METHODS, KITS, AND COOLING DEVICES FOR DISRUPTING FUNCTION OF ONE OR MORE SEBACEOUS GLANDS

Abstract

One aspect of the invention provides a cooling device including a cooling unit and a control unit. The control unit is programmed to control operation of the cooling unit in order to cool one or more sebaceous glands within a local region for a period of time and to a temperature sufficient to disrupt function of the one or more sebaceous glands without permanently injuring epidermal tissue or cooling subcutaneous adipose tissue to 25 C. or below 25 C.

Claims

1-60. (canceled)

61. A cooling device comprising: a cooling unit; and a control unit programmed to control operation of the cooling unit in order to cool one or more sebaceous glands within a local region for a period of time and to a temperature sufficient to disrupt function of the one or more sebaceous glands without permanently injuring epidermal tissue or cooling subcutaneous adipose tissue to 25 C. or below 25 C.

62. The cooling device of claim 61, wherein the control unit is programmed to control operation of the cooling unit to cool the one or more sebaceous glands below a temperature selected from the group consisting of: about 15 C., about 14 C., about 13 C., about 12 C., about 11 C., about 10 C., about 9 C., about 8 C., about 7 C., about 6 C., about 5 C., about 4 C., about 3 C., about 2 C., about 1 C., about 0 C., about 1 C., about 2 C., about 3 C., about 4 C., about 5 C., about 6 C., about 7 C., about 8 C., about 9 C., and about 10 C.

63. The cooling device of claim 61, wherein the control unit is programmed to control operation of the cooling unit to cool the sebaceous glands at a rate selected from the group consisting of: between about 20 C. per minute and about 30 C. per minute, between about 30 C. per minute and about 40 C. per minute, between about 40 C. per minute and about 50 C. per minute, and between about 50 C. per minute and about 60 C. per minute.

64. The cooling device of claim 61, wherein the control unit is programmed to control operation of the cooling unit to cool the sebaceous glands at a rate selected from the group consisting of: between about 1 C. per second and about 2 C. per second, between about 2 C. per second and about 3 C. per second, between about 3 C. per second and about 4 C. per second, between about 4 C. per second and about 5 C. per second, between about 5 C. per second and about 6 C. per second, between about 6 C. per second and about 7 C. per second, between about 7 C. per second and about 8 C. per second, between about 8 C. per second and about 9 C. per second, and between about 9 C. per second and about 10 C. per second.

65. The cooling device of claim 61, wherein the period of time is selected from the group consisting of: less than about 10 seconds, between about 10 seconds and about 20 seconds, between about 20 seconds and about 30 seconds, between about 30 seconds and about 40 seconds, between about 40 seconds and about 1 minute, between about 1 minute and about 2 minutes, and between about 2 minutes and about 5 minutes.

66. The cooling device of claim 61, wherein the control unit is programmed to control operation of the cooling unit to: cool the one or more sebaceous glands for a first period of time; actively or passively warm the one or more sebaceous glands; and cool the one or more sebaceous glands for a second period of time.

67. The cooling device of claim 66, wherein the control unit is further programmed to: tighten one or more constriction elements in order to reduce blood flow to the local region during the cooling steps; and loosen the one or more constriction elements in order to restore normal blood flow to the local region during the warming step

68. The cooling device of claim 61, wherein the control unit is programmed to control operation of an energy source to warm the sebaceous glands at a rate selected from the group consisting of: between about 20 C. per minute and about 30 C. per minute, between about 30 C. per minute and about 40 C. per minute, between about 40 C. per minute and about 50 C. per minute, and between about 50 C. per minute and about 60 C. per minute.

69. The cooling device of claim 61, wherein the control unit is programmed to control operation of an energy source to warm the sebaceous glands at a rate selected from the group consisting of: between about 1 C. per second and about 2 C. per second, between about 2 C. per second and about 3 C. per second, between about 3 C. per second and about 4 C. per second, between about 4 C. per second and about 5 C. per second, between about 5 C. per second and about 6 C. per second, between about 6 C. per second and about 7 C. per second, between about 7 C. per second and about 8 C. per second, between about 8 C. per second and about 9 C. per second, and between about 9 C. per second and about 10 C. per second.

70. The cooling device of claim 61, wherein the period of time is selected from the group consisting of: less than about 5 minutes, between about 5 minutes and about 10 minutes, between about 10 minutes and about 15 minutes, between about 15 minutes and about 20 minutes, between about 20 minutes and about 25 minutes, between about 25 minutes and about 30 minutes, between about 30 minutes and about 35 minutes, between about 35 minutes and about 40 minutes, between about 40 minutes and about 45 minutes, between about 45 minutes and about 50 minutes, between about 50 minutes and about 55 minutes, and between about 55 minutes and about 60 minutes.

71. The cooling device of claim 61, wherein the control unit is programmed to control operation of the cooling unit to cool the one or more sebaceous glands below a temperature selected from the group consisting of: about 11 C., about 12 C., about 13 C., about 14 C., about 15 C., about 16 C., about 17 C., about 18 C., about 19 C., about 20 C., about 21 C., about 22 C., about 23 C., about 24 C., about 25 C., about 26 C., about 27 C., about 28 C., about 29 C., and about 30 C.

72. The cooling device of claim 61, wherein the control unit is programmed to control operation of the cooling unit to subject the one or more sebaceous glands to a plurality of cooling-warming cycles.

73. The cooling device of claim 72, wherein the plurality of cooling-warming cycles is selected from the group consisting of: 2, 3, 4, 5, 6, 7, 8, 9, and 10.

74. The cooling device of claim 61, further comprising: a treatment interface in thermal communication with the cooling unit, the treatment interface adapted and configured for application to the local region, wherein the treatment interface is a mask having a complementary surface that substantially approximates a human face, the treatment interface has a convex surface or a concave surface.

75. The cooling device of 74, further comprising: a vacuum in communication with the treatment interface, the vacuum being adapted and configured to generate reduced pressure in order to hold the treatment interface against the local region, and one or more feedback devices in communication with the control unit.

76. The cooling device of claim 61, wherein the control unit contains computer program code including instructions for controlling operation of the cooling unit in order to cool one or more sebaceous glands within a local region for a period of time and to a temperature sufficient to at least temporarily disrupt function of the one or more sebaceous glands without permanently injuring epidermal tissue or cooling subcutaneous adipose tissue to 25 C. or below 25 C.

77. A kit comprising: the cooling device of claim 61; an intermediary material; and instructions to apply the intermediary material to the local region prior to application of the cooling device.

78. The kit of claim 77, wherein the intermediary material enhances thermal conductivity between the cooling device, wherein the intermediary material is a cryoprotectant.

79. The kit of claim 78, wherein the intermediary material includes one or more selected from the group consisting: water, heavy water, oil, peanut oil, glycerol, glycol, polypropylene glycol (PPG), polyethylene glycol (PEG), propylene glycol, ethylene glycol, dimethyl sulfoxide (DMSO), alcohol, ethanol, propanol, iso-propanol, carboxyl polyethylene polymer, hydroxyethyl xylose polymer, carboxyl methylcellulose, and hydroxyethyl cellulose (HEC).

80. A method for disrupting function of one or more sebaceous glands within a local region of a subject, the method comprising: applying a cooling device to the local region of the subject; and modulating operation of the cooling device or periodically removing the cooling device in order to cool one or more sebaceous glands within the local region for a period of time and to a temperature sufficient to disrupt function of the one or more sebaceous glands without permanently injuring epidermal tissue or cooling subcutaneous adipose tissue to 25 C. or below 25 C.

81. The method of claim 80, wherein a rate of sebum secretion is temporarily reduced after performance of the method relative to a rate of sebum secretion prior to performance of the method, wherein a rate of sebum secretion is reduced by a percentage selected from the group consisting of: less than about 10%, between about 10% and about 20%, between about 20% and about 30%, between about 30% and about 40%, between about 40% and about 50%, between about 50% and about 60%, between about 60% and about 70%, between about 70% and about 80%, between about 80% and about 90%, and between about 90% and about 100%.

82. The method of claim 80, wherein a quantity of sebum secretion is temporarily reduced after performance of the method relative to a quantity of sebum secretion prior to performance of the method, wherein the quantity of sebum secretion is reduced by a percentage selected from the group consisting of: less than about 10%, between about 10% and about 20%, between about 20% and about 30%, between about 30% and about 40%, between about 40% and about 50%, between about 50% and about 60%, between about 60% and about 70%, between about 70% and about 80%, between about 80% and about 90%, and between about 90% and about 100%.

83. The method of claim 80, wherein function of the one or more sebaceous glands is permanently disrupted after performance of the method.

84. A method for reducing an abnormally elevated rate of sebum secretion from one or more sebaceous glands within a local region of a subject, the method comprising: applying a cooling device to the local region of the subject; and modulating operation of the cooling device or periodically removing the cooling device in order to cool one or more sebaceous glands within the local region for a period of time and to a temperature sufficient to disrupt function of the one or more sebaceous glands without permanently injuring epidermal tissue; thereby reducing the abnormally elevated rate of sebum secretion from the one or more sebaceous glands within the local region of the subject.

Description

DESCRIPTION OF THE FIGURES

[0040] For a fuller understanding of the nature and desired objects of the present invention, reference is made to the following detailed description taken in conjunction with the following figures.

[0041] FIG. 1 depicts an exemplary hair follicle structure 100 to which aspects of the invention can be applied.

[0042] FIG. 2 depicts a method 200 of disrupting sebaceous glands according to an embodiment of the invention.

[0043] FIG. 3 depicts a cooling device 300 according to an embodiment of the invention.

[0044] FIG. 4 depicts a kit 400 according to an embodiment of the invention.

[0045] FIG. 5 depicts a cooling device 500 according to an embodiment of the invention.

[0046] FIGS. 6A-6D depict various cooling profiles according to embodiments of the invention.

[0047] FIG. 7 provides a schematic of a cooling apparatus utilized in murine experiments described the Working Examples section.

[0048] FIG. 8 provides gross and histologic views of murine ears at various time points following cooling at 7 C. for 10 minutes. Retraction of lipid-laden sebocytes from peripheral cells is visible at day two. By day three, there is an eosinophilic collection of material within the gland lumen, which is a remnant of the dead and dying sebocytes induced by the treatment. By day five in this case, the gland exhibits normal architecture and lipid accumulation appears to be at baseline by day seven. The gross appearance of the ear remains relatively unchanged at all time courses.

[0049] FIGS. 9A and 9B provide graphs illustrating cooling damage of sebaceous glands in a murine model. Glands from sections from three different experiments with the same experimental condition (7 C. for 10 minutes) were manually counted. Glands were deemed damaged if three or more cells necrotic cells were visualized within the sebaceous gland. Over 60% of glands were damaged at 72 hours as depicted in FIG. 9A. The total number of glands was decreased at 72 hours and 1 week following treatment as depicted in FIG. 9B. Glands from three different animals treated with the same conditions were evaluated at each time point for each figure. An asterisk (*) denotes a significant difference (p<0.001) compared to the control group.

[0050] FIG. 10 depicts the disruption of cellular membrane and enzymatic activity and reduced lipid content of sebocytes by cooling. In panels (a), (b), (d), and (e), propidium iodide (PI) staining in treated (panels (d) and (e)) and control glands (panels (a) and (b)) is visible two hours after cooling treatment. Lipids are stained by BODIPY lipid dye and shown in green. Nuclei are stained by HOECHST stains and shown in blue. PI is shown in red. As seen in panels (a) and (b), PI staining was minimal in untreated controls and, when present, was restricted to the region immediately adjacent to the hair follicle. Treated glands showed extensive PI staining, which co-localized with both nuclei and sebum lipids as seen in panels (d) and (e). The width of images is 100 m. In panels (c) and (f), alkaline phosphatase was highly active (shown in red) in untreated glands in panel (c), but was substantially diminished in treated glands of panel (f) 3 days after cooling. Panels (g)-(i) depict the reduction of lipid content in sebaceous glands by cooling. Representative z-projection images from 4 different animals are shown. In panels (g) and (h), keratin 5 (red), lipids (green) and nuclei (blue) were labeled by whole-mount staining. In panel (g), untreated sebaceous glands (annotated with white arrows) show expected morphology, namely, basal epithelium expressing keratin 5 and a lipid-filled gland interior. In panel (h), keratin 5 expression was retained in the treated glands 3 days after cooling, but lipid content was substantially reduced, and in some cases almost completely abolished (as annotated with the white arrow). Panel (i) provides high-power views of various protein markers in sebaceous glands labeled by whole mount staining, shown in red. Lipids are shown in green, and nuclei in blue. Cooling did not disrupt the expression of any of these markers, but lipid content was diminished. The scale bars in panels (a)-(f) and (i) represent 25 m. The scale bars in panels (g) and (h) represent 100 m.

[0051] FIG. 11 depicts the susceptibility of porcine sebaceous glands cryo-injury through histology of the swine ear. Panel (A) is a control section showing normal sebaceous glands. Panel (B) provides a representative slide from the treatment area 1 week post-treatment, showing both diminished gland density and size. Panel (C) is a representative slide from the treatment area 2 weeks post-treatment showing restoration in gland number but diminished gland size when compared to baseline.

[0052] FIG. 12 depicts the reduction of surface area and density of porcine sebaceous glands after cooling. Sebaceous glands on digitized whole slides were manually counted. Gland density was decreased at one week post treatment, when compared to control, and gradually increased at two weeks and four weeks post treatment.

[0053] FIG. 13 depicts a cooling curve of representative murine procedure. Arrows represent the start and end of contact time.

[0054] FIGS. 14A-14C depict microneedle-based cooling devices 1400 according to embodiments of the invention.

[0055] FIGS. 15A and 15B depicts the disruption of cellular architecture in human sebaceous glands. FIG. 15A depicts a sebaceous gland from an untreated area, showing normal sebaceous gland architecture including lipid-laden sebocytes filling the gland interior and sebocytes nearest the gland duct containing increased lipid content and pyknotic nuclei and about to undergo cell lysis as part of the holocrine secretion process. In FIG. 15B, degenerative changes are seen in the sebaceous glands 3 days after cooling at 10 C. for 20 minutes, including loss of intracellular lipid granules, pyknotic nuclei throughout the gland body, and the presence of an eosinophilic precipitate. The scale bars in both figures represent 50 m.

[0056] FIG. 16 depicts the suppression of sebum output in humans after cooling. Sebum output was measured at various time points using a SEBUMETER system. Data from the 15 C. and 10 C. groups were combined because there was no statistical difference between the two groups. Following cooling treatment of either one 20-minute cycle (Treatment A) or two 10-minute cycles (Treatment B), sebum output was significantly lower than baseline at 1 (p<0.002) and 2 (p<0.0005) weeks post-treatment. In contrast, there was no significant difference in sebum output from untreated sites (Control) across the different time points. Error bars denote 95% confidence intervals. An asterisk (*) denotes a significant difference between post-treatment and baseline sebum output.

[0057] FIG. 17 provides clinical photos from a representative subject taken before and up to 4 weeks after cooling treatment. This particular subject was randomized to the 15 C. treatment. The left lateral site was the untreated control, the left paraspinal site was treated with two separate 10-minute cycles, and the right paraspinal site was treated with one continuous 20-minute cooling cycle. Panel (a) is a photograph taken before the cooling treatment. Panel (b) consists of two photographs taken immediately after the cooling treatment for the left and right paraspinal sites, respectively. Panels (c), (d), (e), and (f) are photographs taken at 3 days, 1 week, 2 weeks, and 4 weeks, respectively, after the cooling treatment.

[0058] FIGS. 18A-18C depict various fractional cooling configurations according to embodiments of the invention.

[0059] FIG. 19 depicts a one-dimensional heat transfer model utilized for modeling temperatures produced by cooling a subject's skin.

[0060] FIGS. 20, 21, and 22 depicts a mathematical model of temperature profiles in tissue in response to cooling with a cooling element at 10 C., 20 C., and 30 C., respectively. Temperature ( C.) is plotted on the x axis and tissue depth (in mm) is plotted on the y axis. The different curves (colored in the original application) represent temperature profiles at different times in 10 second increments from time 0 on the left to 60 seconds on the right. The dashed arrow pointing up (red in the application as originally filed) at a depth of 4 mm represents 20% of the typical subcutaneous fat thickness. The dashed arrow pointing down (green in the application as originally filed) at a depth of 2 mm represents the typical boundary between the skin and the subcutaneous fat.

[0061] FIGS. 23A and 23B depict cross-sections of cooling elements having flat and corrugated cooling surfaces, respectively.

DEFINITIONS

[0062] The instant invention is most clearly understood with reference to the following definitions.

[0063] As used in the specification and claims, the singular form a, an, and the include plural references unless the context clearly dictates otherwise.

[0064] Unless specifically stated or obvious from context, as used herein, the term about is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about.

[0065] As used in the specification and claims, the terms comprises, comprising, containing, having, and the like can have the meaning ascribed to them in U.S. patent law and can mean includes, including, and the like.

[0066] Unless specifically stated or obvious from context, as used herein, the term or is understood to be inclusive.

[0067] The term permanent (and related words such as permanently) refers to changes in a subject's epidermis that will not return to a previous state over time. Examples of permanent injury to epidermal tissue include scarring and ulceration. Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 (as well as fractions thereof unless the context clearly dictates otherwise).

[0068] The term reversible (and related words such as reversibly) refers to changes in a subject's epidermis that will return to a previous state over time. In some embodiments, such reversible changes will return their original state without the need for any medical intervention. Suitable periods of time over which the reversal can occur include less than 1 hour, less than 1 day, less than 1 week, less than 1 month, and the like.

[0069] The term sebaceous gland disorder is intended to include any condition caused by, exacerbated by, or associated with abnormally high sebum production by sebaceous glands within a local region. Examples of such sebaceous gland disorders include, but are not limited to, acne vulgaris, nevus sebaceous, sebaceous gland hyperplasia, and acne rosacea.

[0070] A subject shall be understood to include any mammal including, but not limited to, humans. The term subject can include all subsets of individuals within a particular class of subjects, e.g., males, females, infants, children, adolescents, adults, male adolescents, female adolescents, male adults, female adults, and the like.

DETAILED DESCRIPTION

[0071] Aspects of the invention provide methods, kits, and cooling devices for at least temporarily disrupting function of one or more sebaceous glands.

[0072] Sebaceous glands contain 30% lipid content including triglycerides, wax esters, and squalene. Human sebum reportedly freezes at 15 C. ex vivo. Aspects of this invention exploit these properties to selectively target and disrupt function of sebaceous glands. This disruption can be temporary (e.g., lasting for a period of hours, days, weeks, months, or years) or can be permanent.

Methods of at Least Temporarily Disrupting Sebaceous Glands

[0073] Referring now to FIG. 2, a method 200 of disrupting sebaceous glands is provided. The method 200 includes applying a cooling device to skin surface 115 of a local region of a subject (S202) and modulating operation of the cooling device or periodically removing the cooling device in order to cool one or more sebaceous glands 130 within the local region to a temperature sufficient to at least temporarily disrupt function of the one or more sebaceous glands 130 (S204).

[0074] Method 200 can utilize any cooling device capable of achieving a sufficient cooling of one or more sebaceous glands within a local region. Without being bound by theory, it is believed that freezing of one or more types of cells within the sebaceous glands 130 retard cell growth and/or cause apoptosis of one or more types of cells within the one or more sebaceous glands 130. Suitable temperatures are believed to be between about 15 C. and about 10 C., between about 10 C. and about 5 C., between about 5 C. and about 0 C., between about 0 C. and about 5 C., between about 5 C. and about 10 C., between about 10 C. and about 15 C., between about 15 C. and about 20 C., between about 20 C. and about 25 C., and between about 25 C. and about 30 C.

[0075] As discussed in further detail herein, suitable cooling devices can include topical cooling devices that be held against the epidermis of a local region. The cooling device can be held substantially stationary over a local region or can be translated over the subject's epidermis in order to affect sebaceous glands within a plurality of local regions and facilitate multiple cooling-warming cycles.

Cooling Devices

[0076] Referring now to FIG. 3, a cooling device 300 can include a cooling unit 302 and a control unit 304.

[0077] The cooling unit 302 can be any device capable of achieving a sufficient cooling of one or more sebaceous glands within a local region. Examples of suitable cooling units 302 include thermoelectric (Peltier) coolers, adiabatic cooling devices, fluid-cooled units that communicate with an external heat exchanger, and cryogenic devices that utilize cooled gases such as nitrogen or carbon dioxide to produce the desired low temperatures. Examples of such devices are provided in U.S. Patent Application Publication Nos. 2005/0251120 and 2007/0010861 and in Pascal Laugier et al., In Vivo Results with a New Device for Ultrasonic Monitoring of Pig Skin Cryosurgery: The Echographic Cryoprobe, 111(2) J. Inves. Derm. 314-19 (1998). In some embodiments, the cooling unit 302 can include one or more nozzles to facilitate a controlled spray of compressed fluid onto the subject's skin. The safety of such a structure can be enhanced by inclusion of physical and/or control structures adapted and configured to ensure that the cooling unit 302 is an appropriate distance from the subject's skin in order to prevent aerosol burns and/or inappropriate discharge of the compressed fluid.

[0078] In some embodiments, the cooling unit 302 contains a material that undergoes a phase change (e.g., solid to liquid, solid to gas, liquid to gas, and the like) at a temperature at or near a desired cooling temperature. For example, Sonoco ThermoSafe of Arlington Heights, Ill. produces gels having a phase transition at a specified temperature point (e.g., at 23 C. for Model #418).

[0079] In some embodiments, the cooling unit 302 can be pre-cooled to a temperature below a desired epidermal temperature. For example, if the cooling unit 302 is cooled to 10 C. lower than the desired dwell temperature of the epidermis, a faster initial cooling rate can be achieved without permanently and/or reversibly harming the epidermis because the cooling unit 302 temperature will quickly increase upon contacting the skin.

[0080] Cooling unit(s) 302 can have a variety of geometries as discussed in U.S. Patent Application Publication Nos. 2005/0251120 and 2007/0010861. In some embodiments, the cooling unit has a contact or cooling surface that is substantially flat, convex, or concave. In other embodiments, the contact or cooling surface has a patterned, dimpled, or corrugated surface to increase contact area and heat transfer with the subject's skin. Cross-sectional views of cooling elements having flat and corrugated cooling surfaces, respectively, are depicted in FIGS. 23A and 23B. In both FIGS. 23A and 23B, a thermoelectric heat pump (e.g., a Peltier element) is used to remove heat from the cooling surface in contact with the skin and optionally output heat to cooling fluid on the opposite side of the thermoelectric heat pump (which can optionally be circulated to an additional thermoelectric heat pump, cooling device, heat sink, or heat exchanger for cooling and recirculation.

[0081] Additionally or alternatively, the cooling unit(s) 302 can be fractional cooling devices having a plurality of posts or other geometries that contact the epidermis to cause a plurality of cooled regions. Fractional cooling devices are described in U.S. Patent Application Publication Nos. 2010/0036295, 2011/0313411, and 2013/0066237 and International Publication No. WO 2013/075006. A two-dimensional array of fractional cooling elements can simultaneously heat and cool adjacent regions and can optionally oscillate between heating and cooling of particularly posts as depicted in FIGS. 18A-18C, utilize translation of the cooling unit(s) 302 across the epidermis, or remain in a fixed location.

[0082] Cooling units 302 of the present invention can contain cooling agents in the form of a solid, liquid or gas. Solid cooling agents can comprise, for example thermal conductive materials, such as metals, metal plates, glasses, gels and ice or ice slurries. Liquid cooling agents can comprise, for example, saline, glycerol, alcohol, water/alcohol mixtures, liquid nitrogen, and the like. Where the cooling element includes a circulating cooling agent, preferably the temperature of the cooling agent is constant. Salts can be combined with liquid mixtures to obtain desired temperatures. Gasses can include, for example, cold air, compressed air, and the like.

[0083] Control unit 304 can be an electronic device programmed to control the operation of the cooling unit 302 to achieve a desired result. The control unit 304 can be programmed to autonomously carry out a cooling regimen without the need for input (either from feedback devices or medical professionals) or can incorporate such inputs. The principles of how to use feedback (e.g., from a temperature sensor) in order to modulate operation of a component are described, for example, in Karl Johan strm & Richard M. Murray, Feedback Systems: An Introduction for Scientists & Engineers (2008).

[0084] Control unit 304 can be a computing device such as a microcontroller (e.g., available under the ARDUINO OR IOIO trademarks), general purpose computer (e.g., a personal computer or PC), workstation, mainframe computer system, and so forth. Control unit 304 can include a processor device (e.g., a central processing unit or CPU) 306, a memory device 308, a storage device 310, a user interface 312, a system bus 314, and a communication interface 316.

[0085] Processor 306 can be any type of processing device for carrying out instructions, processing data, and so forth.

[0086] Memory device 308 can be any type of memory device including any one or more of random access memory (RAM), read-only memory (ROM), Flash memory, Electrically Erasable Programmable Read Only Memory (EEPROM), and so forth.

[0087] Storage device 310 can be any data storage device for reading/writing from/to any removable and/or integrated optical, magnetic, and/or optical-magneto storage medium, and the like (e.g., a hard disk, a compact disc-read-only memory CD-ROM, CD-ReWritable CD-RW, Digital Versatile Disc-ROM DVD-ROM, DVD-RW, and so forth). Storage device 310 can also include a controller/interface for connecting to system bus 314. Thus, memory device 308 and storage device 310 are suitable for storing data as well as instructions for programmed processes for execution on processor 306.

[0088] User interface 312 can include a touch screen, control panel, keyboard, keypad, display or any other type of interface, which can be connected to system bus 314 through a corresponding input/output device interface/adapter.

[0089] Communication interface 316 can be adapted and configured to communicate with any type of external device, including cooling unit 302. Communication interface 316 can further be adapted and configured to communicate with any system or network, such as one or more computing devices on a local area network (LAN), wide area network (WAN), the Internet, and so forth. Communication interface 316 can be connected directly to system bus 314 or can be connected through a suitable interface.

[0090] Control unit 304 can, thus, provide for executing processes, by itself and/or in cooperation with one or more additional devices, that can include algorithms for controlling cooling unit 302 in accordance with the present invention. Control unit 304 can be programmed or instructed to perform these processes according to any communication protocol and/or programming language on any platform. Thus, the processes can be embodied in data as well as instructions stored in memory device 308 and/or storage device 310 or received at user interface 312 and/or communication interface 316 for execution on processor 306.

[0091] Control unit 304 can control the operation of the cooling unit 302 in a variety of ways. For example, control unit 304 can modulate the level of electricity or cooling liquid provided to the cooling unit 302. Alternatively, the control unit 304 can transmit instructions and/or parameters to the cooling unit 302 for implementation by the cooling unit 302.

[0092] In addition or as an alternative to modulating the temperature of the cooling unit 302, the control unit 304 can initiate other mechanisms to prevent thermal damage to non-targeted structures. For example, the control unit 304 can initiate an alarm or alert that the cooling unit 302 should be moved to a new location or removed from the subject's skin. In another example, the control unit 304 can control a mechanical, electrical, or electromechanical device to either withdraw a cooling element within a housing of the cooling unit 302 or deploy a mechanical device to lift the cooling unit away from the subject's skin.

[0093] Control unit 304 can interface with one or more feedback devices 318 in order to monitor the temperature of one or more components of the subject's skin. A variety of suitable feedback devices are described in U.S. Patent Application Publication Nos. 2005/0251120 and 2007/0010861.

[0094] Cooling device 300 can also include one or more energy sources 522. Energy sources 522 can be used to heat particular regions of the subject's skin during operation of the cooling unit 202 and/or to warm one or more sebaceous glands in between cooling cycles.

[0095] Suitable energy sources 522 includes radiofrequency (RF) energy generating units that can be adapted, configured, and/or programmed to generate monopolar, bipolar, capacitively-coupled, and/or conductively-coupled RF energy. The RF energy can have a frequency between about 0.3 MHz and about 100 MHz.

[0096] Other suitable energy sources 522 include coherent light sources, incoherent light sources, heated fluid sources, resistive (Ohmic) heaters, microwave generators (e.g., producing frequencies between about 915 MHz and about 2.45 GHz), and ultrasound generators (e.g., producing frequencies between about 300 KHZ and about 3 GHz).

Kits and Consumable Products

[0097] Referring now to FIG. 4, cooling device 300 can be provided as part of a larger kit 400 in order to achieve the desired cooling of sebaceous glands 130.

[0098] Kit 400 can include one or more consumable products such as an intermediary material 402. Intermediary material 402 can advantageously enhance thermal conductivity between the cooling device 300 and the local region. Additionally or alternatively, intermediary material 402 can be a cryoprotectant that protects the epidermis from permanent and/or reversible damage while the sebaceous glands 130 are cooled.

[0099] Intermediary material 402 can be a liquid or a gel. Suitable intermediary materials 402 include water, heavy water, oil, peanut oil, glycerol, glycol, polypropylene glycol (PPG), polyethylene glycol (PEG), propylene glycol, ethylene glycol, dimethyl sulfoxide (DMSO), alcohol, ethanol, propanol, iso-propanol, carboxyl polyethylene polymer, hydroxyethyl xylose polymer, carboxyl methylcellulose, hydroxyethyl cellulose (HEC), the like, and combinations thereof.

[0100] Suitable intermediary materials 402 are available from ZELTIQ Aesthetics, Inc. of Pleasanton, Calif. and described in U.S. Patent Application Publication Nos. 2007/255362, 2010/0280582, and 2011/0300079 and U.S. Pat. No. 6,041,787.

[0101] Intermediary materials 402 can be provided in a variety of formats. Intermediary material 402 can be provided in a dispenser (e.g., a squeeze bottle) for direct application to the subject's epidermis prior to application of the cooling device 300. In another embodiment, intermediary material 302 is impregnated within a pad, textile, foam, sponge, or other porous surface that can be placed on the subject's epidermis prior to application of the cooling device 300. In still another embodiment, the intermediary material 402 is hermetically sealed in a pouch as described for example, in U.S. Patent Application Publication No. 2007/0270925.

[0102] System 400 can also include one or more tokens 404 that can be enable licensing and monetization of the systems, methods, and kits on a per use basis. Exemplary tokens 404 are described in U.S. Patent Application Publication No. 2010/0280582.

Dual-Modality Devices

[0103] Referring now to FIG. 5, some embodiments of the invention utilize one or more energy sources 522 in order to introduce energy to one or more locations and/or layers of the local region. Such energy can protect particular locations and/or layers from cold-induced damage while one or more sebaceous glands are cooled to a desired temperature.

[0104] The device 500 depicted in FIG. 5 includes a plurality of energy sources 522a-522d that are adapted, configured, and/or programmed to direct energy to a particular location at a particular depth within the local region. For example, energy sources 522a-522d can produce a coherent energy beam such as a laser beam. Energy sources 522 can be positioned in order to achieve a desired angle of the beam with respect to a normal to the skin. As seen in FIG. 4, larger angles with respect to normal such as in the context of energy sources 522a, 522b will generally focus the energy on a shallower layer of the local region such as the epidermis 115. Smaller angles with respect to normal such as in the context of energy sources 522c, 522d will generally focus the energy on deeper layers of the local region such as subcutaneous adipose tissue 145.

[0105] In some embodiments, energy sources 522 are positioned with a sufficiently high resolution such that a subset of energy sources 522 can be selectively modulated depending on their location relative to a sebaceous gland 130. For example, the local region can be imaged to identify the location of hair follicles 110, adjacent to which sebaceous glands will be present. Energy sources 522 proximate to the hair follicle 110 can be deactivated or modulated to emit less energy relative to energy sources distal to the hair follicle 110 so that the energy sources 522 proximate to the sebaceous gland 130 do not interfere with cooling.

[0106] Cooling unit 502 and/or cooling interface 524 can include a plurality of openings 526 in order to permit energy from energy sources 522 to exit device 500 and reach the local region. In some embodiments, cooling unit 502 and/or cooling interface 504 is made from a material that is substantially transparent with respect to the wavelength of energy applied, thereby rendering holes in cooling unit 502 and/or cooling interface 524 unnecessary.

Cooling Profiles

[0107] Control unit 304 can be programmed to implement various cooling algorithms in order to selectively disrupt sebaceous gland function.

[0108] Various cooling profiles are depicted in FIGS. 6A-6D, wherein cooling intensity is shown on the y axis and time is shown on the x axis.

[0109] The rates of cooling and rewarming can be controlled and/or modulated in order to achieve desired cooling of the sebaceous glands. For example, a defined number of joules per unit of area or volume per unit of time can be extracted.

[0110] Without being bound by theory, it is believed that aggressive cooling regimes achieve are most disruptive to sebaceous glands 130. For this reason, it may be preferred to apply a pre-cooled cooling unit (e.g., at a temperature of about 7 C., about 8 C., about 9 C., about 10 C., and the like) to the skin.

[0111] Using another example, control unit 304 can actuate cooling unit 302 to cool the local region until the sebaceous glands within the local region reach a desired temperature (e.g., about 7 C. or lower). Once this temperature is achieved, the control unit 304 can reduce the cooling power of the cooling unit 302 in order to hold the sebaceous glands at a substantially steady temperature (e.g., about 7 C. or lower). Alternatively, control unit 304, can utilize a more discrete method of control in which cooling unit 302 is repeatedly cycled between on and off in order to maintain the substantially steady temperature (e.g., about 7 C. or lower).

[0112] As discussed above, control unit 304 can receive inputs from one or more feedback devices 318. For example, control unit 304 can control the operation of the cooling unit 302 based on temperature sensors in the cooling unit 302, on a surface of the cooling unit 302, in a treatment interface 524, on a surface of a treatment interface 524, on the surface of a subject's skin 115, or within the subject's skin.

[0113] In one embodiment, the control unit 304 can be programmed to monitor for increases in temperature and/or increases in cooling activity by cooling unit 302 (which may be manifested in increased power or coolant draw). Such increases in temperature can be associated, for example, with the latent heat of crystallization one or more portions of the skin freezes as discussed further in U.S. Patent Application Publication No. 2009/0149929. Depending on the degree of the temperature increase and the desirability or undesirability of freezing a particular portion of the skin, cooling can be maintained, increased, decreased, or suspended upon detecting a temperature increase.

[0114] In still another embodiment, freeze events can be detected by monitoring for changes in optical properties (e.g., scattering, reflection, transmission, coherence, color, and the like) using one or more optical probes (e.g., an optical coherence tomographic device, an optical spectropic device, an IR-reflectoscopic device, a diffuse optical tomographic device, and the like), one or more electrical properties (e.g., resistance, impedance, and the like), and/or one or more acoustic properties (e.g., acoustic velocity, ultrasound acoustic velocity, impedance, and the like) using one or more acoustic probes (e.g., an ultrasound probe, an echographic cryoprobe, and the like) as described in U.S. Patent Application Publication Nos. 2005/0251120 and 2007/0010861.

Microneedle-Based Cooling

[0115] Referring now to FIG. 14A, another aspect of the invention provides a cooling device 1400 including a plurality of microneedles 1402 adapted to penetrate the epidermis 115. Microneedles 1402 can have a working length L adapted to reach the depth of sebaceous glands 130 (typically about 1 mm deep).

[0116] Microneedles 1402 can be adapted and configured to apply cooling in the immediate vicinity of the needle, and preferably in the immediate vicinity of the tip of the needle. Various architectures to promote cooling proximate to the microneedle tip are depicted in FIGS. 14B and 14C.

[0117] In FIG. 14B, microneedle 1402b includes internal channels 1404 that allow for the recirculating flow of a cooling fluid within the microneedle.

[0118] In FIG. 14C, microneedle 1402c has a solid body 1406 with a beveled tip 1408. An insulating layer 1410 surrounds at least a portion of the microneedle shaft in order the minimize cooling of the epidermis. Insulating layer 1410 can optionally extend onto cooling block 1412 in order to minimize cooling of epidermis 115 when microneedles 1402 are fully inserted. Suitable materials for insulating layer 1410 include polymers that are biocompatible, have low thermal conductivity, and/or have low coefficients of friction. Microneedles 1402 preferably have high thermal conductivity in order to quickly cool the layer of the skin containing the sebaceous gland 130.

[0119] Microneedles 1402 can be cooled using any cooling means described herein including thermoelectric coolers, cooling liquids, and the like.

[0120] Microneedles 1402 can be advanced through the epidermis by pressing cooling device 1400 against the skin. Alternatively, microneedles 1402 can be mechanically, electrically, magnetically, or fluidically extended and retracted, for example, by operation of control unit.

[0121] Microneedles 1402 can have a variety of dimensions. For example, microneedles 1402 can have a maximum cross-section of about 160 m, about 245 m, about 465 m, and the like.

[0122] Suitable microneedles are described in U.S. Patent Application Publication Nos. 2008/0200910, 2012/0265278, 2013/0184696, 2013/0190745, and 2013/0324990.

Protection of Epidermal Tissue and/or Subcutaneous Adipose Tissue

[0123] In some embodiments, the methods and devices can be controlled in order to produce the desired cooling of one or more sebaceous glands without permanently and/or reversibly injuring epidermal tissue or subcutaneous adipose tissue. The absence of permanent and/or reversible injury to epidermal tissue or subcutaneous adipose tissue can be assessed or verified through a variety of approaches prior to, during, or after development of a particular, method, device, or kit. For example, the absence of epidermal damage can be assessed by visual examination of the epidermis for blistering 48 hours after treatment. The absence of injury to the subcutaneous adipose tissue can be assessed visually by detecting the presence or absence of indentation or contouring seen in U.S. Pat. No. 8,840,608 when cryolipolysis is performed, biopsy, ultrasound imaging, and the like. The absence of injury to subcutaneous adipose tissue can also be assessed quantitatively, for example, by assessing whether a certain percentage of adipose tissue (either numerically, dimensionally, or volumetrically) directly below a cooled region was damaged after a period of time (e.g., one month). For example, if the subcutaneous adipose tissue below the application site is reduced (e.g., in adipose cell count, thickness, or volume) by less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% one month after treatment, the procedure can be deemed to have not injured the subcutaneous adipose tissue.

[0124] In some embodiments, optimal time and temperature combinations can be augmented as needed to avoid injury to the subcutaneous adipose tissue, for example, by employing periods of passive rewarming, active rewarming, and/or control of cooling rates.

Thermal Modeling

[0125] Temperature profiles in skin, subcutaneous fat, and muscle at the skin surface, were estimated using a 1-dimensional finite element model. Changes in temperature in response to cooling at the skin surface to various target temperatures and durations were simulated, with the goal of delivering sufficient cooling (at or below 0 C.) in the skin (particularly the sebaceous glands), while maintaining the subcutaneous fat at a temperature above 25 C. Other thresholds such as maintaining subcutaneous fat above about 20 C., about 15 C., about 10 C., and about 5 C. could be utilized as well. Additionally, this modeling assumes for the sake of simplicity that temperature is the only determinant of sebaceous gland disruption, disregarding other factors such as cooling/thawing rates and cooling duration.

[0126] Tissue temperature profiles were estimated using a 1-dimensional, time-dependent, finite element model of bioheat transfer, computations were performed using COMSOL MULTIPHYSICS software (Version 5.0, Comsol AB, Stockholm, Sweden).

[0127] The model tissue consisted of four different layers (epidermis, dermis, subcutaneous fat, and muscle), each with the following characteristics specified in Table 1 below.

TABLE-US-00001 TABLE 1 Thermal Specific Thickness Density conductivity heat Blood perfusion Epidermis 0.1 mm 1200 kg/m.sup.3 0.21 W/m/K 3600 J/kg/K 0 kg/m.sup.3/s/.sub.blood Dermis 1.9 mm 1300 kg/m.sup.3 0.53 W/m/K 3800 J/kg/K 2 kg/m.sup.3/s/.sub.blood Subcutaneous fat 10 mm 850 kg/m.sup.3 0.16 W/m/K 2300 J/kg/K 0.6 kg/m.sup.3/s/.sub.blood Muscle 10 mm 1270 kg/m.sup.3 0.53 W/m/K 3800 J/kg/K 0.5 kg/m.sup.3/s/.sub.blood
In addition, the following global parameters applied.

TABLE-US-00002 TABLE 2 Initial temperature for all layers 37 C. Specific heat of blood 4180 J/kg/K Density of blood 1000 kg/m.sup.3 Metabolic heat 400 W/m.sup.3

[0128] This was a one-dimensional model, i.e., the only concern was with heat transfer in one directiondepthas depicted in FIG. 19. Heat transfer in the lateral directions was assumed to be negligible. In addition, the cold plate was assumed to be an infinite heat sink operating at constant temperature.

[0129] These simulations suggest that cooling with lower temperatures for short periods of time is effective in reducing skin temperatures to sufficient levels to selectively disrupt sebaceous glands, while keeping subcutaneous fat above 25 C.

[0130] Referring to FIGS. 20 and 21, when cooling elements at 10 C. or 20 C., respectively, are applied to the skin surface for 10 seconds, the subcutaneous fat (typically beginning 2 mm below the skin surface) can be maintained at or above 25 C. (and the subcutaneous fat can be maintained above 5 C. during 10-, 20-, 30-, and 40-second applications of a 10 C. cooling element and 10- and 20-second applications of a 20 C. cooling element).

[0131] Referring to FIG. 22, when a cooling element at 30 C. is applied to the skin surface, the subcutaneous fat can be maintained above 5 C. during 10- and 20-second application).

[0132] The modeling results indicate that lower cooling temperatures can be combined with short (e.g., less than or equal to 10, 15, or 20 seconds) cooling durations to achieve cooling to target temperature within the skin, while maintaining subcutaneous fat above desired temperatures. If longer total cooling durations and/or repeat freeze-thaw cycles are desired, then a treatment combining short bursts of cooling, each followed by periods of rewarming (e.g., about 2 or 3 times the cooling time) could be used to minimize damage to subcutaneous fat. Additionally, dual-modality devices can provide additional protection to the subcutaneous fat.

[0133] In general, sharper temperature profiles are obtained with lower surface cooling temperatures, such that lower temperatures could be achieved within the skin while fat tissue remains relatively warm. Topical cryoprotectants (e.g., glycerol, propylene glycol, and other cryoprotectants described herein) could be applied to at least partially reduce non-specific damage during more aggressive cooling regimens.

Working Examples

Materials and Methods

Animals

[0134] Female C57BL/6J mice (16 weeks) were obtained from Jackson Lab (Bar Harbor, Me.). Female Yorkshire (40 kg) and Yucatan pigs (30 kg) were obtained from Tufts University School of Veterinary Medicine (North Grafton, Mass.) and Sinclair BioResources (Windham, Me.), respectively. All animal procedures were performed in compliance with the Public Health Service Policy on Humane Care and Use of Laboratory Animals, and approved by the Massachusetts General Hospital Institutional Animal Care and Use Committee.

Murine Cooling Experiments

[0135] Mice were anesthetized with ketamine-xylazine (90, 9 mg/kg, IP). After anesthesia was obtained, the ear was placed in contact with a cooling plate and held in place with a foam block and a 100-gram weight. The contralateral ear was left untreated for control. A schematic of the cooling equipment and experimental set-up is shown in FIG. 7. Various target temperatures, duration, rates of cooling and re-warming were tested as discussed below. At various time points following the cooling procedure, animals were euthanized and the ear was harvested for histologic analysis. At least 3 independent repeats were performed for each experimental condition.

Porcine Cooling Experiments

[0136] Yorkshire and Yucatan swine were anesthetized with TELAZOL (tiletamine/zolazepam), xylazine, and atropine (4.4, 2.2, 0.04 mg/kg, IM), followed by maintenance with inhaled isoflurane (1-3%). A thermoelectric cooler measuring 3 cm3 cm was placed directly on the ear ridges either with or without the use of a gel pad saturated with propylene glycol (EZ PAD, Zeltiq Aesthetics, Inc., Pleasanton, Calif.). Ear ridges were chosen for this experiment because they contain a high density of sebaceous glands. The cooling plate was brought to the target temperature and placed onto the ear tissue for 10 minutes. Target temperatures ranged from 5 C. to 15 C. A needle thermocouple (Mini-Hypodermic Thermocouple Probe, Model Hyp-0, Omega Engineering, Stamford, Conn.) was implanted intradermally at a depth of approximately 0.1 mm to monitor tissue temperature. Following cooling, the borders of the treatment area were tattooed. The contralateral ear was left untreated for control. Animals were euthanized at various time points (1 week, 2 weeks, and 4 weeks) and tissue was harvested and processed as described below.

Clinical Trial

[0137] A prospective, site randomized trial was approved by the Institutional Review Board of the Massachusetts General Hospital. Eleven male volunteers between the ages of 18-25 with healthy skin and measurable sebum production were recruited for the study. Subjects with a history of vitiligo, keloid formation, and cold sensitive disorders were excluded from the trial.

[0138] Two 22.25 inch rectangles were marked on either side of the scapula and designated as the treatment sites. Subjects were randomized to one of two treatment temperatures (10 C. and 15 C.). Treatment areas on either side of the spine were randomized to receive one 20-minute cycle, or two 10-minute cycles with rewarming up to 10 C. surface temperature in between. A site on the lateral scapula was chosen as an untreated control. The treatment sites were marked with surgical markers and photographed before and immediately after treatment, as well as in each subsequent post-treatment time point. Each subject was evaluated before, immediately after, and at 72 hours and 1, 2, and 4 weeks after treatment. At each evaluation, the treatment sites were photographed, sebum production was measured using a SEBUMETER system available from Courage+Khazaka electronic GmbH of Cologne, Germany following the manufacturer's instructions, and the subjects were questioned regarding symptoms that occurred at the treatment sites. Biopsies where collected 72 hours after treatment and processed for histology, as described in the Histological Processing section below.

Histological Processing

[0139] Tissue was harvested at given time points and fixed in 4% paraformaldehyde in PBS. The tissue was bisected longitudinally and embedded along the long axis. Paraffin-embedded tissues were sectioned in 5 m thickness and stained with hematoxylin and eosin (H&E).

Quantitative Analysis

[0140] For murine sebaceous gland counts, four step sections 100 microns apart from each ear were quantified. Porcine specimens were bisected, embedded cut side down, and step sections were collected 250 microns apart. H&E-stained tissue sections were scanned using a whole mount slide scanner (NANOZOOMER, Hamamatsu Photonics, Japan). The total number of sebaceous glands in each section was counted by a blinded observer and normalized by the surface length of the corresponding tissue section, and the cross-sectional area of individual sebaceous glands in each section was measured using NDPVIEWER software (Hamamatsu Photonics). Over 100 sebaceous glands per sample were evaluated for quantitative analysis.

[0141] Specific staining for propidium iodide, alkaline phosphatase, lipids, and immunohistochemical markers were performed following previously established protocols described in B. K. Handjinski et al., Alkaline phosphatase activity and localization during the murine hair cycle, 131 The British Journal of Dermatology 303-10 (1994); I. Martinez-Corral et al., In vivo imaging of lymphatic vessels in development, wound healing, inflammation, and tumor metastasis, 109 P.N.A.S. 6223-28 (2012); and A. Uemura et al., Recombinant angiopoietin-1 restores higher-order architecture of growing blood vessels in mice in the absence of mural cells, 110 J. Clin. Invest. 1619-28 (2002).

Statistical Analysis

[0142] In the murine model, repeated measures analysis of variance (ANOVA) was applied to account for multiple measurements per animal in comparing absolute gland number and percentage damaged glands at 72 hours and 1 week compared to control with the Wald test used to test for differences. In addition, a multivariate logistic regression analysis was utilized to evaluate the effects of temperature, rate, duration and cycles of cooling on selective damage of sebaceous glands as a binary outcome. Sebaceous gland size and density were analyzed using ANOVA with the F-test used to compare the effect of treated pig ears at 1, 2, and 4 weeks versus control ears. Statistical analysis was performed using IBM SPSS Statistics software (version 21.0, International Business Machines, Armonk, N.Y.). All p-values less than 0.05 were considered statistically significant.

[0143] For sebum output measurements in the clinical trial, a mixed-model repeated-measures ANOVA was used in order to account for the two treatment temperatures and the two different treatment durations with longitudinal assessments at 72 hours and 2 weeks. The three sebum measurements for each site per subject were incorporated as replicates in the model. Two-sided p<0.05 was considered statistically significant with Bonferroni adjustment as appropriate to minimize Type I errors due to multiple comparison. Statistical analysis was performed using IBM SPSS Statistics software. P-values less than 0.05 were considered statistically significant.

Propidium Iodide Staining

[0144] Propidium iodide staining was performed following a previously-established protocol discussed in A. Uemura, Recombinant angiopoietin-1 restores higher-order architecture of growing blood vessels in mice in the absence of mural cells, 110(11) J. Clin. Invest. 1619-28 (2002), with minor modifications. Propidium iodide is a membrane-impermeable dye useful for labeling cells with compromised membranes. Briefly, the mice were given intraperitoneal injections of 400 l of 1 mg/ml propidium iodide (PI, Sigma-Aldrich, St. Louis, Mo.) 2 hours after cooling treatment (7 C. for 10 minutes), then euthanized after another 2 hours. The ears were collected and immediately frozen in Tissue-Tek O.C.T. compound (Sakura Finetek USA, Torrance, Calif.), and stored at 80 C. until use. This procedure was repeated in three animals, with one ear on each animal treated with cooling, while the contralateral ear was left untreated for control. For visualization, 50 m sections of the frozen tissue were prepared using a cryotome, washed, stained with BODIPY 493/503 lipid dye (1:1000 dilution in phosphate buffered saline, Life Technologies, Grand Island, N.Y.), fixed in 4% formaldehyde for 10 minutes, mounted in PROLONG GOLD antifade reagent with DAPI (Life Technologies), and imaged with confocal microscopy.

Results

Murine Model

[0145] Histologically-selective damage to sebaceous glands, with minimal non-specific injury to surrounding tissues, was observed after exposing mouse ears to appropriate combinations of cooling parameters (Table 3). Each set of experimental conditions was repeated independently on at least 3 animals.

[0146] Temperature, cold exposure time, cooling rate, and rewarming rate influenced the outcome. Of the various parameters, one such combinationcooling at 7 C. for 10 minuteswas explored more extensively and is presented below.

[0147] In the days following the cooling procedure (7 C. for 10 minutes), sebaceous glands in the treated regions exhibited a progressive loss of cellular structures, including intracellular lipid granules, nuclei, and intercellular septa, concurrent with accumulation of an eosinophilic precipitate. These histologic signs of cellular damage were first identifiable 2 days after treatment and peaked at 3 days after treatment, presenting in over 60% of the glands. The glands gradually recovered their normal morphology 5-7 days after treatment procedure, and by one week, less than 10% of the glands remained damaged as depicted in FIG. 9A.

[0148] There was transient swelling in the ear after the cooling procedure, but otherwise no gross or histologic evidence of non-specific damage to surrounding skin or ear cartilage tissues were observed (FIG. 8).

[0149] The number of sebaceous glands with and without histological evidence for damage was assessed blindly in treated and control ears, in three independent experiments at 72 hours and 1 week after cooling. A gland was deemed as damaged when the aforementioned histologic signs of cellular damage were observed in two or more adjacent sebocytes. Because sebaceous glands secrete sebum via a holocrine mechanism, necrotic cells are normally seen within the glands. In the control specimens, less than 5% of the glands had histologic evidence of damage. At 72 hours post-cooling, an average of 60% of the glands had histologic evidence of damage (p<0.001 compared with controls). At one week, less than 10% of the glands (p<0.001) displayed histologic evidence of cellular damage (FIG. 9A).

[0150] Referring now to FIG. 9B, there was a small but significant reduction in the absolute number of glands in the treated samples at 72 hours after cooling (p<0.001). This significant reduction in the number of glands present was sustained at the one week time point despite nearly complete recovery of the histological appearance of the remaining glands (p<0.001), suggesting a prolonged attenuation of gland number due to irreversible damage by cooling.

[0151] Referring now to FIG. 10, panels (a), (b), (d), and (e), propidium iodide (PI), a membrane-impermeable dye, was used to label cells with compromised membranes. PI staining was minimal in untreated controlsmost glands showed no staining, while a small number of glands had positive staining in the area immediately adjacent to the hair follicle, as depicted in panels (a) and (b), consistent with the normal degeneration of secreted sebocytes. In contrast, post-cooling glands showed extensive propidium iodide (PI) staining throughout the glands, co-localized with both the nuclei and lipids in the glands, as depicted in panels (d) and (e).

[0152] Referring now to FIG. 10, panels (c) and (f), alkaline phosphatase activity, normally prominent in sebaceous glands as depicted in panel (c), was substantially diminished as depicted in panel (f) at the time point corresponding to maximal histologic damage (3 days post-cooling). At the same 3-day time point, cooling did not disrupt the expression of key protein markers associated with sebaceous glands as depicted in panels (g)-(i), including keratins 5 and 15, Ki67, MUC1 (Mucin 1, Cell Surface Associated), MC5R (melanocortin receptor-5), and PPAR (Peroxisome Proliferator-Activated Receptor Gamma). However, lipid contents in the treated glands were substantially reduced, and in some cases completely abolished as depicted in panel (h).

Porcine Model

[0153] The porcine ear has gross and micro-scale anatomy that is similar to human sebaceous skin as discussed in F. H. Sakamoto et al., Porphyrin distribution after topical aminolevulinic acid in a novel porcine model of sebaceous skin, 41 Lasers Surg. Med. 154-60 (2009).

[0154] Similar to the murine model, sebaceous glands in the porcine ear model were selectively damaged by a controlled cycle of cooling. One week after cooling at the skin surface, sebaceous glands in the treated areas were markedly decreased in size, and in some treated sites were absent. When present, the sebaceous glands often exhibited loss of cellular structures and the accumulation of eosinophilic precipitates as depicted in FIG. 11, panel (B). At two weeks following treatment, the number of glands had largely returned to normal, however gland size was still decreased as depicted in FIG. 11, panel (C). Gland density, expressed as gland number per mm of skin surface length in cross-section, showed a significant decrease 1 week post-treatment (p<0.001). There was a gradual increase in gland density towards baseline at two weeks post treatment (p<0.005) as depicted in FIG. 12. Desquamation was occasionally observed about 3 days after cooling; otherwise, there were no gross or histological signs of non-specific tissue damage outside of the sebaceous glands.

Human Study

[0155] A pilot clinical trial was conducted in 11 healthy male volunteers with measurable sebum output. Fair-skinned subjects of Fitzpatrick skin phototypes I-III were enrolled, to limit the potential risk of cold-induced hypopigmentation, which was not observed in any subject. Subjects were randomized to one of two cooling device target temperatures (10 C. or 15 C.). Two treatment areas of their backs were cooled either for a single 20-minute cycle, or two 10-minute cycles with re-warming in between. Each subject also had one untreated area in the same anatomic region to serve as control. Biopsies taken 72 hours after treatment showed obvious yet selective damage in sebaceous glands, with necrosis and architectural distortion of lipid-laden sebocytes within the sebaceous gland as depicted in FIGS. 15A and 15B. No histologic evidence of non-selective tissue damage was observed. Similar to the murine results, expression of the proliferative marker ki67 and the progenitor basal cell marker keratin 15 was not disrupted by cooling, as determined by immunohistochemistry, which is consistent with the ability of sebaceous glands to eventually recover after treatment. Sebum production decreased significantly at 1 and 2 weeks after treatment, then recovered to baseline levels by week 4 as depicted in FIG. 16. There were no significant differences in any of the measured outcomes between the different treatment temperatures and cycles. The treatment was very well tolerated. There was localized, mild, transient erythema, edema, and dysesthesia after cooling, all of which resolved spontaneously within 72 hours as depicted in FIG. 17. During treatment the subjects reported an average score of 3 on a 10-point Visual Analog Scale for pain, which fell to less than 1 immediately post-treatment, and was 0 by 72 hours.

Discussion

[0156] This study showed that sebaceous glands can be preferentially damaged by controlled cooling at the skin surface, with minimal (in mice) to no (in humans) non-specific injury to surrounding tissues under the conditions studied. Cooling temporarily disrupted cellular architecture, membrane integrity, enzymatic activity, and reduced lipid content in sebaceous glands. In humans, cooling using a 10 C. or 15 C. device for 20 minutes resulted in an approximate 20% suppression of sebum output that persisted for over 2 weeks.

[0157] Temporary histologic damage of murine sebaceous glands with controlled cooling at a variety of temperature and cooling time combinations was achieved. In the porcine model, a transient although significant reduction in sebaceous gland density and surface area was observed for two weeks following cooling and is presented in Table 4.

[0158] The murine experiments illustrate the relative importance of the cooling parameters of temperature, rate of cooling, and duration of cold exposure. The most important factors to achieve consistent and selective damage to sebaceous glands were the rate of cooling, and the target temperature. In the murine model, there was a 2 C. temperature gradient across the ear, between the cooling plate and the non-contact side of the ear as measured by thermocouples (FIG. 7). The ear temperature and this temperature gradient were stable within 60 seconds of placing the cooling plate on the ear (FIG. 13).

[0159] Interestingly, the ear tissue temperature associated with preferential damage to sebaceous glands was far lower than would be predicted based only on the freezing point of human sebum. Without being bound by theory, it is possible that (i) both a water-freeze and a lipid-freeze contribute to the histologic endpoint and that the relatively low water content of sebocytes may predispose these and similar cells to lethal injury by combined lipid and water crystallization and/or (ii) the different physiologic properties of murine and human sebum, including molecular composition and lipid content, accounts for this difference. For example, human sebum has higher proportions of triglycerides, wax esters, and squalene, but lower proportion of cholesterol when compared to murine sebum.

[0160] Increased numbers of freeze-thaw cycles were more destructive, and resulted in non-specific injury to tissue. Slower thawing was also more destructive, and increased non-specific tissue injury.

[0161] The porcine ear has gross and micro-scale anatomy that is similar to human sebaceous skin. The mouse data was extrapolated to the porcine model, which revealed reduction in sebaceous gland density after controlled cooling. Using various cooling parameters in both the murine and porcine models, preferential and prompt injury of sebaceous glands was achieved. In mice, there was nearly complete recovery of normal sebaceous gland histology only one week after cooling at 7 C. for 10 minutes, but the number of glands was reduced. In swine, which have much larger and deeper sebaceous glands, the glands were smaller even after recovering their normal histological appearance.

[0162] The rapid onset of histologic damage following cooling has implications for potential mechanism. Controlled cryoinjury to adipocytes, which leads to intracellular lipid crystallization, induces a pro-apoptotic cascade that results in a delayed panniculitis and fat reduction two to three months following the procedure. The rapid formation of an eosinophilic precipitate within the sebaceous glands in the experiments is similar to what is seen in traditional cryosurgery. In vitro data supports the preferential temperature sensitivity of sebocytes and the induction of apoptosis after prolonged cold exposure.

[0163] There were few or no side effects and no scarring after controlled cooling that preferentially affected sebaceous glands. Side effects were limited to transient edema in the murine model. In the swine ears at 15 C., desquamation was observed at 72 hours followed by transient hypopigmentation at 1 week. This hypopigmentation resolved with perifollicular repigmentation of the treatment area. There were no gross or histological signs of permanent, non-specific tissue damage outside of the sebaceous glands.

[0164] The extensive PI staining that occurred in sebaceous glands shortly (within hours) after cooling treatment indicates that the integrity of the sebocytes' cell and nuclear membranes was compromised by cooling. PI staining was minimal in untreated controls and, when present, was restricted to the area immediately adjacent to the hair follicle, which corresponds to the location where mature sebocytes normally undergo cell death and release their lipid contents as part of the holocrine secretion mechanism. Sebaceous glands normally have a high level of endogenous alkaline phosphatase (ALP) activity. This activity was substantially diminished 3 days after cooling, indicating that the treatment had at least temporarily disrupted some enzymatic machinery of the sebaceous glands. Interestingly, ALP activity remained high in untreated glands, even after prolonged storage at 80 C., which suggests that the post-treatment reduction in ALP activity was not merely a consequence of exposure to low temperatures, but rather a cellular response to the cooling treatment.

[0165] Lipid content was also reduced in the treated glands, which is consistent the reduced post-treatment sebum output seen in the clinical trial in FIG. 16.

[0166] The co-localization of PI with sebum lipid is an unexpected finding, as PI is generally considered a nuclear stain, although cytoplasmic staining has been reported previously. This atypical staining pattern may be caused by the compromised cell membrane following cooling, which may have simply allowed PI to enter and accumulate within the intracellular lipid depots.

[0167] The pilot clinical trial showed the feasibility of selectively damaging human sebaceous glands with controlled cooling, at parameters that were safe and easily tolerated by all eleven subjects in the study. The reduced sebum excretion following a single application of cooling in healthy human subjects strongly suggests that the histologic damage observed correlates with decreased sebum output.

[0168] The severity of cooling-induced injury to sebaceous glands achieved in the study appears to be temporary. However, because the complete absence of sebum could potentially have undesirable effects on skin health, the temporary suppression of sebum output may be preferable to permanently destroying sebaceous glands. The sebum reduction in the human study lasted for at least 2 weeks following a single cooling treatment. These results suggest that a treatment interval of about 2 weeks may be able to provide prolonged suppression of sebaceous gland activity.

TABLE-US-00003 TABLE 3 Constant Variable Temperature 0 C. 5 C. 7 C. 10 C. Duration = + + SG 10 minutes + NS Maximal cooling rate Duration 0 minutes 5 minutes 10 minutes 15 minutes Temperature = + + SG 7 C. NS Maximal cooling rate Rate Cooling Rate Max 2 minutes Max 2 minutes Thawing Rate Ambient Ambient 2 minutes 2 minutes Temperature = + + SG 7 C. + NS Duration = 10 minutes Freeze-Thaw Cycles One 10 Two 10 Two 5 Five 2 minute minute minute minute cycle cycle cycle cycle Temperature = + + + + SG 7 C. + + + NS NS denotes non-specific injury. SG denotes sebaceous gland injury. Plus signs (+) denotes histologic signs of injury. Minus signs () denotes no sign of injury.

TABLE-US-00004 TABLE 4 Porcine Ear Measurements for Control and Treated Ears in Size, Surface Area, and Gland Density Time Cross-sectional area (mm.sup.2) Gland Density (number/mm) Control 0.073 0.082 0.79 0.32 1 Week 0.016 0.015* 0.35 0.24* 2 Weeks 0.039 0.041* 0.52 0.17.sup.# ANOVA F = 20.6.84, P < 0.001 F = 19.20, P < 0.001 Data are mean SD. Asterisks (*) denote P < 0.001 compared to control. Number signs (#) denote P = 0.003 compared to control.

EQUIVALENTS

[0169] The functions of several elements may, in alternative embodiments, be carried out by fewer elements, or a single element. Similarly, in some embodiments, any functional element may perform fewer, or different, operations than those described with respect to the illustrated embodiment. Also, functional elements (e.g., modules, databases, computers, and the like) shown as distinct for purposes of illustration can be incorporated within other functional elements, separated in different hardware, or distributed in a particular implementation.

[0170] While certain embodiments according to the invention have been described, the invention is not limited to just the described embodiments. Various changes and/or modifications can be made to any of the described embodiments without departing from the spirit or scope of the invention. Also, various combinations of elements, steps, features, and/or aspects of the described embodiments are possible and contemplated even if such combinations are not expressly identified herein.

INCORPORATION BY REFERENCE

[0171] The entire contents of all patents, published patent applications, and other references cited herein are hereby expressly incorporated herein in their entireties by reference.