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`IMIWAAL|MANO
`EP 2856986 A1
`
`11)
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`EUROPEAN PATENTAPPLICATION
`
`(43) Date of publication:
`08.04.2015 Bulletin 2015/15
`
`(51) Int Cl.:
`A61F 7/00 (2006.01)
`
`(21) Application number: 14186964.4
`
`(22) Dateoffiling: 30.09.2014
`
`
`(84) Designated Contracting States:
`AL AT BE BG CH CY CZ DE DK EE ES FI FRGB
`GR HR HU IE ISITLILT LU LV MC MK MT NL NO
`PL PT RO RS SE SISK SMTR
`Designated Extension States:
`BA ME
`
`(72) Inventors:
`¢ Plan, Philippe
`08190 Sant Cugatdel Vallés (ES)
`¢ Pinto, Hernan
`08190 Sant Cugat del Vallés (ES)
`
`(30) Priority: 03.10.2013 ES 201331451
`
`(74) Representative: Gallego Jiménez, José Fernando
`Ingenias Creaciones, Signos e Invenciones
`S.L.P.
`
`Avda. Diagonal 421, 2
`(71) Applicant: Clinipro, S. L.
`08008 Barcelona (ES)
`08190 Sant Cugat del Valles (ES)
`
`
`(54)
`
`Cosmetic method for reducing subcutaneous adipose tissue
`
`invention relates to a cosmetic
`The present
`(57)
`method for reducing the subcutaneous adiposetissue in
`a selective and noninvasive manner by applying a cycle
`of hot-cold-hot on the desired area. This method has a
`
`high efficiency in the immediate reduction of the number
`of adipocytes, as well as a high deferred effect in this
`reduction, and maintaining a satisfactory metabolic func-
`tion in the remaining adipocytes.
`
`EP2856986A1
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`Printed by Jouve, 75001 PARIS (FR)
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`EP 2 856 986 A1
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`Description
`
`Technical field
`
`[0001] The present invention relates to the field of cosmetic methods for reducing subcutaneous adiposetissue.
`
`Technical Background
`
`[0002] Adipose tissue or fat tissue is the tissue formed by the association of cells that store fat inside them: the
`adipocytes. In the human body there are two types: the white adipose tissue and the brown adipose tissue. This adipose
`tissue is located in specific regions, which are knownas adipocyte deposits, and is located under the skin (subcutaneous
`fat), around the internal organs (visceral fat), in the bone marrow (yellow bone marrow)and in the breasts. This tissue
`has both mechanical functions (for example, the protection of internal organs) and metabolic functions (for example,
`responsible for generating energy for the organism).
`[0003] Adipose tissue is one of the most abundant tissues and accounts for about 15-20% of the body weight in men
`and 20-25% of the body weight in women.
`[0004]
`Since ancient times, human beings have been developing methods that would allow reducing the accumulation
`of adiposetissue in unwanted parts of their own body for aesthetic reasons. Treatments can be systemic, such as the
`use of gastric balloons, or localized, which are directed to the reduction of localized body fat. Such localized procedures
`can be divided between those using invasive techniques, and those using noninvasive techniques.
`[0005] Liposuction is one of the most used invasive techniques. Liposuction is a surgical procedure that consists in
`the removal of extra fat or adipose tissue in the affected area of the body, by its aspiration with thin cannulas that are
`inserted through small incisions in the fatty deposits located under the skin. Such cannulas are connected to a suction
`machine for mechanical liposuction, or by ultrasound, for ultrasonic liposuction. With the suction of the extra fat, ashaping
`of the treated area is achieved with a consequent improvement in the figure and outline. However, since it is a surgical
`procedure, this technique is not without risk, since complications mayarise, for example a hemorrhage.
`[0006] Within the noninvasive techniques are, for example, the treatments with ultrasounds, radiofrequencies or mi-
`crowaves, and thermal treatmentsat different temperatures.
`[0007]
`Thus, in the web page http://www.biotecitalia.com/en/fusiomedice-eng. html, a system for treating local fat and
`cellulite is described, in which five different technologies are used: monopolar and bipolar radiofrequency, low frequency
`cavitational ultrasound, sub-dermal vacuum massage, and liposhock. In the latter procedure, the area to be treated is
`suctioned, and the tissue is exposed to a sequenceof high temperature (60° C) and low temperature (-10° C) that triggers
`an apoptosis processin the adipocytes, leading to their physiological natural death.
`[0008]
`Inthe U.S. patent application US-A-2009/0069795, a method is described for destroying a particular tissue, for
`example adipose tissue, by heating, without the surrounding tissue being affected. The methodincludes:
`
`a) heating the target tissue and the surrounding tissue at a temperature lower than the one required for destroying
`them, wherein the target tissue has a longer thermal relaxation time than the surroundingtissue,
`b) letting the tissues cool for a period of time, wherein the target tissue does not cool down as muchas thetissue
`that surrounds it, and
`c) reheating the tissues, wherein the target tissue is destroyed, and the surroundingtissue is not.
`
`Inthe U.S. patent application US-A-2009/0157152, a cosmetic method to improvethe state of the skin in the
`[0009]
`face and/or in the neck is described. This method comprises a step of thermal stimulation at a temperature preferably
`comprised between 41 ° C and 43 ° C for a period of time preferably comprised between 60 and 90 minutes.
`[0010]
`Inthe U.S. Patent US5507790, a noninvasive method for the reduction of subcutaneous adiposetissue deposits
`in humans is described by using an acceleratedlipolysis metabolism. Said method includes the use of heat alone or in
`combination with a suitable lipolytic drug. It is also described that the adiposetissue is heated at a temperature comprised
`between 40° C and 41.590 C, but not above 42° C, and this temperature is maintained for a period of up to 20-25 minutes.
`[0011]
`In the international patent application WO-A-01/32091, a method for noninvasive removal of adipose tissue in
`humans is described. In this method heat is applied to raise the temperature of the adipocytes between 41 ° C and 46°
`C for a period comprised between 5 minutes and 2 hours. The thermal energy can be administrated by ultrasounds,
`radiofrequency or microwaves.
`[0012]
`In the international patent application WO-A-02/087700, a method is described for the treatment of a selected
`area of the skin and/or the subcutaneous tissue comprising heating said area at a temperature comprised between 32
`°C and 50° C for a certain period of time comprised between 20 seconds and 24 hours, preferably between 30 minutes
`and 8 hours, and administering a composition comprising an active ingredient, for example, a lipolytic compound such
`as caffeine.
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`Inthe international patent application WO-A-03/078596, it is described the selective disruption of adipose cells
`[0013]
`by the application of a cooling gradient at the skin surface without causing damage to the other cells.
`[0014]
`In the article Avram et al., Cryolipolysis™ for subcutaneous fat layer reduction, Lasers Surg. Med., 2009, 41,
`703-708, it is disclosed that cryolipolysis is a noninvasive method for the selective reduction of adipocytes by localized
`and controlled cooling thereof. It is also indicated that phagocytosis of the lipids lasts 90 days or more, and that after
`this period a clear reduction of the thickness of the fat layer is observed.
`[0015]
`In the international patent application WO-A 2012/075419, it is described a method for the disruption of sub-
`cutaneous adiposetissue by using heat, but it is accompanied by cooling of the area to prevent damagesto the near
`epithelial or vascular tissues. It is also described that the cooling can occur simultaneously to the heat treatment, and
`that can be extendedafter finishing it in order to reduce inflammation and pain. Hyperthermia treatmentof the adipose
`tissue raises its temperature above 400 C, preferably between 42° C and 46° C, while the temperature of the surrounding
`tissues is maintained below 40° C.
`
`In the international patent application WO-A-2013/026393, a method for losing weight by reducing fat is de-
`[0016]
`scribed comprising locally heating an area that requires the reduction of fat at a temperature comprised between 37° C
`and 60° C until the blood and muscles are separated from the fat, and then locally freeze this area at a temperature
`comprised between -10 ° C and 5° C to solidify the fat.
`[0017] None of the proposed solutions satisfactorily solves the problem of the effective removal of subcutaneous fat
`deposits localized in the human body.
`[0018] Thus, there remains a need for a noninvasive cosmetic method, to effectively reduce the fat localized in such
`deposits and thus improve the aesthetic appearance of people.
`
`Summary of the invention
`
`[0019] The object of the present invention is a cosmetic method for reducing the subcutaneous adiposetissue.
`
`Description of the figures
`
`[0020]
`
`Figure 1
`In Figure 1 a diagram of the internal units and the associated functions thereof is represented, which includes a
`particular embodiment of the device for performing the method of the invention. Elements 1, 2 and 3 are three CPUs
`processing units that control the functioning of the equipment. Elements 10, 11 and 12 are, respectively, a touchscreen
`to establish communication with the device, an internal memory necessary for the execution of all the processes
`and an internal database that generates a register of incidents to facilitate the diagnosis of a possible breakdown.
`Elements 13 and 23 are interfaces that communicate the different CPUs. Elements 20, 21 and 22 are, respectively,
`a vacuum system with feedback sensor, to obtain a better result in the treatment by introducing in the cavity of the
`device the maximum adiposetissue possible, a liquid cooling system to compensate the temperature generated in
`the heat/cold elements, and a switch for the powerflow necessary for CPU3 (3). Elements 30 and 31 are temperature
`sensors to regulate the operating conditions of the internal elements of the equipment. Units 32 and 33 are the heat
`and cool generating units according to the step of the method. Element 34 is an emergency shutdown pushbutton.
`
`Figure 2
`Figure 2 showsthe 22 factorial design with the factors: Pre-heating on the ordinates and post-heating on the abscissa,
`wherein the sign "-" means that the treatment is not performed and the"+" sign means that the treatmentis performed.
`Each of the numbersthat are located within a circle correspond to the test described in Example 1, and the numbers
`that are located outside of the circle correspond to the adipocytes mortality, expressed as a percentage of the total
`adipocytes. A synergistic effect is observed by combining a pre-heating step with a cooling step and a post-heating
`step.
`
`Figure 3
`In Figure 3 it is schematically represented the time and temperatureprofiles for the treatment of the method of the
`invention described in Example 1 as wellas forthe comparative treatments described therein. The ordinate represents
`the temperature, and the abscissa the time.
`
`Detailed description of the invention
`
`[0021] The invention has as object a cosmetic method for reducing the subcutaneous adiposetissue in a localized
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`area of an adult human body comprising:
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`EP 2 856 986 A1
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`a) heating a localized area of the human body with heating means that are at a temperature of not more than 45° C,
`b) subsequently, cooling this area with cooling means that are at a temperature comprised between 2°C and 6°C, and
`c) subsequently, heating this area with heating means that are at a temperature of not more than 45° C.
`
`[0022] The authors of the present invention have developed a noninvasive cosmetic method for reducing the subcu-
`taneous adipose tissue in localized areas of an adult human body that has a high efficacy in the immediate reduction of
`the number of adipocytes, as well as a high effect of this reduction deferred in time, and it maintains a satisfactory
`metabolic functionality of the remaining adipocytes.
`[0023] As already stated, the temperature indicated in the steps of the method of the invention corresponds to the
`temperature of the heating/cooling means that are in contact with the skin, not to the temperature of the adipocytes.
`These means allow heating or cooling, depending on the step of the treatment, the adipocytes at a temperature thatis
`closely related to the temperature thereof. For example, when the temperature of the heating/cooling means is 3.19 C,
`the adipocytes are below 10.4°C, after a period of 25-35 minutes, as described in Pinto et al., A study to evaluate the
`action oflipocryolysis, CryoLetters, 2012, 33 (3), 176-180, and when the meansare at 42° C, the adipocytes are ap-
`proximately at 38° C after a period of time comprised between 5-10 minutes.
`
`The cosmetic method
`
`[0024] The methad of the invention is a noninvasive cosmetic method. This is a non-traumatic method that acts from
`the surface of the skin and does not invade the body through incisions to reduce subcutaneous adiposetissue. In this
`method it is used a temperature range that can be considered substantially safe for human body, since temperatures
`below 2° C or above 45° C are not used. The methcdis applied topically to the area having an excessive amount of
`subcutaneous adiposetissue and for this is not necessary any knowledge of medicine, but it can be carried out by people
`not requiring a degree in medicine. This method does not include any surgical step, and neither any therapeutic effect
`is produced. The result of applying the method of the invention leads to an aesthetic effect by reducing the subcutaneous
`adipose tissue located in specific areas of the body. The effects of this method are purely cosmetic because they can
`only be observedin the superficial layers of adipose tissue. Furthermore, as discussed below, the method ofthe invention
`maintains the functionality of the adipocytes which have not been affected byit.
`[0025] The method ofthe invention differs substantially from invasive methods for reducing fat, such as, for example,
`liposuction, that can have side effects on the human health, since it involves a strong interaction with the human body.
`[0026] The method of the invention can be applied easily;
`it is applied directly to the skin through a device that,
`optionally, makes a vacuum effect in order to better get a skin fold with localized subcutaneous fat tissue and, in turn,
`it allows the heating and cooling of this area at temperatures previously stated.
`[0027] This method includes some temperature profiles with which the lipid-rich cells are altered, and simultaneously
`the non-lipid-rich cells are not altered.
`[0028] The methadof the invention can be applied more than one session. In fact, it is usually applied on more than
`one occasion, being usually the period between sessions of approximately 45 days.
`[0029] The methodof the invention produces an almost imperceptible effect after its application to the localized area
`of the human body. The effects of this treatment begin to appear after a period generally comprised between 15 and 30
`days. This is because the adipocytes that have been object of the methodinitiate a process of apoptosis, being enveloped
`and digested by macrophages and eliminated by natural means during the days following the treatment. The repeated
`application of the method allowsto significantly reduce the subcutaneous fat deposits.
`
`Localized area of the human body
`
`[0030] The method of the invention is used to reduce the subcutaneous adipose tissue of a localized area of an adult
`human body, preferably in the abdomen, waist, back, arms and thighs.
`[0031]
`Inthe context of the invention an adult human body is considered to be the one corresponding to a person with
`an age of at least 16 years approximately.
`[0032] The method of the invention is not suitable for reducing the adipose tissue that is located in a generalized
`manner in the human body.
`[0033]
`Preferably the localized area of the human bodyis introduced into an appropriate elongated cavity for housing
`the fold of skin on which the method of the invention is performed. In a more preferred embodiment, this cavity includes
`an absorption hole for vacuum application in order to achieve a greater efficacy in the reduction of the adipose tissue,
`since this way the blood flow to this area is restricted.
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`Pre-heating step
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`EP 2 856 986 A1
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`[0034] The methad of the invention comprises an initial heating step (pre-heating) of the localized area of the human
`body that shows an excess of subcutaneous adiposetissue.
`[0035] The heating of this area can be performed by conventional heating means such as those described, for example,
`in the U.S. patent US5507790. In the method of the invention the heating of the localized area is carried out by using
`radiofrequency, microwaves, ultrasounds or a thermoelectric device based on the Peltier effect; preferably is used a
`thermoelectric device based on the Peltier effect.
`
`[0036] The Peltier effect is a thermoelectric property that is manifested when electric current flows through a circuit
`formed by two types of conductive substances. In the circuit, one of the parts is colder than the other, and the difference
`of temperature generated dependson theintensity of the current flowing through the circuit.
`[0037] The heating of the localized area is performed untilreaching a temperature of not more than 45° C in the heating
`means that are in contact with the skin. Preferably, the temperature is not more than 440 C, more preferably not more
`than 43 ° C, and still more preferably not more than 42 ° C.
`[0038] This temperature of the heating means is maintained generally a period of time comprised between 2 minutes
`and 10 minutes, preferably between 3 minutes and 8 minutes, more preferably between 4 and 6 minutes, and more
`preferably 5 minutes.
`[0039] Typically the heating meansin the pre-heating step are at room temperature and the heating rate from this
`temperature to the desired temperature is comprised between 1 ° C/min and 8° C/min, preferably between 1
`° C/min
`and 60 C/min, and more preferably between 1 ° C/min and 3° C/min. The estimated time to bring the heating means to
`the stated temperature is comprised between 1 and 20 minutes.
`
`Cooling step
`
`[0040] The process of the invention comprises a step of cooling of the localized area of the human body that has an
`excess of subcutaneous adiposetissue. This cooling step is performedafter the first heating step.
`[0041] The cooling of this area can be carried out by conventional cooling means as those described, for example, in
`the international patent application WO-A-2012/075419, in which the use of a cold air stream, or the contact with a cold
`solution are mentioned, or in the international patent application WO-A-00/44346, where some compositions appropriate
`to reduce the temperature of the skin of a person are described. In the method of the invention the cooling means
`preferably use cold solutions, cold air, or a thermoelectric device based on the Peltier effect; more preferably is used a
`thermoelectric device based on the Peltier effect.
`
`[0042] The cooling of the localized area is performed until reaching a temperature comprised between 2 ° C and 6 °
`C in the cooling means in contact with the skin. Preferably the temperature is comprised between 3° C and 5° C, and
`more preferably between 3° C and 4° C.
`is maintained for a minimum period of time of 5
`it
`[0043] Once the cooling means reach the stated temperature,
`minutes, preferably between 5 minutes and 60 minutes, more preferably between 8 minutes and 50 minutes, more
`preferably between 10 minutes and 40 minutes, and still more preferably between 20 minutes and 30 minutes.
`[0044] Usually the rate of cooling from theinitial temperature, at which the cooling means are, to the desired temperature
`is comprised between 1 ° C/min and 8° C/min, preferably between 1 ° C/min and 60 C/min, and more preferably between
`1 °C and 3° C. The estimated time to bring the cooling means to the stated temperature is comprised between 2? and
`30 minutes.
`
`Post-heating step
`
`[0045] The methadof the invention comprises a heating step (post-heating) of the localized area or the human body
`that has an excess of subcutaneous adipose tissue after the cooling step.
`[0046] As already mentioned above, in the methodof the invention the heating of the localized area is carried out by
`using radiofrequency, microwaves, ultrasounds or a thermoelectric device based on the Peltier effect; preferably is used
`a thermoelectric device based on the Peltier effect.
`
`[0047] The heating of the localized area is performed until reaching a temperature of not more than 45° C. This
`temperature refers to the temperature in the heating means that are in contact with the skin. Preferably the temperature
`is not more than 440 C, more preferably not more than 43° C, and still more preferably not more than 42 ° C.
`[0048] Once the heating means reach the stated temperature, it is generally maintained a period of time comprised
`between 2 minutes and 20 minutes, preferably between 5 minutes and 15 minutes, more preferably between 8 and 12
`minutes, and still more preferably 10 minutes.
`[0049] Usually the heating rate from the initial temperature, at which the heating means are, to the desired temperature
`is comprised between 1 ° C/min and 8° C/min, preferably between 1 ° C/min and 60 C/min, and more preferably between
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`EP 2 856 986 A1
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`1° C/min and 3° C/min. The estimated time to bring the heating meansto the stated temperature is comprised between
`1 and 20 minutes.
`
`In order to control temperatures in the skin, the method of the invention can include the use of a feedback
`[0050]
`device in the form of a sensor, as described in the patent application GB-A-2286660.
`[0051] A particularly preferred embodiment of the method of the invention comprises:
`
`a) heating a localized area of the human body with heating means that are at a temperature of not more than 440
`C, for a period comprised between 2 and 10 minutes,
`b) subsequently, cooling this area with cooling meansthat are at a temperature comprised between 3° C and 5° C
`for a period comprised between 5 and 60 minutes, and
`c) subsequently, heating this area with heating means that are at a temperature of not more than 440 C for a period
`comprised between 2 and 20 minutes.
`
`[0052] A particularly preferred embodiment of the method of the invention comprises:
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`a) heating a localized area of the human body with heating means that are at a temperature of not more than 420
`C, for a period comprised between 4 and 6 minutes,
`b) subsequently, cooling this area with cooling meansthat are at a temperature comprised between 3° C and 4° C
`for a period comprised between 10 and 40 minutes, and
`20
`c) subsequently, heating this area with heating means that are at a temperature of not more than 420Cfor a period
`comprised between 8 and 12 minutes.
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`Surprisingly it has been observed that with the method of the invention it is possible to obtain an immediate
`[0053]
`reduction in the numberof adipocytes bydirect apoptosis, a deferred reduction thereof by crystal formation, and maintain
`the functional viability of the adipocytes so that they maintain a substantially normal level of lipolysis. The combination
`of the steps of pre-heating, cooling and post-heating allows to obtain a significant increase in the direct apoptosis of
`adipocytes in comparison with the methods that only include one or two of such steps, whilst the crystal formation remains
`comparable to that obtained by employing said alternative procedures.
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`[0054] The device suitable for carrying out the method of the invention can be, for example, the one described in the
`patent application GB-A-2286660. Thus, this device comprises one or more thermoelectric units based on the Peltier
`effect, a heat exchangerto dissipate the cold generated in the heating steps and the heat generated during the coaling
`step, a container ofliquid to cool or heat the heat exchanger, a pump for recirculating this liquid between the container
`and the heat exchanger, and a cooling/heating unit to cool this liquid.
`[0055]
`Ina preferred embodiment the heat and cool generation is carried out by using one or more thermoelectric
`units based on the Peltier effect.
`
`[0056] The device can further incorporate a screen as a userinterface, to report in which state is the device when the
`method of the invention is performed. Preferably, it
`is a touch screen and therefore, the process parameters can be
`configured, it is also possible to execute the stop and the start of the treatments, the monitoring of each ofthe variables,
`and information about the status and situation of each cycle of the method.
`[0057] The thermoelectric units are preferably located in a head comprising an elongated cavity suitable for housing
`the fold of skin on which the method of the invention is performed. In a more preferred embodiment this cavity includes
`an absorption hole for vacuum application in order to achieve greater efficacy in the reduction of the adiposetissue.
`This head mayinclude several light indicators of the LED type to inform about the function which is being performed
`during the procedure.
`[0058] The device can advantageously include process control units for controlling the different elements involved in
`the method of the invention. In Figure 1 it is shown a possible embodiment of a diagram that includes the internal units
`of the device and the functions associated therewith. According to this embodiment, the beginning of the procedureis
`performed by the user pressing the "start" button placed in the user screen (10), selecting next the thickness of the
`adiposetissue, and then eventually the command to start the vacuum is given, from the screen (10). From this moment
`the CPU corresponding to the unit (2) receives the command from the interface and activates the suction (20) with a
`pre-assigned regulation level according to the adipose tissue. The implemented software can work on any standard
`operating system as, for example, WindowsCE or GNU/Linux, and automatically managesall the internal processesin
`the memory (12) and stores the necessary information to use the equipment, also generating internal error codesin the
`database (11) to facilitate repair operations in case of the eventual breakdowns.
`[0059] The system (2) detects when the internal cavity of the applicator has the correct vacuum (20) enabling in the
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`EP 2 856 986 A1
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`screen (10) the beginning of the therapy, at that moment a start session commandis sent to the cooling/heating system
`(32)/(33), refrigeration system (21) and vacuum (20). The type of therapy and the sequencesofheat/cool are determined
`by the CPU1 (1) sending the necessary commandsbyinternal communication (13) (23) to the rest of the units according
`to the cycle, at the same time all the internal variables for monitoring and tracking the equipment status and the effec-
`tiveness of the therapy are received. The CPU2 (2) monitors the powerdelivered at the CPU3 (3) during the application
`of heat/cold (32)/(33),
`in case of obtaining a deviation outside the established margins, as a possible failure of CPU3
`(3), the power module (22) is disconnected to stop supplying power to the heat/cold elements (32) (33).
`[0060] The CPU3 (3) is responsible for not exceeding the maximum and minimum temperature values stated, by using
`temperature sensors (30)/(31) in the critical areas, these being necessary to not exceed the maximum values of coaling
`detrimental to the internal components of the equipment or electronic components, such as the ice generation and
`moisture. During the treatment the necessary power for the generation of heat or cold (32)/(33) is applied depending on
`the point of cycle according to the commands received from the upper CPUs (1¥(2). The CPU3 also controls the
`emergency stop pushbutton (34) to release the order of immediately stop the session to all upper control units (1)/(2)
`stopping the heat/cool system, refrigeration system and vacuum, and simultaneously a stop activation message is
`displayed on the screen for validation by the user.
`[0061] Once finished the session, the heat/cold system (32)/(33) and vacuum (20) are stopped. The device returns to
`the idle state and displays on the screen the start button waiting to be pressed by the user from the screen (10).
`
`Tests on adipocytes
`
`[0062] Tests on isolated rat adipocytes were performed applying different thermal profiles:
`
`1. Cooling,
`2. Pre-heating combined with cooling,
`3. Cooling combined with post-heating, and
`4. Pre-heating combined with cooling and with post-heating.
`
`In the tests with adipocytes, the temperatures correspond to the temperature at which the adipocytes were
`[0063]
`maintained at different steps of the procedure.
`[0064] After the tests, it was observed that the adipocytes treated according to the methodof the invention (profile 4)
`showeda significantly higher mortality rate than the mortality rate observed in the other comparative methcds. This cell
`death is an immediate effect that occurs during the application of the method of the invention itself.
`[0065] Aconsiderable formation of beta crystals in the adipocytes is also observed, which is related to the slow death
`by apoptosis of the adipocytes, and that results in a deferred effect of reduction of the numberof adipocytes.
`[0066]
`In Figure 2 the design space of the tests performed is shown, and it corresponds to an experimental matrix of
`a factorial design 22, in which a synergistic effect is observed by combining the pre-treatment with heat, the cooling step,
`and the post-treatment with heat in terms of mortality of the adipocytes. It was observed that a single step of pre- or
`post- heating combined with cooling practically does not modify, rather reduce, the effect of the cold treatment alone.
`[0067] Tests were also carried out to determine the functional viability of adipocytes from the subcutaneous adipose
`tissue after being subjected to the method of the invention. As described in the Examples section, these adipocytes
`maintained normal lipolytic activity. l.e., the adipocytes maintained a lipolytic activity and generated glycerin as a product
`from the hydrolysis of the fats. Lipolysis is the physiological mechanism by which adipose tissue mobilizes the energy
`stored as fat to makeit available to cell metabolism. The fatty acids released in this process are used in many body
`tissues as energy substrate, alternative to glucose.
`[0068]
`It is thus observedthat, surprisingly, the cosmetic noninvasive method of the invention produces a synergistic
`effect by combining its componentsteps, and allows obtaining a superior effect in the immediate removalof the adipocytes,
`while maintaining a significant effect on the deferred elimination thereof, and furthermore the unaffected adipocytes
`continue to be functionally viable.
`[0069] The examples whichfollow are given in order to provide the skilled in the art with a sufficiently clear and complete
`explanation of the present invention, but they should not be considered as limiting the essential aspects of the object of
`the invention, as have been exposedin the previous sections of this description.
`
`Examples
`
`Preparative example 1.- Isolation of the adipocytes
`
`To conduct the method of the invention it was proceeded ta obtain murine white adipose tissue.
`[0070]
`[0071] Seven Sprague Dawley male rats of 56 days of age were anesthetized, euthanized by cervical dislocation, and
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`EP 2 856 986 A1
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`white adipose tissue from the retroperitoneal area of the animals was obtained.
`[0072] Approximately 2 g of white adipose tissue were digested with 10 mg of collagenase (Collagenase Type4,
`Worthington) in 20 ml of Krebs buffer (Hepes 1.25 mM, NaCl 12 mM, KCI 0.6 mM, MgSO,:7H20 0.12 mM, CaCl, 0.1
`mM, 2 g albumin and 0.045 g of glucose). The tissue was incubated at 37 ° C in a bath with mild stirring for 40 minutes.
`The digestion was stopped by addition of 2 ml of EDTA 1 mM, and incubation was continued for 5 additional minutes.
`The sample wasfiltered and the isolated adipocytes were collected in a syringe (without piston), which was stoppled
`with a stopcock. The syringe was keptin a vertical position for 5 minutesto facilitate the flotation of the adipocytes in
`the buffer. The infranatant buffer was removed and the isolated adipocytes were washedtwice with 10 ml of Krebs buffer.
`Finally, the adipocytes were resuspendedin 2.5 mL of Krebs buffer.
`
`Example 1.- Effects of the exposure of adipocytes to different the