Method of assessing skin and overall health of an individual

Method for assessing the overall health of the skin and the individual

The present invention relates to a method of using fluorescence to assess the overall health of the skin and the individual.

The native fluorescence of human and mouse skin has been shown to change in a predictable manner with age and UV exposure. See Brancaleon et al., J. Invest. Dermatol. 1999, 113(6): 977-982; Kollias et al., J. Invest. Dermatol. 1998, 111(5): 776-780; Leffell et al., Arch Dermatol. 124 (10): 1514-1518, 1988; Na et al., J. Invest. Dermatol. 2001, 116(4): 536-540; and Tian et al., J. Invest. Dermatol. 2001, 116 (6): 840-845. Therefore, fluorescence spectroscopy has proven to be an objective quantitative method for studying skin ageing aging and photoaging due to sun exposure.

Has been in vivo (<i>In vivo</i>The major fluorescent bands detected by fluorescence spectroscopy include: a) a band assigned to tryptophan (maximum excitation at 295 nm, radiation at 345 nm), b) partitioned to gastric protein ( Pepsin) Digestible collagen cross-links (maximum at 335 nm, 390 nm), c) Collagenase-digestible collagen (maximum excitation at 370 nm, emission at 460 nm) and d) the most likely band due to elastin and collagen (maximum excitation at 390 to 420 nm, 500 nm emission). See Gillies et al., J. Invest. Dermatol. 2000, 115(4): 704-707. The second fluorescent band has been identified as being peroxidized with collagen (Odetti et al., Lab Invest. 1994 70(1): 61-67) or elastin (Leffell et al., Arch Dermatol). 1988, 124 (10): 1514-1518) related: the two are excited at 356 nm, 420 nm, and excited at 390 nm, 460 Nm radiation.

We have found that when the epidermal proliferation of epithelial cells increases, the part of the tryptophan is assigned to the original place (<i>In situ</i>The measured fluorescent signal will increase. See Kollias et al., J. Invest. Dermatol. 1998 111(5): 776-780 and Zhang et al., Lasers Surg. Med. 1997 20(3): 319-331. This has been verified by inducing epidermal repair after, for example, mechanical damage from the tape is removed. See Brancaleon et al., J. Invest. Dermatol. 1999, 113(6): 977-982. In addition, an increase in the substitution of α-hydroxy-acid-induced cells in the human epidermis increases the 295 nm excitation band in a dose-dependent manner. See Doukas et al., Photochem. Photobiol. 2001 74(1): 96-102. The fluorescence of SKH hairless mice (which is due to a portion of tryptophan) decreases with age, suggesting that the reduction in epidermal cell replacement rate is associated with age. See Kollias et al., J. Invest. Dermatol. 1998 111(5): 776-780.

Non-enzymatic glycolysis of proteins occurs naturally with aging (see Monnier et al., Clin Endocrinol Metab. 1982 11(2): 431-452; Njoroge et al., J. Biol. 1988 263(22): 10646-10652; Sell et al., J. Biol. Chem. 1989 246(36): 21597-21602; and Shaklai et al., J. Biol. Chem. 1984 259(6) :3812-3817), causing an increase in protein absorption and fluorescence (Maillard reaction). This glucose-protein adduct is rearranged and dehydrated to form a brown and fluorescent pigment that forms a complexation that results in reduced protein solubility and altered mechanical properties. Such ligatures are dominant in long-lived proteins such as elastin and collagen. The accumulation of fluorescein in collagen has been used as a marker of accelerated aging rate observed in diabetes. See Monnier et al. at Clin.Endorcrinol.Metab 1982 11 (2): 431-452. The maximum amount of digestible gastric protein fluorescing in SKH mice increases with age, whereas the maximum amount of digestible collagen-decomposing collagen and elastin-related fluorescence is not too large. . See Kollias et al., J. Invest. Dermatol. 1998 111(5): 776-780. A similar trend has been seen in mice (<i>Ex vivo</i>(Odetti et al., Lab Invest. 1994 70(1): 61-67), human hip skin in vivo (Na et al., J. Invest. Dermatol. 2001, 116(4): 536-540), and The human dermal skin taken from the skin was observed in vitro when the patient underwent vascular surgery (Odetti et al., Metabolism 1992 41(6): 655-658).

The inventors have surprisingly found that the natural spontaneous fluorescence of the skin is a tool for assessing the health effects of skin health and aging, such as aging over time and aging due to sun exposure.

In one aspect, the invention features a method of determining skin health in a skin area by (i) exposing the skin area to a first exposure radiation to induce the skin area to emit a first firefly Light radiation, wherein the first exposure radiation comprises a wavelength predominantly from about 290 nm to about 300 nm; (ii) measuring the intensity of the first fluorescent radiation having a wavelength of from about 320 to about 350; (iii) The skin area is exposed to a second exposure radiation to induce the skin area to emit a second fluorescent radiation, wherein the second exposed radiation comprises a wavelength predominantly from about 330 nm to about 420 nm; (iv) measuring the having The intensity of the second fluorescent radiation at a wavelength of from about 380 to about 470; (v) calculating the ratio of the intensity measured in step (ii) to the intensity measured in step (iv); and (vi) the ratio A control ratio is compared.

In another aspect, the invention features a method of determining a therapeutic effect on a skin of a subject by (i) exposing a first skin area to a first exposure radiation to induce The skin region emits a first fluorescent radiation, wherein the first exposed radiation comprises a wavelength predominantly from about 290 nm to about 300 nm, and wherein the first skin region is exposed to the mixture; (ii) measuring the having An intensity of a first fluorescent radiation having a wavelength of from about 320 to about 350; (iii) exposing the first skin region to a second exposure radiation to induce a second fluorescent radiation to the skin region, wherein the first The second exposure radiation comprises a wavelength predominantly from about 330 nm to about 420 nm; (iv) measuring the intensity of the second fluorescent radiation having a wavelength of from about 380 to about 470; (v) calculating the measurement in step (ii) (vi) repeating steps (i) through (v) for a second skin region, wherein the two skin regions are not exposed to the mixture; and (vii) The ratio of the first skin area is compared to the ratio of the second skin area.

In a third aspect, the invention relates to a method for assessing the overall health of a body comprising generating a standard curve for a plurality of healthy individuals by exposing one of the skin regions of each healthy individual to a first exposure Radiation to induce a first fluorescent radiation to the skin region, wherein the first exposed radiation comprises a wavelength predominantly from about 290 nm to about 300 nm; II) measuring the wavelength from about 320 nm to about 350 nm The intensity of the first fluorescent radiation; III) exposing the skin region to a second exposure radiation to induce a second fluorescent radiation to the skin region, wherein the second exposed radiation comprises predominantly from about 330 nm to a wavelength of about 420 nm; IV) measuring the intensity of the second fluorescent radiation having a wavelength from about 380 nm to about 470 nm; V) calculating the intensity measured in step (II) and measuring in step (IV) Ratio of intensity; plot a standard curve of the individual's age relative to step V; perform measurements of steps II and IV on one body; calculate the ratio of the individual step V; and discuss the individual in step V Ratio and the standard For comparison, to determine the overall health of the individual.

Alternatively, the average fluorescence value according to age can be determined by following the same steps as above. Next, the fluorescence value of a body whose overall health is under discussion can be compared to the average fluorescence value of that age. If the individual's fluorescence value is below the age-dependent average fluorescence value, it may represent a health problem such as diabetes.

It is believed that those skilled in the art should be able to use the invention to its limits in accordance with the description herein. The specific embodiments described below are illustrative only and are not intended to be limiting in any part of the disclosure.

Unless otherwise defined, all technical and scientific terms used herein are intended to be understood by those of ordinary skill in the art. In addition, all publications, patent applications, patents, and other references are herein incorporated by reference. Unless otherwise indicated, a percentage (%) represents weight percent (i.e., % (W/W)).

<u style="single">Exposure to radiation</u>

In one embodiment, the skin area is exposed to at least two exposure radiations (eg, from a source of UV radiation such as xenon arc lamps or mercury lamps). In an embodiment, the first exposed radiation comprises a wavelength predominantly from about 290 nm to about 300 nm, and the second exposed radiation comprises a wavelength predominantly from about 330 nm to about 420 nm. "Primary" means the wavelength at which at least half of the radiation is exposed. In a further embodiment, the first exposed radiation comprises a wavelength that is predominantly about 295 nm, and the second exposed radiation comprises a wavelength that is predominantly from about 390 nm to about 410 nm.

The exposed radiation is directed to the skin to emit a fluorescent radiation and measure the intensity of such radiation (e.g., a particular wavelength or range of wavelengths). In one embodiment, the method includes measuring the intensity of the first fluorescent radiation having a wavelength from about 330 nm to about 350 nm (eg, about 340 nm), and measuring the having from about 380 nm to about 470 nm. The intensity of the second fluorescent radiation at a wavelength (e.g., about 440 nm).

The ratio of the two intensities measured above can be calculated and compared to a control ratio. "Control ratio" means an established standard ratio (eg, previously obtained from the same skin area, or obtained from, for example, another skin area that has not been exposed to UV radiation (eg, underarm or hip). Therefore, the method can determine the skin health of the subject. We have found that the difference in the ratio of the exposed skin area to the protected skin area generally decreases with age. This difference is believed to be an indication of the ability of the skin to respond to external stimuli by self-healing. Therefore, it is believed that a higher ratio of exposed areas than unexposed areas represents healthy skin that is capable of self-regeneration. Furthermore, it is believed that a higher ratio of exposed areas than unexposed areas also represents the youthfulness of the skin.

In one embodiment, the method is used to determine the effect on a subject's skin treatment. Such treatments include, but are not limited to, cosmetic and pharmaceutical treatments (eg, topical, parental, or oral), laser treatment, or abrasive treatment (eg, crystal dermabrasion cream) (microderm abrasion)). In one embodiment, the treatment is a topical mixture, such as a topical lotion comprising an anti-aging agent such as retinoid (eg, retinol acid or retinol). Or cream.

Applicants have discovered that when the fluorescence of a part of tryptophan is reduced with age, it is distributed to the fluorescent band of the gastric protein and the digestible collagen decomposing collagen, and the elastin network. The fluorescent tape will increase. We were surprised to find that these trends have nothing to do with geographic regions and seasonal effects. Similar trends have been observed in areas where sunlight is protected. Also based on the ratio of the fluorescence intensity of a portion of tryptophan (centered at 295 nm excitation) to the fluorescence intensity (centered at 390 nm excitation) assigned to collagen and elastin, one was also found to age with skin. Strongly associated markup. This mark has been found to decrease with ageing, and aging caused by sun exposure accelerates the rate of decrease. It has also been found that generalized tryptophan can be used to monitor the effects of anti-aging treatments.

<u style="single">Fluorescence measurement</u>

In vivo fluorescence spectroscopy can be performed, for example, using a fiber optic probe attached to a spectrofluorimeter (e.g., SkinSkan (JY Horiba, Edison, NJ)). This method requires: a) a UV radiation source (such as a xenon arc lamp or a mercury lamp), b) a method of selecting the wavelength of the radiation (for example, a monochromator, a prism or a grating), c a method of delivering the radiation to a tissue (e.g., a fiber bundle), d) a method of collecting radiation emitted from the tissue (e.g., a fiber bundle), e) selecting a wavelength of the radiation of the radiation a method (such as a monochromator, a chirp or a grating), and f) a method of detecting the radiation of the radiation (eg, a photomultiplier, a single photodiode, a photodiode array, Or a charge coupled CCD array). See, for example, Stamatas GN et al., J Invest Dermatol 2002, 118(2): 295-302.

The measurement is carried out by placing a fiber optic probe in contact with the site of the skin under investigation. Prior to each set of measurements, the instrument was calibrated for excitation and emission in the 250 nm to 650 nm range. The chromatic resolution of the fluorescence spectrometer is +/- 1 nm.

Obtaining the excitation spectrum is a preferred method for measuring skin fluorescence in vivo. The reason for choosing the emission spectrum is that the base-emitting spectrum is similar to the absorption spectrum, and the band is usually narrower than in the radiation acquisition. The above two reasons are advantageous for identifying individual fluorophores in a complex spectrum. The excitation spectra used in this study were as follows: a) with excitation from 240 nm to 320 nm in the 340 nm emission group (tryptophan excitation at 295 nm max), b) with 240 in the 390 nm emission group Excitation from nm scan to 380 nm (digestible gastric protein collagen excitation is maximal at 335 nm), c) excitation from 240 nm scan to 410 nm in the 420 nm radiation group (digestible collagen decomposed collagen complex excitation 360 nm max), d) has excitation from 260 nm to 490 nm in the 500 nm emission group (elastin-ligand-iso-chain-excited at 390 nm max).

In order to be able to take into account various changes in the natural pigmentation of the skin, which would attenuate the detected fluorescent signal, the fluorescence intensity is normalized to the diffuse reflection signal at the same skin position at the corresponding wavelength. See, for example, Stamatas GN et al., J Invest Dermatol 2002, 118(2): 295-302. A diffuse reflectance spectrum can be obtained by synchronizing the excitation and emission monochromators to select the same wavelength and scanning from 240 nm to 500 nm. For those with wavelengths greater than 315 nm, corrections are especially necessary. Fluorescence measured in this wavelength region is emitted from the dermis (Gillies et al. 2000, Kollias et al. 1998), meaning that the excitation light must travel through the entire epidermis where it is attenuated by epidermal melanin and protein. The emitted light must then travel through the entire epidermis to the collection fibers. This means that both the excitation and the radiation intensity are damaged. On the other hand, for a fluorophore located in the epidermis, that is, a signal with an excitation wavelength of less than 315 nm (see Gillies et al., J. Invest. Dermatol. 2000, 115(4): 704-707), this The weakening effect is not too serious. Further, the intensity of the light source is less than 300 nm, and the noise is normalized by the fluorescence of the diffuse reflection signal. This problem is only in the tryptophan strip (295 Nm excitation occurs. To overcome this problem, the tryptophan fluorescent signal can be normalized to another fluorescent band rather than to the diffuse reflection value of 295 nm. Since it has been found that 390 nm can change aging to the slowest speed, the tryptophan acid band is normalized to the 390 nm excitation band. Other belts can also be used for standardization. Also, if the intensity of the radiation source is sufficient to normalize the diffuse reflection signal at about 295 nm, the wavelength can be used.

<u style="single">Clinical research</u>

To study the effects of aging on the natural fluorescence of human facial skin, the spectrum of the cheek region of 255 healthy individuals is required. They range from 15 to 75 years old and come from five different geographical locations in Asia Pacific: a) Guangzhou, China , b) Harbin, China, c) Shanghai, China, d) Sendai, Japan, and e) Manila, Philippines. To identify potential seasonal effects, the same individuals were measured for facial fluorescence in summer and winter at two locations (Harbin and Shanghai). The skin types of all subjects are II-IV.

In order to investigate the observed changes in facial skin fluorescence due to ageing over time or exposure to sunlight (aging due to sun exposure), a second set of measurements was performed. The skin of the upper inner arm ("unexposed area") and the cheek area of 45 healthy subjects aged 22 to 63 years old were measured. The skin types of all subjects are II-IV. The study was conducted in Skillman, New Jersey, in October.

In the third group of experiments, the effect of vitamin A on facial skin was studied. A 20-year-old vitamin A cream prescription was applied to 20 healthy Caucasians aged 50 to 70 years old, including a broadband spectrum SPF 15 sunscreen on one side of the face, and a matching SPF 15 vehicle control (no vitamin A) on the other side of the face. Both active and vehicle are randomly assigned to each side of each subject's face. Participants and interns of this study did not know anything about the distribution. The study began in February in Tueson, Arizona. Fluorescence excitation spectra were obtained at baseline, three months and six months from the cheeks and the untreated, upper arm that was protected from sunlight. Diabetic patients are excluded because this condition may affect fluorescence measurements.

<u style="single">date analyzing</u>

The linear regression of this data was calculated using a least square error algorithm. Goodness of fit with correlation coefficient (R²) said. The significance of the statistics was calculated using the student's paired data distribution t-test.

<u style="single">result</u>

It has been found that the intensity of skin fluorescence changes with age. As shown in Figure 1, a series of excitation spectra were obtained from the cheek regions of two individuals, 30 and 60 years old, and skin types II. In general, the fluorescence excitation band attributed to a portion of tryptophan (295 nm) decreases with age, while collagen and elastin (335 nm, 360 nm, and 390 nm) increase.

The age distributions of the fluorescence intensity of the 295 nm, 335 nm, 360 nm and 390 nm excitation bands obtained from 108 individuals in Shanghai, China are shown in Figures 2a, 2b, 2c and 2d, respectively. This data has been adapted to linear regression and is shown in the interval between the mean +/- one standard deviation. It is clear that at the 295 nm excitation band, the standard deviation of this data distribution is higher for younger people. The opposite result was found for all other bands. This 295 nm excitation zone is the only zone that decreases with age (in -0.002 units/year). All of the bands attributed to the binding of collagen to elastin represent an accumulation of extracellular matrix complexes with age. From these bands, the 390 nm band showed the slowest increase with age (0.005 units/year). Correlation coefficient (R²The best is the 390 nm band (0.61), followed by 360 nm (0.55), 335 nm (0.41), and 295 nm (0.32).

We observed that the same trend was not related to geographic areas, skin types or seasonal measurements. The slope of the best linear fit for this data represents the rate of change in fluorescence intensity, which is shown in Table I. Change rate of skin fluorescent band (units/year) and standardized tryptophan fluorescence (I_295nm/I_390nm). All measurements are performed on the face (cheek). These rate of change are calculated from the slope of the best linear fit. These values are expressed in fluorescent units per year in the fluorescent band/in the standardized tryptophan fluorescent system in units of ratio per year. PDCXL = gastric protein digestible collagen, CDCXL = collagenase digestible collagen, NTF = standardized tryptophan fluorescence.

<tables><img file="TW200529792A_D0001.tif" /></tables>

The fluorescence intensity of the tryptophan acid decreased with age in all geographical areas in which the study was conducted, and the intensity of the other three bands increased. Further, the slope values are fairly close within the limits of uncertainty.

The 295 nm band fluorescence intensity value is normalized to the other three bands to create a fluorescent marker that is quite independent of skin pigmentation. Further, since the intensity of the 295 nm band is lowered, and the intensity of the band due to the connection is increased, the above ratio causes a strong dependence on age. The ratio that causes the strongest age dependence is the fluorescence intensity of the 295 nm band versus the 390 nm band. Standardized tryptophan fluorescence in Shanghai, China (I_295nm/I_390nmThe age distribution is shown in Figure 3. All features of this data have been adapted to linear regression and provide an interval between the mean +/- one standard deviation. The standard deviation of this data distribution is higher for younger people, however the coefficient of variation (mean/standard deviation) is not significantly related to age. In addition to Manila (R²In addition to =0.15), the correlation coefficient is 0.4 to 0.5 in all places.

The cheek area is chosen because it is expected to receive sunlight UV radiation that can cause cumulative skin damage throughout life. In order to investigate the exposure to sunlight (aging due to sun exposure) affecting the observed standardization of tryptophan fluorescence as a result of age reduction, 45 volunteers were subjected to upper internal arm (equivalent unexposed areas) and cheeks (exposure) Measurement in the sun). The results are shown in Figures 4a and 4b. According to the information shown in Figure 3, the fluorescence ratio obtained from the face (I_295nm/I_390nm) as the age decreases (Figure 4a). The rate of decrease (0.087 units/year) is similar to the values of the other areas shown in Table I. The standardized tryptophan fluorescence to the protected area of sunlight also decreased with age (Fig. 4b), but at a much slower rate (0.010 units/year).

Skin fluorescence measurements in vivo were used to continue the anti-aging effect of topical treatment with vitamin A. The results of treatment of the cheek site with 0.15% vitamin A or excipient formulation are shown in Figures 5a, 5b and 5c. Both groups showed a decrease in fluorescence at 295 nm (Fig. 5a), but the rate of treatment for vitamin A on the cheeks was significantly reduced (-0.01 units/month for the active treatment group versus the placebo group) 0.04 units/month). The 390 nm band did not change significantly during the study period, but there was a significant slight increase in both active and placebo treatment groups (Fig. 5b). Standardization of the tryptophan fluorescent band in the 390 nm excitation band (Fig. 5c) showed that the decrease in the 295 nm band in the vitamin A treatment group was most likely due to an increase in pigmentation during the study period (note that the study was in the second From month to July). The rate of change of the standardized tryptophan fluorescence value of the morphological treatment group was -0.062 +/- 0.029 ratio units per month. The intensity of the 295 nm band was significantly reduced (p &lt; 0.01) compared to the site of the voxel treatment. At the site of vitamin A treatment, the normalized tryptophan fluorescence value remained vertically constant, which was significantly different from the relative value of the receiving form of the treatment site (p &lt; 0.05).

Measurements obtained from individuals with internal arms (untreated) at 0, 3, and 6 months showed that the standardized tryptophan fluorescence value was reduced, although compared to the data shown in Figure 4b. The placebo treats the skin, which is much slower. The anti-aging results of vitamin A treatment measured by fluorescence are consistent with visual observations of reduced appearance of wrinkles in the treatment area. It is only treated with a voxel cream and has no wrinkle effect.

In a separate study, an age-related curve of facial skin fluorescence (normalized tryptophan fluorescence or "NTF") of a healthy individual was established.

Volunteers were interviewed with 9 healthy and 13 type 2 diabetic patients (self-declared) and volunteers aged 45 to 60 years. For the diabetic population, the HbA1c values were distributed between 5.7 and 8.1. Fluorescence spectra were taken from the ventral arm of all arms to all volunteers.

Fluorescence was obtained by simultaneous scanning of excitation and emission wavelengths in the range of 250 to 600 nm with a constant 50-nm Stokes shift, and was performed with a fluorescence spectrometer (SkinSkan (JY Horiba, Edison, NJ)). . This scan provides information on tryptophan fluorescence (excitation 295 nm, emission 345 nm) and collagen-elastomer-associated fluorescence, including the standardization of tryptophan fluorescence (excitation 390 nm, emission 440 nm) ).

The standardized tryptophan fluorescence values (mean +/- one standard deviation = 2.94 +/- 1.94) measured in the control group were higher than the diabetes group (mean +/- one standard deviation = 0.85 +/- 0.45) . The low NFT number of the diabetic group was associated with a reduced ability to repair the epidermis compared to the control healthy group.

The reduced NFT value of diabetic skin may be associated with poor wound healing and repair capacity in diabetic skin. Compare this information (Table 2) with the previously plotted data. Based on the NTF values of the "exposed" regions collected from previous studies, it was shown that the control group fell within the age-mated "health" curve, while the diabetic patients had lower values.

The association between the NTF value of the participant and the HbAc1 value is shown. A certain trend is that higher HbAc1 values are associated with higher NTF values. Since these two parameters are indicators of diabetes, and there is no necessarily a cause-effect correlation, this association does not have to be very strong.

As shown in Table 3, the difference between the NTF value of the diabetic patient and the mean NTF value of the age-matched control group was slightly correlated with the HbAc1 value of the diabetic patient. Thus the NTF value is an indication of the progression of diabetes.

<tables><img file="TW200529792A_D0002.tif" /></tables>

<tables><img file="TW200529792A_D0003.tif" /></tables>

This information indicates that the overall health of a body can be compared by the ratio of the ratio of the ratio of the ratio of the ratio of a body (the health is in the discussion) to the ratio of the curve. As indicated above, the ratio falling below the standard curve represents the individual's health problems such as diabetes.

It is to be understood that the invention has been described by the foregoing description The scope of the invention is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of this patent.

Figure 1 shows the excitation spectra of two individuals aged 30 years (dashed line) and 60 years old (solid line).

Figure 2a shows the age distribution of the fluorescence intensity at the 295 nm excitation band.

Figure 2b shows the age distribution of the fluorescence intensity at the 335 nm excitation band.

Figure 2c shows the age distribution of the fluorescence intensity at the 360 nm excitation band.

Figure 2d shows the age distribution of the fluorescence intensity at the 390 nm excitation band.

Figure 3 shows the age distribution normalized to the fluorescence intensity of the 295 nm excitation band versus the 390 nm excitation band.

Figure 4a shows the age distribution of normalized fluorescence intensity for areas of sunlight that are exposed to sunlight.

Figure 4b shows the age distribution of normalized fluorescence intensity for sunlight as a protected skin area.

Figure 5a shows the change in fluorescence intensity at 295 nm in the skin treated with retinal (active) and placebo (placebo) over time.

Figure 5b shows the change in fluorescence intensity at 390 nm over time in skin treated with retinal (active) and placebo.

Figure 5c shows the change in normalized fluorescence intensity over time in skin treated with retinal (active) and placebo.