Method for cosmetic treatment of skin hyperpigmentation

Abstract

The present invention relates to a cosmetic composition intended for application to the epidermis for the treatment of skin hyperpigmentation by means of a cryogenic fluid and a device configured to ensure the ejection and spraying of a controlled quantity of said fluid, characterized in that it comprises a dose of cryogenic fluid obtained by a sequence of at least one spraying (N) of said fluid onto the epidermis at a flow rate of 0.61 ml / s, in a negative temperature range on the skin of between 0°C and -3°C, creating oscillations of the temperature of said epidermis, the total duration of which is between 1.5 seconds and 25 seconds, each oscillation consisting of a phase of abrupt cooling at a temperature decrease rate of between 50°C / s and 170°C / s followed by a phase of slower increase in temperature up to a temperature of between 20°C and 34°C.

Description

DESCRIPTION TITLE: Cosmetic treatment method for skin hyperpigmentation

[0001] The invention applies to the field of cosmetics and is more particularly concerned with a method of treating skin hyperpigmentation by cryogenic means.

[0002] Cryogenics is commonly used in cosmetics to remove brown spots. Brown spots, also known as age spots, are small spots of varying sizes on the skin that appear on sun-exposed areas such as the face, hands, shoulders, or arms. Brown spots are considered benign cosmetic imperfections and are often considered unsightly. They are treated with cosmetic methods such as creams applied to the skin or with cryogenics.

[0003] Traditional methods of cosmetic treatment by cryogenics are implemented, for example, by means of a spraying device ensuring local projections of a cryogenic fluid (liquid nitrogen, di-methyl-ether, di-fluoro-ethane, tetra-fluoro-ethane, ...) onto the epidermis at strongly negative temperatures (between -20°C and -180°C) in one or more freezing / thawing cycles.

[0004] Indeed, in general, the sudden drop in the temperature of the epidermis to a negative temperature causes, even before the cellular tissue has solidified, a micro-crystallization of intracellular water which induces, at the same time, membrane alterations, a denaturation of structural proteins and enzymatic systems, all conditions leading to a deleterious effect on the cell (cell lysis by mechanical shock).

[0005] During these cycles, another phenomenon occurs, called osmotic shock. The sudden drop in temperature followed by a slow warming, occurring in several consecutive cycles, leads to the crystallization of extracellular water, followed by changes in the concentrations of extracellular and intracellular elements, creating concentration gradients. Intracellular fluid then moves towards the extracellular environment during the cooling phase. (or just after), then return to the intracellular compartment during the warming phase, and this several times in succession following the cycles. Eventually, the cell will no longer be able to manage these fluid movements and the osmotic shock will cause cell lysis.

[0006] More precisely, during the very rapid cooling phase, microcrystals of ice form in the extracellular environment. This results in the formation of a concentration gradient between the extracellular and intracellular environments, and therefore a movement of water from the intracellular to the extracellular environment. The cell then decreases in volume. After the epidermis has cooled, its temperature rise to at least 20°C and at most 34°C is a slow process (typically 10 to 100°C / min), unlike the temperature decrease. During this phase, the extracellular microcrystals melt, and this time, a new phenomenon occurs: a reversal of the gradient between the extracellular and intracellular environments.Indeed, the water, which is abundant in the extracellular environment, enters the cells en masse to compensate for this gradient, causing intracellular edema, resulting in cell swelling and membrane rupture. This leads to another instance of cell lysis, not through the formation of microcrystals, but through a mechanism of cellular dehydration / rehydration. This is known as osmotic shock.

[0007] Other methods of cryogenic cosmetic treatment are implemented, for example, using a spray device that delivers localized projections of a cryogenic fluid onto the epidermis at sub-zero temperatures (between -5°C and -20°C) in one or more freeze / thaw cycles. This allows for a cyto-selective action, eliminating only the cells (melanocytes) responsible for skin hyperpigmentation such as brown spots, without harming other cells. This cyto-selective mode of action helps preserve keratinocytes, in particular, because these cells are less sensitive to cold than melanocytes.

[0008] Cryogenic devices such as the one described in patent application WO 2016 / 113305 exist for the implementation of cyto-selective treatment. brown spots. This device ensures the controlled spraying of a cryogenic fluid intended to lower and maintain, for a few seconds, the temperature of the epidermis between -5°C and -20°C then to carry out a slow rise in temperature to obtain a lysis, i.e. a destruction, of the melanocytes and, consequently, an elimination of the brown spots.

[0009] To this end, the device comprises a pressurized cryogenic gas cartridge, a nozzle ensuring the delivery of a precise dose of cryogenic gas from the cartridge coupled with automated timing means, and a nozzle designed to concentrate, diffuse, and apply the gas flow precisely and homogeneously to a defined skin area. Such a nozzle is described, in particular, in FR3076200A1.

[0010] The effect of cold on the epidermis has already been widely studied and analyzed, notably by the article [Bunsho Kao et al. Evaluation of Cryogen Spray Cooling Exposure on In Vitro Model Human Skin. Lasers in Surgery and Medicine 34: 146-154 (2004)] which showed that the mechanical shock produced by very rapid freezing causes the formation of ice microcrystals inside and outside melanocyte cells, resulting in the rupture of the cell wall and the death of the cell.

[0011] Osmotic shock, through differences in osmotic pressure gradient between the intracellular and extracellular environments, will also cause the lysis of melanocytes. Subsequently, within 2 to 4 weeks, which corresponds to the average cell turnover time, the body will progressively eliminate the cellular components and melanin until the hyperpigmentation disappears completely.

[0012] Other scientific studies, including that of Gage et al. [Gage AA et al. Effect of varying freezing and thawing rates in experimental cryosurgery. Cryobiology. 1985 Apr;22(2):175-82], present the results of cryotherapy trials performed on the epidermis at various negative temperature ranges. The protocol usually used for these trials involves a rapid freezing phase and a slow thawing phase. The observed cellular damage is due to the formation of ice crystals and is associated with microcirculatory failure. blood flow after the thawing phase. Indeed, the interruption of blood circulation deprives epidermal cells of a chance of survival, which leads to a preference for a short cooling phase.

[0013] Histological sections showed the viability of melanocytes for 7 days after the application of a temperature between 0°C and -4°C. At temperatures below -4°C, cellular damage occurs in the melanocytes, and no pigment is observed one week after the application of cold.

[0014] From -4°C to -7°C, some cell types remain viable in the deeper layers of the epidermis, but melanocytes are no longer present. Melanocyte disintegration occurs over several days; the melanin is broken down, and then the melanocytes disappear from the deeper layers. This is because the melanosomes containing melanin are phagocytosed by neighboring keratinocytes, whose enzymes digest the membrane, as demonstrated by electron microscopy of biopsies.

[0015] From -7°C to -30°C, no melanocytes or melanin are observed. However, repigmentation towards the skin's physiological (natural) pigmentation occurs 4 months after the application of cold within a temperature range of -4°C to -30°C. In fact, the repigmentation of the treated area occurs from the melanocytes present at the periphery of the treated area.

[0016] If the temperature applied to the epidermis drops to -40°C, a total disappearance of melanocytes and melanin is observed. This results in permanent depigmentation of the skin.

[0017] Studies [Har-Shai Y et al. Effect of skin surface temperature on skin pigmentation during contact and intralesional cryosurgery of keloids. JEADV 2007, 21, 191-198] further specify that melanocytes remain viable only if they are subjected to a temperature above -4°C. Indeed, between -4°C and -7°C, lysis of the granules containing the pigments (melanosomes containing melanin) is observed, followed by the beginning of enzymatic digestion of melanin within the melanocytes. in keratinocytes (in the deep layer of the epidermis, therefore near the basal layer).

[0018] RAIDAN et al. [Raidan M. et al. Effect of Cooling to Low Temperatures on Viability of Human Skin Keratinocytes at Different Stages of Differentiation. Cell and Tissue Biology, 2010, Vol. 4, No. 6, pp. 1-10] conducted cell viability studies of keratinocytes treated with cold in a temperature range of -10°C to -70°C. Keratinocyte preservation is essential because these cells have the capacity to regenerate the different layers of the epidermis.

[0019] These studies indicate that keratinocyte destruction occurs at temperatures below -35°C, with a significant elimination of melanocytes. Therefore, temperatures between -5°C and -20°C would act on melanin and melanocytes without damaging keratinocytes, according to the principle of cytoselectivity.

[0020] BURGE et al. [Burge SM et al. Pigment changes in human skin after cryotherapy. Cryobiology, 1986, 23:422-432] showed that after a brief 5-second cooling of the skin by spraying liquid nitrogen at -196°C over a margin of 1 to 5 mm beyond the lesion to be treated, hypopigmentation persists and that a 15-second prolongation of the application of cold completely eliminates the melanosomes in the keratinocytes.

[0021] According to Andrews [Andrews MK et al. Cryosurgery for common skin conditions. Am fam physician, 2004, 69 (10): 2365-2373], a liquid nitrogen spray can reach a temperature of -50°C at an epidermal depth of between 4 mm and 5 mm. These authors therefore hypothesize that hypopigmentation may be due to a decrease in the number of melanocytes, a decrease in the production of melanosomes, or a blockage of melanosome transfer to keratinocytes, and conclude that cryotherapy impairs epidermal control.

[0022] Furthermore, GAGE ​​AA et al. (1985) showed that a temperature below -50°C leads to tissue necrosis and, conversely, for cooling temperatures above -15°C, the tissue remains viable regardless of the warming program used. However, they also observed that slow thawing of the tissue between 5 and 7 minutes is also harmful because large ice crystals form, causing intracellular dehydration.

[0023] Therefore, a method involving rapid cooling (or even freezing) (between 30 seconds and 2 minutes) to temperatures between -15°C and -50°C with maintenance of this temperature (for 3 minutes) followed by slow warming (between 5 and 7 minutes), represents the most aggressive experimental scheme for the epidermis.

[0024] Conversely, rapid defrosting (less than 2 minutes) results in less necrosis regardless of the program used, with a total absence of necrosis if it follows slow cooling (3 minutes) between -15°C and -24°C.

[0025] Furthermore, a clinical study was conducted by Attia et al. [Attia EAS et al. Cryotherapy versus phenol chemical peeling for solar lentigines: a clinical histology, immunohistochemical and ultrastructural study. J Egypt Women Dermatol SOC, 2010, 7:87-96, 2010] on twenty patients with actinic lentigines on the backs of their hands who were treated with liquid nitrogen. A cooling cycle of 3 to 5 seconds was performed until the lesion froze. Biopsies taken 3 weeks after treatment showed that cryotherapy reduced skin pigmentation. This result is due to a decrease in the number of melanocytes as well as a reduction in the size and aggregates of melanin granules in melanocytes and keratinocytes, corroborated by histological sections and electron microscopy.

[0026] Finally, there are cosmetic treatment methods using a spray device that delivers localized projections of a cryogenic fluid onto the epidermis at positive temperatures (between 0°C and 14°C). These methods demonstrate that it is possible to achieve, with minimal irritation, a significant reduction in melanocyte viability while preserving keratinocytes (and thus effective elimination of cutaneous hyperpigmentation). This is achieved through a cryogenic and cyto-selective cosmetic treatment characterized by a series of epidermal temperature oscillations over very short periods, while maintaining the epidermis within a positive temperature range.

[0027] In view of these previous studies, the results of which are set out above, a person skilled in the art could therefore deduce that the optimal effectiveness of a cryogenic process intended for the cosmetic treatment of cutaneous hyperpigmentation, and thus aimed at the elimination of melanocytes while preserving keratinocytes, lies in a temperature range of the epidermis between -5° and -20°C and, preferably, between 0 and 14°C.

[0028] Indeed, studies show that when the epidermis is exposed to very low temperatures (between -20°C and -180°C), epidermal control is impaired and, concomitantly, inflammatory processes or severe irritations occur.

[0029] Furthermore, applying a cryogenic fluid to the epidermis at very low temperatures also presents the risk of simultaneously destroying cells other than melanocytes. This risk is particularly critical with regard to keratinocytes, which play an essential role in regulating melanogenesis. All these side effects are problematic in the context of cosmetic treatment for skin hyperpigmentation because they can cause pain during application and post-treatment, inflammation, crusting, edema, blisters, hypopigmentation, and scarring.

[0030] However, experiments carried out during the development of the invention revealed that, contrary to a dose-dependent effect (dependent on the amount of cryogenic fluid applied) which could be expected, the melanocyte mortality curve as a function of the temperature reached (called target temperature or arrival temperature) is made up of several parts which are not all dose-dependent and one in particular of which is stable over a temperature range of -5°C to 0°C.

[0031] For example, when comparing the viability of melanocytes between the application of a dose of sprayed cryogenic gas allowing the epidermis to reach a target temperature of -5°C (sequence referenced EC-05) from its normal starting temperature (34°C) in an ambient temperature environment (23°C), and the application of a lower dose allowing a At a temperature of 0°C (sequence referenced ECOO), we observe that this viability remains the same (63 and 61% respectively), whereas we could have expected it to be quite different (the dose of cryogenic gas in the ECOO sequence being lower).

[0032] It has been demonstrated previously by trials of multiple sprays of the same cryogenic gas on the epidermis to cool it while maintaining it at positive temperatures (these sprays being repeated in a given time to produce temperature oscillations), that the decrease in the viability of melanocytes increased compared to a single spray.

[0033] However, further experiments have also shown that it is also possible to decrease the viability of melanocytes with sequences of oscillations of the target temperature of the epidermis in a range of slightly negative temperatures.

[0034] In this context and according to a first object, the invention relates to a cosmetic treatment method for cutaneous hyperpigmentation using a cryogenic fluid and a device configured to ensure the ejection and spraying of a controlled quantity of said fluid onto the epidermis, characterized in that a sequence is carried out comprising at least one spraying of said fluid onto the epidermis at a flow rate of 0.61 mL / s to create, in a range of negative temperatures on the skin between 0°C and -5°C, a series of temperature oscillations of said epidermis, the total duration of which is between 1.5 seconds and 25 seconds, each consisting of a phase of rapid cooling at a rate of temperature descent between 50°C / s and 170°C / s followed by a phase of slower temperature rise to a temperature between 20°C and 34°C.

[0035] Here, "total duration" refers to the temperature drops and rises for the entire sequence.

[0036] According to an advantageous feature of the process of the invention, the duration of each spray is between 0.10 seconds and 0.35 seconds.

[0037] According to another feature of the method of the invention, the duration of each of the oscillations is between 1.5 seconds and 16 seconds and is preferably between 1.5 seconds and 5.8 seconds.

[0038] According to yet another feature of the process of the invention, the warming phase of the oscillations corresponds to a rise in the temperature of the epidermis of between 20°C and about 34°C.

[0039] According to other characteristics, the number of successive oscillations of epidermal temperature in a sequence is between 1 and 6 and the cooling phase of the epidermis is carried out according to a gradient of at least 15°C from an initial temperature of about 34°C.

[0040] Preferably, the cryogenic fluid used is a gas whose boiling point is between -196°C and -0.5°C under a normal pressure of 1 atmosphere or 1.013 bars.

[0041] This cryogenic gas is chosen from the group consisting of 1,1-difluoroethane, methoxymethane, 1,1,1,2-tetrafluoroethane, trans-1,3,3,3-tetrafluoropropene, butane, propane, isobutane, nitrogen or mixtures of these gases.

[0042] According to a first embodiment of the process of the invention, the sequence comprises a single spray of 1,1-di-fluoro-ethane at 0.61 mL / second for a duration of 0.30 seconds so as to achieve a target temperature of the epidermis of 0°C.

[0043] According to a second embodiment of the process of the invention, the sequence comprises 6 successive sprays of 1,1-di-fluoro-ethane at 0.61 mL / second, each having a duration of between 0.11 seconds and 0.31 seconds so as to reach a target temperature of the epidermis of 0°C after each spray.

[0044] According to a third embodiment of the process of the invention, the sequence comprises a single spraying of 1,1-difluoroethane at 0.61 mL / second for a duration of 0.345 seconds in order to reach a target epidermal temperature of -5°C.

[0045] According to a fourth embodiment of the process of the invention, the sequence comprises 6 successive sprays of 1,1-di-fluoro-ethane at 0.61 mL / second, each having a duration of between 0.14 seconds and 0.345 seconds so as to reach a target temperature of the epidermis of -5°C after each spray.

[0046] Other features and advantages of the invention will become apparent from the description that follows, with reference to the attached and detailed drawings below.

[0047] [Fig. 1] is a graph illustrating various modes of implementation of the cosmetic treatment process of the invention with the variations in the mortality rate of melanocytes as a function of the temperatures applied according to different sequences.

[0048] [Fig. 2] is a graph illustrating a method of implementing a cosmetic treatment process of the invention with temperature variations according to cycles.

[0049] [Fig. 3] is a graph illustrating various modes of implementation of a cosmetic treatment process of the invention with the variations of melanocyte viability as a function of a slightly negative target temperature applied according to several oscillations.

[0050] [Fig. 4] is a graph representing a preferred sequence of the cosmetic treatment process of the invention with temperature variations as a function of time in the form of a series of oscillations.

[0051] [Fig. 5] are graphs representing, respectively, the variations in epidermal temperature as a function of time (curves in the lower part) in relation to a preferred sequence of sprays (top diagram) for the implementation of a cosmetic treatment process of the invention.

[0052] For clarity, identical or similar elements are identified by the same reference symbols in the description and in the figures. Naturally, the methods of preparing the cosmetic composition and the various implementations of the cosmetic processing procedure using this composition, illustrated in the figures above and described below, are given only as non-limiting examples. It is explicitly intended that different methods may be proposed and combined to create further solutions.

[0053] The invention relates to the field of cosmetic and cryogenic treatment of cutaneous hyperpigmentation and, in particular, the elimination of so-called brown spots that appear on the epidermis.

[0054] In general, analysis of the scientific literature reveals that the destruction or "lysis" of melanocytes responsible for this skin hyperpigmentation is obtained at temperatures below -4°C, a temperature measured at the level of the epidermis.

[0055] Furthermore, the majority of publications describe mechanisms leading to cell death in melanocytes at negative temperature ranges. This is the case with osmotic shock in cryobiological processes involving the freezing of cells in liquid nitrogen (-196°C).

[0056] As is known, mechanical and / or osmotic shocks occur following the formation of ice crystals, which cause cell membrane rupture. Therefore, these shocks only appear possible within a range of sub-zero temperatures.

[0057] Furthermore, numerous scientific publications emphasize that lowering the temperature within a range of positive temperatures on the epidermis (even down to 0°C) does not affect cell viability (General Guide to Cryopreservation of Animal Cell Cultures, Corning).

[0058] Although other studies indicate that a slight decrease in cell viability can be observed by maintaining the epidermis at positive temperatures, they specify, however, that this result can only be obtained on the long term (several days). However, these conditions are not compatible with the goal sought by the invention which aims to achieve this objective almost instantaneously, by means of a treatment requiring only a very short exposure (a few seconds) of the cells to the effects of the cryogenic fluid to cool them to temperatures close to 0°C when implementing the process by means of a device as described in patent application WO 2016 / 113305 with a nozzle corresponding in particular to that of FR3076200A1.

[0059] At around 20 to 25°C, most cells can survive, and even grow and metabolize for at least 3 or 4 days, or even longer. However, some cells eventually die upon exposure to moderate cold (but only after several days) when the exposure time is sufficiently long, while others remain alive and can multiply again if re-incubated at eugenic temperatures. Indeed, many cells survive in the +4°C to +5°C range for periods ranging from 24 to 48 hours. They then suffer no apparent damage other than an increased latency period, and when returned to an optimal temperature, they are slower to resume multiplication.

[0060] It is indeed possible to preserve a wide variety of cell types at +4°C for 6 to 9 weeks, provided certain methodological rules are followed: (a) The cells must be in good condition at the time of cooling; (b) they must adhere to a support; (c) they must be cultured in their usual culture medium; (d) the culture medium must not be changed during the storage period at 4°C; (e) the culture medium must not be changed during the few days following the return to eugenic temperature. All these conditions were met in our experiments.

[0061] Given the state of the art, there was therefore no reason to think that significant mortality of melanocytes could be observed when they were only kept at slightly negative temperatures and far from the thermal threshold of their viability and, moreover, for very short periods of time.

[0062] Therefore, experiments (in vitro cell cultures) were conducted within the framework of the invention to determine the effects of different temperatures (+10, +5, 0, -5, -10, -15, and -16°C) on melanocyte mortality. Sequences were developed from the test bench (reference sequences, for example, EC-5 to reach a target temperature of -5°C). A control group of cells was tested at +23°C. After 24 hours of treatment, cell mortality was quantified by measuring protein expression (see Figure 1).

[0063] The results of these experiments showed that the variation in the mortality rate of melanocytes as a function of temperature was not always linear over the entire temperature range studied, contrary to what could be expected.

[0064] More specifically, the mortality rate curve as a function of temperature, which is represented by the graph in Figure 1, is in three parts. A first part is linear and increasing and includes the sequences EC-16 (at -16°C), EC-15 (at -15°C) and EC-10 (at -10°C), a second part is linear but in the form of a plateau and includes the sequences EC-05 (-5°C), ECO (at 0°C), EC+05 (at +5°C) and EC+10 (at +10°C) then a third part is again linear and increasing and includes the sequences going from EC+10 to EC+20 (+20°C).

[0065] It was observed that the mortality rate for the EC-05 and ECO sequences was very different from that obtained for the control group. These results show that different phenomena occur, even at slightly negative epidermal temperatures, for extremely short sequence durations (less than 30 seconds).

[0066] Furthermore, the modification of cell viability occurs for very small temperature gradients (for example, a gradient of 28°C to go from 23°C to -5°C in the case of experiments on cell cultures where the experimental conditions place the cells at a temperature of about 23°C for the duration of the experiment).

[0067] Further experiments were conducted in this slightly negative temperature range (from -5°C to 0°C) to confirm these results. These data were complemented by a study demonstrating the effect of temperature on melanocyte cell mortality for sequences where the target temperatures reached were closer together, in order to more precisely observe the influence of the applied cryogenic fluid dose.

[0068] These observations have made it possible to design cryogenic gas spray sequences that allow the temperature of the epidermis to be lowered abruptly to slightly negative temperatures, with the aim of limiting as much as possible the inflammatory processes, irritations and transient hyperpigmentation (hyperpigmentation following the inflammatory process) encountered, in particular, with the EC-16 sequence (sequence inducing negative temperatures of about -16°C).

[0069] Furthermore, it was found that lowering the cell temperature along a 28°C gradient, beyond the ranges tested so far, did not lead to cell destruction. Indeed, it was observed that lowering the incubation temperature of melanocytes and keratinocytes (+37°C) to a lower temperature (-5°C) did not alter cell viability. These data therefore contradict the results of the study conducted over a range of +0°C to -5°C.

[0070] The rate of change in epidermal temperature is also not a factor. In cryogenics, three temperature decrease rates are generally distinguished. A slow decrease corresponds approximately to a drop of 5°C / min, while a rapid decrease occurs at 25°C / min and a very rapid decrease at 100°C / min.

[0071] The method of applying the cosmetic composition of the invention comprises, as illustrated in Figure 2, a sequence of N successive sprays of the cryogenic fluid intended to cause, upon contact with the skin, an ultra-rapid decrease (> 3000°C / min) in its temperature from a starting temperature denoted (TO) (generally 34°C) to a target temperature denoted (Tl), i.e. a gradient (T0-T1). This temperature drop is carried out in a given time period or duration (t) and therefore at a certain speed or kinetic (denoted Cl).

[0072] Due to the relatively short pause between successive sprays, the epidermal temperature tends to rise naturally during this interval. Consequently, this temperature decrease is accompanied by a series of (n) oscillations (i.e., periodic temperature variations) within a range of epidermal temperatures (T2, T3, T4...) over a given total time (t1). All these temperatures are slightly negative, and these oscillations may or may not have a uniform amplitude and / or period.

[0073] A first example of a sequence (EC-03 and 4 oscillations) allowing a decrease of approximately 66.8% in the viability of melanocytes consists of the following parameter values ​​with a number (n) of oscillations (and therefore of sprays) equal to 4 (the precision of the experimental model being ±2°C) and a time (tl) of 12 seconds: TO: 34.0°C / T1: -4.15°C / T2: 18.13°C / T3: -3.64°C / T4: 20.16°C / T5: -2.37°C / T6: 20.29°C / T7: -3.39°C.

[0074] Furthermore, a supplementary study confirmed that the number (n) of epidermal temperature oscillations also contributed to increasing the melanocyte mortality rate; therefore, the treatment process using the cosmetic composition is expected to include a series of 1 to 6 oscillations. Thus, a given sequence of N sprays corresponds to a number n of oscillations, the respective durations and amplitudes of which may vary.

[0075] The cryogenic properties of the fluid used in the cosmetic composition of the invention, as well as the functionalities of the device envisaged for preparing this composition in situ, make it possible to perform, in the laboratory, a temperature decrease from 23°C (which is the temperature of cell cultures when they are handled) to -15.8°C in 0.6 seconds (in an experiment), i.e., a rate of 76°C per second or 4560°C / min. This is therefore a cooling process described as ultra-rapid.

[0076] However, there was no reason to think that a drop in temperature of the epidermis with, for example, a gradient of the order of 23°C to 28°C in less than a second, reaching slightly negative temperatures and outside the zone of non-viability of melanocytes, could cause significant melanocyte mortality and disappearance of brown spots.

[0077] Consequently, the cosmetic treatment process of the invention consists of delivering a dose of cryogenic fluid, by means of the device described above, in order to obtain a sudden drop in the temperature of the epidermal cells with a gradient enabling the creation of a thermal shock (for example, of 28°C), while maintaining these cells in a range of positive or slightly negative temperatures.

[0078] However, this treatment procedure was carried out by applying several doses, typically between one and six, depending on the chosen formulation. These doses are delivered and applied to the epidermis via a sequence of very short, successive sprays. This sequence produces oscillations in the epidermal temperature that unexpectedly reduce the viability of melanocytes, thus treating skin hyperpigmentation.

[0079] Thus, neither the importance of the temperature gradient, nor its proximity to the optimal temperature for cell viability (+37°C), nor the fact of proceeding only within a range of slightly negative temperatures of the epidermis, nor the speed of temperature reduction, nor the number of oscillations, nor the very short duration of exposure of the cells to the effects of the cryogenic fluid, could lead the person skilled in the art to conclude that such a cosmetic treatment would result in a significant reduction of cutaneous hyperpigmentation on the epidermis.

[0080] In summary, the invention thus proposes a cosmetic composition whose doses (micro-volumes) are formulated in situ by means of a sequence of very short sprays (of duration typically between 0.01s and ls) comprising successive sprays of a cryogenic fluid for its immediate application to the epidermis.

[0081] Thus, through a series of ultra-rapid temperature drops, each followed by a brief temperature rise (each drop and rise curve forming a temperature oscillation), this composition allows for the periodic lowering of the epidermal temperature, initially at 34°C (and therefore that of the melanocytes), along a temperature gradient exceeding 20°C, while maintaining the epidermis within a slightly negative temperature range during these temperature drops. Due to the structure of the epidermis and the thermal gradient that can form when cold penetrates its different layers, it is possible that for certain sequences (for example, those with a target temperature of 0°C), the temperature reached at the melanocyte level may be slightly positive.

[0082] Naturally, the duration of the sprays, their number N, their frequency, and the intervening pause times depend on the nature of the cryogenic fluid used, the flow rate of the solenoid valve, and other critical parameters. Theoretically, the duration of the sequence comprising N sprays separated by pauses is between 0.01 seconds and 30 seconds (for N=6 sprays). It is clear that, in all cases, and due to the temperature increases after each spray, the sequence durations will be longer than the spray times. According to the invention, the duration of the oscillation series for each selected sequence is therefore expected to be between 0.01 s and 30 s.

[0083] A suitable device intended for the in situ preparation of the composition of the invention comprises, as described above, a pressurized cryogenic gas cartridge having specific physical properties, a nozzle ensuring the delivery of a precise micro-dose of cryogenic gas from the cartridge by being coupled to automated timing means and a nozzle intended to concentrate, diffuse and apply the gas flow precisely and homogeneously on a determined skin area.

[0084] One of the preferred methods of implementing the cosmetic treatment process of the invention, enabling the desired cosmetic result with respect to cutaneous hyperpigmentation, comprises a sequence (referenced EC-05) which consists of targeting, starting from a normal starting temperature of the epidermis of 34°C, A target epidermal temperature of -5°C was achieved each time during N=4 sprays of cryogenic fluid. This sequence therefore comprises a series of 4 oscillations of epidermal temperature.

[0085] In this sequence, the opening times of the solenoid valve (EV) integrated into the cryogenic device used (which correspond exactly to the spraying times) are those indicated in the following Table 1:

[0086] [Table 1]

[0087] In Table 1, the solenoid valve state labeled "0" corresponds to a sealed, closed position, preventing the passage of any gas, while state "1" corresponds to an open position for pressure relief and ejection of the cryogenic gas. Table 1 shows that the spray phase lasts between 0.5 and 7.84 seconds, with the oscillation duration ranging from 0.5 to 14 seconds. The volumes of gas sprayed with each opening of the solenoid valve can be calculated based on the opening times given in Table 1 (above) and Table 2 (below), using an average of the cryogenic gas flow rates at the solenoid valve outlet as a reference.

[0088] The dosage, or in other words, the doses of cryogenic fluid applied to the epidermis to achieve the desired cosmetic effect, will thus (cumulative volumes of successive fluid sprayed during a sequence), from 0.10 to 0.7 ml with 1,1-difluoroethane gas. These doses are given in Table 2.

[0089] This result is obtained by always starting from an initial epidermal temperature of +34°C, reaching the target temperature, then rising to a temperature of +20°C, and repeating this process as many times as necessary depending on the number of oscillations, and using an average solenoid valve output flow rate of 0.61 ml / second.More generally, these doses depend on the nature of the cryogenic fluid used (and therefore on its boiling point and pressure), as well as the solenoid valve's exhaust flow rate, the structural and technical characteristics of the components (e.g., the solenoid valve, the gas cartridge, the timing system) and the parts of the device that influence the flow rate of the cryogenic gas and therefore the dose applied (e.g., the nozzle, the application nozzle, the spray distance between the point from which the cryogenic gas begins to expand, producing the cryogenic effect, and the point of application to the skin surface).

[0090] [Table 2] Quantities (Q in ml) of gas applied to the skin surface as a function of the target temperature (Te in °C) that we wish to reach and the number n of oscillations (ose) that we wish to perform.

[0091] In these referenced sequences (EC), the opening times (in seconds) of the solenoid valve (EV) integrated into the cryogenic device used with 1,1-difluoroethane gas are associated with a solenoid valve flow rate of 0.61 mL / second.

[0092] In Table 3 below, each sequence labeled (ECXX_YYosc) is associated with a code indicating the target temperature (XX) and the number of oscillations (YY). The sequences start with the solenoid valve (EV) in the closed position. The term (Pu) This corresponds to the duration of the solenoid valve opening (in seconds), and therefore to a spray for a given time at a fixed flow rate of 0.61 mL / s. The term (Pa) corresponds to the time the solenoid valve closes between two sprays (in seconds). This time corresponds to a pause (Pa) resulting in a natural rise in skin temperature above 20°C.

[0093] The development of the invention involved numerous experiments focused on optimizing spray sequences. These sequences were tested on cell culture models to determine, in particular, their impact on the cell viability of melanocytes and keratinocytes. Key markers of irritation were also measured.

[0094] The first objective of these experiments was to select cyto-selective sequences, that is, those which only act on melanocytes by decreasing their viability while preserving keratinocytes (concept of performance).

[0095] A second objective was to determine the sequences that generated the lowest possible variation in irritation markers (the concept of risk). In summary, the sequences with the optimal performance / risk ratio were selected.

[0096] Among the sequences tested during these experiments, 4 sequences presented an optimal performance / risk ratio. These are the sequences referenced (EC-05_losc), (EC-03_losc) and (EC-03_3osc), (ECOO_losc) and (EC00_3osc) which are explicitly mentioned in Table 3.

[0097] Among the other sequences tested, some variants approached this optimal ratio and should be considered as falling within the scope of the invention. Generally, all sequences with target temperatures between 0°C and -5°C and a number of oscillations between 1 and 6 are of interest for formulating the cosmetic composition of the invention. Further studies conducted on skin treated with the cosmetic composition of the invention confirm these results.

[0098] [Table 3] '

Claims

DEMANDS 1. A cosmetic treatment method for cutaneous hyperpigmentation using a cryogenic fluid and a device configured to ensure the ejection and spraying of a controlled quantity of said fluid onto the epidermis, characterized in that a sequence is performed comprising at least one spray (N) of said fluid onto the epidermis at a flow rate of 0.61 mL / s to create, within a range of negative temperatures on the skin between 0°C and -3°C, a series of temperature oscillations of said epidermis, the total duration of which is between 1.5 seconds and 25 seconds, each consisting of a phase of rapid cooling at a rate of temperature descent between 50°C / s and 170°C / s followed by a phase of slower temperature rise to a temperature between 20°C and 34°C.

2. A method according to claim 1, characterized in that the duration of each spray is between 0.10 seconds and 0.35 seconds.

3. A method according to any one of the preceding claims, characterized in that the duration of each of the oscillations is between 1.5 seconds and 5.8 seconds.

4. A method according to any one of the preceding claims, characterized in that the heating phase of the oscillations corresponds to a rise in the temperature of the epidermis of between 20°C and about 34°C.

5. A method according to any one of the preceding claims, characterized in that the number of successive oscillations of the epidermis temperature in a sequence is between 1 and 6.

6. A method according to any one of the preceding claims, characterized in that the cooling phase of the epidermis is carried out according to a gradient of at least 15°C from an initial temperature of about 34°C.

7. A process according to any one of the preceding claims, characterized in that said cryogenic fluid is a gas whose boiling point is between -196°C and -0.5°C under a normal pressure of 1 atmosphere or 1.013 bars.

8. A process according to the preceding claim, characterized in that said cryogenic gas is selected from the group consisting of 1,1-difluoroethane, methoxymethane, 1,1,1,2-tetrafluoroethane, trans-1,3,3,3-tetrafluoro- propene, butane, propane, isobutane, nitrogen or mixtures of these gases.

9. A method according to any one of the preceding claims, characterized in that said sequence is carried out by means of a single spray of 1,1-di-fluoro-ethane at 0.61 mL / second for a duration of 0.30 seconds so as to achieve a target epidermal temperature of 0°C.

10. A method according to any one of claims 1 to 8, characterized in that said sequence is carried out by means of 6 successive sprays of 1,1-di-fluoro-ethane at 0.61 mL / second, each having a duration of between 0.11 seconds and 0.31 seconds so as to achieve a target epidermal temperature of 0°C after each spray.

11. A method according to any one of claims 1 to 8, characterized in that said sequence is carried out by means of a single spray of 1,1-di-fluoro-ethane at 0.61 mL / second for a duration of 0.335 seconds so as to achieve a target epidermal temperature of -3°C.

12. A method according to any one of claims 1 to 8, characterized in that said sequence is carried out by means of 6 successive sprays of 1,1-di-fluoro-ethane at 0.61 mL / second, each having a duration of between 0.14 seconds and 0.335 seconds so as to achieve a target epidermal temperature of -3°C after each spray.

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