in the art upon careful reading of this specification, and are intended to be within the scope and spirit of the invention as defined by the claims. Methods Used in the Examples References made in this section are incorporated herein by reference. Histology and morphometry. Duplicate biopsies with a 4-mm perforation were obtained from the skin of the buttock of each individual. Fixed tissue pieces with formalin were sectioned, stained with hematoxylin and eosin, alloyed and harvested. The sections were examined using an Olympus BX40 microscope together with a Sony DCX-151 high resolution camera. The blocked areas of 200 μm on one side were isolated using an NIH Imager software and the epidermal height was evaluated in four places (25 μm separation) in each of the two areas. The same two blocked areas were used for the epithelial cell count. The number of interstitial cell nuclei (ie, nuclei below the clermo-epidemic junction not associated with the capillaries) throughout the histological section was determined as a measure of dermal cellularity. The same mowed sections were classified for collective tissue fiber separations, thickness, degree of disorganization and depth of disorganization, using a scale of 1-9 for each parameter. Preparation of skin supernatants for biochemical analysis. The skin samples were ground in a mortar with liquid nitrogen, and a pH buffer containing 10 M Hepes, 1 mM EDTA, 5 mM EGTA, 10 mM MgCl2, 50 mM glycerophosphate, 5 M were homogenized in a Dounce fabric grinder. NaV04, 2 mM DTT, 0.5 PMSF, 10 μg / ml aprotinin, 10 μg / ml leupeptin, and 10 μg / ml pepstatin, and 0.5% NP-40. The homogenates were centrifuged at 14,000 g for 15 minutes, and the supernatants were collected and used for biochemical determinations as described herein. Growth of cells ex vivo. The biopsies were cut to form small fragments (approximately 15 fragments per piece of tissue) and the tissue fragments were transferred to plastic cell culture flasks. The culture medium consisted of an Eagle Dulbeco Modified Minimum Essential Medium with Earle salts, non-essential amino acids and 10% fetal bovine serum. The tissue fragments were incubated at 37 ° C and 5% C02 / 95% air for up to one month. Each fragment was evaluated to know if keratinosites and / or fibroblasts grew outside the tissue and from this, the percentage of fragments of which keratinocytes and fibroblasts were isolated according to the method of Varani, J. et al. al , J. Cl in. Inves t. , 96, 1747-1756 (1994). Metalloproteinase matrix assay. The pieces of tissue were frozen in liquid nitrogen immediately after the biopsy, homogenized in 20 mM Tris HCl (pH 7.6) plus 5 mM CaCl2 and centrifuged at 3000 x g for 10 minutes to remove the particles. The ability to release soluble radioactive fragments of fibrillar type I collagen type 3H (described by Fisher, GJ, et al., Na ture, 379. 335-339 (1996) and Hu, CL et al., Ana lyt ic. Bi ochem, 88, 638-643 (1978)), was used as a measure of collagenolytic activity. The tissue extracts were incubated for 3 hours with 1 mM aminophenyl mercuric acetate (APMA) to convert the inactive form of the metalloproteinase matrix into its active form. Subsequently, a collagen substrate of 0.2 μCi (NEN-DuPont, Boston, MA) was incubated for 24 hours with 50 μl of tissue extract. At the end of the 24 hour incubation period, the samples were centrifuged at 12,000 x g for 10 minutes to form granules of the intact protein. The remaining radioactivity in the supernatant fluid was measured and from this, the percentage of hydrolyzed substrate was determined. Gelatin zymography (Varani et al., Op.cit.) Was used to evaluate the activity of MMP-2 (gelatinase 72-kD); gelatinase A) and the activity of MMP9 (gelatinase 92-kD; gelatinase B). The tissue extracts underwent electrophoresis in an 8.5% SDS polyacrylamide gel containing 1 mg / ml of gelatin. After electrophoresis, the SDS was removed by three sequential washes in 1% Triton X-100. The first two washes were for 20 minutes each and the last was during the night. The quantification of the width of the hydrolysis zone was made by laser densitometry. Activity and phosphorylation assays of ERK. The ERK1 and ERK2 in skin supernatants were immunoprecipitated with antibodies from Santa Cruz Biotechnology Inc. and assayed for enzyme activity using myelin basic protein as a substrate, as described by J.D. Weber et al. ("Sustained activation of kinase I (ERK1) regulated by an extracellular signal is required for the continued expression of cyclin Di in the Gl phase," Biochem, J., 326: 61-68 (1997)). The phosphorylated and total ERK1 and ERK2 in the supernatants were determined by Western analysis, using antibodies from the New England Biolabs Inc. (Beverly, MA). Assay of kinase activity c-Jun. The activity of c-Jun in the supernatants was determined by the solid phase kinase assays (as described, for example, by M. Hibi et al., "Identification of an oncoprotein and UV-responsive protein kinase that binds and potentiates the c-Jun activation domain "Genes Dev., 7. - 2135-21 48 (1 993)). Northen analysis of RNA. Total RNA (for example, for c-Jun, procollagen to (III)) was isolated from the skin sample by lysis of guanidinium hydrochloride and ultracentrifugation (as described by GJ Fisher et al., "Cellular, Immunologic and Biochemical Characterization of topical retinoic acid-trated human skin ", J. Inves tig Derma tol., 96: 699-707 (1991)). Northern analysis of total RNA (40 μg / path) with cDNA samples randomly labeled with the 3 P prime for the mRNA to be determined were performed as described by G.J. Fisher et al., (in "All trans retinoic acid induces cellular retinol-binding protein in human skin in vivo", J. Inves tig Derma tol., 105-80-86 (1995)). Type III procollagen mRNA was determined using a reverse transcriptase polymerase chain reaction. Western protein analysis. Jun proteins, cyclin D2 (both antibodies from Santa Cruz Biotechnology Inc.), and phosphorylated c-Jun (antibodies from New England Biolabs Inc.) were detected in nuclear extracts of human skin by means of host analysis as described by GJ Ficher et al. (in "Immunological identification and fuctional quantitation of retinoic acid and retinoid X receptor proteins in human skin" J. Biol. Chem, 269: 20629-20635 (1994)). The immunoreactive proteins were visualized by an improved chemotherapeutic detection and quantified by laser densitometry, or by the detection of improved chemofluorescence and quantified by a Storm imager (Molecular Dynamics, Palo Alto, CA).

Claims (25)

1. REI INDICATIONS 1. A method for rejuvenating chronologically aged skin, characterized in that it comprises the application of an effective non-toxic amount of at least one active ingredient that inhibits MMPs and promotes the synthesis of procollagen.
2. 2. The method of compliance with the claim 1, characterized in that the active ingredient is a retinoid that both inhibits MMPs and promotes the synthesis of procollagen.
3. 3. The method of compliance with the claim 2, characterized in that the retinoid is selected from retinol, retinal, retinoic acid, a retinoic acid salt, a derivative or analogue thereof or a mixture thereof.
4. 4. The method of compliance with the claim 3, characterized in that the retinoid is a retinol or a retinoic acid.
5. 5. The method according to claim 3, characterized in that the skin is skin protected from the sun.
6. 6 The method according to claim 1, characterized in that the MMP inhibitor is selected from aspirin, E5510, glucocorticoids, Vitamin D3, GI12947, TIMPs, hydroxates and hydroxyurea derivatives, and tetracycline and derivatives thereof and the various salts thereof. .
7. 7. A method for improving the proliferation of keratinocytes and / or fibroblasts in a chronologically aged skin, characterized in that it comprises the application of an effective non-toxic amount of a retinoid on the skin.
8. 8. The method of compliance with the claim 7, characterized in that the skin is protected from the sun.
9. 9. A method for preventing chronological aging of the skin, characterized in that it comprises providing a non-toxic, topically administrable amount of a retinoid in a cosmetically suitable vehicle and applying said retinoid on the skin at least once a week in an effective amount to normalize the synthesis of procollagen and inhibit the degradation of collagen.
10. 10. The method according to claim 9, characterized in that the skin is skin protected from the sun.
11. The method according to claim 9, characterized in that the retinoid is selected from retinol, retinal, retinoic acid and retinoic acid salt, a derivative or analogue thereof, or a mixture thereof.
12. 12. The method according to claim 11, characterized in that the retinoid is retinol.
13. 13. The method according to claim 9, characterized in that the retinoid is applied daily.
14. 14. A method for normalizing the production of procollagen in chronologically aged skin, characterized in that it comprises the steps of providing a topically administrable, non-toxic retinoid and administering said retinoid on the skin in an amount effective to normalize the production of procollagen.
15. 15. The method according to claim 14, characterized in that the retinoid is retinol.
16. 16. The method according to claim 14, characterized in that the retinoid is applied on a regular basis.
17. 17. The method according to claim 16, characterized in that the retinoid is applied daily.
18. 18. The method according to claim 14, further characterized by comprising the step of simultaneously inhibiting collagen degradation, the method further comprising the steps of providing a topically administrable non-toxic amount of an MMP inhibitor and administering an effective amount to inhibit MMP on chronologically aged skin on a regular basis.
19. 19. The method according to claim 18, characterized in that the MMP inhibitor is selected from aspirin, E5510, glucocorticoids, vitamin D3, GI12947, TIMPs, hydroxamates and hydroxyurea derivatives and tetracycline and derivatives thereof and the various salts thereof, and compatible mixtures thereof.
20. 20. The method according to claim 18, characterized in that the retinoid and the MMP inhibitor are present in a single topically administered formulation.
21. 21. The method according to claim 18, characterized in that the chronologically aged skin is skin protected from the sun.
22. 22. A method for improving the level of ERK activity in aged human skin characterized in that it comprises the topical application of an effective non-toxic amount of a retinoid on the skin.
23. 23. The method according to the claim 22, characterized in that the retinoid is retinoid or retinoic acid.
24. 24. The method for reducing the level of c-Jun protein and / or the activity of the cJUN kinase in aged human skin characterized in that it comprises the topical application of an effective non-toxic amount of a retinoid on said skin.
25. 25. The method according to claim 22, characterized in that the retinoid is retinoid or retinoic acid.