MX2012012235A - Genetic signatures and gene chips associated with administration of electrically conducted radio frequency current to skin and methods and treatments relating thereto. - Google Patents

Abstract

A gene panel comprising genes regulated in mammalian skin in response to generation of a radio frequency current in a tissue volume of the mammalian skin sufficient to heat the tissue volume to a treatment temperature, wherein at least one gene is selected from Table 1 or Table 2 and at least one gene is selected from Table 3. Further there is a method for providing a benefit to mammalian skin, the benefit comprising inducing collagen formation and/or dermal remodeling in a dermal layer of the mammalian skin in the absence of a skin-damaging inflammatory cytokine response, the method comprising generating a radio frequency current in a tissue volume of the mammalian skin for a treatment cycle sufficient to heat the tissue volume to a treatment temperature while avoiding an upregulation in expression of genes listed in Table 3.

Description

GENETIC SIGNATURES AND GENETIC MATRIX ASSOCIATED WITH THE ADMINISTRATION OF RADIUS FREQUENCY ELECTRICALLY DRIVEN TO THE SKIN AND METHODS AND TREATMENTS RELATED TO IT FIELD OF THE INVENTION The invention relates to cosmetic treatment regimens for the skin comprising the administration of radiofrequency current electrically driven through the skin to provide benefits in it and, further, refers to the evaluation based on genomics of the benefits and treatments to control, analyze and optimize the treatment regimens of the skin.

BACKGROUND OF THE INVENTION The treatment of the skin to avoid or reduce the effects of extrinsic intrinsic and environmental chronological aging on the skin is a multi-billion dollar commercial industry supported by an even higher dollar investment for the development and validation of new technologies. The administration of electromagnetic energy (EM) to the skin by means of the application of EM on the surface of the skin has been known for several decades and has been implemented in a wide variety of ways and through various supply devices. Generally, EM-based skin treatment methods can be divided into ablative and nonablative procedures, although both take advantage of the thermolytic effect of EM energy application.

A variety of products are available to consumers to improve the condition of the skin and to delay or prevent the typical signs of aging. These signs include, for example, fine lines, wrinkles, hyperpigmentation, pallor, flabbiness, dark circles, eye puffiness, uneven skin tone, open pores, reduction in the rate of epidermal cell renewal, and abnormal peeling or peeling. However, for some consumers, the wide variety of available products and advances in skin care technology still fail to produce the desired results, and others feel the need to resort to more invasive medical procedures.

Ablative procedures, such as ablative laser, have proven effective for general morphological skin removal or renewal with laser, such as in procedures for removing scars and tattoos and, in addition, have proven effective for the treatment and improvement of appearance of aged and photodamaged skin. While ablative procedures are effective in improving the appearance of fine lines and wrinkles in the periorbital and perioral areas of the cosmetically vulnerable skin of the face, the main disadvantages include prolonged periods of healing and recovery that impose a highly compromised cosmetic appearance on the consumer. for undesirably long periods.

In addition, consumers often consider that the potential side effects of infection, scar formation and irregularities in pigmentation that may occur are cosmetically unacceptable, particularly when related to facial skin.

Therefore, recent research and development efforts focused on providing consumers with non-ablative anti-aging and skin rejuvenation treatments that are more economical, more comfortable and safer. Cosmetic regimens involving the administration of thermal energy to the skin in order to promote a better appearance of this are well known in the industry. The technology of supplying electromagnetic energy in the form of handheld devices intended for the consumer to use at home has been available in the market for practically a decade. Improvements and advances in technology are aimed at maximizing a thermally induced benefit in deeper target cutaneous tissues and, at the same time, minimizing undesirable damage in both the target tissue and peripheral and superficial tissues. It is believed that the selective thermal treatment induces the formation of new collagen and the selective thermal damage induces, among others, the remodeling of the dermal matrix. However, it is known that currently available technologies produce prolonged and undesirable negative side effects of troublesome damage including overheating, burns, erythema and pigmentary irregularities.

Previously, the effectiveness of these devices and compositions was determined only by external methods. That is, a visible improvement in the outer layer of the skin was an indication of the efficiency of the power supply device. However, there are few methods to determine the time, temperature and optimal composition for these power supply devices. In addition, it is generally accepted that many of the visible changes produced on the surface of the skin are the result of changes that occur below the surface in the dermal and epidermal layers of the skin. It is difficult to know precisely how to control and maximize the performance of a device if the effect it has on the underlying layers of the skin is not known.

Selective photothermolysis in the skin tissue is a widely used form of cosmetic treatment, particularly, in treatments comprising the administration of monochromatic laser energy and intense pulsed light energy (IPL) of broad spectrum. In these technologies, optical energy is applied directly to the surface of the skin and the penetration is based on the transmission through the epidermis and absorption in the dermis. Dark skin and hyperpigmented spots in the epidermis can prevent transmission and impede the effectiveness of the treatment by means of energy absorption and, in addition, overheating of the pigmented areas and, consequently, the formation of blisters, can occur. burns and other cosmetically undesirable effects.

In addition, as an alternative to EM-based thermolysis, electrically driven radiofrequency (RF) current has been investigated as a modality of cosmetic treatment for the skin. In the medicinal industry, the use of RF current and pulsed radiofrequency (pRF) is known, even though the use of RF current as a non-ablative skin rejuvenation technology for self-application by the consumer is still relatively new. When RF current is applied to the skin, a bipolar RF current transfer occurs through two electrodes that are placed directly on the skin. Therefore, the current passes directly through the dermal layer led from one electrode to the other electrode, and is distinguished from the application of EM energy that passes through the epidermis and is limited by factors that affect the depth of the penetration of the length of wave.

In theory, RF current management offers more significant advantages for the treatment of the skin than the application of EM energy. Unlike electromagnetic energy, for example, electrically driven RF energy is independent of chromophores and this avoids complications and efficiency problems related to the existence of an absorption differential between pigmented and non-pigmented skin and the problems that are encountered. They produce in the treatment of darker skin that contains more energy absorbing melanin and lighter skin that can reflect optical wavelengths. In both cases the regularity of the results is jeopardized and the thermal control in the skin containing chromophores is still a problematic factor.

However, the inability of researchers to optimize the parameters necessary to obtain a desired benefit without producing undesirable skin damage can prevent the delivery of RF current to the skin at adequate energy levels for effective heat treatment. The RF current is delivered through the dermal tissue below the surface of the skin, while the control of the effect by means of temperature or humidity sensors is limited to the accessible surface of the skin. The impedance of the RF current is a function of the composition of the tissue and various attributes of the skin tissue that include the density and integrity of the collagen, the level of hydration and the similar. Although the distance between electrodes and the control of parameters, such as pulse length and frequency can theoretically be adjusted to optimize the effect and avoid security issues, it is practically impossible to make such adjustments without taking advantage of some device or other means to control the changes in the condition of the tissue. In the case of EM energy-based delivery, the surface of the skin typically reaches the highest treatment temperatures so that control of the surface temperature can prevent unwanted damage to the subepidermal tissue. However, in the case of the RF current the subepidermal tissues reach a treatment temperature higher than the surface skin so that the damage can occur in the deeper tissues without manifesting on the surface in a measurable way. Therefore, outside of a clinical setting and under the supervision of highly trained medical personnel and the use of sophisticated instrumentation, devices and regimens for personal use by consumers based solely on power supply have generally been avoided. RF, since they still exist considerations related to safety at effective treatment levels without an appropriate subepidermal control mechanism. Handheld devices for the supply of energy that provide RF current as a single treatment modality and intended for consumers to use at home are virtually unknown. One of these devices (STOP ™, Ultragen Ltd) is marketed in Europe for personal use by consumers, but the tolerances set for treatment with the device are so low to avoid the undesirable damage that objective evidence of the clinical efficacy under controlled conditions.

Consequently, the action of RF in the treatment of the skin is practically limited to a coadyuvant or preparative function in conjunction with other thermolytic processes. For example, in 2002, Bitter and Mulholland ("Report of a new technique for enhanced non-invasive skin rejuvenation using a dual mode pulsed light and radio frequency energy sources: selective radiothermolysis," J. Cosmet Dermatol 2002; 1: 142-145 ) proposed a treatment protocol based on a combination of RF and IPL current and reported the results based on the facial treatment of 100 test subjects, although the authors could not describe specific treatment design parameters. In that study, RF was reported to increase the effects of IPL treatment. Some reported side effects included cosmetically undesirable pigmentation effects and the pain perceived by the consumer was controlled by surface cooling.

In addition, RF has been proposed and researched as useful for the cosmetic treatment of the skin together with the application of directed optical energy. Generally, according to the design of this treatment protocol, the RF is used as a complement to the optical energy and is applied in accordance with some parameter of the optical energy. For example, in Hammes et al. ("Electro-optical synergy (ELOS ™) for nonablative skin rejuvenation: a preliminary / prospective study," Journal of European Academy of Dermatology and Venereology 2006, 20, 1070-1075), authors focus on a coordinating pulse frequency between the RF current and the optical energy and suggest that between these forms of energy there is a synergy that can allow the use of lower and less invasive levels of optical energy and, in addition, suggest that the side effects associated with the exclusive application of RF are reduced or avoided by means of the combined protocol. In addition, the regimens employing these devices include means for mechanically cooling the skin in response to overheating or to prevent overheating.

In another example, the published application of the US. UU no. 2008/0033516 Altshuler's A1 describes the "controlled photobioestimulation of the temperature" of skin tissue that involves a combination of heating the skin to a target depth and irradiating a target area with electromagnetic radiation. Altshuler highlights the existing technologies for skin treatment methods based on photostimulation with a low level of light, low laser level, monochromatic and quasi-monochromatic that is thought to generally increase ATP production, cell proliferation and protein production, in addition to triggering a response from growth by induction of a low-level inflammatory response, but highlights reports of inconsistent results and lack of clinical confirmation of efficacy. Altshuler suggests that the application of thermal energy can improve the photoestimulatory response. Altshuler teaches that a skin volume can experience hyperthermia produced by any known source to raise the temperature of the volume, preferably, up to 37 ° C to 45 ° C and, specifically, exemplifies the heating by means of hot air, AC or DC electrical current, the use of a conductive heat source, ultrasound or microwave radiation or any length or wavelengths of EM radiation in the range of 380-2700 nm. However, in all Altshuler modalities, the desired effect for the treatment is obtained by means of EM energy. Altshuler teaches that heat provides a synergistic improvement in the desired photostimulation effects, but, in addition, suggests that heat in the absence of EM can produce undesirable biostimulation, for example, reducing the rate of DNA damage repair induced by radiation, the production of heat shock proteins that generate tolerance to subsequent heat applications and the modification of enzymatic processes that include those involved in the regeneration and repair of skin tissue and, generally, discourages the use of heat in the absence of light as a modality for the treatment of the skin. Altshuler does not recommend ways to overcome the deficiencies related to the inability to evaluate or control subepidermal skin conditions.

In addition, the effectiveness of currently available devices they always depend on compliance by the consumer. Consumers have little time per day for their beauty care. While device manufacturers would like to recommend consumers to use the devices for extended periods of time to obtain maximum benefit, consumers are unlikely to comply. Therefore, there is a certain balance between recommending prolonged use of the device and recognizing that consumers have little free time per day to use the device.

In addition, the consumer experience is important when designing a device, a composition and a regime. For example, sonograms are procedures that are commonly performed and provide an important medical benefit. But the gel used in the procedures of sonograms is thick and difficult to eliminate and this is uncomfortable for the consumer. In addition, many energy supply devices heat the external part of the skin very quickly or excessively and, therefore, the consumer feels some discomfort.

Therefore, there is a constant need to achieve methods to sufficiently improve the condition of the skin to avoid the need for more invasive procedures and the risks associated with them. And, there is a need to achieve better methods to determine the efficiency of the power supply devices and such methods can then be used to develop more acceptable experiences for the consumer, and at the same time, achieve the desired results that include a better appearance of the skin In the industry, there is a continuing need for safe, effective non-ablative skin treatment, rejuvenation, and treatment devices, therapies, and regimens suitable for personal use by consumers. Particularly, there remains a need for a means to treat subepidermal skin tissue by improving collagen synthesis and dermal remodeling without causing undesirable damage to the target tissue treated or the surrounding tissue. There is a specific need in the industry for methods to assess and control the effects of consumer-based treatments based on RF current to optimize the delivery of RF current to provide desired benefits and the need for optimized RF current-based therapies persists They avoid the problems associated with EM-based therapies and are not based on mechanical cooling in conjunction with the treatment.

BRIEF DESCRIPTION OF THE INVENTION Accordingly, by using recent advances in the biotechnology of genomics and proteonomics that allow an accurate and multiple feedback of the cellular response to the manipulations of physical and chemical environments, the inventors of the present invention investigated and determined a genetic fingerprint for the effect of electrically driven RF current on the skin tissue and have developed novel gene arrays and methods of using those gene arrays to provide safe and effective RF-based treatment regimens and treatment analysis protocols. Surprisingly, the inventors of the present invention discovered that the optimization of RF current-based treatments provides benefits related to collagen synthesis and certain damage-induced benefits that stimulate skin remodeling and, at the same time, prevent a response of the inflammatory cytokine induced by the most problematic damage. Furthermore, surprisingly, it was found that RF current-based treatment regimes in accordance with the present invention do not require supplemental power supply and, specifically, do not include the administration of electromagnetic (EM) energy outside the length portion of radio wave of the EM radiation spectrum and do not require mechanical cooling of the surface. Therefore, the benefits of the invention are provided without the presence of known problems associated with the administration of EM energy to the skin.

One embodiment of the invention is directed to genetic panels comprising genes regulated in the skin of mammals in response to the generation of a radiofrequency current in a volume of mammalian skin tissue sufficient to heat the tissue volume to a temperature of treatment. The genes of the panels are selected from Table 1 and / or Table 2 with the condition that at least one gene is selected from Table 3. The aspects include microarrays and genetic signatures based thereon.

Another broad embodiment is directed to methods for providing a benefit to the skin of a mammal; the benefit comprises inducing the formation of collagen and / or dermal remodeling in a dermal layer of the skin of the mammal without there being a response of the inflammatory cytokine that damages the skin; the method comprises generating a radiofrequency current in a tissue volume of the skin of a mammal during a treatment cycle sufficient to heat the tissue volume to a treatment temperature and, at the same time, prevent an upregulation of the expression of the genes listed in Table 3.

In accordance with another embodiment, methods are provided to evaluate the efficiency of the treatment of a power supply device. The power supply device is apparently designed to provide a benefit to the skin by heating the skin and can be evaluated by a method comprising: treating the skin by applying the power supply device; extract the mRNA from a sample of the treated skin; and generating an expression profile for a genetic panel according to the invention. The facial skin treatment regimens can also be analyzed or evaluated to determine the efficacy in providing a benefit of skin remodeling and / or collagen to the skin of a mammal without stimulating the response of the inflammatory cytokine that damages the skin. skin in accordance with another modality. The method comprises: treating the facial skin in accordance with a treatment regimen; extract the mRNA from a sample of the skin of the treated face; generate a gene expression profile for a genetic panel according to the invention; compare the profile of gene expression with a reference profile; and determining that the treatment regimen of the face is effective, wherein the expression profile reflects the up-regulation of selected genes from Table 1 and / or Table 2 and a considerable lack of regulation of selected genes from Table 3. In Certain aspects of the invention, cosmetic actives and / or compositions can be similarly analyzed or evaluated to provide benefits in accordance with the invention or to determine synergism with the methods of treatment of the invention.

BRIEF DESCRIPTION OF THE FIGURES While the description concludes with the claims that indicate, particularly, and distinctly claim the present invention, it is believed that it will be better understood from the following description taken in conjunction with the accompanying figures and in which: Figure 1 is a schematic representation of the damage of natural origin and the repair cycle in human skin; Figures 2A, 2B illustrate the properties of collagen in the human being; Figure 3 is a schematic representation of the biological model of the efficiency of RF current management; Figures 4A, 4B represent the times of average change in 24 hours for the genes of the collection; Figures 5A, 5B represent the times of average change in 24 hours for the illustrative genes; Figures 6A, 6B represent the times of average change in 1 month for the genes of the collection; Figures 7A, 7B represent the times of average change in 1 month for the illustrative genes; Figures 8A, 8B are two graphs of simple heat transfer by RF; Figure 9 is a schematic representation of simple heat transfer by RF; Figure 10 is a schematic representation of the area of crow's feet around the eyes of a consumer; Figure 11 is the area A of the skin of a consumer; Figure 12 is the area B of the skin of a consumer; Figure 13 is the area C of the skin of a consumer; Figure 14 is the area D of the skin of a consumer; Figure 15 is the area E of the skin of a consumer; and Figure 16 is the area F of the skin of a consumer.

DETAILED DESCRIPTION OF THE INVENTION Unless defined otherwise, the scientific and technical terms used in connection with the invention described in the present description will have the meanings commonly understood by persons of ordinary skill in the industry. In addition, unless the context requires it in any other way, the terms in the singular will include the plural and the terms in the plural will include the singular. Generally, the nomenclatures used in connection with and the techniques of cell and tissue culture, molecular biology and protein chemistry and oligo or polynucleotides and hybridization described in the present disclosure are those well known and commonly used in the industry. Standard techniques are used, for example, for the purification and preparation of genetic material (nucleic acid), chemical analysis and the synthesis of oligonucleotides. Enzymatic reactions and purification techniques are carried out in accordance with the manufacturer's specifications or as commonly carried out in the industry or as described in the present description. The techniques and methods described in the present description are generally performed in accordance with conventional methods well known in the industry and as described in several general and more specific references which are cited and discussed throughout the present description. The nomenclatures used in connection with and the laboratory procedures and techniques described in the present description are those well known and commonly used in the industry.

In order to explain and illustrate various aspects of the invention, several specific details are included below. However, those with experience in the relevant industry will understand that the embodiments of the present invention can be practiced without these specific details. In other cases, in order to effectively illustrate the invention and not overlook aspects of the invention, known structures and devices are shown or discussed more generally. It should be noted that inventions described and the examples that illustrate them can be applied to many different configurations, devices, software and technologies and existing alternatives. The full scope of the various embodiments and the inventions themselves are not limited to the specifically described examples.

When a range of values is provided, it is understood that said interval comprises each intermediate value up to one tenth of the unit of the lower limit, unless the context clearly indicates otherwise, between the upper and lower limits of that interval and any other value mentioned or intermediate within the specified interval. The upper and lower limits of these smaller ranges can be independently included in the smaller ranges and, further, are comprised within the invention, subject to any limits specifically excluded from the aforementioned range. When the aforementioned range includes one or both limits, the ranges that exclude one or both of the included limits are, furthermore, included in the invention. Unless indicated otherwise, all numbers that express quantities of reactants, reaction conditions, etc. used in the description and claims will be understood as modified in all cases by the term "approximately".

It is to be understood that the steps of the methods described in any of the claims appended hereto may be performed in any order unless otherwise specified. For example, in a claimed method that enumerates steps (a), (b) and (c), step (c) may be carried out before steps (a) and (b) or between them. Moreover, the individual stages, although described as distinct stages, can be carried out during periods that partially or completely overlap.

In the present description, "regulating the condition of the skin" means improving the condition of the skin or prophylactically regulating the condition of the skin, and includes, for example, protecting the tissue against ultraviolet radiation, and regulating the signs of aging of the skin. the skin. In the present description, "improving the skin condition of mammals" means a positive change perceptible to sight or touch in the appearance and feel of the tissue. Conditions that can be regulated or improved include, but are not limited to, one or more of the following: reduce the appearance of wrinkles and deep and thick lines, fine lines, cracks, protrusions and open pores; Thickening of the skin (eg, the formation of epidermal, dermal or subdermal layers of the skin and, where applicable, the keratinous layers of the nails and the hair shaft, to reduce atrophy of the skin, hair or the nails); increase the circumvolution of the dermal-epidermal borders (also known as interpapillary bridges); prevent the loss of elasticity of the skin or hair, for example, by loss, damage or inactivation of the functional elastin of the skin, which produces conditions such as elastosis, flaccidity, loss of skin retraction or hair with deformation; reduction of adiposis; changes in the coloring of skin, hair or nails, for example, dark circles, spots (such as red patches due to, for example, rosacea), paleness, discoloration caused by telangiectasia or arachnoid vascularization, dryness, fragility and graying.

As used in the present description, "signs of skin aging" include, but are not limited to, the external visual and tactile perceptible manifestations, as well as other macro or microeffects due to the aging of the skin. These signs may be the consequence of processes that include, but are not limited to, development of texture interruptions, such as wrinkles and deep and coarse wrinkles, fine lines, skin lines, cracks, bumps, open pores, roughness or rugosity; peeling; dryness; loss of skin elasticity; discoloration (including dark circles); spots; pallor; regions of hyperpigmented skin, such as senile spots and freckles; keratosis; abnormal differentiation; hyperkeratinization; elastosis; collagen rupture and other histological changes in the stratum corneum, dermis, epidermis, vascular system (eg, telangiectasia or arachnoid vascularizations) and underlying tissues (eg, fat or muscles), especially the next To the skin.

As used in the present description, "hyperpigmentation" refers to an area of the skin on which the pigmentation is greater than that of the adjacent area (eg, a pigmented area, an aging stain and the like).

In the present description, "personal care composition" means compositions suitable for topical application in the skin of mammals. The personal care compositions described in present description may contain one or more active ingredients for skin care. As used in the present description, "active skin care" or "active" means compounds that contribute to regulating the skin and skin condition of other mammals, for example, by providing a benefit or improvement to the skin.

As used in the present description, "energy supply device" means any device used to supply energy to the skin or hair of mammals. In the present description, "energy supply" means that the surface or layers of the skin are exposed to energy from a power supply device, which can penetrate the desired layers of the skin, including the hair shaft or hair follicle.

As used in the present description, "continuous level" means that the energy supplied by the device, or energy output, remains at a substantially constant level between the time of activation of the device and the time of deactivation of the device.

As used in the present description, "pulsed" means that between the time of activation of the device and the moment of deactivation of the device, the energy output varies in a predictable manner, characterized by periods of higher output (pulses) alternating with periods of lower output. The start of the pulses can be abrupt or gradual. "Predictable" means that the intensities of the pulse peaks, the pulse shapes, the pulse durations and the temporal separations between the pulses are practically identical. The pulse duration and the intervals between the pulses may vary.

As used in the present description, "hand-held" means that the device has a suitable weight and size so that it can comfortably be held in the hand of a normal adult human being.

Human skin can be divided into two main structural layers: the epidermis and the subepidermal or underlying dermis. The epidermis with the stratum corneum serves as a biological barrier to the environment. Pigment-forming cells, called melanocytes, are present in the basilar layer of the epidermis and are considered to be the main determinants of skin color.

The underlying dermis provides the main structural support of the skin. It is composed, mainly, by an extracellular protein called collagen. Collagen is produced by means of fibroblasts and is synthesized as a triple helix with three polypeptide chains connected with heat-labile and heat-stable chemical bonds. When the tissue containing collagen is heated, alterations in the physical properties of this protein matrix occur at a characteristic temperature. The structural transition of collagen contraction and remodeling of the collagen matrix occurs with heat.

Some repair activity occurs within the skin to promote the continuous production of collagen. During aging, the damage can occur faster than the repair activity or the speed at which the damage occurs can be practically constant, but the repair activity becomes slower. In either case, the result is reduced collagen and a worse appearance manifested by signs of aging that include the presence of surface defects, such as fine lines, wrinkles and hyperpigmented spots. The normal repair activity cycle and the impact produced on the appearance over time are illustrated in Figures 1 and 3.

In Figure 1 the tables represent the normal causes of collagen production. For many reasons, the increase of collagen in the skin and the speed of repair and replacement of this contribute to the skin exhibiting a more voluminous, healthy and attractive appearance. Regular dermal proliferation and remodeling occurs naturally, but, unfortunately, these processes slow down as we get older. In addition, collagen is produced when the skin is damaged, for example, after inflammation or attack, for example, from solar radiation. The circles in Figure 1 represent some of the mechanisms of collagen formation and control of its formation. The activity of the MMPs and the cytokine are only two measurable amounts that help control the cycle of repair and renewal of collagen.

Figure 3 is a schematic representation of the bi-action biological model for the effectiveness of Elure 20. Skin 21 is divided into three layers, stratum corneum 30, epidermis 32 and dermis 34. Collagen remodeling and collagen production new occurs in the dermal layer. Normal attack 22 on skin 21 occurs constantly and includes normal aging, UV attack, changes in pH, chemical attacks and others. Normal attacks 22 cause damage to collagen 24. Similarly, normal inflammation of low level 26 occurs that generates activity of the cytokine, HSP and HSF. Ultimately, repeated low-level thermal energy produces a higher activity of MMP 36 in the production of collagen fragments. The increase in heat causes an up-regulation 38 of cellular activity that generates the formation and repair of collagen. Both mechanisms 36 and 38 generate the formation and remodeling of healthy collagen 28.

While the measurement of collagen indicates that its melting temperature is up to 50 ° C, the repair cycle can be altered and improved if heat is added at lower levels. The thermal cleavage of the crosslinking of the intramolecular hydrogen bond is produced by the equilibrium between the cleavage events and the relaxation events (hydrogen bond reformation). For this process to occur, no external forces are required. Consequently, the thermal cleavage of intramolecular hydrogen bonds produces intermolecular stress. The contraction of the tertiary structure of the cross-linked molecule creates the initial intermolecular contraction vector. The heating curves of the RF model are illustrated in Figures 8A-8B.

The dermal structure is mostly comprised of collagen 1. Fibroblasts express collagen as procollagen, a single chain protein. When the expression occurs, the procollagen is assembled to collagen 1 and folded into a triple helix conformation called "tropocollagen". The heating curves of the RF model are illustrated in Figures 8A, 8B and 9.

The dermal structure is mostly comprised of collagen 1, 50, Figures 2A and 2B. Fibroblasts express collagen as procollagen 52, a single chain protein. When expression occurs, procollagen 52 is assembled to collagen 1 50 and folded into a triple helix conformation termed "tropocollagen" 54. This process is illustrated in Figures 2A and 2B.

The cross-linking of collagen can be intramolecular (hydrogen bond or covalent) or intermolecular (ionic or covalent bonds). The causes of collagen denaturation as a function of age include the attack of thermal energy, mechanical attack, the effects of pH on collagenase and the speed of MMP, the state of hydration and the general alteration in the natural balance of collagen microfibrils that can "interlace" or "separate" and, therefore, are more vulnerable to degradation by the action of MMP. Although these represent multiple types of attacks, the speed of all of them can be controlled by means of temperature. In addition, the normal collagen turnover cycle can be regulated within a temperature range of < 37 ° C - 43 ° C.

The cleavage of collagen bonds occurs, moreover, at lower temperatures, but at a lower rate. Low-level thermal cleavage is often associated with the relaxation phenomenon in which the bonds re-form without much change in molecular length.

Dermal remodeling is a biophysical phenomenon that occurs at cellular and molecular levels. Molecular contraction or partial denaturation of collagen involves the application of an energy source that destabilizes the longitudinal axis of the molecule by cleavage of the heat labile bonds of the triple helix. In consecuense, an effort is created to break the intermolecular links of the matrix. This is practically an extracellular process, while cellular contraction requires a period of time for the migration and multiplication of fibroblasts in a damaged area. A curative response usually involves an initial inflammatory process that consists of the infiltration of white blood cells or leukocytes that eliminate cellular debris. After that, the fibroblasts proliferate at the site of the injury and / or an increase in turnover occurs with a final increase in the collagen available for the deposition. The fibroblast cells differentiate into contractile myofibroblasts that are the source of contraction of soft cell tissue. After cellular contraction, the collagen is deposited as a static support matrix in the soft tissue structure tensed. The deposition and subsequent remodeling of this incipient scar matrix provides the means to alter the consistency and soft tissue geometry for aesthetic purposes.

In industry, the application of thermal energy is known to initiate the damage and repair cascade and, ultimately, to achieve an improvement in the surface appearance of the skin; however, currently available technologies are associated with known deficiencies. For example, laser delivery devices use specific wavelengths of light that penetrate the skin, bind to specific chromophores and, through a process called selective photothermolysis, remove various colors and pigments from the skin. Lasers are large and expensive equipment, only dealing with specific problems or colors on the skin, they are prone to cause laser burns and scars, they can cause hyper and / or hypopigmentation and can cause eye injuries to the user and patients. Intensive broadband light systems emit multiple wavelengths of light and, through selective photothermolysis, also improve skin discoloration and, through heating of the skin, produce a non-specific improvement in texture of the skin. The systems are also larger and more expensive, the improvements in the textures and wrinkles of the skin are minimal and there is also the risk of skin burns, hiccups or hyperpigmentation and scars. Generally, electromagnetic energy is applied through the epidermis and penetration is limited by pigmentation factors on the surface and composition of the dermal layer.

In the skin treatment industry, radiofrequency technologies are also known. RF technology uses electric current to heat the dermis and stimulate the production of collagen and elastin fibers that firm and tighten the skin. However, state-of-the-art technology exhibits considerable disadvantages due to the inability to optimize treatment parameters. The application of RF current creates a thermal gradient in the skin that is inverse to other technologies of thermal energy supply. The administration of RF current is carried out between electrodes placed on the skin at a distance between them. The current is conducted between the electrodes, through the dermis, so that the temperature of the dermis rises faster than the temperature on the surface of the skin. Since most devices for measuring skin parameters are designed for measurement on the surface of the skin, the dermis can become excessively warm before measurement.

Accordingly, treatment in the industry has been avoided by means of RF current in the absence of thermal cooling, mechanical cooling or other technologies designed to control internal heating.

However, the use of recent advances in the biotechnology of genomics and proteonomics has allowed the inventors to develop methods to evaluate the effects of dermal administration of RF current. Particularly, the inventors of the present invention analyzed a group of potential genes identified as involved in the dermal collagen matrix, dermal inflammation and remodeling and in epidermal differentiation. The genetic signatures and constituents of gene matrices based on the resulting gene expression profiles were determined by analyzing and reviewing the differential regulation of potential genes when exposed to parameters, conditions and treatment regimes with RF current.

The energy delivered in the layers of the skin and / or within them may be in the form of RF energy which includes, for example, radiofrequency waves and microwaves. Illustrative RF energy devices are described in U.S. Pat. UU Nos .: 6,889,090; 6,702,808; 6,662,054; 5,569,242; 5,755,753; 6.241, 753; 6,430,446; 6,350,276; 5,919,219; 5,660,836; 6,413,255; 6,228,078; 5,366,443; and 6,766,202.

The application of RF and treatment regimens comprising the administration of RF current through the dermis can be designed, for the first time, so that they optimize the desired effects for RF treatment. When analysis and review of gene expression data was performed, it was surprisingly discovered that the pool of potential gene data could be reduced to three subsets specifically useful for optimizing treatment regimens and providing an increase in dermal collagen and the dermal remodeling induced by the desired hormonal effort, without producing a response to the most problematic inflammatory cytokine damage. As the inventors of the present invention are well aware, this is the first time that genomics has been applied to cosmetic treatment employing RF current technology. The expression profiles reveal that the controlled and optimized administration provides a hormonal effort that initiates and maintains the desirable dermal remodeling and at the same time avoids the traditional damage associated with the undesirable biological effects. The present invention provides gene arrays, genetic signatures, analytical methods and novel optimizable regimes based on these findings.

The genes investigated as potential genes include genes associated with the integrity of the dermal matrix (FBN1, FBLN1, TNXB, FN1, L0XL2, C0L3A1, C0L1A1, ELN and L0XL1), genes associated with remodeling initiated by dermal inflammation (TIMP2, IL1A, TIMP1, TNF, MMP1, MMP9, MMP3, SOD2 and IL1 B) and genes associated with epidermal barrier function (KRT2, KRT6A, CLDN1, LOR, FLG, IVL, DRT10, AQP3 and KRT14). The subsets derived from the analysis of the expression data for these genes are indicated in Table 1 (dermal markers), Table 2 (markers of matrix remodeling, ie, positive stress hormone primers) and Table 3 (markers of one response to inflammatory atocin, ie, damage outside the repair response).

A gene expression profile provides information on the cellular response to a set of conditions. The genes contain the instructions for making messenger RNA (mRNA). However, at any time, each cell makes mRNA only from a fraction of the genes it contains. It is said that a gene is "activated" if it is used to produce mRNA and, in any other case, it is said to be "deactivated". The term "regulation" refers to the unleashing of a transcriptional condition that is different from the control state of a gene. For example, "up-regulation" may only include activation or may refer to the increase of a transcriptional velocity with respect to an initial velocity value obtained from a control or reference condition.

In the identification of the expression profile, the relative amount of mRNA expressed in two or more experimental conditions is measured. The altered levels of mRNA suggest a modified need for protein encoded by the mRNA. For example, greater transcription of enzyme catalysts or cofactors is observed in response to higher levels of the enzyme substrate in the cellular environment.

Generally, a gene expression profile includes those genes that exhibit significant differences under modified experimental conditions. This is typically a subset of some data sets that can include the entire genome. For a cell type, a group of genes whose combined expression pattern is exclusively characteristic for a given condition constitutes a genetic signature of the condition. Genetic signatures can be used, for example, to select patients who can benefit from a specific treatment or to design treatment protocols aimed at maximizing a desired signature.

The gene expression profiles for the potential gene data set can be determined with the use of a microarray. Illustrative cDNA microarrays are commercially available and can be purchased from companies such as Agilent Technologies, Affymetrix Inc. (Santa Clara, Calif.), Nanogen (San Diego, Calif.) And Protogene Laboratories (Palo Alto, Calif.). Hybridization technology specifies that it can be practiced, furthermore, to generate the expression profiles used in the methods of the present disclosure includes the technology described in US Pat. UU num. 5,143,854; 5,288,644; 5,324,633; 5,432,049; 5,470,710; 5,492,806; 5,503,980; 5,510,270; 5,525,464; 5,547,839; 5,580,732; 5,661, 028; 5,800,992; whose descriptions are incorporated in the present description as a reference; Y also in WO 95/21265; WO 96/31622; WO 97/10365; WO 97/27317; European patent no. 373203; and EP 785 280. Generally, in these methods, a "probe" nucleic acid matrix that includes a probe for each phenotype determining gene whose expression is tested is contacted with the target nucleic acids as indicated above. The contact is carried out under hybridization conditions, for example, stringent hybridization conditions and, then, the unbound nucleic acid is removed. The resulting pattern of hybridized nucleic acid provides information related to the expression for each probe gene, wherein the information of the expression indicates whether the gene is expressed or not and, typically, at what level, and where the expression data, that is, the expression profile, can be qualitative and quantitative. Alternatively, the expression profile is determined by means of quantitative PCR or other quantitative methods to measure the mRNA.

One embodiment of the invention provides a genetic panel comprising genes regulated in the skin of mammals in response to the generation of a radiofrequency current in a volume of mammalian skin tissue sufficient to heat the volume of tissue to a treatment temperature . At least one gene from Table 1 or Table 2 and at least one gene from Table 3 is selected. In specific embodiments, at least one gene is selected from each of the three tables. It is contemplated that all genetic panels according to the invention include at least one gene from Table 3, since the change in lack of expression in these genes indicates the lack of a "bad" response to damage. The probes can be designed to target each gene that constitutes a gene matrix of the invention to construct very specific microarrays useful for designing, analyzing, adapting or controlling treatment regimens to obtain the desired effect or validation of the treatment regimen in treated subjects. Microarrays are contemplated comprising a set of immobilized nucleic acid probes capable of hybridizing to and detecting genes that constitute a genetic panel according to the invention.

As used herein, a "probe" refers to an oligonucleotide, polynucleotide or DNA molecule regardless of whether it is natural or synthetic that is capable of specifically hybridizing to a nucleic acid with sequences complementary to the probe. The probes of the present invention specifically refer to oligonucleotides attached to a solid support in the substrate of the DNA microarray. A probe can be single-stranded or double-stranded. The probe typically contains 15-25 or more nucleotides, although it may contain fewer nucleotides. The probes of the present disclosure are selected to be complementary to different chains of a particular target nucleic acid sequence and, therefore, must be sufficiently complementary to be able to hybridize specifically with their respective target chains in a set of predetermined conditions. Therefore, it is not necessary for the sequence of probes to reflect the exact complementary sequence of the destination. For example, a non-complementary nucleotide fragment can be attached to the 5 'or 3' end of the probe with the remainder of the sequence of the probe complementary to the target chain. Alternatively, non-complementary bases or longer sequences may be intermixed in the probe, provided that the sequence of probes is sufficiently complementary to the target nucleic acid sequence to hybridize specifically therewith.

Another embodiment of the invention includes methods for providing a benefit to the skin of a mammal. The benefit comprises inducing the formation of collagen and / or dermal remodeling in a dermal layer of the skin of the mammal without an inflammatory cytokine response that damages the skin, and the method comprises generating a radiofrequency current in a volume of tissue of the mammalian skin during a cycle of treatment sufficient to heat the volume of tissue to a treatment temperature and at the same time avoid up-regulation of the expression of the genes listed in Table 3. The RF current can be generated a plurality of times during a treatment cycle.

As used in the present description, the terms "treat" and "treatment" and the like generally refer to obtaining a desired cosmetic or aesthetic effect backed by a directed biological response. As used in the present description, "treatment" refers to treatment in a mammal, particularly, a human being and includes: (a) preventing or preventing the development of a cosmetically undesirable condition in the skin, for example, fine lines, wrinkles, hyperpigmented spots and other irregulaes of the skin produced by aging or chronological or environmental impact on the skin, (b) inhibiting, improving or delaying the development of a cosmetically undesirable skin condition; (c) reversing or causing regression of the cosmetically undesirable condition in the skin.

During one treatment period, one or more treatment cycles according to the invention can be applied. The treatment cycles can be as short as necessary to achieve the desired response. In specific modalities the treatment cycle is about one minute or less, while in other specific modalities the treatment cycle is greater than about one minute. In other specific embodiments, a treatment cycle lasts from about 1 to about 6 minutes and in other modalities, from about 2 to about 6 minutes. A treatment period according to the invention comprises one or more treatment cycles and, in specific embodiments, is at least one week and comprises at least one treatment cycle. In other specific modalities, the treatment period is from one week to 12 weeks. In more specific modalities, the treatment period is 3 to 8 weeks. In certain modalities, each week of a treatment period comprises from one to six treatment cycles, although it is understood in the industry that this may vary depending on the unique characteristics of the individual treated.

Generally, although the administration of RF current generates a higher temperature in the dermis than on the surface of the skin, the temperature of the skin is measured more conveniently and non-invasively on the surface. Accordingly, in certain embodiments, a treatment temperature, defined in the present description as the temperature of the tissue volume through which the RF current passes, generates a skin surface temperature in the tissue volume smaller than about 45 ° C. In more specific embodiments, the treatment temperature generates a surface temperature of the skin in the tissue volume of about 37 ° C to about 43 ° C.

The desired benefits according to the invention can be evaluated by extracting mRNA from a sample obtained from the volume of tissue through which the RF current passes; and determining an expression profile of a genetic panel consisting of at least one gene selected from Table 1 and / or Table 2 and at least one gene selected from Table 3. An expression profile that reflects up-regulation of genes selected from Tables 1 and / or 2 and, practically, no change in the expression of selected genes from Table 3 is indicative of the provision of a benefit.

Generally, the regulation of genes according to the invention is reflected in the information of change times of the expression. Several methods available for analyzing experiments based on microarrays are known in the industry in order to identify genes that are expressed differently in different conditions and the choice of methods can affect the set of genes identified. It is believed that the value of the times of change provides the most reproducible results. The value of the times of change can be defined as the ratio between the average observations of the control and the treatment (or as the difference between the average logarithmic data of the control and the treatment). Therefore, a value of times of change of 1 indicates that no change occurred during the observation of the control. A value of times of positive change indicates an increase in the expression in a period that is mentioned in the present description as up-regulation and a value of times of negative change indicates a reduction in the expression in a period of time mentioned in the present description as down regulation. The significance of a value of times of change can be determined by common statistical methods.

In accordance with certain aspects of the invention, the benefit can be optimized by adjusting the treatment temperature and / or other treatment parameters in response to the expression profile wherein the up-regulation of the selected genes in Table 1 and / or Table 2 is maximized while the expression of selected genes of Table 3 is maintained, practically, without change. The treatment regimes of the invention may comprise maintenance treatments wherein a benefit is maintained beyond the treatment period by one or more maintenance treatments.; each maintenance treatment comprises at least one maintenance cycle.

The skin treatment regimes of the invention contemplate the application of thermal energy by means of RF current conducted electrically through a handheld device for the supply of RF current. Preferred RF delivery devices in accordance with the invention provide RF as an electrically driven current between two electrodes placed on the skin at some distance therebetween. The delivery device that achieves the benefits of the invention conducts the radiofrequency current through the tissue volume without the presence of electromagnetic radiation at frequencies of visible or infrared light of the electromagnetic spectrum and without the use of monochromatic or polychromatic supplementary light sources. directed towards the tissue volume and, in addition, without applying mechanical cooling.

Certain embodiments of the invention are capable of evaluating the effectiveness of any power supply device or combination of power supply technologies to provide benefits in accordance with the invention. Accordingly, specific methods for evaluating the effectiveness of the treatment of an energy delivery device designed to provide the skin with a benefit by heating the skin include: treating the skin by applying the power supply device; extract the mRNA from a sample of the treated skin; and generating an expression profile for a genetic panel according to the invention. As illustrated in Example 3, below, there are RF current supply devices comprising tolerances of parameter specifications that can be adjusted to produce the expression profile according to the invention. A suitable device is manufactured by Syneron. An example of a device not designed to achieve the genomic response goals of the present invention is a personal handheld device for RF supply manufactured by Ultragen Ltd for distribution in Europe under the STOP ™ brand. With this device that is illustrated, moreover, in Example 3 for comparative purposes, the up-regulation of the genes of Tables 1 and 2 is not obtained. The specifications of the device are designed to obtain a regime based on the application of a practically lower amount of thermal energy in a larger area of the skin and, possibly, does not reach the levels required to achieve the positive homeotic effect reached by the devices and regimes in accordance with the present invention.

In accordance with another embodiment of the invention, methods are provided to analyze a treatment regime of facial skin. The methods are analyzed to determine the efficacy in the provision of a benefit of dermal remodeling and / or collagen to the skin of a mammal without stimulating the response of the inflammatory cytokine that damages the skin in accordance with another modality. The methods generally comprise treating the facial skin in accordance with a treatment regimen; extract the mRNA from a sample of the skin of the treated face; generate a gene expression profile for a genetic panel according to the invention; compare the profile of gene expression with a reference profile; and determining that the treatment regimen of the face is effective, wherein the expression profile reflects the up-regulation of selected genes from Table 1 and / or Table 2 and a considerable lack of regulation of selected genes from Table 3. In Specific modalities, the facial skin treatment regimen comprises generating a pulsed radiofrequency current through a first volume of tissue of the facial skin during a treatment cycle with a radiofrequency current generating device. The treatment regime may comprise moving the radiofrequency current generating device and generating a pulsed radiofrequency current through a volume of a second tissue of the facial skin during the treatment cycle.

In the analysis methods, skin areas are selected to verify the concordance with characteristics of target treatment areas, particularly the face. The cosmetically vulnerable areas of facial skin include the periorbital and perioral areas. Therefore, the structure of the skin of the face around the ear, known as the periauricular skin, is close to the target areas of the treatment, but in that area the spots that can be produced due to minor biopsy procedures are not easily visible. Therefore, in aspects of the invention that require biopsy the necessary sample is obtained by means of the biopsy of treated periauricular skin and the reference is obtained by means of the biopsy of pre-treated or untreated periauricular skin. Generally, the pretreated or untreated periauricular skin of reference comprises the skin practically adjacent to the treated periauricular skin. In very specific modalities, the expression profile of selected genes of Table 1 and / or Table 2 exhibits a value of fold change over the reference statistically greater than one.

Notably, the present invention can provide benefits to the skin from other parts than the face; however, in the examples illustrative highlights the skin of the face because it imposes greater challenges of cosmetic treatment because the effects of damage and chronological and environmental aging are manifested more easily and is initially thinner and more vulnerable than the skin of other areas of the body. In addition, the skin of the face is an area of great aesthetic importance for consumers of cosmetic treatment technologies.

In certain embodiments, the benefit provided by the application of RF current may be increased or enhanced by the application of a composition or cosmetic active in the facial skin in combination with the generation of the pulsed radiofrequency current. The active or composition can have an enhancing or synergistic effect on the gene expression profiles according to the invention. Non-limiting examples of suitable cosmetic actives include retinol propionate and derivatives thereof, caffeine, hyaluronic acid and, generally, plant extracts.

The methods of analysis according to the invention comprise, practically, the same parameters as the treatment methods according to the invention with respect to treatment cycles, treatment periods, treatment temperatures and delivery devices, adjusted for the tolerances of the desired analyzes.

In accordance with other embodiments of the invention, a genetic signature of genes that are expressed in a different form suitable to identify a cosmetic benefit to the skin is provided. The benefit includes the induction of collagen formation and / or dermal remodeling in a a dermal layer of the skin of a mammal without an immatory cytokine response that damages the skin; the genetic signature comprises at least one gene selected from Table 1, Table 2 and Table 3. Generally, a genetic signature is a subset of genes obtained from a set of gene data related to a specific feature, trait or biological function . Genetic signatures can be obtained from a whole set of genetic data or a part of it and the signatures according to the invention can comprise information of at least about two genes or any number of genes up to the number that constitutes the data set total. When a subset of the data set is used, the subset may comprise up-regulated genes, down-regulated genes, practically unregulated genes, or combinations of these.

The method of the present invention comprises the step of applying a first personal care composition and, optionally, a second personal care composition on an area of the skin of the mammal. The first and second personal care compositions can be found in a variety of forms including, but are not limited to, lotions, creams, serums, foams, gels, sprays, ointments, masks, bars, moisturizers, patches, powders or wipes. In one embodiment, the first composition for personal care is applied before or during the power supply. In an alternative embodiment, the second composition for personal care is applied after the application of the first composition and the energy supply.

Optionally, the method of the present invention may comprise the step of applying a third personal care composition on the skin of the mammal, wherein the third composition comprises a conditioning agent. In one embodiment, the third composition for personal care is applied before the application of the first composition for personal care. Preferably, the third composition for personal care is applied at least 24 hours before the power supply. In an alternative modality, the first composition for personal care is applied twice a day, and the energy is supplied once a day; alternatively, once a week and, alternatively, once a month. In one embodiment, the first personal care composition is applied to the skin twice a day, and energy is delivered to the skin per week.

The first, second and third personal care compositions may contain a variety of ingredients; non-limiting examples of them can be found in the CTFA International Cosmetic Ingredient Dictionary and Handbook, Tenth Edition (2004).

The compositions of the present invention may comprise from 50% to 99.9% of a dermatologically acceptable carrier. The carrier of the present invention may be in the form of an emulsion. In the present description, "emulsions" generally contain an aqueous phase and an oily phase. The oils can be derived from animals, plants or oil, can be natural or synthetic, and can include silicone oils. Emulsion carriers include, but are not limited to, oil in water, water in oil, water in oil in water, and oil in water in silicone emulsions. In one embodiment, the dermatologically acceptable carrier comprises an oil in water emulsion and, alternatively, a silicone emulsion in water. The emulsion may further comprise a humectant, for example, glycerin and a nonionic, cationic and / or anionic emulsifier. Suitable emulsifiers are described, for example, in U.S. Pat. UU no. 3,755,560 issued to Dickert et al., US Pat. UU no. 4,421, 769 issued to Dixon et al. and in McCutcheon's Detergents and Emulsifiers, North American edition, pages 317-324 (1986).

A wide variety of amounts of the compositions of the present invention can be used to improve the condition of the skin. The amount of personal care composition that is applied to the skin may vary depending on the location of the body and the desired benefit. Illustrative amounts include from 0.1 mg / cm2 to 40 mg / cm2. A useful application amount is 0.5 mg / cm2 to 10 mg / cm2.

A temperature change can be induced simultaneously in the skin or alternatively, in a composition applied to the surface of the skin. This change in temperature is additional to any change in temperature induced by the same energy supplied. For example, the skin may be heated before the energy supply or, alternatively, the skin may be cooled before, during or after the energy supply.

The use of genetic panels and genetic signatures after Combined treatment with extensive consumption research allowed the development of regimen areas that optimize the size of the treatment area and also determine the optimal treatment time. In contrast to the teachings of the industry it was discovered that the use of the device over large areas is not the best method of treatment. When treating an area that is too large, the initial portion may be cooled and recovered before the consumer passes the device over it again. If the area is too small, there is a risk that the consumer will treat the area excessively and cause unnecessary damage. In addition, the treatment itself requires a lot of work on the part of the consumer and the division of the treatment into different sections makes it easier for the consumer to comply with the regime. Equally, for the consumer it is much more pleasant if the personal care composition used with the device can be applied in a small area, treat the area and then remove any remaining composition for personal care. On the other hand, the experience of the treatment can be unpleasant for the consumer if he places the composition for personal care on the whole face or even in the middle of the face. This, in turn, has a negative effect on compliance by the consumer.

With reference to Figure 10 where the crow's feet area 60 of the consumer 62 is identified with a dashed circle, this area is located above and below the eyes 64 and adjacent to the outer corner of each eye. It is known that the crow's feet areas 60 are prone to form wrinkles and fine lines as the human gets older and are subject to environmental attacks. Figure 1 1 shows Zone A 70 where arrows within Zone A demonstrate an illustrative treatment path for the device (not illustrated). The treatment times are indicated above. Figure 10 illustrates Zone B 72 that is on the same consumer's eye 62 and overlaps the crow's feet area 60. Illustrative arrows show a possible treatment path. By defining Zone A 70 and Zone B 73 in this way the crow's feet area 60 is treated twice as much as the non-crowned portion of Areas A and B. This is an important discovery because most of the Fine lines and wrinkles on the face of a consumer who needs treatment is in the area of crow's feet.

Figures 13 and 14 illustrate Zones C 74 and D 76 in the other eye 64 of the consumer 62. As expected, these treatment areas are practically similar and treated in the same manner as Zones A and B.

An alternative treatment protocol is illustrated in Figures 15 and 16 wherein Zones E 80 and F 82 are illustrated at consumer 62. These "C" shaped treatment areas treat the crow's feet area 60, Figure 10 , only once in each pass, but the larger "C" shaped treatment area allows each area of the treated skin to cool slightly before making another pass with the device. Therefore, this is more comfortable for the consumer, but the crow's feet area 60, Figure 10, is treated only during one treatment cycle.

Optionally, a second personal care composition can be used in conjunction with the method described above. The The second composition for personal care can be used between successive treatment periods, which employ the first composition for personal care and the thermal device. The second composition for personal care preferably comprises at least one active for skin care that is not present in the first personal care composition.

The following examples are provided to illustrate certain features and advantages of various embodiments of the invention and should not be construed as limiting the scope thereof.

Examples Example 1: Treatment protocol This example illustrates the treatment of the periauricular region of the facial skin in order to perform genomic and histological evaluations. Therefore, all treatments are administered by a technician in a hospital setting for control purposes. The treatments are administered in four areas within the treatment area for a total of 16 minutes with 4 minutes per area, 3 times a week for six weeks. The protocol is based on a face / neck study divided into a control design within the parameters of the subject, where the treatment is applied only in the periauricular region on one side and a placebo treatment consisting of gel combined with a device No power is applied in the corresponding periauricular region on the other side. Treatments are controlled with thermal imaging chambers to ensure that the target skin surface temperature of 40-43 ° C is reached within the first minute and maintained, but not exceeded during each 4 minute treatment.

The treatment site is located and centered just below the ear. The site has a width of approximately 3.81 cm (1.5 inches) and a length of approximately 10.2 cm (4 inches). If an area of treated skin is too dry or too hot, an additional amount of gel can be applied. The subject's face and neck are clean and free of lotions, perfumes and the like. The hair is fastened with bras back from the treatment area.

Treatment with the device: 1 A pump (approximately 1.4 g of gel) is applied to the treatment site and dispersed evenly without rubbing. An additional gel pump may be applied to the cheek above the treatment area as needed during the treatment to cool the skin if it becomes too hot, depending on what the subject says. 2 The device turns on and the timer is set to 4 minutes 3 The applicator head of the device is applied to the skin and moves along the treatment site with a back-and-forth movement and a light touch. Connectivity is maintained while the head of the device is operated through the treatment area. Appropriate contact with the skin is indicated by a lens on the applicator that should flash red to indicate contact.

The image of the thermal camera is controlled during the treatment. Within 30-60 seconds, the skin in the treatment area reaches the optimum temperature of 40-43 ° C. The color patterns in the thermal image indicate temperature. Generally, as the device heats up, the applicator moves rapidly through the skin to avoid the discomfort caused by the increase in temperature. As the treatment progresses, the movement slows down to maintain the ideal temperature. The subjects are warned that they will feel that the device is hot, but that it will not burn them. The temperature is higher than 43 ° C if the thermal image shows areas of solid red color. The temperature is controlled by increasing the speed of the treatment in the movement from back to front, by means of a slight increase in the size of the treatment area, by means of the variation of the movement from back to front with a pattern in eight. or by adding more gel by touching the head of the applicator so that extra gel is deposited on the cheek for this eventuality. 6. If the treatment area fails to reach the optimum temperature or if it cools, the movement from back to front should slow down until the volume of the treatment area is at the target temperature.

Example 2: Biopsy protocol This example illustrates the relatively moderate biopsy protocol used to collect samples for evaluation and control purposes in accordance with certain aspects of the invention and confirms data described in the present disclosure.

A clinical biopsy study was performed with an RF power supply device in 30 adult women. Treatments were given in the periauricular region (around the ear) of the facial skin. In the cosmetics industry, periauricular skin is known as a suitable perorbital substitute. The treatments were administered clinically with a Syneron V8 device (see Example 3) and a four minute warm-up profile as the target profile. The treatment was applied three times a week for a total of six weeks. Biopsies were performed to review the histology of the skin as a function of the treatment and to generate a genomic profile. Biopsy procedure: A 4-mm biopsy was taken from the periauricular area on both right and left sides of the neck (this area is located just below the ear). With a sterile 30 gauge needle an anesthetic was injected contained 2% xylocaine with epinephrine just below the skin from which the biopsy was to be taken. Once the subject indicates that the area is numb, a puncture biopsy of the appropriate size is taken by standard aseptic techniques followed by closure of the suture. A 20% aqueous solution of aluminum chloride is used from homeostasis, as needed. After the biopsy the puncture site is checked to determine normal healing and the sutures are removed after 7 days.

Handling the sample after the biopsy: The biopsied tissue is divided into two separate samples, one for the genetic expression test and the other for the histological evaluation. The sample is divided into equal halves in a line parallel to a line drawn from the stratum corneum to the dermis. The handling of the sample is carried out in accordance with industrial standards.

Example 3: Extraction and analysis of mRNA This example illustrates the identification and analysis of the RNA biomarker that supports certain embodiments of the invention that include gene arrays, genetic signatures and the discovery of the biological model that guides the design of the RF delivery regime.

Biopsies of 4 mm were taken in accordance with Example 2 on both sides of the face / neck just below the ear, as described above 2 times, once at 24 hours after the final treatment and once at 4 weeks after to the treatment.

The biopsied samples were transferred to 2 ml centrifuge tubes containing 1.5 ml of tissue storage reagent (RNAIater® solution, invitrogen, Life Technologies, Carlsbad, CA). The tubes were refrigerated overnight at 2-8 ° C. The storage reagent was removed and the samples were placed in a freezer at -80 ° C until processing. Immediately prior to processing, the samples were removed from the freezer and 1.5 ml of monophasic solution of phenol and guanidine isothiocyanate (TRIzol® reagent, Invitrogen, Life Technologies, Carlsbad, CA) and a 3 mM tungsten carbide bead ( 3 mM Tungsten Carbide beads, Qiagen, catalog number 69997) in each tube. The samples were immediately homogenized in a mixer mill (Qiagen Inc.) with four 3 minute agitations at 30 / second, rotating the adapter after each. The samples were centrifuged for 10 minutes at 12,000 rpm to remove dirt. Then, the supernatant was transferred to pre-centrifuged Phase Lock gel heavy tubes (Phase Lock gel heavy tubes, Eppendorf, New York, NY, catalog No. 0032-005-152), 300 pL of chloroform (Sigma) and the tubes were added. they were shaken vigorously without excess vortex. The samples were centrifuged for 10 minutes at 12,000 rpm and then the supernatant was transferred to new 2 ml centrifuge tubes.

The agglutination conditions were adjusted by the addition of 800 pL of 70% ethanol and the tubes were mixed by vortexing and then centrifuged for a short time. 830 ml of the sample was transferred to a mini-column of RNeasy centrifugation in a vacuum collector and vacuum was applied. The remaining sample (approximately 21.1 mm (830 mil)) was transferred to the same RNeasy column and vacuum applied. To remove the contaminants, 700 μl and 500 μl were pipetted in sequence. of regulator RW1 in the RNeasy column and, after each rinse, vacuum was applied.

The RNeasy centrifugation column was transferred to a new 0.05 mm (2 mil) collection tube, the tube was centrifuged for 2 minutes at 14,000 rpm and the column was transferred to a new 1.5 mL collection tube. The residual ethanol was sucked from the inside edge of each column. Thirty pL of pre-warmed ribonuclease-free water was pipetted directly onto the RNeasy membrane and the membrane was incubated for 5 minutes and then centrifuged for 2 minutes at 14,000 rpm. Then, the eluate was collected and, thus, RNA was provided in water ready for use.

After isolation, RNA yield was determined with the RNA 6000 Nano LabChip® kit no. 5065-4476 available from Agilent Technologies, Inc. of Santa Clara, CA. RNA was evaluated by one-step RT-PCR. For the analysis of the RT-PCR biomarker the RNA was diluted to a final concentration of 5 ng / well.

The purified RNA was converted to cDNA with a RT-PCR kit (QScript ™ One-Step Fast MGB) RT-PCR available from Quanta BioScience, Inc., Gaithersburg, MD). Then, five hundred nanograms of RNA were mixed with the QScript enzyme / regulator mixture and the mixture was processed in a thermal cycler in accordance with the instructions of the kit. Then, 1 pL of the resulting cDNA was mixed with the master mix Quanta Perfecta Master Mix and aliquoted through a common matrix plate (Custom RT Profiler ™ PCR Array available from SABiosciences, Corp., Federick, MD) containing primers. previously validated for the following genes: KRT2, KRT6A, CLDN1, LOR, FLG, IVL, KRT10, AQP3, KRT14, FBN1, FBNL1, TNXB, FN1, LOXL2, COL3A1, COL1A1, ELN, LOXL1, TIMP2, IL1A, TIMP1, TNF , MMP1, MMP9, MMP3, SOD2 and IL1 B. The matrices were then sealed and processed in a thermal cycler (StepOnePlus ™ Real-Time PCR System Upgrade from Applied Biosystems, Inc. Foster City, CA).

The data was analyzed with the data analysis program provided by SABiosciences of Frederick, MD.

Data on the times of expression change for the 27 compilation genes listed above at the 24-hour and 4-week target times are indicated in Figures 4A, 4B and Figures 6A, 6B, respectively, above. The genes associated with the dermal matrix are grouped as 90 and 91, the genes associated with the remodeling of dermal inflammation are grouped as 92 and 93 and the genes associated with epidermal differentiation are grouped together as 94 and 95. The data from the Times of change for the illustrative genes are indicated in Figures 5A collagen 1A1, 5B elastin and Figure 7A collagen 1A1 and 7B elastin. The joint regulation between many of the collagen1A1 genes and elastin genes must be observed.

The dimensions and values described in this description should not be construed as strictly limited to Exact numerical values mentioned. Instead, unless otherwise specified, each of these dimensions will mean both the aforementioned value and a functionally equivalent range that includes that value. For example, a dimension described as "40 mm" refers to "approximately 40 mm." All documents cited in the present description, including any cross-reference or related application or patent, are incorporated in their entirety by reference herein unless expressly excluded or limited in any other way. If any document is mentioned it should not be construed as admitting that it constitutes a prior art with respect to any invention described or claimed in the present description, or that independently or in combination with any other reference or references, instructs, suggests or describes such invention. In addition, to the extent that any meaning or definition of a term in this document contradicts any meaning or definition of the term in a document incorporated as a reference, the meaning or definition assigned to the term in this document shall govern.

Although particular embodiments of the present invention have been illustrated and described, it will be apparent to those skilled in the art that various changes and modifications can be made without departing from the spirit and scope of the invention. Therefore, it has been intended to encompass in the appended claims all changes and modifications that are within the scope of this invention.

The data of the times of change were reviewed and analyzed in the groupings. Surprisingly, it was discovered that when the genes that showed a very low regulation in response to treatment were segregated (IL1A, IL1 B and TNF), these included the genes of response to the inflammatory cytokine, while the genes related to the synthesis and replacement of collagen and thermal remodeling were regulated upwards in the treatment conditions. The genes selected according to the information of times of change are therefore indicated in Tables 1, 2 and 3 below.

In addition, surprisingly, it was found that the positive impact of the treatment regimen on gene expression at 24 months remained virtually unchanged until 1 month before the target date. Maintenance treatments may be desirable to maintain genomic effects for periods longer than this.

It is contemplated that the gene expression profile reflects downstream regulation in translation products. Although the value of times of observed change may be relatively low, it has been found that the sustained up regulation in the synthesis of certain proteins and enzymes over a prolonged period gives the skin a practically lasting cosmetic benefit.

Example 4 Composition for personal care An illustrative personal care composition for use with the devices of the present invention is included below.

Table 1 Dermal markers Table 2 Marker remodeling markers MMP3 Metalloproteinase family of Degrada fibronectin, laminin, matrix collagens (MMP) proteins III, IV, IX and X, and the proteoglycans of the (SEQ ID NO: 14) cartilage. It is thought that the enzyme is involved in the repair of wounds, advance of atherosclerosis and initiation of tumors.

Table 3 Markers of the response to the inflammatory cytokine IL1A A member of the family of Stimulates the proliferation of thymocytes by the atocinas interleucina 1; produced induction of IL-2 release, maturation (SEO, ID NO: 17) by monocytes and macrophages such as and proliferation of B cells and the activity of a proprotein that is processed fibroblast growth factor. Proteolytically and released into IL-1 proteins are involved in the response response to cell injury and, because of the inflammatory and are identified as both pyrogens, induces apoptosis. endogenous and it is reported that they stimulate release of prostaglandin and collagenase from synovial cells

Claims (11)

NOVELTY OF THE INVENTION CLAIMS

1. A genetic panel comprising genes regulated in the skin of mammals in response to the generation of a radiofrequency current in a volume of mammalian skin tissue sufficient to heat the volume of tissue to a treatment temperature, wherein at least one The gene is selected from SEQ ID NOs: 1-9 or SEQ ID NOs: 10-14 and at least one gene is selected from SEQ ID NOs: 15-17.

2. The genetic panel according to claim 1, further characterized in that at least one gene is selected from SEQ ID NOs: 1-9, at least one gene is selected from SEQ ID NOs: 10-14 and at least one gene is selected from SEQ ID NOs: 5-17.

3. A microarray comprising a set of immobilized nucleic acid probes capable of hybridizing to and detecting genes that constitute a genetic panel of claims 1 or 2.

4. A method for evaluating the effectiveness of the treatment of a power supply device designed to provide the skin with a benefit by heating the skin; the method comprises: extracting the mRNA from a sample of the previously treated skin by means of the application of the power supply device; and generating an expression profile for a genetic panel of claims 1 or 2.

5. A method for analyzing a treatment regimen of facial skin to determine the efficacy in providing a dermal remodeling and / or collagen benefit to the skin of a mammal without stimulating the response of the inflammatory cytokine that damages the skin; the method comprises: extracting the mRNA from a sample of the face skin previously treated according to a treatment regimen; generating a gene expression profile for a genetic panel of claims 1 or 2; compare the profile of gene expression with a reference profile; and determining that the treatment regimen of the face is effective, wherein the expression profile reflects the up-regulation of selected genes of SEQ ID NOs: 1-9 and / or SEQ ID NOs: 10-14 and a considerable lack of regulation of selected genes of SEQ ID NOs: 15-17.

6. The method according to claim 5, further characterized in that the treatment regime of the facial skin comprises generating a pulsed radiofrequency current through a first tissue volume of the facial skin during a treatment cycle with a generating device. of radiofrequency current.

7. The method according to claim 6, further characterized in that the treatment regime further comprises moving the radiofrequency current generating device and generating a pulsed radiofrequency current through a volume of a second tissue of the facial skin during the treatment cycle.

8. The method according to claim 7, further characterized in that the skin of the face comprises the periauricular skin and the sample was obtained by means of the biopsy of treated periauricular skin and the reference was obtained by means of the periauricular skin biopsy pretreated or not treated.

9. The method according to claim 8, further characterized in that the pre-treated or untreated periauricular skin of reference comprises the skin practically adjacent to the treated periauricular skin.

10. The method according to claim 9, further characterized in that the genes selected from SEQ ID NOs: 1-9 show a statistically greater increase than one time in expression with respect to the reference.

11. A genetic signature of differentially expressed genes to identify a cosmetic benefit for the skin; the benefit comprises the induction of collagen formation and / or dermal remodeling in a dermal layer of the skin of a mammal without a response of the inflammatory cytokine that damages the skin; the genetic signature comprises at least one gene selected from SEQ ID NOs: 1-9, SEQ ID NOs: 10-14 and SEQ ID NOs: 15-17.