[Nle4-D-Phe7]-alpha-Melanocyte-Stimulating Hormone Significantly Increased Pigmentation and Decreased UV Damage in Fair-Skinned Caucasian Volunteers
Abstract
Epidermal melanin reduces some effects of UV radiation, the major cause of skin cancer. To examine whether induced melanin can provide protection from sunburn injury, 65 subjects completed a trial with the potent synthetic melanotropin, [Nle4-D-Phe7]-alpha-melanocyte-stimulating hormone ([Nle4-D-Phe7]-alpha-MSH) delivered by subcutaneous injection into the abdomen at 0.16 mg/kg for three 10-day cycles over 3 months. Melanin density, measured by reflectance spectroscopy, increased significantly in all [Nle4-D-Phe7]-alpha-MSH-treated subjects. The highest increases were in volunteers with lowest baseline skin melanin levels. In subjects with low minimal erythemal dose (MED) skin type, melanin increased by an average of 41% (from 2.55 to 3.59, P<0.0001 vs placebo) over eight separate skin sites compared with only 12% (from 4.18 to 4.70, P<0.0001 vs placebo) in subjects with a high-MED skin type. Epidermal sunburn cells resulting from exposure to 3 MED of UV radiation were reduced by more than 50% after [Nle4-D-Phe7]-alpha-MSH treatment in the volunteers with low baseline MED. Thymine dimer formation was also shown to be reduced by 59% (P=0.002) in the epidermal basal layer. This study has shown for the first time the potential ability of a synthetic hormone that augments melanin production to provide photoprotection to people who normally burn in direct sunlight.
Introduction
Melanin is synthesized in a multistep biochemical pathway within melanocytes situated in the epidermis, the most superficial layer of the skin (Chaturvedi and Hadley, 1988; Hadley et al., 1993). UVR, especially UVB, causes the release of alpha- melanocyte-stimulating hormone (alpha-MSH) from cutaneous keratinocytes and melanocytes. Unfortunately, in this process, significant UVR-induced skin damage needs to occur in order to trigger this release (Gilchrest et al., 1996). alpha-MSH binds to melanocortin-1-receptor on the melanocytes to induce synthesis of melanin and produce tanned skin (Chaturvedi and Hadley, 1988; Hadley et al., 1993). The increased melanin subsequent to UVR may provide protection from the further damaging effects of solar radiation (Pathak, 1995).
Several studies have suggested that individuals whose skin tends to burn easily on exposure to the sun and does not tan readily are at a higher risk of both non-melanoma skin tumors and of cutaneous melanoma (Stenback, 1978; Fitzpatrick, 1988; Kricker et al., 1995b). Such poorly tanning individuals, termed types I and II in the Fitzpatrick scale (Fitzpatrick, 1988), typically possess defects in the melanocortin-1-receptor gene (Valverde et al., 1995) and are generally thought to be at a greater risk of developing skin cancer (Epstein, 1983; Sober et al., 1991; Palmer et al., 2000; Box et al., 2001).
Stimulating melanin production without the associated cell damage caused by UVR may prove an extremely important addition to the photoprotective strategies being advocated to try and reduce the increasing incidence of skin cancer in the world, especially in most fair-skinned populations (Armstrong and Kricker, 1994). Several derivatives of alpha-MSH have been synthesized (Sawyer et al., 1980; Castrucci et al., 1984; Hruby et al., 1993). One of these molecules, [Nle4-D-Phe7]-alpha-MSH, was used in this study. [Nle4-D-Phe7]-alpha-MSH is more potent and long lasting than alpha-MSH (Sawyer et al., 1980). Several studies on human volunteers have been performed and have clearly demonstrated that [Nle4-D-Phe7]-alpha-MSH can induce safe and effective tanning in humans at an optimum dose of 0.16 mg/kg/day for 10 days (Levine 1991, 1999; Dorr 2000). However, these studies have been generally limited to small cohorts of volunteers who are known to tan easily in response to sunlight (Fitzpatrick scale III-IV) and to a dosage regimen of only 10 days. Previous results have shown increased pigmentation predominantly in the sun-exposed areas of skin such as the forehead, cheek, and shoulder.
In the current placebo-controlled study, we aimed to establish the safety and tolerability in Caucasian subjects of a prolonged dosage treatment (three monthly courses of [Nle4-D-Phe7]-alpha-MSH at a fixed subcutaneous dose of 0.16 mg/kg/day for 10 days in each course), determine whether 30 days of dosing increased the tanning effect compared to 10 days, study the degree of tanning at different anatomical sites and in subgroups of Caucasians with UV-sensitive (low-threshold minimal erythema dose (MED)) compared with relatively UV-insensitive (high-threshold MED) skin, and determine whether cultivating suntan could protect against UVR-induced skin damage.
Results
A total of 79 subjects were enrolled in the study, 59 randomized to [Nle4-D-Phe7]-alpha-MSH and 20 to placebo. All subjects were Caucasian (46 male and 33 female subjects) with a meanplusminusSD age in the [Nle4-D-Phe7]-alpha-MSH group of 37.6plusminus15.5 years and in the placebo group of 44.5plusminus17.6 years. In the [Nle4-D-Phe7]-alpha-MSH group, 47/59 (80%) completed the study, whereas 18/20 (90%) subjects receiving placebo completed the study. Injections of [Nle4-D-Phe7]-alpha-MSH were associated with four major side effects that have been previously observed, namely nausea (experienced by 85% of subjects), facial flushing (74%), fatigue (44%), and vomiting (26%). Injection site reactions occurred in 13% of the subjects. There were no significant changes in vital signs, hematological parameters, or blood biochemistry (including liver and renal function tests). Fourteen subjects withdrew during the 3-month duration of the study (12 active and two placebo) with the most common reason in the [Nle4-D-Phe7]-alpha-MSH group (n=9) being problems with nausea, three others were either lost to follow-up, or withdrew owing to side effects associated with bruising at the site of injections. Therefore, 65 subjects completed the full protocol including skin biopsy assessment. The most common skin type found was the low-MED subgroup (easily burn) assigned to 44 (68%) subjects (30 [Nle4-D-Phe7]-alpha-MSH and 14 placebo). The average MED in this subgroup was 311plusminus11 mJ/cm2 (range 167-426). Twenty-one subjects (32%) were assigned to the high-MED subgroup (rarely burn) (17 [Nle4-D-Phe7]-alpha-MSH and four placebo). The average MED in this subgroup was 611plusminus22 mJ/cm2 (range 457-840). Pigmentation change
The change from baseline to day 90 for melanin density (MD) and luminance (L*) was compared between the two treatment groups, [Nle4-D-Phe7]-alpha-MSH and placebo, using an analysis of variance model with skin type and treatment group as factors and an interaction factor (skin type times treatment group). At all skin sites, MD increased in [Nle4-D-Phe7]-alpha-MSH-treated subjects and decreased in placebo controls. An increase in darkening, signified by a decrease in L*, was also seen in [Nle4-D-Phe7]-alpha-MSH-treated subjects, whereas skin color lightened in placebo controls. Importantly, the P-value for the difference between treatment groups was highly significant at all times, for all sites and overall (P<0.0001). The effect of [Nle4-D-Phe7]-alpha-MSH on skin sites designated as sun-exposed (namely, forehead, cheek, neck, and forearm) compared with non-sun-exposed (namely, shoulder, inner upper arm, abdomen, and calf) in the two groups ([Nle4-D-Phe7]-alpha-MSH vs placebo) is shown graphically (expressed as a percentage MD change (upper) and L* value change (lower) from baseline) in Figure 1. There was no significant difference between sun-exposed and non-sun-exposed skin sites. For MD, the site where the largest differences occurred between treatment groups in the change from baseline to day 90 was in the abdomen, forehead, and inner upper arm. The adjusted (least squares) meanplusminusSEM change in MD from baseline to day 90 for the abdomen was 1.06plusminus0.13 in the [Nle4-D-Phe7]-alpha-MSH group and -0.15plusminus0.08 in the placebo, and mean (95% confidence interval) for difference between treatment groups was 1.17 (0.78, 1.56), P<0.0001. For the forehead, the values were 1.00plusminus0.10 and -0.10plusminus0.09 ([Nle4-D-Phe7]-alpha-MSH and placebo), a difference of 1.09 (0.76, 1.42), P<0.0001. For the inner upper arm, there was a change of 0.85plusminus0.09 with [Nle4-D-Phe7]-alpha-MSH compared to a decrease of 0.3plusminus0.11 in placebo, a difference of 1.00 (0.81, 1.44), P<0.0001.
Figure 1 Mean change in MD and luminance (L*) for sun-exposed and non-sun-exposed skin sites in [Nle4-D-Phe7]-alpha-MSH-treated and placebo subjects. MD and skin darkening (L *) were determined at eight skin sites by skin reflectance using a Minolta spectrophotometer at different times (days) throughout the study. Four skin sites (forehead, cheek, neck, and forearm) were combined as sun-exposed skin sites and four (shoulder, inner upper arm, abdomen, and calf) combined as unexposed skin sites. The mean of 47 [Nle4-D-Phe7]-alpha-MSH (filled square) and 18 placebo (circle) is shown as the change from baseline measurement at day 0. Sun-exposed skin sites are shown by the solid lines and unexposed skin sites by the dashed line. The same sites have the same symbols in the two (a and b). Symbols represent the mean for each group and the bars represent SEM. (a) MD increases in [Nle4-D-Phe7]-alpha-MSH- but not in placebo-treated subjects in both sun-exposed and unexposed skin sites. Unexposed sites showed a smaller increase, but the difference was not statistically significant. Differences between treated and placebo were highly significant at all sites (P<0.0001). (b) Decrease on the L * scale (indicating skin darkening) occurred in the [Nle4-D-Phe7]-alpha-MSH-treated group at all skin sites and an increase occurred in placebo-treated subjects. The difference between [Nle4-D-Phe7]-alpha-MSH-treated and placebo was highly significant (P<0.001), whereas differences between exposed and unexposed skin sites in either group were not significant.
The average L* (inclusion of all eight skin sites) decreased by 1.16+0.25 (from 61.8+1.15 to 60.64+1.17) in the treated group over the 90 days, whereas the placebo group increased by 1.34 (60.51+0.77 to 61.85+0.70), giving an overall significant difference between active and placebo of 2.50 (P=0.0283). The largest changes were in the neck and abdomen (-1.95+0.53 and -1.89+0.53, respectively).
A comparison of the effects of [Nle4-D-Phe7]-alpha-MSH on subjects categorized as low and high MED is shown in Figure 2. For simplicity, only the MD values are illustrated although similar but less demonstrable changes were seen with the L* criteria. Overall, the change in MD in the low-MED skin type population (active; n=30) was approximately double that of the high-MED skin type (active; n=17) (1.04plusminus0.13 vs 0.52plusminus0.09, respectively; P<0.0001). This represents an average increase over all skin sites in the low-MED group of about 41% (1.04/2.55) and only 12% (0.52/4.18) in the high-MED group. Conversely, the placebo groups showed a decrease of 6 and 12% in MD (high- and low-MED groups, respectively). In contrast to the low-MED group, the overall treatment effect in the high-MED group was comprised of approximately equal but opposite small changes in the active and placebo subjects. An example of the pigmentation change in the face of a subject from the low-MED group is shown in Figure 3.
Figure 2 Comparative change in overall MD in low- and high-MED skin types with and without [Nle4-D-Phe7]-alpha-MSH. The increase in MD in volunteers treated with [Nle4-D-Phe7]-alpha-MSH was greater in subjects with a low MED than in subjects with a high MED. MD was determined by skin reflectance using a Minolta spectrophotometer at different times (days) throughout the study. The subjects were divided into two groups with low and high MED]-alpha-MSH-treated low-MED subjects (n=30, circles, full line), placebo-treated low-MED subjects (n=14, circle), [Nle4-D-Phe7]-alpha-MSH-treated high-MED subjects (n=17, triangle) and placebo-treated high-MED subjects (n=4, triangle).
Figure 3 Skin pigmentation with [Nle4-D-Phe7]-alpha-MSH. Subject no. 39 in the low-MED group (a) before and (b) 60 days after the start of [Nle4-D-Phe7]-alpha-MSH treatment. The MD of the forehead increased from 1.68 to 2.89, and left cheek from 2.99 to 3.64.
To assess the change in pigmentation in the treatment group overall, a plot was generated of the maximum change in MD at the inner upper arm compared with the equivalent baseline MD. The inner upper arm was chosen to represent the most likely value for the constitutive melanin. This is shown in Figure 4 and demonstrates that the lower the starting MD, the greater the effect of [Nle4-D-Phe7]-alpha-MSH. So the persons with a baseline MD of 3 or less generally showed increases of 1 or more, whereas those with 4 or more at starting level exhibited only a small (average 0.3 unit) increase. The results indicate that [Nle4-D-Phe7]-alpha-MSH treatment was associated with an approximate doubling in MD for individuals with the lowest baseline MD.
Figure 4 MD change in the inner upper arm after 90 days, [Nle4-D-Phe7]-alpha-MSH versus baseline values. The increase in MD in volunteers treated with [Nle4-D-Phe7]-alpha-MSH was greater in subjects with low levels of constitutive melanin. MD at the inner upper arm (non-sun-exposed site) was determined by skin reflectance using a Minolta spectrophotometer for 47 subjects who completed the course of treatment with [Nle4-D-Phe7]-alpha-MSH. Baseline measurements at day 0 are plotted against the increase in MD on day 90 at the end of the study (MD at day 90-MD at day 0). Each subject is shown as an individual point. The line of best fit generated by linear regression analysis is shown as a solid line. Statistical analysis is by linear regression.
Biopsy of melanin-containing cells
Sections from skin biopsy samples taken at day 0 (baseline) and day 90 were stained for melanin. Figure 5 shows the relative levels for low- and high-MED skin types at these times. At the start of treatment, melanin levels were higher in the high-MED group than in the low-MED group (as expected). In both placebo groups, the melanin skin content fell slightly from baseline to day 90 (829plusminus191 to 726plusminus150 and 2,270plusminus166 to 2,260plusminus112 melanin-positive cells per mm2, for low- and high-MED groups, respectively), in agreement with the spectrophotometric data. Skin melanin content increased significantly in the low-MED subjects by approximately 61% from 901+103 to 1,453+137 melanin-positive cells per mm2 (P<0.001). In contrast, melanin levels remained unchanged in the high-MED subjects (1,917+148 to 1,917+134 melanin-positive cells per mm2). The data are illustrated in Figure 6 in which photomicrographs of biopsies taken at baseline (Figure 6a) and day 90 (Figure 6b) for a subject in the low-MED group are shown. Abundant melanin stain can be seen as supranuclear capping of cells after treatment with only minimal melanin staining observed at baseline.
Figure 5 Epidermal melanin content in treated and untreated subjects. Epidermal melanin detected by Masson-Fontana staining and image analysis was increased by [Nle4-D-Phe7]-alpha-MSH treatment in subjects with low MED. Sections from biopsies were stained by Masson-Fontana for melanin. MD was determined by image analysis (arbitrary units). The mean+SEM is shown for groups of subjects with low and high MED who were treated with placebo (square) or [Nle4-D-Phe7]-alpha-MSH (filled square). Statistical comparisons between samples taken at baseline on day 0 and at day 90 upon completion of the study in the same subjects are shown in the figure (paired t-test). Low-MED placebo, n=14; low-MED [Nle4-D-Phe7]-alpha-MSH, n=30; high-MED placebo, n=4; high-MED [Nle4-D-Phe7]-alpha-MSH, n=16*. *Biopsy specimen from one subject in the high-MED [Nle4-D-Phe7]-alpha-MSH group was lost. Figure 6 Photomicrographs of sections of epidermis labelled to visualize melanin. Representative photomicrographs of one subject with a low MED who had been exposed to 3 times the subject's MED on (a) day 0 and (b) again on day 90 upon completion of the study. Paraffin-embedded section stained with Masson-Fontana stain for melanin (brown pigment counter-stained with Sasranin). Melanin, indicated by arrows, can be seen as supranuclear capping in many cells in (b) but only sparsely in the basal layer of (a). Bar=0.10 mm.
Sunburn injury estimation
For those subjects who showed a large increase in melanin, namely the low-MED group of subjects, the possible protective effect of this increased melanin was assessed by measuring the number of sunburn (apoptotic) cells/mm length of epidermis resulting from exposure to 3 times individual's MED at the beginning and end of the study period (day 90). This was determined by light microscopy and the results are shown in Figure 7a. There was a highly statistical (P<0.001) (paired t-test) reduction in the number of sunburn cells in the treated population with no significant change in the placebo group (P=0.806). This represents a reduction by more than 50% in sunburn cell injury at the end of the study after [Nle4-D-Phe7]-alpha-MSH treatment compared with 26% in the equivalent placebo group. The variable response in the placebo individuals resulted in the difference between [Nle4-D-Phe7]-alpha-MSH and placebo being non-significant (P=0.28) (unpaired t-test). A slide showing sunburn cells is shown in Figure 7b following 3 times MED, and before treatment with [Nle4-D-Phe7]-alpha-MSH.
Figure 7 (a) Effect of [Nle4-D-Phe7]-alpha-MSH on sunburn cell response to 3 MED of solar-simulated UVR in the low-MED group. [Nle4-D-Phe7]-alpha-MSH decreases the number of 3 MED-induced sunburn cells in subjects with low MED. Subjects with a low MED were treated with placebo (n=14) or [Nle4-D-Phe7]-alpha-MSH (n=30). Areas on the lower back/buttock were exposed to 3 times the MED of that individual on day 0 and again on day 90 upon completion of the study. Sections from biopsies were stained with hematoxylin and eosin and sunburn cells identified by light microscopy were counted per mm length of epidermis. Sunburn cells were not detected in matched unirradiated samples from each subject. The mean+SEM is shown for each group. Statistical comparisons between samples taken at baseline on day 0 and at day 90 upon completion of the study in the same subjects showed no significant difference (paired t-test). (b) Sunburn cells in the epidermis. Sunburn cells, as indicated by the arrows, shown in a trial subject following 3 times MED and before treatment with [Nle4-D-Phe7]-alpha-MSH. Bar=0.10 mm.
Thymine dimer (T–T) assessment
The number of nuclei stained for the presence of thymine dimers in the epidermis of eight low baseline MED subjects 24 hours after exposure to 3 times MED before and after [Nle4-D-Phe7]-alpha-MSH treatment is shown in Figure 8. T-T dimers were reduced from a mean of 344+25 to 161+34 or by 53% (P=0.0036, paired t-test; Figure 8) after 90 days of [Nle4-D-Phe7]-alpha-MSH compared to baseline levels in the whole epidermis. The dimer levels were also measured specifically in the basal layer and an even greater reduction was observed in this region (97+10 to 40+12 or by 59%; P=0.002). A representative photo micro-graph of the epidermis before and after 3 times MED in one subject is indicating the reduction in the brown-stained nuclei after treatment.
Figure 8 Effect of [Nle4-D-Phe7]-alpha-MSH on thymine dimer count in response to 3 MED of solar-simulated UVR. [Nle4-D-Phe7]-alpha-MSH decreases 3 MED-induced thymine dimer formation. Biopsies from eight subjects with a low MED who had been exposed to 3 times MED of that individual on day 0 and again on day 90 upon completion of the study were used for assessment of thymine dimers. Sections were stained by immunohistochemistry and thymine dimers were quantitative by counting thymine dimer-positive nuclei throughout the whole thickness of the epidermis, or only in the basal layer of the epidermis and expressed as positively stained cells per mm length. The mean+SEM is shown for each group. Statistical comparisons (paired t-test) were performed between samples taken at baseline on day 0 and at day 90 upon completion of the study in the same subjects. Figure 9 Photomicrographs of sections of epidermis labelled to visualize thymine dimers. Representative photomicrographs of one subject with a low MED who had been exposed to 3 times the subject's MED on (a) day 0 (83% cells positive) and (b) again on day 90 upon completion of the study (14% cells positive). Thymine dimers are identified as brown staining in the nuclei of cells of the epidermis. Reduction of staining is evident in (b), particularly in the basal layer. Bar=0.10 mm.
In this study, we have shown a highly significant increase in melanin in [Nle4-D-Phe7]-alpha-MSH-treated individuals compared to placebo, particularly in the fairer skin types, that is, those with a low MED threshold. In this group, an average increase of about 40% in skin melanin levels compared to baseline was shown. This was associated with a reduction in solar-simulated UVR skin damage of approximately 50%.
The two major forms of pigment present in human skin are the dark brown/black eumelanin and the yellow/red, sulfur-containing pigment, pheomelanin (Prota, 1980). The lowest concentration of eumelanin and the highest phaeomelanin/eumelanin ratio are found in the epidermis of Caucasians with lightly pigmented skin and red or blond hair (Fitzpatrick skin types I and II) when compared to individuals with darkly pigmented skin and black hair (Fitzpatrick skin types III and IV) (Thody et al., 1991; Hunt et al., 1995a; Dwyer et al., 2000).
Individuals with a predominance of phaeomelanin in their skin and/or a reduced ability to produce eumelanin may explain why they fail to tan and are at risk from UVR. Importantly, it has been shown in recent years that it is eumelanin that is preferentially produced when alpha-MSH or [Nle4-D-Phe7]-alpha-MSH initiates melanogenesis in human melanocytes (Hunt et al., 1995b; Furumura et al., 1996; Abdel-Malek et al., 1999, 2000; Thody, 1999; Dorr et al., 2000). Indeed, Dorr et al. (2000) showed that administration of 10 daily injections over 2 consecutive weeks of [Nle4-D-Phe7]-alpha-MSH at a similar dose to that used in the current study induced a preferential increase in eumelanin]
Overwhelming epidemiological data demonstrate that melanin in the skin confers protection from sunlight-induced skin cancer, and the greater the level of melanin, the greater the level of protection (Armstrong and Kricker, 1993; Kricker et al., 1995a; Dwyer et al., 2002). It is interesting to note that those with negroid skin (Fitzpatrick skin type VI) have a built-in sun protection factor of 13.4 (Kaidbey et al., 1979). There is no doubt that melanin in human skin attenuates the penetration of UV light (Chedekel, 1995), the higher the concentration of melanin, the more attenuation]
One of several possible mechanisms of UV-induced tanning is that sunlight damages the skin cells and triggers both keratinocytes and melanocytes to produce alpha-MSH (Gilchrest et al., 1996). Tanning then occurs in response to the damage and the skin now has increased protection from sunlight. In nature, this protection has come too late to prevent the skin damage (sunburn or skin cancer) that occurred before the instigation of tanning. [Nle4-D-Phe7]-alpha-MSH stimulates the skin to produce the protective tan before the sunlight can damage the skin.
The effect of systemic alpha-MSH administration on human (Negroid) pigmentation was first published by Lerner and McGuire (1961) more than 40 years ago. More recently, studies (Dorr et al., 2000, 2004; Levine et al., 1991, 1999) using the synthetic analogue of alpha-MSH, [Nle4-D-Phe7]-alpha-MSH, administered over 10 days have demonstrated increased tanning of the skin, but this has been generally confined to sun-exposed areas of the skin and to predominantly people of skin type III/IV. Interestingly, in this study, the major side effects of [Nle4-D-Phe7]-alpha-MSH were similar to those previously reported (Levine et al., 1991, 1999; Dorr et al., 2000, 2004). Interestingly, there were no reports of spontaneous erections, which indicates that the linear analogue of alpha-MSH, at least at the dosage used in this study, has a different pharmacodynamic profile to the cyclic peptide, which is currently being tested for the treatment of erectile dysfunction. Adverse events in the third month of treatment were similar and slightly less predominant than in the first month. This is the first study in which [Nle4-D-Phe7]-alpha-MSH has been given over such a prolonged period of time and the fact that this did not produce cumulative, more intense or new side effects attests to the relatively good safety record for this drug.
It is possible that the high incidence of nausea, vomiting, and peripheral vasodilation, which mainly occurred in the immediate post-dose period, may have been the result of the rapid rate of absorption from the administration site and the consequently relatively high but short-lived (half-life is 30 minutes) blood level. The incidences of these adverse events could potentially be reduced by reformulation of the drug as a sustained release injection that will result in a markedly lower plasma concentration.
It is anticipated that this will attenuate most side effects associated with the first few hours of bolus injection.
In previous studies, the increased darkening of the skin has been reported using the L* and b* indices of chromaticity matching the Commission Internationale de l'Eclairage standard observer response (Westerhof et al., 1986; Weatherall and Coombs, 1992; Alaluf et al., 2002). We used both chromaticity and a new method to monitor skin darkening. The reduction of 1–2 on the L* chromaticity scale in this study is similar but slightly lower than previous reports of skin tanning using [Nle4-D-Phe7]-alpha-MSH at this dosage (Levine et al., 1991; Dorr et al., 2004). In this study, participants were encouraged to avoid the sun where possible and to use sunblock when exposed, and this may account for the relatively small change in luminance units. It is noteworthy that Dorr et al. (2004) obtained a mean 10-unit decrease in luminance when [Nle4-D-Phe7]-alpha-MSH administration was combined with sunlight exposure. Our results indicate that the estimation of MD by the method of Dwyer et al. (1998) appears to provide a good estimation of melanin changes as shown by the actual biopsy measurements. The actual increase in histological measurement of melanin in the biopsies of the lower back showed changes that correlated extremely well with the MD spectrophotometric assessment at a similar, non-sun-exposed skin site. That is, melanin increased by 60 and 0%, respectively, in the low-MED skin type and the high-MED skin types when measured microscopically at the lower back and by 60 and 11% when assessed non-invasively by spectrophotometry at the lower abdomen. Although there was no direct comparison at the same anatomical site, the results are very supportive that MD measurement is an accurate reflection of the actual tissue melanin content. We believe that this MD measurement should be used in future studies to measure changes in human skin pigmentation. The advantages over the chromaticity values are that the results appear more consistent and also give an actual representation of the melanin change rather than an indirect hue or luminicity value.
[Nle4-D-Phe7]-alpha-MSH had the greatest impact on those people with the lowest constitutive melanin and therefore the greatest risk of sunburn injury. In some people with the lowest baseline skin melanin, a doubling of their constitutive melanin was achieved. Importantly, the increase in MD appeared to be just as pronounced in non-sun-exposed skin sites (abdomen, inner upper arm) as in sun-exposed sites (forehead and neck). The increased melanin in the skin was maintained for at least 3 weeks after the drug regime had finished. Previous studies of just 10 days of a similar drug dosage regimen have shown maintained skin darkening for up to 3 or 4 weeks after the end of treatment (Levine et al., 1991, 1999). Extended duration of melanization after the end of treatment is very likely because of the residence time that melanin will remain in the keratinocytes until the normal process of desquamation removes them; there may also be an extended duration of increased melanin production within the melanocytes. Also in a recent paper (Dorr et al., 2004), it was shown that [Nle4-D-Phe7]-alpha-MSH in combination with a minimal amount of sun exposure extended the skin tanning effect from about 4 weeks with [Nle4-D-Phe7]-alpha-MSH alone to at least 11 weeks with 30-minute sun exposure a day for 3–5 days.
Of particular interest is the increase seen in the inner upper arm (increase of 0.85plusminus0.09 with [Nle4-D-Phe7]-alpha-MSH vs decrease of 0.3plusminus0.11 in placebo), as this is the area designated as being most representative of constitutive melanin at birth and the least likely affected by environmental factors (Dwyer et al., 2002).
The decrease in average skin melanin levels in the placebo group at all skin sites measured was an unexpected result but was most probably due to seasonal variation (van der Mei et al., 2002). The study was conducted from mid-summer through Autumn to early winter and suggests that skin melanin built up over summer may decline by about 6–12% during the winter months, at least in people residing in the relative sunny climes of southern Australia. At the two investigative sites (Sydney and Adelaide), the monthly UV index at the start of the study in January (extreme high) was approximately five-fold greater than at the end of the study in July (low) (Australian Government Bureau of Meteorology, 1997–2001). More dramatic changes could be expected in countries with relatively sunny summers and long sunless winters.
The estimation of skin cell damage using 3 times MED of UVR exposure is well accepted (Dorr et al., 2004). Although two different solar simulators were used in this study, the MED estimations at both study centers were remarkably consistent with no statistical difference between the two sites. Therefore, values could be combined in our analysis. Our results would tend to support the concept that increased melanin levels can attenuate UV damage. However, the variable effect in the placebo group resulted in an unexpected although not significant reduction in sunburn cells. This result cannot be explained, except to state that there may be a wide variation in the response to 3 times MED in different people and larger numbers may be needed to adequately determine this effect. It also provides a cautionary note when extrapolating increased melanin levels to photoprotection. Increased melanin levels per se may not necessarily be associated with increased photoprotection. Epidemiological data would suggest that an increase of two- or three-fold in the constitutive melanin level may be needed to afford protection (Dwyer et al., 2002). This would depend on the constitutive melanin level, for example, those individuals with a low MED threshold who have constitutive melanin levels around 1–2 in our study would need to increase to 3 or more to provide significant extra defense against UV skin damage. When coupled with adequate sunscreen use and sensible exposure times, this could lead to greater freedom for outdoor activities to a vast number of individuals who are currently limited to indoor lifestyles. Further studies are planned to explore this important potential.
Results of our thymine dimer assessment indicated that the [Nle4-D-Phe7]-alpha-MSH-treated group showed a real reduction in DNA damage. This is important for the interpretation of the reduction in apoptotic (sunburn) cells in our study, which could otherwise be interpreted as a reduction in the skin's protective mechanism to induce cell death on UV-induced DNA-damaged cells. However, it must be noted that because these studies were performed 24 hours after UV exposure, the burden of DNA photoproducts reflects both the rate of repair and the extent of initial protection. Consequently, sunburn cells and photoproducts were assessed at the same time. It also demonstrated that a protective effect is seen in the epithelial basal layer, which may contain the long-lived stem cells and it is these cells in which the damage may persist and increase the risk of cancer (Morris, 2000). The basal layer is damaged mainly by UVA radiation and this finding is important in reference to a recently published paper indicating that UVA may play a far greater role in human skin carcinogenesis than was previously realized (Agar et al., 2004).
This study has, for the first time, indicated the potential to provide photoprotection to melanocompromised Caucasians by converting them into melanocompetent persons (Fitzpatrick and Bolognia, 1995). In the face of increased deterioration of the ozone layer, especially in Australia (Ewan et al., 1991), and the increasing incidence of skin cancer (Armstrong and Kricker, 1994), the ability to stimulate the skin's own "protective mechanism" of tanning may prove extremely important as an additional photoprotective strategy.
Study design
This was a double-blinded, randomized, placebo-controlled study conducted in accordance with the Declaration of Helsinki Principles and ICH guidelines for Good Clinical Practice including written informed consent from all subjects. Ethical approval to conduct the study was obtained from the Central Sydney Area and the Royal Adelaide Hospital Human Ethics Committees. The population consisted of male and female Caucasian subjects accrued in a 3]-alpha-MSH to placebo ratio. [Nle4-D-Phe7]-alpha-MSH was prepared by solid-phase chemistry under GMP conditions at Bachem, Peninsula Laboratories (San Carlos, CA). Single-use sterile 6 ml vials each containing 16 mg of [Nle4-D-Phe7]-alpha-MSH in 1 ml sterile saline for injection and placebo vials were prepared under GMP conditions by Octoplus (Leiden, The Netherlands).
Before treatment, each volunteer's baseline skin pigmentation was measured and the volunteers' MED response determined on the lower back. A small area on the back was then exposed to 3 times each individual's MED and punch biopsies collected from the exposed and an adjacent unexposed area 24 hours later for histological evaluation. Subjects were assigned to a low- or high-MED subgroup. This was based on a prospectively assigned cutoff level. Sensitive skin types (easily burning) were defined as having an MED
Abstract
Solar urticaria is a rare photosensitivity disorder demonstrating a range of action spectra, which can inflict a very large impact on life quality despite available treatments. Melanin broadly reduces skin penetration by ultraviolet-visible wavelengths, thus increased melanization may protect in solar urticaria.
To examine quantitatively for impact of the potent ?-melanocyte stimulating hormone analogue afamelanotide ([Nle(4) -d-Phe(7) ]-?-MSH) on the solar urticaria response and skin melanization.
Five patients with solar urticaria received a single dose of 16mg subcutaneous afamelanotide implant in winter time. Melanin density was assessed spectrophotometrically from day 0 to day 60. Detailed monochromated light testing to geometric dose series (increment ) of wavelengths 300-600nm was performed at 0, 30 and 60?days, with assessment of weal and flare area and minimum urticarial dose (MUD).
Mean melanin density increased by day 7, peaked at day 15 and remained elevated at day 60 (P=0·03, 0·01, 0·02 vs. baseline, respectively). Baseline phototesting revealed action spectra of 320-400 (n?=1), 320-500 (n=2), 300-600 (n=1) and 370-500?nm (n=1), and on afamelanotide mean rises in MUD of 1-12 and 1-3 dose increments were seen at the individual wavelengths tested, at 30 and 60 days, respectively. A significant fall in weal area occurred across responding wavelengths from 300 to 600nm at 60?days post-implant (P=0·049 vs. baseline), accompanied by greater than twofold overall increase in MUD (P=0·058 vs. baseline).
Melanin development following afamelanotide exposure is accompanied by reduction in solar urticaria response across a broad spectrum of wavelengths. Further study is warranted to assess clinical benefit under ambient conditions in summer.
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