WO2006119971A1

WO2006119971A1 - Methods for peeling and increasing turnover of skin with high-fluency, intense pulsed light

Info

Publication number: WO2006119971A1
Application number: PCT/EP2006/004339
Authority: WO
Inventors: Careen A. Schroeter
Original assignee: Individual
Current assignee: Individual
Priority date: 2005-05-09
Filing date: 2006-05-09
Publication date: 2006-11-16
Anticipated expiration: 2007-11-09
Also published as: EP1904176A1; WO2006119971A8

Abstract

The present invention relates to the use of a high- fluency, intense pulsed light source for inducing the expression of TGF-β in skin cells, resulting in the release of extracellular matrix proteins such as tenascin, and an increase in the concentration of fibroblasts and plasma cells. As the high-fluency, intense pulsed light source methodology disclosed herein produces an artificial wound which stimulates the immune system and extracellular matrix formation, the present invention relates to the use of a high-fluency intense pulsed light source alone, or in combination with laser or radiof requency, for dermatologic treatment and plastic surgery treatment.

Description

METHODS FOR PEELING AND INCREASING TURNOVER OF SKIN WITH HIGH-FLUENCY, INTENSE PULSED LIGHT Background of the Invention

Light therapy is an emerging field, wherein light emitting diodes and other emitters of electromagnetic radiation are used to treat various medical conditions such as acne, hair growth stimulation, hair growth inhibition, scar reduction and removal, wrinkle reduction, etc.

Dermabrasion, chemical peeling, and laser and flashlamp skin resurfacing have been established to attain their therapeutic effects by stimulating a wound-healing response that leads to an increase in dermal collagen (Tsai, et al . (1995) Dermatol. Surg. 21:539-4; Nelson, et al . (1995) J. Am. Acad. Dermatol. 32:472-8; Menaker, et al. (1999) Dermatol. Surg. 25:440-4; Fournier, et al . (2001) Dermatol. Surg. 27:799-806; Goldberg and Cutler (2000) Lasers Surg. Med. 26:196-200; Zelickson and Kist (2000) Lasers Surg. Med. 12:17) . For example, the nonablative laser is able to generate dermal temperature increase of 30-40⁰C while limiting thermal injury to the epidermis (Lask, et al. (1997) SPIE Proc . 2970:338-49). The dermal heat may eventually result in activation of fibroblasts to produce a long term healing response and subsequent collagen remodeling (Lask, et al . (1997) supra; Fitzpatrick, et al . (1996) Arch. Dermatol. 132:395-405). Shorter wavelength technologies have been hypothesized to induce a heat-mediated cytokine activation (Goldberg (2000) J. Cutan. Laser Ther. 2:59-61).

Further, U.S. Patent Application Serial No. 10/119,772 describes the manipulation of collagen, fibroblast, and fibroblast-derived cell levels in mammalian tissue using a plurality of pulses from at least one source of narrowband, multichromatic electromagnetic radiation (e.g., from a light emitting diode, a laser, a fluorescent light source, an organic light emitting diode, a light emitting polymer, a xenon arc lamp, a metal halide lamp, a filamentous light source, an intense pulsed light source, a sulfur lamp, and combinations thereof) having a dominant emissive wavelength of from about 300 nm to about 1600 nm, and wherein said pulses have a duration of from about 0.1 femtoseconds to about 100 seconds, the interpulse delay between said pulses is from about 0.1 to about 1000 milliseconds, and the energy fluence received by said tissue is less than about 10 joule per square centimeter.

Summary of the Invention The present invention is a method for increasing the proliferation or turnover of at least one layer of skin by exposing skin to a high-fluency, intense pulsed light source.

The present invention is also a method for stimulating sloughing of the stratum corneum of the epidermis by exposing skin to a high-fluency, intense pulsed light source .

Brief Description of the Drawings Figure 1 illustrates the relative expression of TN and TGF- β in the follicular bulb (Figure IA) , germinative epidermis (Figure IB) and in plasma cells (Figure 1C) up to approximately 4 months after treatment with an intense pulsed light source.

Detailed Description of the Invention

It has now been shown that a high-fluency, intense pulsed light source can induce the expression of TGF-β in skin cells, resulting in the release of extracellular matrix proteins such as tenascin, and an increase in the concentration of fibroblasts and plasma cells. Further, increased expression of Ki67 is observed in both dermal and epidermal layers, indicative of increased proliferation of these skin cells. As the high-fluency, intense pulsed light source methodology disclosed herein produces an artificial wound (i.e., not occurring by natural means such as by accident) thereby stimulating the immune system and extracellular matrix formation, the present invention relates to the use of a high-fluency intense pulsed light source for dermatologic treatment and plastic surgery treatment. Such dermatologic treatment includes increasing the turnover of at least one layer of human or mammalian skin and stimulating sloughing of the outer layer of the epidermis .

To demonstrate the utility of high-fluency intense pulsed light in dermatologic treatment, pigs were treated with 40 flashes (6.5-21 ms) of light having a wavelength ranging from 565 to 695 nm and a fluency of 35-60 J/cm². With higher fluency, erythema was observed. Using a wavelength of 640 nm, pulse time between 4.2 to 4.8 milliseconds (ms) and energy of 35 J/cm², negligible erythema was observed. While no blisters were identified, hair on the pigs was burnt and transient depigmentation was observed, but disappeared after some time.

Biopsy samples taken from the irradiated pigs were stained for the presence of TGF- βl and tenascin. One day after radiation, the appearance of TGF- βl in the basal epidermal layer and the follicular bulb was detected.

Fibroblasts and plasma cells were also strongly positive. After 1 week, the outermost epidermis, endothelial vessels, follicular bulb, fibroblasts and plasma cells were intensely stained (Figure 1) . TGF-βl immunoreactivity decreased after 2 weeks, reaching a level similar to that found on the first day of treatment. After one month, the expression of TGF-β reached a peak value and then began to slowly fall in the second and third month. Subsequently, TGF-βl levels began to increase and on day 120 elevated levels were observed.

Tenascin was visible below the epidermal germ layer and around the hair follicles. At about 7-10 days after radiation, the biopsies showed a strong increase in tenascin expression (Figure 1) . Tenascin was found to be localized in the papular dermis, the epidermal layer and also in the depths of the dermis, while in the endothelial vessels and the follicular bulb there was a sharp decrease. Biopsies showed a sharp decrease in tenascin immunoreactivity after one month, but this gradually recovered in the germinative epidermis, papular dermis and the follicular bulb in the second month, but not in the endothelial vessels and plasma cells, where it was absent. In the third month, tenascin levels gradually increased.

The Ki67 protein is expressed in all phases of the cell cycle except GO and serves as a marker for cell proliferation (Schluter, et al . (1993) J. Cell Biol. 123:513-522). Upon exposure to high-fluency, intense pulsed light, both dermal and epidermal layers were found to exhibit an increase in the expression of Ki67, indicative of an increase in cell proliferation.

In view of the role of TGF-β in cellular proliferation (Lee, et al . (1993) J. Burn Care Rehabil. 14:319-35; Mustoe, et al . (1997) Science 237:1333-6; Beck, et al . (1991) Growth Factors 5:295-304) and the instant findings which demonstrate that high-fluency, intense pulsed light increases TGF-β levels and Ki67 levels in the dermal and epidermal layers of skin of a subject, the present invention embraces the use of a high-fluency, intense pulsed light source in a method for increasing the proliferation and turnover of at least one layer of skin in a subject. In carrying out such a method, the skin of the subject is exposed to an effective amount of high-fluency, intense pulsed light so that TGF-β and Ki67 expression is increased thereby stimulating proliferation and turnover of skin cells. Depending on the wavelength of light, the proliferation and turnover of one or more layers of skin is contemplated. For example, the use of a dominant wavelength emission in the range of about 500 nm will be useful for increasing the proliferation and turnover of epidermal layers of skin, whereas light having a wavelength of about 600 nm to about 660 nm can be used for increasing the proliferation and turnover of epidermal and dermal layers of skin.

An effective amount of light, in the context of this method of the invention, is an amount which stimulates or increases the turnover of skin, e.g., from 28-30 days as found in a normal human subject to less than 28 days (e.g., 20-25 days) upon exposure to high-fluency, intense pulsed light. Suitable effective amounts or parameters associated with high-fluency, intense pulsed light which achieve the desired effect are disclosed herein and can vary with the desired layers to be treated.

The epidermis is composed of many layers including the horny layer (stratum corneum) , the clear layer (stratum lucidum) , granular layer (stratum granulosum) , prickle-cell layer (stratum spinosum) ; and basal layer (stratum basale) . In the basal layer of the epidermis, new cells are constantly being reproduced, pushing older cells to the surface. As skin cells move farther away from their source of nourishment, they flatten and shrink. They lose their nuclei, move out of the basal layer to the stratum corneum which is composed of about 15 to 20 cell layers, and turn into keratin. After serving a brief protective function, the keratinocytes are imperceptibly sloughed off. This process is called keratinization and takes about 4 weeks. By increasing proliferation and turnover of skin cells, high-fluency, intense pulsed light is also useful for accelerating the natural process of sloughing. Accordingly, the present invention is also a method for stimulating sloughing of the stratum corneum of the epidermal layer using an effective amount of high-fluency, intense pulsed light. An effective amount of said light is an amount which results in an increase in the amount or rate of sloughing of stratum corneum cells. For example, an adult human sheds approximately 10 grams of skin cells a day, and therefore an increase in the amount of skin cells sloughed per day would be indicative of high-fluency, intense pulsed light exposure stimulating skin cell sloughing.

As used herein, fluency is defined as the power level of the radiation reaching target tissue. In the context of the instant methods, a high-fluency radiation source produces about 3 to 90 joule per square centimeter (J/cm²) of tissue. In one embodiment of the present invention, high-fluency of about 10 to 50 J/cm² is employed. In particular embodiments, high-fluency of about 35 J/cm² is employed.

For purposes of the present invention, any device that emits intense pulsed light in a bandwidth of ± about 100 nanometers around a dominant wavelength can be can be used in accordance with the methods disclosed herein. Generally, any intense pulsed light source capable of exposing the target tissue with from about 3 to 90 J/cm² of energy in the desired wavelength (generally within the range of from about 435 nm to about 1900 nm) will be able to achieve the desired result. It is contemplated that the tissue penetration depth for intact skin may be different than the tissue penetration depth for ulcerated or burned skin and may also be different for skin that has been abraded or enzymatically peeled or that has had at least a portion of the stratum corneum removed. Thus, the wavelength used for any particular application can vary.

By way of illustration, light having a dominant wavelength emission in the range of about 500 nm may be more suitable for exposing epidermal layers of skin as such a short wavelength has limited depth of penetration, whereas light having a wavelength of about 600 nm to about 660 nm can more easily penetrate to a greater depth, if treatment of the lower dermal layers or even deeper is desired. Accordingly, the selection of the dominant wavelength of the radiation emitter is also dependent on the depth of treatment desired. Suitable wavelengths for inducing TGF-β are in the range of 590 to 690 nm. As a wavelength of 640 nm was found to minimize erythema, a dominant wavelength of 640 nm is particularly suitable for use in the methods of the instant invention. Exposure time is another aspect of intense pulsed light which can vary with the desired effect and the target cell, tissue or organ. In general, pulse lengths can vary from less than one picosecond to several seconds with interpulse intervals of a few picoseconds to a few hundred milliseconds. In particular embodiments, the pulse length is in the range of 0.1 to 500 milliseconds and the interpulse interval in the range of 20 to 60 milliseconds. To maximize skin turnover and sloughing, while minimizing erythema, pulse durations of from about 4.2 to 4.8 milliseconds with an interpulse interval of about 20 milliseconds are particularly suitable.

Similarly, the number of pulses (also referred to as repetitions or flashes) per treatment can also be varied. For example, large numbers of pulses may be less effective at inducing cell proliferation than a smaller numbers of pulses. In general, a treatment regime can include between 10 and 100 pulses. In particular embodiments, the number of pulses per treatment is in the range of 40. Further, a treatment can be repeated multiple times over a given period of time (e.g., weeks or months) to achieve the desired result.

Further, light penetration into the target tissue can be optimized by varying the treatment head of the intense

, pulsed light source. For example, a large spot size diminishes the scattering of the light beam. Treatment head size can vary from 8-10 mm x 20-45 mm. A particularly suitable treatment head is in the range of 8 mm x 34-35 mm. By changing the different parameters of the flashlamp, namely energy, wavelength, pulse duration etc., efficient treatment for a variety of diseases or conditions can be achieved with a high degree of selectivity and safety. In a particular embodiment of the present invention, a high- fluency, intense pulsed light source used in accordance with the instant methods has an emissive wavelength of from about 435 nm to about 1900 nm, wherein the pulses have a duration of from about 2 ms to about 30 ms, and the energy fluence received by the tissue is about 3 to 90 joule per square centimeter.

Suitable flashlamp systems which can be used for carrying out the methods disclosed herein include, but are not limited to, the IPL™ Quantum, VASCULIGHT™, and LUMENIS ONE™, PHOTODERM¹" and EPILIGHT™ systems as well as those disclosed in GB 2,293,648; EP 0 565 331; U.S. Patent No. 5,405,368; and WO 91/15264. For example, the VASCULIGHT™ (ESC Medical Systems Ltd., Yokneam, Israel) and IPL™ Quantum (ESC Sharplan, Yokneam, Israel) systems are based on the principal of selective photothermolysis . In contrast to laser systems, these flashlamps use non-coherent light of a broad-band spectrum in the range of 435-1900 run. By applying different cut-off filters (e.g., 515, 550, 570, 590, 615, 645, 695, and 795 run), a specific part of the shorter wavelengths can be cut off. The pulse duration ranges between 2-5 ms (VASCULIGHT™) and 6-26 ms (IPL™ Quantum) . Further, fluence ranges between 3-90 J/cm² (VASCULIGHT™) and 5-45 J/cm² (IPL™ Quantum) can be achieved. Other suitable intense pulsed light sources or flashlamps which can be used in accordance with the parameters disclosed herein are well-known to those of skill in the art.

In particular embodiments, a water-containing gel or solution is used between the skin and the intense pulsed light source probe during treatment to facilitate optical coupling of the light and facilitate heat transfer from the epidermis to the gel.

Advantageously, the methods of the present invention can be carried out without blistering, significant erythema or damage to the underlying tissues. Thus, it is contemplated that the methods of the instant invention will be useful for facilitating epidermal renewal in subjects with acne, large pores, age spots, sun-damaged skin, burns, etc. The methods of the present invention will also be useful in the treatment of psoriasis, eczema

(neurodermitis) , viral warts, precancerous solar keratosis or skin lesions, skin ulcers (diabetic, pressure, venous stasis), and the like.

It is further contemplated that the high- fluency, intense pulsed light source as disclosed herein can be combined, either serially or simultaneously with another light or wavelength source (e.g., laser or radiofrequency) to carry out the methods disclosed herein. The high- fluency, intense pulsed light source and other light or wavelength source can be used together to work synergistically, or combined, wherein each has its own effect. Suitable parameters for radiofrequency include a fluence in the range of about 0.5 J/cm² to about 500 J/cm²; a wavelength in the range of about 300 nm to about 1200 nm; a frequency in the range of 500 kHz to 300 MHz; and a spot size in the range of 1 mm² to 10 cm².

The invention is described in greater detail by the following non-limiting examples.

Example 1 : Animal Model Male, dark-haired pigs were randomly selected for analysis. An intra muscular injection of Azopyrone (0.5 mg/kg) was administered to initiate anesthesia. The anesthesia was maintained with halothane (3-4%) and nitrous oxide. The back region of the pigs, i.e., between their forelegs and hind legs, and the abdomen was shaved to achieve a maximum penetration depth. The area was washed with hibiscub. Treatment sites were outlined by West Indian ink applied to the skin with hypodermic needles. Four marks outlined a 40 x 20cm² treatment area. No ointment was applied pre- or post-operatively in the treatment area. No chemical or mechanical debridement was performed during follow up examinations. Buprenorphine, 0.05-0.1 mg/kg i.p., was given soon after the treatment to minimize pain. The animals were euthanized 120-360 days after treatment using intravenous nembutal (100-150 mg/kg) .

Example 2 : High-Fluency, Intense Pulsed Light Method

A stencil was placed on the back of the pigs to treat the same surface every time. The pigs underwent general anesthesia for 15-30 minutes (5 times for the treatment and 5 times for the biopsies) . In total there were 40 flashes per pig, per treatment. A VASCULIGHT™ system with 590 and 695 nm filters and a QUANTUM™ system with 565 and 640 nm filters were used. Pulse times were 6.5 to 21 milliseconds and the fluence was 35 to 60 J/cm². An entire surface of 40 x 2.8 cm² was treated per pig. The treatment took 4 to 5 minutes with an interval of 4 weeks between treatments. Treatment regimes are listed in Table 1.

TABLE 1

Biopsies were performed immediately following light treatment and again on the second, seventh, fourteenth and twenty-eighth post-operative day. In the second month, one week after the last biopsy, the pigs were radiated again and after 3 weeks biopsies were performed. This was continued for four months. A total amount of 240 biopsies were thus taken. Full-thickness, 3-mm punch biopsies were obtained from each treated site. Tissue specimens were fixed in formalin, embedded in paraffin, sectioned and stained with hematoxylin and eosin for light microscopic examination.

Pigs were followed daily during the first week. After that they were seen and evaluated after 1, 2, 3, 6, 12, and 30 weeks. They were examined for presence of crust formation, swelling, bleeding, erythema, wound healing, scar formation, depigmentation and loss of hair. Photographs were taken before and after treatment.

Example 3 : Immunohistochemistry

Tissue blocks of pig skin biopsy were fixed in 4% neutral-buffered paraformaldehyde at 4⁰C for 4 to 12 hours. The specimens were then routinely processed for paraffin embedding at 56°C. Paraffin sections measuring 4 μm were used for light microscopy and immunohistochemical analysis.

Paraffin sections were deparaffinized and rehydrated. Immunohistochemical demonstration of tenascin and TGF-β antigens was performed on these sections with the avidin- biotin peroxidase (ABC) method. Briefly, these sections were processed through the following incubation steps: hydrogen peroxide 3% in methanol for 30 minutes to block endogenous peroxidase; normal goat serum for TGF-βl and normal horse serum for tenascin, diluted 1:75 v/v, for 30 minutes to 1 hour to reduce non-specific background staining; incubation with primary antibody anti-TGF-βl (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and with primary antibody anti-tenascin (Sigma, St Louis, MO) overnight at 4°C; biotinylated secondary goat anti-rabbit IgG (for TGF- βl ), and horse anti-mouse IgG (for tenascin), diluted 1:200 v/v, for 30 minutes to 1 hour (Vector Labs, Burlingame, CA) ; ABC complex for 1 hour (VECTASTAIN® ABC kit, Vector Labs); and histochemical visualization of peroxidase using 3 ' , 3 ' -diaminobenzidine hydrochloride as chromogen (Sigma, St. Louis, MO) for 5 minutes in a dark room. Sections were then rinsed in tap water, counterstained with hematoxylin, dehydrated and mounted with EUKITT® (Kindler GmbH & Co . , Freiburg, Germany) .

For the above immunohistochemical procedures controls included omission of the primary or secondary antibody which resulted in negative staining and the use of human placenta as positive control for tenascin and TGF- βl antibodies.

Example 4: Statistical Analysis

The statistical analysis of the data was descriptive.

The data is presented as contingency tables to detect association between technical and clinical parameters.

Exact Fisher tests were used to evaluate the homogeneity of the tables, p-values below 0.05 are considered significant.

Claims

What is claimed is;

1. A method for increasing the proliferation or turnover of at least one layer of skin comprising exposing skin to a high-fluency, intense pulsed light source thereby increasing the proliferation or turnover of at least one layer of the skin.

2. The method of claim 1, further comprising exposing the skin to laser or radiofrequency.

3. A method for stimulating sloughing of the stratum corneum of the epidermis comprising exposing skin to a high-fluency, intense pulsed light source thereby stimulating sloughing of the stratum corneum of the epidermis .

4. The method of claim 3, further comprising exposing the skin to laser or radiofrequency.

5. An apparatus for carrying out the method of any of claims 1 to 4.