Method for selective disruption of fatty tissue by controlled cooling

ES 2 390 598 T3

DESCRIPTION

Devices for the selective disintegration of adipose tissue by controlled cooling

FIELD OF THE INVENTION

The present invention relates to devices for use in the disruption of lipid-rich cells by controlled cooling. The present invention also relates to a device for use in the implementation of procedures for the selective disintegration of lipid-rich cells by controlled cooling. Other aspects of the invention are described in, or are obvious from, the following specification (and within the scope of the invention).

BACKGROUND

The subcutaneous fat tissue of newborns is extremely sensitive to cold. In newborns, the intracellular lipid content of subcutaneous fat cells, or "adipocytes", comprises high levels of triglycerides with a high degree of saturation. Even moderately cold temperatures can be detrimental to cells with highly saturated lipid content, making the newborn's subcutaneous fatty tissue vulnerable to adipocyte necrosis after exposure to cold. Hypothermia of the subcutaneous fatty tissue can lead to associated inflammation of the dermis and / or epidermis. For example, cold panniculitis disorders in newborns are known to cause painful skin lesions.

As newborns mature, the ratio of saturated to unsaturated fatty acids among adipocyte intracellular triglycerides gradually decreases. By having a higher content of unsaturated fatty acids, they are more protected against the cold, and the incidence of cold panniculitis gradually decreases. For articles on the topic of cold panniculitis, see Epstein et al. (1970) New England J. of Med. 282 (17): 966-67; Duncan et al. (1966) Arch. Derm. 94: 722-724; Kellum et al. (1968) Arch. Derm. 97: 372-380; Samuel L. Moschella and Harry J. Hurley (1985) Diseases of the Corium and Subcutaneous Tissue, in Dermatology (WB Sanders Company): 1169-1181; John C Maize (1998) Panniculitis In Cutaneous Pathology (Churchill Livingstone): 327-344; Edward E. Bondei and Gerald S. Lazarus (1993) Disorders of Subcutaneous Fat (Cold Panniculitis). In Dermatology in General Medicine (McGraw-Hill, Inc.): 1333-1334.

In adults, the intracellular lipid content varies between cell types. For example, cells in the dermis and epidermis are relatively low in unsaturated fatty acids compared to the underlying adipocytes that make up subcutaneous fatty tissue. For a detailed article on the composition of fatty tissue in mammals, see Albert E. Renold and George F. Cahill Jr. (1965) Adipose Tissue. In Handbook of Physiology (American Physiology Society): 170-176. Consequently, different cell types, eg lipid-rich and non-lipid-rich cells, have different degrees of susceptibility to cold. In general, non-lipid-rich cells can withstand colder temperatures than lipid-rich cells.

It would be very convenient to damage the adipocytes of the subcutaneous fatty tissue, selectively and non-invasively, without causing damage to the surrounding dermal and epidermal tissue. It is known that health and cosmetic benefits are obtained from the reduction of fat tissue; however, in current procedures such as liposuction, invasive procedures are employed with potential life risks (eg, excessive bleeding, pain, septic shock, infection, and inflammation).

Current procedures for the non-invasive removal of subcutaneous fat tissue include the use of radiant energy and cooling solutions. In US Patent Nos. 5,143,063, 5,507,790, and 5,769,879, procedures for the use of radiant energy to reduce subcutaneous fatty tissue are described; however, the energy levels applied are difficult to control and collateral damage often occurs in the dermis and / or epidermis. The cooling solutions proposed in WO 00/44346 do not stabilize the surface temperatures of the skin and therefore fail to adequately protect against collateral damage to the dermis and / or epidermis.

A previous study carried out with guinea pigs described the removal of subcutaneous fatty tissue by cryoinjury. S. Burge and R. Dawber (1990) Cryobiology 27: 153-163. However, this result was achieved using relatively aggressive cooling modalities (eg liquid nitrogen), which induced damage to the epidermis. Ideally, the removal of subcutaneous fatty tissue by cooling would not cause associated damage to the epidermis.

Until now, there were no known temperature-controlled methods and devices to selectively damage lipid-rich cells (for example, adipocytes that form subcutaneous fatty tissue) without causing damage to non-lipid-rich cells (for example, dermis and / or or epidermis).

In GB 22 86 660, a skin cooling device is described.

ES 2 390 598 T3

ABSTRACT

The invention is defined in the independent claims. It has now been shown that adipose tissue comprising lipid-rich cells can be disintegrated without causing injury to the surrounding non-lipid-rich tissue (e.g. dermal and / or epidermal tissue) by controlling the temperature and / or pressure applied to the respective tissues.

A cooling method for the selective disintegration of lipid-rich cells in a non-lactating human subject is disclosed which comprises applying a cooling element proximal to the subject's skin to create a temperature gradient within a local region that is sufficient to to selectively disintegrate and, therefore, reduce the lipid-rich cells of said region, and, simultaneously, maintaining the subject's skin at a temperature where non-lipid-rich cells in the vicinity of the cooling element do not disintegrate.

Also disclosed is a method for treating a region of a subject's body to achieve a desired reduction in subcutaneous adipose tissue, comprising a) applying a cooling element to a point near the subject's skin in the region in the one in which the reduction of the subcutaneous adipose tissue is desired to create a temperature gradient within said region, sufficient to selectively disintegrate the lipid-rich cells found there, and simultaneously maintain the subject's skin at a temperature where the non-lipid-rich cells in the vicinity of the cooling element do not disintegrate; b) repeating the application of the cooling element to the skin of the subject of step (a) a plurality of times until the desired reduction in subcutaneous adipose tissue has been achieved.

A device is disclosed for selectively disaggregating lipid-rich cells in a non-lactating human subject by cooling, comprising: a means for creating a temperature gradient within a local region of the subject's skin to selectively disaggregate, and, thus reducing lipid-rich cells in the region, while simultaneously maintaining the subject's skin at a temperature where non-lipid-rich cells do not disintegrate.

An apparatus for the local reduction of lipid-rich cells is described, comprising a treatment device operable to receive a cooling agent; a source of cooling agent connected to the treatment device to supply said cooling agent; a control unit connected to the treatment device and the cooling agent source for controlling a cooling temperature of said cooling agent, wherein said treatment device exposes target tissue to said cooling agent, selectively inducing cell damage rich in lipids of said target tissue.

An apparatus for the local reduction of lipid-rich cells is described, comprising means for bringing a cooling agent to a predetermined temperature; and a means for applying said cooling agent to the target tissue, whereby the cooling agent selectively induces damage to lipid-rich cells of said target tissue.

These and other objects and embodiments are described or obvious from the following detailed description and within the scope of the invention.

DESCRIPTION OF THE DRAWINGS

Figure 1A illustrates a treatment system.

Figure 1B represents a diagram illustrating a configuration of the control unit.

Figure 1C represents a diagram showing a cooling / heating element.

Figure 1D illustrates a planar cooling treatment system with a probe controller.

Figure 2A illustrates a treatment system for cooling lipid-rich cells within a skin fold.

Figure 2B illustrates a treatment system for cooling lipid-rich cells within a skin fold with a probe controller.

Figure 3A illustrates a treatment system that includes a suction unit.

Figure 4 illustrates a treatment system that is combined with a suction system to provide treatment of an isolated area.

Figure 5A, B illustrates a treatment system that can circumferentially enclose a mass of target tissue.

ES 2 390 598 T3

Figure 6 represents an image of the skin surface showing a cleft after 17 days in some areas that coincide with the cold exposure sites.

Figure 7 represents the histology of the subcutaneous adipose tissue 17 days after exposure to cold (pig II, site E). Figure 7A shows the low magnification view and Figure 7B shows the high magnification view.

Figure 8A, B represents site C; 8C, D represents site E; and 8E, F represents the F site; each of which shows the histology of subcutaneous adipose tissue 17 days after cold exposure (pig II, sites C, E and F).

Figure 9 represents an image of the device used to administer the chill to pig III.

Figure 10A, B, C, D, E, F, G, H, I and J represents the temperature graphs of the exposure sites 1, 2, 7, 11, 12, 13, 14, 15, 16 and 18 of pig III at various depths of the tissue.

Figure 11 depicts an ultrasound image of test site 11, 3.5 months after exposure.

Figure 12A, B depicts the histology of the test site 8.6 days after challenge. Figure 12C, D depicts the histology of test site 9 (control).

Figure 13A, B, C, D and E depict macroscopic cross sections through the center of test sites 1, 3, 11, 12 and 18, 3.5 months after exposure.

DETAILED DESCRIPTION

The present specification refers to a procedure for the local reduction of adipose tissue, which comprises the application of a cooling element to a subject at a temperature sufficient to selectively disintegrate cells rich in lipids, in which the temperature does not produce effects. unwanted in non-lipid-rich cells. Preferably, the cooling element contains or is connected to a cooling agent.

A cooling method is disclosed for the selective disintegration of lipid-rich cells in a non-lactating human subject, which comprises applying a cooling element to a point near the subject's skin to create a temperature gradient within a local region. that is sufficient to selectively disintegrate and, therefore, reduce the lipid-rich cells of said region, and, simultaneously, maintaining the subject's skin at a temperature where non-lipid-rich cells in the vicinity of the cooling element do not disintegrate.

Also disclosed is a method for treating a region of a subject's body to achieve a desired reduction in subcutaneous adipose tissue, comprising a) applying a cooling element to a point near the subject's skin in the region in the one in which the reduction of the subcutaneous adipose tissue is desired to create a temperature gradient within said region, that it is sufficient to selectively disintegrate the lipid-rich cells found there, and, simultaneously, maintain the subject's skin at a temperature at which the non-lipid-rich cells near the cooling element do not disintegrate; b) repeating the application of the cooling element to the skin of the subject of step (a) a plurality of times until the desired reduction in subcutaneous adipose tissue has been achieved.

The cooling elements of the present invention may contain cooling agents in solid, liquid or gas form. Solid cooling agents can comprise, for example, materials that act as thermal conductors, such as metals, metal plates, glasses, gels, and ice or liquid ice. Liquid cooling agents can comprise, for example, saline, glycerol, alcohol, or water / alcohol mixtures. When the cooling element includes a circulating cooling agent, the temperature of the cooling agent is preferably constant. Salts can be combined with liquid mixtures to obtain the desired temperatures. Gases may include, for example, cold air or liquid nitrogen.

In one embodiment, the cooling elements can be applied in such a way as to establish direct contact with a subject, through either the agent or the element. In another embodiment, direct contact is established solely through the agent. In yet another embodiment, no direct contact is made through the agent or element; cooling is accomplished by proximal placement of the element and / or the cooling agent.

Preferably, the temperature of the cooling agent is less than about 37 ° C, but not less than -196 ° C (ie, the temperature of liquid nitrogen).

Preferably, the temperature range of the administered cooling element is between about 40 ° C and -15 ° C, even more preferably between 4 ° C and -10 ° C if the cooling agent is a liquid or a solid. Generally, the cooling element is preferably maintained at an average temperature of between about -15 ° C and about 35 ° C, 30 ° C, 25 ° C, 20 ° C, 15 ° C, 10 ° C, or 5 ° C. ; about -10 ° C and about 35 ° C,

ES 2 390 598 T3 ° C, 25 ° C, 20 ° C, 15 ° C, 10 ° C or 5 ° C; about -15 ° C and about 20 ° C, 15 ° C, 10 ° C or 5 ° C.

The element and / or cooling agent can be applied for a period of up to two hours. Preferably, the cooling element is applied between 1 and 30 minutes. The cooling element can be applied for at least one hundred milliseconds (eg, shorter durations are contemplated, for example with sprays). For example, liquid nitrogen can be applied in very short intervals (for example, about 1 second), repeatedly (for example, about 10 to 100 times) and between applications, a temperature is maintained that does not cause epidermal damage (for example , from approximately 0 ° C to -10 ° C, depending on the duration of the exposure). In a moderate cooling regime, for example, liquid nitrogen can be sprayed from a distance (for example, from about 10 to 30 cm), where some of the liquid nitrogen droplets evaporate during spraying and / or are mixes with the surrounding air.

The elements and / or cooling agents of the present invention are applied, for example, to the surface of the skin through direct or indirect contact. The skin of a subject comprises the epidermis, the dermis, or a combination of both. The cooling element and / or agent is a non-toxic cooling agent when applied directly to the surface of the skin.

The element and / or cooling agent can be applied more than once, for example, in repeating cycles. The cooling agent can be applied in pulses or continuously. The cooling element and / or agent can be applied by all conventional procedures known in the art, including topical application by means of a spray if in the form of a liquid, gas or solid particulate material. Preferably, the application is carried out by external means; however, the elements and / or cooling agents of the present invention can also be applied subcutaneously by injection or other conventional means. For example, the cooling agent can be applied directly to the subcutaneous tissue and then removed after contact or left in the subcutaneous tissue to achieve thermal equilibrium and therefore cooling of the lipid-rich tissue (for example, a subcutaneous injection liquid cooling agent or small cooling particles, such as granules or microspheres).

Preferably, the procedures herein are non-invasive (eg, superficial, laparoscopic, or topical procedures that do not require invasive surgical techniques).

The element and / or cooling agent can be applied to a defined zone or to multiple zones. The spatial distribution of the element and / or cooling agent can be controlled as required. In general, the dimension of the surface (eg, where the cooling agent is in contact with the skin) should be at least three times the depth of the subcutaneous fatty tissue that is targeted for cooling. Preferably, the minimum diameter of the surface is at least 1 cm². Even more preferably, the minimum diameter of the surface is between 3 and 20 cm.². Determination of the optimal surface will require routine variation of several parameters. For example, larger surfaces can be cooled, such as those that exceed 3500 cm², in accordance with the methods of the present invention if hypothermia is prevented by other means. Hypothermia can be prevented by compensating for heat transfer from the body at other sites (for example, by applying hot water to one or more additional sites). Multiple cooling elements can be used, for example, to make contact with larger surfaces (for example, greater than 3500 cm²).

The element and / or cooling agent can follow the contour of the area to which it is applied. For example, a flexible apparatus can be used to follow the contour of the surface to which the cooling is applied. The apparatus can also modify the shape of the contacted surface such that the surface is delimited around or within the cooling agent or the apparatus containing the cooling agent upon contact. The element and / or cooling agent may contact more than one surface at a time, for example, when the surface is folded and in contact on each side with the element and / or cooling agent to increase cooling efficiency.

Preferably, the solid cooling element and / or agent has a shape that enhances thermodynamic heat exchange ("heat exchange") at the contacted surface (eg, the surface of the skin). To improve conduction, a liquid can be used on the intermediate surface between the solid cooling agent and the contacted surface.

When necessary, the application of the cooling element and / or agent can be combined with the use of pain management agents, such as an anesthetic or analgesic (cooling, by itself, has analgesic properties, so the use pain management agents is optional). For example, local anesthetics can be applied topically to the point of contact before or after or during the application of the cooling agent. When necessary, systemic administration of the anesthetic can be provided through conventional procedures, such as injection or oral administration. The temperature of the cooling agent can be changed during treatment, for example, so that the rate of cooling is decreased in order to provide a treatment that causes less discomfort. Furthermore, the procedures of the present invention can be carried out in combination with other fat reduction procedures known in the art, such as liposuction.

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Preferably, the lipid-rich cells of the present invention are adipples contained in subcutaneous fatty tissue or cellulite. Thus, the lipid-rich cells that make up the subcutaneous adipose tissue are targeted for disintegration by methods of the present invention. Furthermore, targeting the disruption of lipid-rich cells that constitute the adventitia surrounding organs or other anatomical structures is included within the scope of the present invention.

The intracellular lipids of adipocytes are confined within the paraplasmic vacuole. There are unilocular and plurilocular adipocytes within the subcutaneous fatty tissue. Most are unilocular, and greater than 100 pm in diameter. This size can drastically increase in obese subjects due to an increase in intracellular lipid content.

Preferably, the lipid-rich cells of the present invention possess a total intracellular lipid content of between 20 and 99%. Preferably, the lipid-rich cells of the present invention possess an Intracellular lipid content that comprises between about 20 and 50% saturated triglycerides, and, even more preferably, between about 30 and 40% saturated triglycerides. Intracellular triglycerides include, but are not limited to: saturated fatty acids, eg, myristic, palmitic, and stearic acid; monounsaturated fatty acids, for example palmitoleic acid and oleic acid; and polyunsaturated fatty acids, for example, linoleic acid and linolenic acid.

Preferably, the lipid-rich cells of the present invention are found within subcutaneous adipose tissue. The saturated fatty acid composition of subcutaneous adipose tissue varies in different anatomical positions in the human body. For example, human subcutaneous adipose tissue in the abdomen may have the following saturated fatty acid composition: myristic acid (2.6%), palmitic (23.8%), palmitoleic (4.9%), stearic (6.5%) %), oleic (45.6%), linoleic (15.4%) and linolenic (0.6%). The subcutaneous adipose tissue of the abdominal area can comprise approximately 35% saturated fatty acids. This percentage is comparatively higher than the buttock area, which can comprise approximately 32% saturated fatty acids. At room temperature, the saturated fatty acids in the abdominal area are in a semi-solid state as a result of the higher content of fatty acids. The buttock area is not similarly affected. G. Malcolm et al., (1989) Am. J. Clin. Nutr. 50 (2): 288-91. One skilled in the art can modify the temperature ranges or the number of applications as necessary to compensate for anatomical differences in response to the cooling procedures of the present invention.

Preferably, the non-lipid-rich cells of the present invention have a total intracellular lipid content of less than 20%, and / or are not disaggregated by the cooling procedures of the present invention. Preferably, the non-lipid-rich cells of the present invention include cells that possess an intracellular lipid content that comprises less than about 20% triglycerides with a high degree of saturation, and, even more preferably, less than between about 7 and 10% triglycerides with a high degree of saturation. Non-lipid-rich cells include, but are not limited to, those surrounding subcutaneous fatty tissue, such as: cells of the vascular system, peripheral nervous system, epidermis (eg, melanocytes), and dermis (eg, fibrocytes).

Damage to the dermis and / or epidermis that is avoided by the methods of the present invention may include, for example: inflammation, irritation, swelling, lesion formation, and hyper- or hypopigmentation of melanocytes.

Without being bound by theory, it is believed that the selective disintegration of lipid-rich cells is due to the localized crystallization of fatty acids with a high degree of saturation after cooling to temperatures that do not induce the crystallization of fatty acids with a high degree of saturation. saturation in non-lipid-rich cells. The crystals break the bilayer membrane of lipid-rich cells, causing necrosis. In this way, damage to non-lipid-rich cells, such as dermal cells, is avoided at temperatures that induce crystal formation in lipid-rich cells. Cooling is also believed to induce lipolysis (eg, metabolization) of lipid-rich cells, further enhancing reduction in subcutaneous adipose tissue. Lipolysis can be enhanced by local exposure to cold that induces stimulation of the sympathetic nervous system.

In one embodiment, the temperature of the lipid-rich cells is not Lower than about 10 ° C. Preferably, the temperature of the lipid-rich cells is between -10 ° C and 37 ° C. More preferably, the temperature of the lipid-rich cells is between -4 ° C and 20 ° C. Even more preferably, the temperature of the lipid-rich cells is between -2 ° C and 15 ° C. Preferably, the lipid-rich cells are cooled to less than 37 ° C, for a period of up to two hours. In general, lipid-rich cells are preferably kept at an average temperature of between about -10 ° C and about 37 ° C, 35 ° C, 30 ° C, 25 ° C, 20 ° C, 15 ° C, 10 ° C or 4 ° C; about -4 ° C and about 35 ° C, 30 ° C, 25 ° C, 20 ° C, 15 ° C, 10 ° C or 4 ° C; about -2 ° C and about 35 ° C, 30 ° C, 25 ° C, 20 ° C, 15 ° C, 10 ° C or 5 ° C.

In yet another embodiment, the temperature range of lipid-rich cells is between 37 ° C and -10 ° C. Pulse cooling procedures followed by brief warm-up periods can be used to minimize collateral damage to non-lipid-rich cells. More preferably, the temperature range of the lipid-rich cells is between -8 ° C and 33 ° C. Even more preferably, the temperature range of the cells

ES 2 390 598 T3 rich in lipids ranges between -2 ° C and 15 ° C. The temporal profile of skin cooling can be carried out in a continuous cooling action or in multiple cooling cycles or even in a combination of cooling with active heating cycles.

The cooling methods of the present invention advantageously eliminate unwanted effects on the epidermis. In one embodiment, the temperature of the epidermis is not less than about -15 ° C. Preferably, the temperature of the epidermis is between about -10 ° C and 35 ° C. More preferably, the temperature of the epidermis is between about -5 ° C and 10 ° C. Even more preferably, the temperature of the epidermis is between about -5 ° C and 5 ° C.

The cooling procedures of the present specification advantageously eliminate unwanted effects on the dermis. In one embodiment, the dermis temperature is not less than about -15 ° C. Preferably, the temperature of the dermis is between about -10 ° C and 20 ° C. Most preferably, the dermis temperature is between about -8 ° C and 15 ° C. Even more preferably, the temperature of the dermis is between about -5 ° C and 10 ° C. In a preferred embodiment, the lipid-rich cells are cooled to between approximately -5 ° C and 5 ° C for a period of up to two hours and the cells of the dermis and epidermis maintain an average temperature of approximately 0 ° C. . In one of the more preferred embodiments, the lipid-rich cells are cooled to between about -5 ° C and 15 ° C for periods ranging from about one minute to about two hours.

The methods of the present specification can be applied in short intervals (for example, time intervals of 1 minute, 5 minutes, 15 minutes, 30 minutes and 60 minutes) or long intervals (for example, time intervals of 12 hours and 24 hours). Preferably, the intervals are between 5 and 20 minutes. Optionally, heat can be applied between cooling intervals.

Feedback mechanisms can be used to monitor and control temperatures in the subcutaneous adipose tissue of the skin (ie, the dermis, epidermis, or a combination of both). A feedback mechanism can monitor the temperature of a subject's skin to ensure that the temperature recorded within it does not drop below a predetermined minimum temperature, eg, from about -10 ° C to about 30 ° C. A non-invasive device can be applied externally to measure the surface temperature at the point of contact and / or the surrounding region. An invasive device, such as a thermocouple, can be used to measure internal temperatures.

Feedback mechanisms can include all those known in the art for monitoring temperature and / or crystal formation. Crystal formation can be measured, for example, by ultrasound imaging systems and acoustic, optical and mechanical measurements. Mechanical measurements can include, for example, tensile strength measurements.

In one embodiment, a multilayer model can be used to estimate temperature profiles over time and within different depths. Temperature profiles are designed to produce a temperature gradient within the tissue, with a lower temperature on the surface. In a preferred embodiment, the temperature profiles are designed to minimize blood flow during cooling. To achieve optimal temperature gradients, feedback mechanisms comprising for example thermocouples, ultrasound wave propagation (eg to detect phase changes of subcutaneous adipose tissue) or shock can be used.

Substantial cooling of the subcutaneous fat layer, for example to a desired temperature of between -5 ° C and 15 ° C, by cooling at the skin surface has several requirements. The heat extracted from the skin's surface establishes a temperature gradient within the skin, which in turn cools, first, the epidermis, the dermis, and finally the subcutaneous fat layers. Dermal blood flow carries heat from inside the body to the dermis. Therefore, dermal blood flow can severely limit the cooling of the deep dermis and subcutaneous fat. Therefore, temporary limitation or elimination of cutaneous blood flow is highly preferred, for example by locally applying pressure to the skin greater than systolic blood pressure, while cooling as a treatment to achieve a reduction in subcutaneous fat. A general requirement is that the cooling time on the skin surface must be long enough to allow heat to flow from the dermis and subcutaneous fat layers in order to achieve the desired temperature for treating them. When subcutaneous fat is cooled to a temperature below the crystallization temperature of its lipids, the latent heat of freezing for these lipids must also be removed, by diffusion. The cooling temperature of the skin surface and the cooling time can be adjusted to control the depth of the treatment, for example the anatomical depth to which the subcutaneous fat is affected. The diffusion of heat is a passive process, and the temperature inside the body is almost always close to 37 ° C. Therefore, another general requirement is that the temperature of the skin surface during cooling must be lower than the temperature of the desired target (e.g. adipocytes) for treatment of the region, for at least part of the time during which cooling is carried out.

When a skin diameter greater than about 2 cm is cooled, and without blood flow, thermal diffusion

One-dimensional ES 2 390 598 T3 offers a good approximation to estimate temperature profiles in the skin over time during cooling. Thermal diffusion obeys the general diffusion equation, óT / ót = κ or²T / oz², where T (z, t) is the skin temperature as a function of depth z and time t, and κ is the thermal diffusivity, which is approximately 1.3 x 10^-3 cm²s^-1 for skin tissue. Solutions and approximate solutions to the general diffusion equation have been obtained for the plane geometry of a semi-infinite plate (or slab), which approximates the situation of the skin. When the surface of the skin (z = 0) is kept at a lower given temperature, a useful approximation is that the flow of heat from a depth z requires a time of approximately t = z² to achieve a temperature difference of ¹Z is the initial difference, where t is in seconds and z is in millimeters. Thus, z² it can be considered an approximate value for a thermal time constant. For example, if the initial skin temperature is 30 ° C, and 0 ° C ice is applied firmly against the skin surface, it takes about 1 second for the temperature at a depth of 1 mm to reach about 15 ° C. The subcutaneous fat layer usually begins at approximately z = 3 mm, and extends to a thickness of many centimeters. The thermal time constant for heat transfer from the upper part of the subcutaneous fat layer is therefore approximately 10 seconds. To achieve substantial cooling of the subcutaneous fat, at least several and preferably more than 10 cooling time thermal time constants are required. Therefore, cooling must be maintained for approximately 30 to 100 seconds on the surface of the skin, and in the absence of dermal blood flow, so that the temperature of the upper part of the subcutaneous fat approaches that of the surface of the skin. cool skin. The latent heat of crystallization for lipids, mentioned above, must also be removed when the temperature of the fat falls below that of crystallization. Therefore, in general, cooling times greater than 1 minute are desirable, and cooling times greater than about 1 minute can be used to adjust the depth to which adipocytes will be affected, for periods of time up to more than one hour.

Accordingly, in yet another embodiment, the dermis is cooled at a rate sufficient to induce vasoconstriction. The circulation of blood within the dermis stabilizes the temperature of the dermis to a temperature close to body temperature. To cool subcutaneous adipose tissue to temperatures below body temperature, blood flow can be minimized. Rapid cooling of the epidermal surface can achieve reflex vasoconstriction that limits blood circulation in an appropriate manner.

In yet another embodiment, a vasoconstrictor drug is administered to induce vasoconstriction. Vasoconstrictor drugs, for example, can be applied topically to the point of contact before or after or during application of the cooling agent. When necessary, systemic administration of the vasoconstrictor drug can be provided through conventional procedures, such as injection or oral administration. The vasoconstrictor drug can be any of those known in the art. Preferably, the vasoconstrictor drug is an EMLA cream or epinephrine.

In yet another embodiment, pressure is applied to a surface, either at the point of contact with the cooling agent or in the vicinity thereof, so as to limit lateral blood flow. Pressure can be applied, for example, to a surface of the skin by compressing the surface of the skin into a skin fold comprising a single fold or multiple folds. Pressure can also be applied by applying a vacuum either at the point of contact with the cooling agent or in its vicinity.

Without being bound by theory, it is believed that the rate of crystal formation in lipid-rich cells can be altered by applying pressure during the cooling process. Sudden crystallization, rather than a slow accumulation of crystals, would cause further damage to lipid-rich cells. It is also believed that the application of pressure can force the movement of crystals within lipid-rich cells, intensifying damage to the bilayer membrane. Furthermore, the different compartments of the subcutaneous adipose tissue have different viscosities. In general, the viscosity increases at colder temperatures (for example, those that are particularly close to the point of phase change). Because the phase change for lipid-rich cells occurs at higher temperatures than for non-lipid-rich cells, non-uniform lines of tension are formed within the subcutaneous adipose tissue upon application of pressure. Pronounced damage is believed to occur within these power lines.

In yet another aspect, the temperature of the dermis and / or epidermis ranges between 35 ° C and -15 ° C. More preferably, the temperature of the dermis and / or epidermis ranges from -10 ° C to 10 ° C. Even more preferably, the temperature of the dermis and / or epidermis ranges from -8 ° C to 8 ° C. Oscillating temperatures on the skin's surface can provide intermittent heating to counteract potential side effects of the cooling process (eg, crystal formation in dermal or epidermal cells).

In yet another aspect, the application of the cooling agent is combined with the application of electric or acoustic fields, constant or oscillating in time, located in the dermis and / or epidermis to reduce or eliminate the formation of crystals inside.

Figure 1A illustrates a treatment system 100 for cooling a target zone. As shown in Figure 1A, the treatment system 100 can include a control unit 105 and a treatment unit 107, which can

ES 2 390 598 T3 include a cooling / heating element 110 and a Treatment Interface 115.

The control unit 105 may include a power source, for example, the control unit may be connected to a power source, to supply power to the treatment unit 107. The control unit 105 may also include a computing device provided control hardware and / or software to control, depending on the properties and / or parameters entered, the cooling / heating element 110 and the treatment interface 115. Treatment interface 115 may include detector 120.

FIG. 1B is a diagram illustrating a configuration of the control unit 105 in accordance with an embodiment of the invention. As shown in Figure 1B, the control unit 105 may comprise a computing device 125, which may be a general purpose computer (such as a PC), workstation, central computing system, and so on. The computing device 125 may include a processing device (or central processing unit "CPU") 130, a memory device 135, a storage device 149, a user interface 145, a system bus 150, and a communications interface. 155. CPU 130 can be any type of processing device for executing instructions, processing data, and so on. Memory device 135 can be any type of memory device, including any one or more of: random access memory ("RAM"), read-only memory ("ROM"), fast memory (flash), memory programmable read-only with electrical erasure (“EEPROM”), etc. The storage device 140 can be any storage device for reading / writing to / from any optical, magnetic and / or magneto-optical, removable and / or integrated storage media, and the like (e.g., a hard disk, a memory of only reading on compact disc “CD-ROM”, CD rewritable “CDRW”, ROM on digital versatile disc “DVD-ROM”, DVD-RW, etc.). Storage device 140 may also include a controller / interface (not shown) for connecting to system bus 150. Thus, memory device 135 and soul device 140 are suitable for storing both data and instructions for processes scheduled to be executed on a CPU 130. The user interface 145 may include a touch screen, control panel, keyboard, numeric keypad, display device, or any other type of interface, which can be connected to the system bus 150 through a respective interface / adapter of the device. input / output (not shown). Communications interface 155 can be adapted to communicate with any type of external device, including processing unit 107. Communications interface 155 may also be adapted to communicate with any system or network (not shown), such as one or more computing devices on a local area network ("LAN"), wide area network ("WLAN" ), Internet, etc. Interface 155 may be directly connected to system bus 150, or it may be connected through a suitable interface (not shown). In this way, the control unit 105 can provide execution processes, by itself and / or in collaboration with one or more additional devices, which can include algorithms for controlling the processing unit 107 in accordance with the present invention. The control unit 105 can be programmed or instructed to carry out these processes according to any communication protocol, programming language on any platform. In this way, the processes can be materialized in data, as well as in instructions, stored in the memory device 135 and / or the storage device 140 or be received in the interface 155 and / or user interface 145 to be executed in a CPU 130.

Referring back to FIG. 1A, the treatment unit 107 can be a portable device, an automated apparatus, and the like. Cooling / heating element 110 can include any type of cooling / heating component, such as a thermoelectric cooler, and the like.

Figure 1C is a diagram showing the cooling / heating element 110. As shown in Figure 1C, the cooling / heating element 110 may include a network of conduits through which a cooling / heating fluid flows. The conduits can be formed by any heat conductive tubing and the like. Cooling / heating fluid can be directed into element 110 through inlet 175 and expelled through outlet 180. The cooling / heating fluid can be any liquid that has a controlled temperature, such as cooled air / gas or a liquid. For example, a salt water or acetone bath can be used that is cooled using ice or frozen carbon dioxide as the source of chilled liquid pumped through element 110. In this way, a circulation system can be formed in which fluid expelled from outlet 180 is re-cooled at the fluid source and redirected to inlet 175. Control unit 105 can monitor and control the temperature of the fluid. fluid source and / or element 110, which may include the rate at which cooling fluid is pumped through element 110. In this way, the temperature of the cooling / heating element 110 can be controlled or programmed using the control unit 105. As also shown in FIG. 1C, there may be a temperature difference, AT, between regions of the element 110. For example, heat from the target tissue can be transferred to the cooling fluid during treatment, causing the fluid near the outlet 180 to have a higher temperature than the cooling fluid near the inlet 175. Said AT can be reduced by reducing the size of element 110. In accordance with one embodiment of the invention, the configuration of the conduits in element 110 and the corresponding application of element 110 to the target tissue can compensate for any temperature differences necessary to treat various target tissues. For example, the region of element 110 that is near outlet 180 can be applied to treatment zones that require a higher treatment temperature, and so on. Thus, the passageways of element 110 can be configured according to the size, shape, formation, etc., of the target tissue required by the various treatment temperatures. Cooling / heating fluid can also be pumped through element 110 in a pulsatile manner.

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Referring back to Figure 1A, treatment interface 115 can be any type of interface between cooling / heating element 110 and epidermis 160 to carry out treatment on epidermis 160, dermis 165, and fat cells 170. . For example, treatment interface 115 may include a cooling (conductive) plate, a container filled with cooling fluid, a free-form membrane (for a complementary interface with an irregular epidermis), a convex cooling element (for example, such as the shown in Figure 3), and the like. Preferably, treatment interface 115 comprises a heat conductive material that complements epidermis 160 to obtain maximum heat transfer between cooling / heating element 110 and epidermis 160, dermis 165, and / or fat cells 170. For example, treatment interface 115 can be a fluid-filled container or membrane, so that the change in pressure from cooling element 110 caused by a pulsatile flow of cooling fluid can be transferred to the target tissue. Furthermore, the treatment interface 115 can simply be a chamber in which the cooling / heating fluid can be applied directly to the target tissue (epidermis 160, dermis 165 and fat cells 170), using for example a spray device and the like.

The detector 120 can be a temperature monitor, eg, a thermocouple, a thermistor, and the like. Detector 120 can include any type of thermocouple, including types: T, E, J, K, G, C, D, R, S, B, to monitor tissue cooling. Detector 120 may also include a thermistor, which may comprise thermosensitive resistors whose resistances change as the temperature changes. The use of thermistors can be particularly advantageous due to their sensitivity. According to one embodiment of the invention, a thermistor with a large negative temperature coefficient of resistance ("NTC") can be used. Preferably, a thermistor used for detector 120 may have an operating temperature range of -15 ° C to 40 ° C. In addition, detector 120 may include a thermistor with active polymer or ceramic elements. A ceramic thermistor may be most preferable, as they can have the most reproducible temperature measurements. A thermistor used for detector 120 can be encapsulated in a protective material, such as glass. Of course, other temperature monitoring devices can also be used, depending on the desired size, geometry and temperature resolution. The detector 120 may also comprise an electrode that can be used to measure the electrical resistance of the skin surface. The formation of ice within the superficial structures of the skin such as the epidermis or the dermis causes an increase in electrical resistance. This effect can be used to monitor ice formation inside the dermis. Detector 120 can also consist of a combination of several measurement procedures.

In this way, the detector 120 can extract, among other things, information about the temperature of the epidermis 160, dermis 165 and / or fat cells 170 by way of feedback information to control the control unit 105. The control unit 105 can analyze the sensed temperature information based on entered properties and / or parameters. For example, the temperature of the fat cells 170 can be determined by calculations based on the temperature of the epidermis 160 detected by the detector 120. In this way, the treatment system 100 can non-invasively measure the temperature of the cells. fat 170. This information can then be used by the control unit 105 to carry out continuous feedback control of the treatment unit 107, for example, by adjusting the energy / temperature of the cooling / heating element 110 and the treatment interface 115, maintaining thus an optimal treatment temperature of the targeted fat cells 170, while the surrounding epidermis 160 and dermis 165 are left intact. As described above, the cooling / heating element 110 can provide adjustable temperatures in the range of about -10 ° C to 42 ° C. An automated temperature measurement and control sequence can be repeated to maintain that temperature range until a procedure is completed.

It should be noted that the reduction of adipose tissue by cooling lipid-rich cells can be even more effective when tissue cooling is accompanied by physical manipulation, eg massage, of the target tissue. In accordance with one embodiment of the present invention, treatment unit 107 may include a device for massaging tissue, such as a vibrating device and the like. Another possibility is to use a piezoelectric transducer within the treatment unit 107 in order to provide an oscillation or mechanical movement of the cooling / heating element 107 (or, better, treatment unit?). Detector 120 may include feedback devices to detect changes in skin viscosity to monitor the effectiveness of treatment and / or prevent surrounding tissue from being damaged. For example, a vibration detection device can be used to detect any change in the resonance frequency of the target tissue (or surrounding tissue), which may indicate a change in tissue viscosity, which is mechanically moved or vibrated. by means of a vibrating device contained in the treatment unit 107.

To further ensure that the epidermis 160 and / or the dermis 165 are not damaged by the cooling treatment, an optical sensing / feedback device can be used to monitor the change in the optical properties of the epidermis (increased scattering). if ice formations appear); An electrical feedback device can be used to monitor the change in electrical impedance of the epidermis caused by ice formation on the epidermis; and / or an ultrasonic feedback device can be used to monitor for ice formation (actually, to prevent it) on the skin. Any such device may include a signaling control unit 105 to stop or adjust treatment to prevent skin damage.

In accordance with one embodiment of the invention, treatment system 100 may include various

ES 2 390 598 T3 configurations and instruments. Algorithms that are designed for different types of procedures, settings, and / or instruments may be included for the control unit 105.

As shown in FIG. 1D, treatment system 100 may include a probe controller 175 and probe 180 to perform a minimally invasive measurement of the temperature of fat cells 170. Advantageously, probe 180 may be capable of measuring a more accurate temperature of the fat cells 170, thereby improving the control of the treatment unit 107 and the efficiency of the treatment.

It should be noted that the treatment system 100 can be remotely controlled. For example, the link between the control unit 105 and the treatment unit 107 may be a remote link (wired or wireless) that provides the control unit 105 with remote control over the cooling / heating element 110, the interface treatment 115, probe controller 175, and probe 180.

While the above exemplary treatment system 100 illustrates the basic components of a suitable system for use with the present invention, the architecture should not be considered restrictive as there are many possible variations of the hardware configuration without departing from the present invention. .

Figure 2A illustrates a treatment system 200 for cooling fat cells 170 by folding the target tissue in accordance with one embodiment of the invention. As shown in Figure 2A, treatment system 200 may include control units 105 and corresponding treatment units 107, on two sides, connected to a compression unit 205. The compression unit 205 may be adapted to bring the treatment units 107 towards each other, thereby folding (or "pinching") the target tissue (epidermis 160, dermis 165, and fat cells 170) between the treatment units 107. The treatment interface 115 of the respective treatment units 107 on both sides of the target tissue can thus cool the fat cells 170 from multiple sides with greater efficiency, as described above. Detectors 120 may be included to measure and monitor the temperature of the target tissue. As shown in Figure 2A, the control units 105 may be connected to form an integrated system. In accordance with one embodiment of the present invention, the various components of system 200 can be controlled using any number of control unit (s).

As described above, physical manipulation of the target tissue can improve the effectiveness of the cooling treatment. In accordance with one embodiment of the present invention, the compression unit 205 can vary the force with which the treatment units 107 move towards each other around the target tissue (epidermis 160, dermis 165 and fat cells 170 ). For example, the compression unit 205 may apply a pulsating force to alternately tighten and loosen the fold (or "pinch") of the target tissue. The squeeze strength can also be monitored for any changes in the characteristics (eg, viscosity) of the target tissue, thus ensuring the efficacy and safety of the treatment.

Figure 2B illustrates the system 200 with a probe 180 similar to that of the system 100 shown in Figure 1C for minimally invasive measurement of the temperature of fat cells 170. As described above, the probe 180 may be capable of measuring a more accurate temperature of the fat cells 170, thereby improving the control of the treatment unit 107 and the efficiency of the treatment.

Figures 3A and 3B are diagrams showing a treatment system 300 in accordance with one embodiment of the present invention. As shown in Figure 3A, the system 300 may include a suction unit 305, and a treatment unit 107 may include a treatment interface 115 provided with a curved surface, which, for example, forms a dome, to form and contain a chamber 310 above the epidermis 160. As shown in Figure 3B, the suction unit 305 can be activated to draw air out of chamber 310 so that the target tissue (epidermis 160, dermis 165, and fat cells 170) is lifted into contact with the treatment interface 115. Advantageously, treatment interface 115 can surround targeted fat cells 170 for more efficient cooling. Treatment interface 115 may consist of a rigid or flexible solid material that is in contact with the skin or a thermal coupling agent between the skin surface and the treatment unit. Interface surface 115 may also have multiple openings connected to aspiration unit 305. The skin is partially inserted into these multiple openings, which can increase the total surface area of ​​the epidermis 160 that is in thermal contact with the treatment interface (eg, skin tightening). Stretching the skin reduces the thickness of the epidermis and dermis, which facilitates the cooling of fat 170. A number of detector (s) 120 and / or probe (s) 180 may be included in treatment system 300 to monitor tissue temperature during treatment, as described above with reference to Figures 1A, 1C, 2A. and 2B, the detailed description of which will not be repeated here.

Figure 4 illustrates a treatment system 400 in accordance with one embodiment of the invention. As shown in Figure 4, the suction unit 305 may be connected to a ring-shaped opening around the treatment interface 115 so that, when activated, a suction seal 410 is formed with the epidermis. 160 around treatment interface 115. As a result, treatment can be applied at treatment interface 115 to an isolated area of ​​the target tissue.

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Advantageously, the subject or part of the subject can be immersed in a heating bath and the treatment at interface 115 can be unaffected. Accordingly, the treatment area can be enlarged while a surrounding warming environment can prevent general hypothermia.

Figures 5A and 5B are diagrams showing a treatment system 500 in accordance with one embodiment of the present invention. As shown in Figures 5A and 5B, treatment system 500 can form a band (or cylinder) around a mass of target tissue 515. Treatment system 500 can comprise any flexible or rigid material. Cooling / heating fluid can be pumped through treatment system 500 through inlet 175 and outlet 180, as shown in Figure 5B. The cooling / heating element 110 can be formed by an internal container or a network of conduits, such as pipes and the like. Heat transfer with target tissue mass 515 can be accomplished through treatment interface 115, which can include any heat conductive material. The treatment system 500 may also include a clamping mechanism 510, such as a hook and loop clamp, and the like, to clamp and wrap the mass of tissue 515. In addition, the treatment interface 115 may include a flexible material of such that the pressure of the cooling fluid pumped through the treatment system 500 can be transferred to the target tissue 515. For example, referring to FIG. 5A, treatment system 500 can apply negative pressure to target tissue mass 515. Target tissue mass 515 can be any section, body part, or limb of a subject. For example, the target tissue mass 515 can be an arm, upper or lower leg, waist, etc., of a subject. The control unit 105 can control the pressure and flow of the cooling fluid in the system 500 until an optimum treatment temperature and / or pressure is obtained. A tight fit around tissue mass 515 and an increase in negative pressure may also allow the subject to be immersed in a heating bath. As described above, the flow of the fluid can be a pulsatile flow.

The description of the present invention is completed on the basis of the following illustrative and non-restrictive examples, which provide a better understanding of the present invention and its many advantages.

EXAMPLES

Example 1

Selective damage to fat tissue by controlled in vivo cooling.

The procedures of the present specification were carried out with a 6-month-old female, white, Hanford miniature pig ("Pig I") and a 6-month-old female, black, Yucatán miniature pig ("Pig II ”). Pigs were anesthetized using telazol / xylazine (4.4 mg / kg IM + 2.2 mg / kg IM). Inhalable anesthetic (halothane or isoflurane (1.5 to 3.0%) with oxygen (3.0 L / min) was administered through a mask and filtered with an F / Air cartridge, only if the injectable anesthetic did not provide adequate Sufficient somatic analgesia Several test sites were marked with micro-tattoos by applying India ink to the corners of each of the test sites. After mapping the test sites, cold exposures were carried out using a cooling device as described in Figure 1A. The treatment interface area was a flat area with a size of 2 x 4 cm²with an integrated temperature sensor. The interface was in thermal contact with a thermoelectric cooler controlled electronically by a control unit, so that the temperature at the interface surface was kept constant at a preset temperature. During exposure to cold, the cooling device was applied to the skin with mild to moderate pressure that did not cause significant mechanical compression of blood flow. The cooling element was applied to the skin without any manipulation of the surface profile.

Various combinations of cooling interface temperatures and preset exposure times were tested. For some sites, a heat conducting lotion was applied between the skin and the cooling interface. This heat conducting lotion was mainly composed of glycerol. Pig I was observed for 61 days until excisional biopsies were obtained from all sites and the pig was sacrificed. On day 2, an additional punch biopsy was obtained from site C.

Biopsies were processed for routine light microscopy and stained with hematoxylin-eosin. The indicated temperature is that of the applied cooling element. Table 1 represents the parameters of the application of cooling and the results obtained in the various sites of pig I:

<td>Place</td><td>Temperature</td><td>Weather</td><td>Lotion</td><td>Results</td>

<td>TO</td><td>-6 ° C</td><td>1 minute</td><td> +</td><td>At 61 days: There was no epidermal damage. There was no dermal damage. There was no obvious indentation. There were no obvious histological changes.</td>

<td>B</td><td>-6 ° C</td><td>1 minute</td><td></td><td>At 61 days: There was no epidermal damage. There was no dermal damage.</td>

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<td></td><td></td><td></td><td></td><td>There was no obvious indentation. There were no obvious histological changes.</td>

<td>C</td><td>-6 ° C</td><td>5 minutes</td><td> +</td><td>At 61 days: There was no epidermal damage. There was no dermal damage. Cleft due to loss of subcutaneous adipose tissue (1 week to 61 days). Decrease in the average size of adipocytes at a depth of between approx. 3 to 6 mm. Obvious histological damage to adipose tissue. After 2 days: Inflammation of the tissue and panniculitis.</td>

<td>D</td><td>-3.5 ° C</td><td>5 minutes</td><td> +</td><td>At 61 days: There was no epidermal damage. There was no dermal damage. There was no obvious indentation. At the limit of histological damage in adipose tissue. Decrease in the average size of adipocytes.</td>

<td>AND</td><td>Control</td><td></td><td></td><td>Normal - unchanged within the epidermis, dermis, and subcutaneous adipose tissue.</td>

Pig II was observed for 50 days until blops were obtained by excision of all sites and the pig was sacrificed. On day 17, an additional biopsy was obtained from site E. Biopsies were processed for routine light microscopy and stained with hematoxylin-eosin, as described above. The indicated temperature is that of the applied cooling element. Table 2 represents the parameters of the application of cooling and the results obtained in the various sites of pig II:

<td>Place</td><td>Temperature</td><td>Weather</td><td>Lotion</td><td>Results</td>

<td>C</td><td>-6 ° C</td><td>5 minutes</td><td></td><td>At 50 days: Pronounced cleft (2 to 3 mm) due to loss of subcutaneous adipose tissue. There was no epidermal damage. There was no dermal damage. There were no pigmentation changes; however, there was a decrease in the size of adipocytes and histological damage to the adipose tissue.</td>

<td>D</td><td>-8 ° C</td><td>5 minutes</td><td></td><td>At 50 days: Pronounced cleft (2 to 3 mm) due to loss of subcutaneous adipose tissue. There was no epidermal damage. There was no dermal damage. There were no pigmentation changes; however, there was damage to the adipocytes to a depth of approx. 6 mm. Decrease in the size of adipocytes and histological damage to adipose tissue.</td>

<td>AND</td><td>-9 ° C</td><td>5 minutes</td><td></td><td>At 50 days: Pronounced cleft (2 to 3 mm) due to loss of subcutaneous adipose tissue. There was no epidermal damage. There was no dermal damage. There were no pigmentation changes; however, there was damage to the adipose cells to a depth of approx. 6 mm. Decrease in the size of adipocytes and histological damage to adipose tissue. At 17 days:</td>

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<td></td><td></td><td></td><td></td><td>Signs of panniculitis.</td>

<td>F</td><td>-22 ° C</td><td>5 minutes</td><td></td><td>At 50 days: Pronounced epidermal damage with pronounced hypopigmentation. Scar formation with dermal contraction and complete ablation of subcutaneous adipose tissue.</td>

Figure 6 represents a skin surface image of pig II test sites D, E and F, 17 days after challenge. A cleft can be seen in 1 that corresponds to the sites of cold exposure, which corresponds to the D site, and in 2, which corresponds to the E site. Abnormal epidermal changes can not be observed in these sites of test. In 3, which corresponds to test site F, where aggressive test methods were applied, the damage to the epidermis is pronounced (eg loss of pigmentation and central crusting).

Figure 7 depicts the histology of test site E (pig II), 17 days after cold exposure at -9 ° C for 5 minutes, in samples taken from an area below the cold exposure site.

Figure 7A depicts a low magnification (1.25x) and Figure 7B depicts a close-up with a medium magnification (5x) of the same specimen. Epidermis 701, 702, subcutaneous fat 703, and muscle layer 704 are shown. Histology reveals signs of lobular and septal panniculitis within subcutaneous fat 703, which is an inflammation of adipose tissue. The average size of the fat cells has been reduced compared to the sample from the unexposed area. No evidence of tissue alteration has been observed in the epidermis, dermis, or muscle layer.

A reduction in subcutaneous adipose tissue was demonstrated by clinical observation of the cleft on the skin surface at the precise site of cooling, as well as by histology (hematoxylin and eosin staining). Figures 8A, B, C, D, E and F represent a histology 50 days after exposure with a low magnification of 2.5x (Figures 8A, 8C and 8E) and with a mean magnification of 5x (Figures 8B, 8D and 8F) from test site C (Figures 8A and 8B), from test site E (Figures 8C and 8D) and from test site F (Figures 8E and 8F). Epidermis 801 and dermis 802 were not damaged at test sites C and E while the more aggressive cooling regimen applied to test site F resulted in damage to the epidermis and dermis (for example, formation of of scars and inflammation). Subcutaneous fat 803 shows a reduction in the size of adipocytes and structural changes (eg, an apparent condensation of the fat cell layer with fibrous septa is included in the condensed fat layer). As a result of the aggressive cooling regime applied to test site F, the layer was practically completely removed, leaving only a few residual clumps of fat cells. Therefore, where an aggressive cooling regime is applied (test site F), pronounced non-selective damage to the epidermis and dermis is observed.

Taken together, the results demonstrate that selective disintegration of subcutaneous adipose tissue is achieved using the cooling procedures of the present invention without causing damage to the epidermis and dermis.

The temperature measurement was carried out during the cooling of the skin surface to -7 ° C applied with enough pressure to stop the blood flow of the skin, to illustrate the dependence of the time and the depth of the cooling in a live pig. Thermocouples inserted at depths of 0, 2, 4 and 8 millimeters were used to record the temperature. Although the conditions of this experiment were not ideal (the skin cooler did not strictly maintain -7 ° C on the surface), it is clear that the cooling of the dermis (2 mm) and fat (4 and 8 mm) it generally occurred as expected (see, for example, Figure 10).

Example 2

Temperature profile measurements at various tissue depths.

This study was carried out using a 6-month-old, black, hairless female Yucatán miniature pig (Sinclair Research Center, Columbia, MO). The pig was anesthetized using telazol / xylazine (4.4 mg / kg IM + 2.2 mg / kg IM). Inhalable anesthetic (halothane or isoflurane (1.5 to 3.0%) with oxygen (3.0 L / min) was administered through a mask and filtered with an F / Air cartridge, only if the injectable anesthetic did not provide adequate sufficient somatic analgesia. Micro-tattoo test sites were marked by applying India ink to the corners of each of the test sites and inserting hypodermic needles into those corners of the test site. Exposure to cold was carried out with a round and convex copper plate attached to a heat exchanger, which was cooled by circulating a tempered cooling agent to -7 ° C. The exposure time varied between 600 and 1200 s. Table 3 represents the parameters of the application of cooling and the results obtained in the various sites of pig III. The cold plate had three central openings approximately 1mm in diameter through which thermocouples were placed to monitor the temperature profile at various depths of the tissue during cold exposure. The cold exposure device, shown in Figure 9, was held firmly against the test site during cold exposure. Cold exposures were carried out on two experimental days

ES 2 390 598 different T3, with a week interval. On the first experimental day, the thermocouples occasionally shifted during cold exposure, resulting in a 0.5 mm variability of the thermocouple depth measurement. Another set of thermocouple exposures was carried out on the second experimental day at well-defined depths with minimal to zero variability in thermocouple depths. The location of the thermocouples on the first experimental day for test sites 1, 2, 3, 7, 11 and 12 was 2.5, 4.5 and 10 mm deep (+ / 0.5mm). Test samples 14, 15, 16 and 18 were treated on the second experimental day at a thermocouple depth of 2, 4 and 8 mm, with a displacement between minimum and zero. There may still be some variability in thermocouple depth due to tissue compression during cold exposure. A solution containing glycol was used to ensure good thermal contact on the skin surface. The pig was observed for 3 and months after treatment, until it was sacrificed and the tissue was collected from the test sites for analysis. Table 3 represents the parameters of the application of cooling and the results obtained in the various sites of pig III:

<td>Place</td><td>Temp. (agent cooler)</td><td>Exhibithion time</td><td>Location</td><td>Temp. min. to depth</td><td>Temp. min. to depth</td><td>Temp. min. to depth</td><td>Cleft 3 and months</td><td>Relative decrease in fat layer superficial at 3 and months</td>

<td> 1</td><td>-7 ° C</td><td>5 minutes</td><td>Side stand</td><td>0 ° C to 2.5mm</td><td>7 ° C at 5mm</td><td>24 ° C at 10mm</td><td> +</td><td> 66 %</td>

<td> 2</td><td>-7 ° C</td><td>5 minutes</td><td>Side stand</td><td>-2 ° C at 2.5mm</td><td>N / A</td><td>21 ° C at 10mm</td><td> +</td><td></td>

<td> 3</td><td>Control</td><td></td><td>Side stand</td><td> -</td><td></td><td></td><td> -</td><td> 9 %</td>

<td> 7</td><td>-7 ° C</td><td>10 minutes</td><td>Abdomen</td><td>3 ° C to 2.5mm</td><td>7 ° C at 5mm</td><td>19 ° C at 10mm</td><td> +</td><td></td>

<td> 9</td><td>Control</td><td></td><td>Abdomen</td><td> -</td><td></td><td></td><td></td><td></td>

<td> 11</td><td>-7 ° C</td><td>10 minutes</td><td>Buttock</td><td>N / A</td><td>N / A</td><td>12 ° C at 10mm</td><td> + +</td><td> 79 %</td>

<td> 12</td><td>-7 ° C</td><td>10 minutes</td><td>Buttock</td><td>4 ° C to 2.5mm</td><td>N / A</td><td>13 ° C at 10mm</td><td> +</td><td> 57 %</td>

<td> 13</td><td>-7 ° C</td><td>10 minutes</td><td>Buttock</td><td>-4 ° C to 2mm</td><td>N / A</td><td>7 ° C to 10mm</td><td> +</td><td></td>

<td> 14</td><td>-7 ° C</td><td>21 minutes</td><td>Buttock</td><td>-4 ° C to 2mm</td><td>3 ° C at 4mm</td><td>12 ° C to 8mm</td><td> +</td><td></td>

<td> 15</td><td>-7 ° C</td><td>11 minutes</td><td>Buttock</td><td>-4 ° C to 2mm</td><td>1 ° C at 4mm</td><td>12 ° C to 8mm</td><td> +</td><td></td>

<td> 16</td><td>-7 ° C</td><td>10 minutes</td><td>Buttock</td><td>-4 ° C to 2mm</td><td>0 ° C at 4mm</td><td>14 ° C to 8mm</td><td> + +</td><td></td>

<td> 18</td><td>-7 ° C</td><td>15 minutes</td><td>Side stand</td><td>-3 ° C to 2mm</td><td>N / A</td><td>15 ° C to 8mm</td><td> +</td><td> 66 %</td>

The test sites were exposed to the device, set to a cooler temperature of -7 ° C and exposed for a period of between 600 and 1200 s. The dermis hardened immediately after exposure to cold, as determined by palpation, and became viscous as it returned to its normal temperature, approximately one minute after exposure. There was no evident epidermal alteration or damage on close inspection with a polarized magnifying lens minutes after exposure. There was no blistering and the Nikolsky sign was negative. During the entire survival period, there was no significant damage to the epidermis. No crusting, blistering, or pronounced pigmentation changes were observed. Some sites showed a slight increase in epidermal pigmentation. This slight hyperpigmentation could be removed after a few months, gently rubbing the epidermis.

The temperature measurements of the thermocouples depended on the depth, the area of ​​the body, and the pressure with which the cooling was applied. Plots of temperature at different tissue depths during cold exposure are shown in Figures 10A-J for various test sites and are also summarized in the table.

3. At some test sites, temperature swings were observed that could be related to a nearby blood vessel. Some temperature graphs were not considered due to movement or incorrect thermocouple placement (indicated as “error” in Table 3). The temperature inside the deep dermis or the superficial fat layer is in the range of approximately 0 ° C to 7 ° C, depending on the variations in contact pressure and the anatomical area. This location presented a high variability of the different temperature graphs. The temperature within a depth of 8 to 10 mm, which corresponds to a depth of the interior of the subcutaneous fat layer, had a temperature in the range of 7 to 24 ° C.

ES 2 390 598 T3

Histology was obtained from a control (site 9) and cold-exposed site (site 8) (-7 ° C, 600 s) 6 days after exposure and analyzed by a dermatopathologist. The following description of what happened in the control and in the site exposed to the cold was made:

The epidermis of both samples is normal and presents a basket horny layer with a normal thickness, normal interpapillary ridges compared to the control. Within the cold-exposed site there was a slight presence of perivascular lymphocytic infiltrate. However, there were no overt signs of vasculitis in both samples.

Control subcutaneous fat has normal morphology. The subcutaneous fat at the site exposed to cold shows clear signs of lobular and septal panniculitis. Most adipocytes are surrounded by lymphocytic infiltrate with occasional lipids containing macrophages. Increases the thickness of the subcutaneous septum. There are slight vascular changes; however, there are no overt signs of vasculitis. Three and a half months after cold exposure, the pig was sacrificed and tissue was harvested from the challenge sites by full-thickness excision, after performing 20 MHz ultrasound visualization in vivo at selected test sites. In vivo ultrasound images clearly demonstrated the loss of fatty tissue in the cooling treatment area compared to the surrounding tissue not exposed to cold. Figure 11 shows an image obtained in vivo by ultrasound, of 3 ¹Z months after exposure to cold.

The collected tissue was macroscopically cut through the test sites and macroscopic tissue cross-sections were imaged. Macroscopic tissue cross-sections from sites 1, 3, 11, 12, and 18 are shown in Figure 13A-E. Decreased thickness of the subcutaneous fat layer was observed for all cold-exposed sites compared to the adjacent fat layer not exposed to cold. The macroscopic sections matched well with the ultrasound images. Two different compartments could be identified within the subcutaneous fat, a superficial fat layer and a deep fat layer. The thickness of the superficial fat layer was drastically reduced at cold treatment sites, while the deep fat layer did not undergo significant changes. Table 3 shows the percentage of the decrease in the surface fat layer within the test zone compared to the outside, for some test sites. A change in the subcutaneous fatty layer was observed at cold exposed sites 1, 11, 12 and 18. The mean decrease in the thickness of the superficial fatty layer within the evaluated test sites was 47%. On the unexposed control side, no significant decrease in thickness was found in either fat layer.

These examples confirm that selective subcutaneous adipose tissue damage can be achieved in a porcine model by external cooling within a specific range of external cooling temperatures and exposure time, without significant damage to the epidermis and dermis. . The removal of subcutaneous fat was also demonstrated by an obvious indentation in the surface of the treated skin, which exactly coincided with the exposure to cold, and by measurements of the fat layer in relation to the site of cold exposure by ultrasound and macroscopic cross sections after sacrifice. Pronounced histological changes, selectively affecting subcutaneous adipose tissue, were observed 6 days after cold exposure. Histologically, a panniculitis with a decrease in the size of fat cells was observed. There was evidence that the response to cold can vary at different sites and that the uppermost fat layer is more affected by tissue loss than the deeper fat layer. However, the results from pig III imply that there is an increase in fat removal in the superficial fat layer compared to the deeper layer. The explanation for this is that a) the surface fat layer is exposed to cooler temperatures because the gradient and or b) the deeper fat layer of pigs may be less susceptible to selective cold damage.

Figure 9 represents an image of the device for cold exposure of pig III. The cold copper plate 91 is brought into contact with the skin. The temperature profile of the interior of the skin during exposure to cold is measured by thermocouples 92 inserted into the tissue at different depths. The device is provided with a spring mechanism 93 to provide pressure during cold exposure.

Figure 10 represents the temperature profile at various depths during cold exposure of pig III from different test sites: 10A (site 1), 10B (site 2), 10C (site 7), 10D (site 11), 10E (site 12), 10F (site 13), 10G (site 14), 10H (site 15), 10I (site 16), and 10J (site 18). Temperature at various depths is indicated as T3-E (surface), T0-B (2 to 2.5 mm), T1-C (4 to 5 mm), and T2-D (8 to 10 mm).

Figure 11 represents an ultrasound image of test site 11 taken 3 ¹Z months after exposure. The section below 1105 is outside the cold zone and the section below 1106 is inside the cold zone. The dermis 1102 can be clearly distinguished from the fat layer 1103 and the muscle layer 1104. Within the fat layer 1103, two different layers can be distinguished: the superficial fat layer 1103a and the deep fat layer 1003b. The ultrasound image matches well with the macroscopic cross section of the same tissue in Figure 13c.

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Figure 12 represents the histology of test site 8 (Figures 12A and 12B) six days after cold exposure (-7 ° C, 600 s) and test site 9, which is an unexposed control (Figures 12C and 12D). The micrographs show a low magnification image (1.25x) in Figures 12A and 12c and a medium magnification (5x) in Figures 12B and 12D. The images show the epidermis 701, the dermis 702 and the subcutaneous fat 703.

While the unexposed control shows normal tissue morphology, the cold-exposed tissue shows clear signs of panniculitis in the subcutaneous fat. Inflammatory cells have migrated to this area and the average size of the cells has decreased.

Figures 13A-E represent macroscopic cross sections through the center of different test sites after slaughter of the pig, 3 ¹Z months after cold exposure: 13A (site 1), 13B (site 3), Figure 13C (site 11), Figure 13D (site 12), and Figure 13E (site 18). Each figure has a 1300 scale, which has 1 cm units and 1 mm subunits. The epidermis 1301, the dermis 1302, the superficial fat layer 1303 and the deep fat layer 1304. In Figure 13B of the unexposed control, no change in thickness of the different layers is observed. Figures 13A, 13C, 13D and 13E show the cross-section of cold exposed areas, which is compared to the central 4 to 5 cm of the surrounding tissue and non-cold areas. In all samples exposed to cold, a decrease in thickness is observed within the superficial fat layer of areas exposed to cold compared to areas not exposed to cold. Table 3 indicates the change in% of the thickness for each of the samples.