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Figure 1.
Comparison of mean (n = 3) reflectance changes over the 7 weeks in 3 control subjects receiving sunlight only tohalf of the back (protocol 3). The contralateral nonexposed back site did not change in terms of L-scale and had slight b-scale changes from baseline.In contrast, the sun-exposed site demonstrated a significant drop in L-scalevalues and a significant increase in b-scale values , both indicative of tanning.The L-scale values returned to baseline at 7 weeks, whereas b-scale valueswere still slightly elevated at week 7. Arrows indicate days of sun exposure.

Comparison of mean (n = 3) reflectance changes over the 7 weeks in 3 control subjects receiving sunlight only tohalf of the back (protocol 3). The contralateral nonexposed back site did not change in terms of L-scale and had slight b-scale changes from baseline.In contrast, the sun-exposed site demonstrated a significant drop in L-scalevalues and a significant increase in b-scale values , both indicative of tanning.The L-scale values returned to baseline at 7 weeks, whereas b-scale valueswere still slightly elevated at week 7. Arrows indicate days of sun exposure.

Figure 2.
Comparison of mean (n = 3) reflectance changes to the back over 10 weeks for subjects receiving sunlight to the backfor 3 to 5 days at the start of melanotan-1 (MT-1) dosing (protocol 3). Tanning is indicated by reduced L-scale and increased b-scale values for the sun-exposedback site and nonexposed back site. Arrows indicate days of sun exposure; x, days of MT-1 injection.

Comparison of mean (n = 3) reflectance changes to the back over 10 weeks for subjects receiving sunlight to the backfor 3 to 5 days at the start of melanotan-1 (MT-1) dosing (protocol 3). Tanning is indicated by reduced L-scale and increased b-scale values for the sun-exposedback site and nonexposed back site. Arrows indicate days of sun exposure; x, days of MT-1 injection.

Figure 3.
Comparison of mean reflectance changes on the back over 10 weeks for subjects (n = 2) receiving sunlightto half the back starting 1 week after finishing melanotan-1 (MT-1) treatment (protocol 3). Tanning is indicated by reduced L-scale and increased b-scalevalues for the sun-exposed back and the nonexposed back. Arrows indicate daysof sun exposure; x, days of MT-1 injection.

Comparison of mean reflectance changes on the back over 10 weeks for subjects (n = 2) receiving sunlightto half the back starting 1 week after finishing melanotan-1 (MT-1) treatment (protocol 3). Tanning is indicated by reduced L-scale and increased b-scalevalues for the sun-exposed back and the nonexposed back. Arrows indicate daysof sun exposure; x, days of MT-1 injection.

Table 1. 
Skin Reflectance Changes From Baseline 1 Week After Completing Protocol 1*
Skin Reflectance Changes From Baseline 1 Week After Completing Protocol 1*
Table 2. 
Effect of Melanotan-1 on Sunburn Cell Response to 3 MED ofUV-B Radiation to the Neck (Protocol 1)*
Effect of Melanotan-1 on Sunburn Cell Response to 3 MED ofUV-B Radiation to the Neck (Protocol 1)*
Table 3. 
Skin Type Characteristics for Subjects in Protocol 2*
Skin Type Characteristics for Subjects in Protocol 2*
Table 4. 
Reflectance Changes in 11 Subjects Given Melanotan-1, 0.16 mg/kg per Day for 10 Injections (Protocol 2)*
Reflectance Changes in 11 Subjects Given Melanotan-1, 0.16 mg/kg per Day for 10 Injections (Protocol 2)*
Table 5. 
Increased Tanning at UV-B–Irradiated Buttock Sites in Melanotan-1(MT-1)−Treated Subjects (Protocol 2)
Increased Tanning at UV-B–Irradiated Buttock Sites in Melanotan-1(MT-1)−Treated Subjects (Protocol 2)
Table 6. 
Characteristics of Subjects Receiving Sunlight With or Without a Prolonged Course Melanotan-1 (Protocol 3)
Characteristics of Subjects Receiving Sunlight With or Without a Prolonged Course Melanotan-1 (Protocol 3)
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Study
July 2004

Effects of a Superpotent Melanotropic Peptide in Combination With Solar UV Radiation on Tanning of the Skin in Human Volunteers

Author Affiliations

From the Department of Medicine, Cancer Center Division (Drs Dorr, Powell, and Alberts, and Ms Brooks), and Dermatology Section (Drs Ertl, Levine,and Bangert), University of Arizona, Tucson; Department of Radiation Oncology, Stanford University, Stanford, Calif (Dr Powell); and EpiTan Ltd, Melbourne,Australia (Dr Humphrey). Dr Ertl in now with Palm Canyon Dermatology, Yuma, Ariz. Dr Dorr is a consultant to EpiTan Ltd, and Drs Alberts, Dorr, and Levineare shareholders in EpiTan Ltd.

Arch Dermatol. 2004;140(7):827-835. doi:10.1001/archderm.140.7.827
Abstract

Objective  Three phase 1 clinical trials of a superpotent melanotropic peptide, melanotan-1 (MT-1, or [Nle4-D-Phe7]α-melanocyte-stimulatinghormone) were performed to demonstrate safety for MT-1 therapy combined with UV-B light or sunlight.

Design  Open-label studies at 2 dose levels of MT-1 combined with small doses of UV-B to the neck or buttock or full sunlight to half of the back.

Setting  Dermatology clinics at the Arizona Health Sciences Center, Tucson.

Interventions  The first study randomized 4 subjects to MT-1 (0.08 mg/kg per day subcutaneously) and 4 subjects to injections of isotonic sodium chloride (9%) solution for10 days, followed by neck irradiation with 3 times the minimal erythema dose (MED) of UV-B light. In the next study (n = 12), the MT-1 dosage was increasedto 0.16 mg/kg per day for 10 days, with UV-B radiation (0.25-0.75 MED) given to a buttock site for 5 days during (n = 7) or after (n = 5) MT-1 administration.A final study randomized 8 subjects to 3 to 5 days of sunlight to half ofthe back or to sunlight plus 0.16 mg/kg of MT-1 for 5 days per week for 4weeks.

Results  Tanning in the first study was achieved in 3 of 4 subjects receiving MT-1, and these subjects also had 47% fewer sunburn cells at the irradiated neck site. More skin sites darkened with the higher dose of MT-1 in the second study. In the third study, there was significantly enhanced tanning of theback in the MT-1 group, and this was maintained at least 3 weeks longer than the tanning in the sunlight-only controls, who required 50% more sun-exposuretime for equivalent tanning.

Main Outcome Measure  There were no pathologic findings at any UV-B or sun-exposed sites in any subject. Toxic effects due to MT-1 were minor, consisting of nausea andtransient facial flushing.

Conclusion  Melanotan-1 can be safely combined with UV-B light or sunlight and appears to act synergistically in the tanning response to light.

The melanocortins include a family of peptide hormones that induce pigmentation by interaction with melanocortin-1 receptor s (MC1R) in the epidermis.1 α-Melanocyte–stimulating hormone (α-MSH)1 is the primary pigmentary hormone that is releasedfrom the pars intermedia of the pituitary gland in some animals and from UV-B–exposed keratinocytes and melanocytes in human skin. This 13–amino acid peptidebinds to MC1R to induce cyclic AMP-mediated signal transduction, leading to the synthesis of melanin polymers from dopa precursors.1 Twotypes of melanin can be expressed in humans. The brownish-black pigment, eumelanin, is believed to convey protection from sun damage, whereas the reddish, sulfur-containingpigment, pheomelanin, is often expressed in light-skinned individuals who report poor tanning and burning in response to sunlight.2 Suchpoorly tanning, easily sun-burning populations (termed type I-II by the Fitzpatrick scale3) typically posses mutations in the MC1R gene4 and are generallythought to be at greater risk of developing skin cancer.5,6

We have previously demonstrated that a superpotent derivative of α-MSH,7 melanotan-1 (MT-1, or [Nle4-D-Phe7]α-MSH), can induce tanning in human volunteers who are known to tan easily in response to sunlight (Fitzpatrick scale III-IV).8 In humans, MT-1 primarily induces eumelanin synthesisin the skin coincident with its tanning effect.9 Although melanotropins have been postulated to affect immunologic function,1012 all of our priortrials reported only facial flushing and transient gastrointestinal upset, unless doses greater than those needed for tanning were administered.13

All of the previously reported clinical trials with MT-1 were performed in human volunteers who were instructed to avoid sunlight and use sunscreenswith an sun-protective factor rating of 30 on all sun-exposed skin sites.8,9,13 Thus, the effect of MT-1 when combined with either sunlight or simulated UV-B radiation has notbeen tested. In the present report, we describe 3 pilot phase 1 clinical trialswith MT-1 combined first with small doses of UV-B radiation delivered froma solar simulator, and then with direct sunlight. The intent of these studies was to examine whether MT-1 could be safely combined with small amounts ofUV-B. A secondary end point that was investigated was whether there was additive stimulation of pigmentation or an alteration in the biological response ofskin to UV-B, measured by the presence of sunburn cells. In addition, a subset of patients receiving MT-1 and localized UV-B radiation underwent detailedanalysis of 17 different B- and T-lymphocyte populations to evaluate any effect of MT-1 on immune function.

METHODS
DESIGN

A series of 3 phase 1 clinical trials were performed to evaluate the response of human skin to MT-1 when combined with sunlight or simulated UV-Bradiation. Healthy subjects with an easily tanning skin type by history (Fitzpatrick type III-IV) were treated with subcutaneous MT-1 at 1 of 2 dose levels for10 days. The effects on skin pigmentation were evaluated by serial reflectance measurements at 8 anatomic sites taken before and at the end of MT-1 treatmentand up to 8 weeks after treatment. Placebo controls were used for protocol 1 (MT-1, 0.08 mg/kg per day, plus 3 times the minimal erythema dose [MED]of UV-B to a neck area) and protocol 3 (MT-1 plus midday sunlight to halfof the back area). In protocol 2, subjects were randomized to receive UV-Bexposure on the buttock at either the start of MT-1 treatment (group A) or immediately after finishing the 10-day course of MT-1 (group B).

SUBJECTS

Healthy subjects were recruited from newspaper advertisements and were screened to have skin types III or IV by the Fitzpatrick scale.3 Subjectscould not have any history of skin diseases, including skin cancers or atypical moles. All subjects were required to have normal laboratory values for renal,hepatic, and hematologic function as assessed by serial chemistry (sequential multiple automated chemistry [SMAC] 20 system studies), complete blood cellcount, and urinalysis. Any women must have tested negative for pregnancy and agreed to avoid becoming pregnant by means of active contraception. Additionallaboratory tests that were required by the Food and Drug Administration (FDA) to be monitored included serum cortisol, luteinizing hormone (LH), and follicle-stimulatinghormone (FSH), which were measured before treatment and at the end of the 2-week treatment period.

MELANOTAN-1

Melanotan-1 was prepared by solid-phase chemistry under good manufacturing practices at Bachem Inc, Torrance, Calif. The white powder was reconstitutedin bacteriostatic sodium chloride for injection and tested negative for pyrogens by the Limulus amebocyte lysate (LAL) assay.14 Itwas stored frozen prior to thawing immediately before injection. The powdered drug was diluted to a concentration of 20 mg/mL, and therefore all subcutaneousinjection volumes were less than 1.0 mL. All doses were subcutaneously administered into the upper arm or thigh using a 25-gauge needle. The doses were calculatedusing actual body weight to deliver either 0.08 mg/kg per day of MT-1 (protocol 1), or 0.16 mg/kg per day of MT-1 (protocols 2 and 3). Melanotan-1 was administereddaily on Monday through Friday for 2 consecutive weeks in protocols 1 and 2 (10 total injections) and for 4 weeks in protocol 3 (20 total injections).The 0.16-mg/kg dose was used for protocols 2 and 3 following the observation of safety for MT-1 and UV-B in protocol 1 . Melanotan-1 doses were not escalatedbeyond 0.16 mg/kg, since this is the maximally effective daily dose for tanning by MT-1 without sunlight.9

UV RADIATION

UV radiation was delivered by a solar simulator (Model 600 Multiport Solar Ultraviolet Simulator; Solar Light Co, Philadelphia, Pa) for protocols1 and 2 or by a series of timed mid-day sun exposures in Tucson over March through June for protocol 3. The solar simulator instrument was initiallycalibrated against a standard white zinc oxide background. All subjects had their MED of UV-B radiation defined at the outset of each study. The MED wasdefined using a series of graded doses of UV delivered from the solar irradiation simulator. An individual subject's MED was determined visually following gradedUV-B exposure to the opposite buttock prior to MT-1 dosing. The MED was defined as the amount of radiation (15.75-42 mJ/cm2) that produced fainterythema with 4 distinct borders 24 hours after UV-B dosing.

REFLECTANCE MEASUREMENTS

For all 3 protocols, pigmentation was included as a secondary end point.Pigmentation was measured as skin reflectance at 8 different anatomic bodysites: the forehead, cheek, dorsal neck, inner forearm, scapula, abdomen, buttock, and anterior leg. Skin chromaticity was measured by light reflectancerecorded on a Minolta CR200 Chromameter (Minolta Camera, Osaka, Japan). The reflected light is received and split into 3 fractions. Luminance (or L-scale)indicates relative brightness from black to white and decreases with tanning. A 1-unit decrease in luminance represents the threshold for detecting a tanningchange by the naked eye.1517 Luminance decreases with tanning (negative L-scale change from baseline) and increaseswith loss of pigmentation (positive L-scale change). There are 2 color scales: the a-scale for yellow to red, which does not change with tanning, and theb-scale, which indicates blue-yellow hue and increases with tanning.1517 For these studies, only L-scale and b-scale responses were recorded and stored on a computer.Eight measurements per anatomic site were averaged at each time point. Each subject served as his own control and reflectance was serially measured atbaseline (predosing), at the end of dosing, and for several weeks thereafter, usually until the reflectance values returned to the baseline level for eachsubject.

END POINTS

The primary end point in the trials was safety, assessed both clinically and by histopathologic examination of irradiated skin sites. The pathologist(J.L.B.) was kept blinded to the treatment group. Two secondary biological end points were evaluated in protocols 1 and 2. In protocol 1, the numberof UV-B–induced sunburn cells was evaluated in histologic sections from a 5-mm punch biopsy specimen of neck skin that was irradiated 24 hours earlierwith 3 MED of UV-B radiation individualized for each subject. Sunburn cells were quantified by the method of Gilchrest18 asmodified by Cesarini et al.19 Four punch biopsy specimens were obtained: at baseline (no UV-B); on the last day of dosing(at the UV-B site and an adjacent nonirradiated site); and 2 months after dosing at the UV-B site only. In protocol 2, a panel of immunologic parameterswas evaluated by flow cytometry quantitation of immune cell markers from peripheral blood samples. Blood samples drawn from 7 patients treated with MT-1 (0.16mg/kg per day × 10 days) were collected in acid-citrate-dextrose Vacutainer tubes. The analysis of blood leukocytes included the number of lymphocytes,monocytes, and granulocytes as well as subset populations of B cells (CD19+), T cells (CD3+), and natural killer cells (CD16+/CD56+). Ratios of the B- and T-cell populations and T-helper and T-suppressor/cytotoxic cells were determined. The analysis was performedusing the BD Simultest system (Becton Dickinson Bioscience, San Jose, Calif). The HECA 452 antibody for skin-associated T cells kindly provided by LouisPicker, PhD, University of Texas, Austin.20 Identified T-cell subsets included T cells that were positive for the activation markers,HLA-DR, interleukin 2 receptors (CD25+), transfer in receptor (CD71+), memory T cells (CD45R0+), and T lymphocytes that "home" to the skin (expressing the cutaneous lymphocyte-associated antigen (CLA/HECA452+). For these studies, blood was withdrawn by peripheral venipunctureat 2 predosing times, at the completion of the 10 doses of MT-1, and 10 days after the completion of dosing (ie, the time at which the tanning responseis generally maximal). The white blood cell fraction was separated by Ficoll centrifugation, and different subtypes of lymphocytes and monocytes were detectedand quantified by automated fluorescence-activated cell sorting (FacScan; Becton Dicksinson Biosciences).

STATISTICAL ANALYSES

Analysis of variance (ANOVA) was performed on the reflectance (tanning) data in all 3 protocols. A paired t test was performedfor each subject value at baseline and at the 4 different postdosing time points. Differences in the number of sunburn cells between saline (isotonicsodium chloride [9%] solution) placebo-treated subjects, and those receiving MT-1 in protocol 1 were also tested using ANOVA followed by a χ2 test. The time of sunlight exposure to a given level of skin tanningin protocol 3 was also compared using ANOVA. The immunologic parameters were compared at 2 baseline time points and then on the day of dose 5, the dayof dose 10, and 10 days after dosing ended. The 95% confidence intervals were analyzed for the 2 baseline values and between the mean of those baselinevalues and the other time points. The Bonferroni adjustment was used to correct the P value for the multivariate tests, such thata significant difference would be .05 for the 17 immunologic variables or <.003 for significance.

RESULTS
PROTOCOL 1

This pilot study evaluated the effect of a low dose of MT-1, 0.08 mg/kgper day, plus 3 MED of UV-B light to a small site on the neck. The numberof sunburn cells following 3 MED of UV-B radiation was evaluated at the site on the neck 24 hours after irradiation. There were 4 male subjects in eacharm of the study who were randomized to receive UV-B plus MT-1 or saline placebo. Significant tanning was observed in 3 of 4 subjects treated with MT-1, withresponding sites limited to the forehead (2 of 4), cheek (3 of 4), and neck (2 of 4). These are as detailed in Table1. None of the saline-treated controls displayed any changes in pigmentation, and 1 (patient 4) showed significantly lighter pigmentation.This latter observation might suggest that the subjects were avoiding sunlight exposure and using sun-protective factor 30 sunscreen as instructed. Importantly,microscopic examination of the irradiated skin biopsy specimens showed no pathologic interactions (skin irritation, ulceration, or histopathologic change)between MT-1 and UV-B light. In contrast, there was a significant reduction in the number of sunburn cells in those subjects receiving MT-1 plus 3 MEDof UV light. These effects are summarized in Table 2. Despite large variability between groups, the analysis shows that 24 hours after the UV-B dose was delivered, subjects receivingMT-1 had a 47% reduction in the number of sunburn cells at the irradiatedsite compared with the saline-treated controls (P =.001; Table 2). This protective effect was achieved despite the fact that only 1 subject in the MT-1 group(patient 7) achieved a statistically significant change in skin reflectance on the neck. A final follow-up sunburn cell determination was made 2 monthsafter the end of MT-1 or placebo dosing (Table 2, last column). At that time there was no difference in the number of sunburn cells due to UV-B for placebo controls and the MT-1–treatedsubjects.

PROTOCOL 2

Because there were no toxic effects (as determined microscopically) in the skin of subjects given MT-1, 0.08 mg/kg, plus 3 MED of UV-B, a secondgroup of subjects were treated with the prior maximally tolerated dose of MT-1 (0.16 mg/kg), with the addition of UV-B exposure to the buttock. Thegoal of this larger study was 3-fold: (1) determine the safety of UV-B with a high dose of MT-1; (2) evaluate whether there was additive tanning if UV-Bradiation was given at the beginning or at the end of MT-1 dosing period; and (3) evaluate immune function parameters in subjects given MT-1. Therewere 12 subjects with Fitzpatrick skin types III and IV randomized in this study. Subjects in group A (n = 7) received UV-B radiation to the buttockon the first 5 days of MT-1 dosing. Patients in group B (n = 5) received the same 3 dose levels of UV-B radiation to the buttock for 5 consecutive days,starting 3 days after the last dose of MT-1 was administered. When combined with MT-1, the UV-B radiation was delivered daily for 5 consecutive days tothe buttock area at 3 solar simulator settings of 0.25, 0.50, and 0.75 of that subject's MED. This typically represented a range of delivered energy,of 5.25 mJ/cm2, 10.5 mJ/cm2, and 15.75 mJ/cm2, respectively, for the 3 sub-MED UV-B doses. The MT-1 treatments were administeredby subcutaneous injection into the upper arm on Monday through Friday, for 2 consecutive weeks (a total of 10 doses). Each daily dose delivered 0.16mg/kg. Three subjects in group A and 2 in group B dropped out of the studyfor personal reasons prior to receiving the drug. Characteristics of the remaining12 subjects in the 2 groups are summarized in Table 3.

A biopsy of the UV-irradiated sites 24 hours after UV-B dosing ended showed no histopathologic findings in either group. Tanning results for the11 treated subjects are summarized in Table 4. One subject (patient 78) did not complete the entire MT-1 dosingstudy for personal reasons and was dropped from analysis. With the higherdose of MT-1 there was significant skin darkening at some body site in allbut 1 individual (patient 73). This indicates an overall response rate of 90%, but the sites of significant skin pigmentation varied for different subjects.The most responsive skin sites were the forehead, cheek, and scapula, with 6 of 11 subjects responding at all 3 sites. The dorsal neck area was muchless responsive, with only 3 of 11 subjects exhibiting significant changes in both luminance and b-scale reflectance values. The buttock, abdomen, andanterior leg exhibited significant skin darkening in 4 or 5 of the 11 subjects in this trial, and this differs from our prior study.13 Anothermajor difference was the prolonged duration of tanning in this trial. The results in Table 4 show that manysubjects had still not returned to their baseline reflectance values at the final evaluation at week 6 (4 weeks after MT-1 dosing ended). Finally, therewas significant further enhancement of skin tanning at the 3 dose sites for UV-B delivered to the buttock on the early group A schedule or the later groupB schedule. Both schedules of UV-B exposure produced significant darkening at the UV-B treated sites compared with adjacent nonirradiated skin (Table 5). Table 5 also shows no difference in buttock tanning at the UV-Bsite between the early or late addition of UV-B to the 10-day MT-1 regimen. Importantly, there were no toxic effects noted at any of these sites.

Adverse effects in protocol 2 were quantitatively and qualitatively similar to our previous studies.8,9,13 Themost common adverse effect was flushing of the upper body that occurred variably within minutes after MT-1 injection. About half of the subjects experienceda median of 3 instances of this self-limited reaction at some time during the 2-week dosing period. The flushing reactions lasted from a few minutesup to an hour and were not associated with other sequelae. A mild sensation of nausea was reported in 4 of the 11 subjects. This effect was typicallynoted after the second or third injection of MT-1 and lasted for 30 minutes up to several hours. Because of the mild severity, antiemetic therapy wasnot required in any subject, but a few subjects described mild anorexia late in the evening on injection days. Fatigue was also reported in about one thirdof the subjects, usually in the afternoon of the first few days of injections. This was variable in intensity, but was never severe enough to require bedrest or drug discontinuance. Similar to the flushing reactions, the episodes of afternoon fatigue did not recur or increase in intensity with successiveMT-1 doses.

Seven subjects in protocol 2 had 5 serial blood samples collected before, during, and after receiving MT-1 to determine whether the acute drug regimeninduced changes in 17 different types of white blood cells. The first 2 samples were baseline samples, drawn prior to dosing, about 8 weeks apart and werecompared statistically. This analysis showed that for all 17 parameters in the 2 baseline samples, zero was contained in the 95% confidence interval.The average of these 2 baseline samples was then calculated and used for comparison with the other 3 time points: on the day of doses 5 and 10 and 10 days afterdosing ended. The results show that 2 parameters, T-memory cell (P = .05) and T-cell LCA2 cell (P = .01) levels, were approximately doubled at the time of dose 5. At the time of dose 10,the T-helper LCA cell levels were decreased by about 50% (P = .01). There were no significant changes noted at the last time point (10 days after the last dose was delivered). However, these individualstatistical differences did not remain significant after applying the Bonferroni correction for multiple analyses.

PROTOCOL 3

This open-label trial in 8 subjects with type III-IV skin evaluated the effects of a prolonged schedule of MT-1: 0.16 mg/kg per day for 20 injectionsover 4 weeks . This was combined with full sunlight exposure to one half of the back for 3 to 5 days until a visual tan was apparent. The sunlight exposureswere given either at the start of MT-1 dosing (n = 3) or after 10 of the total 20 doses had been administered (n = 3). One subject in the latter group droppedout midway through dosing because of time commitments, and therefore only 2 subjects were available for analysis. A control group of 3 subjects receivedthe same sun-exposure regimen to the back without any MT-1 (Figure 1) to allow for a comparison of the time to achieve comparable tanning of the exposed hemi-back site.

Table 6 summarizes the sun-response characteristics of the subjects in this trial. The mean sun-exposure timerequired for the dermatologists (G.E. or N.L.) to detect a visually perceptible tan on the exposed back site in the MT-1 group was 87 minutes (a mean of 4½sun-exposure periods). This was delivered over 3 to 5 days, with each daily exposure averaging 30 minutes. By comparison, in the sunlight-only controlgroup, 5 to 6 exposures of 25 to 35 minutes each were required to achieve the same degree of tanning at the exposed back site. The total mean sun-exposuretime in this group was 165 minutes (16 exposures), about double that in the MT-1 group (P<.001).

The 3 control subjects developed significant darkening of the sun-exposedback that involved a mean 12.5-unit decrease in luminance values and a mean 6-unit increase in b-scale values (Figure1). This degree of pigmentary change would be equal to that of avery dark tan using the validated standards of reflectance for the laboratory scales.1517 Asexpected, these tans were limited to the sun-exposed site, and the nonexposed back sites actually lightened slightly (increased L-values) over the courseof the study (Figure 1, open square). In addition, the sun-exposed back sites returned to baseline reflectance values7 weeks after the sun exposures (L-value). Figure 2 compares the effect of the combination of sunlight exposurebegun on the first day of MT-1 dosing with the degree of tanning achievedon the same subjects' non–sun-exposed back site. This comparison shows that the combination of MT-1 plus sunlight produced rapid and profound skindarkening at the sun-exposed back site (Figure2, solid symbols). This was significantly greater than the darkeningproduced by MT-1 alone at the contralateral (nonexposed) back site (Figure 2, open symbols). The absolute change in reflectance units for the combination was profound, involving the largestreflectance changes recorded: a mean 15-unit decrease in luminance and a mean10-unit increase in b-scale values.

When sun exposure was added to MT-1 at the end of the first 2 weeks of dosing, there was a similar increase in darkening at the sun-exposed backsite compared with the non–sun-exposed back site (Figure 3). However, in this case, the onset of darkening was delayed by approximately 2 weeks. This represents a significant difference comparedwith sunlight delivered on the first day of MT-1 dosing (Figure 2). Figure 2 and Figure 3 show that the duration of darkening was remarkably prolonged for all subjects receiving sunlight plus MT-1. Inthis case, it did not matter whether sunlight was added at the start or middle of the MT-1 dosing period. Indeed, at the conclusion of reflectance monitoringat 11 weeks, all MT-1–treated subjects were as dark as they were at the onset of maximal tanning at both sun-exposed and non–sun-exposedback sites. This differs dramatically from the sun-only controls, wherein reflectance values had all returned to baseline after 7 weeks (Figure 1). The difference is even more striking when one considersthat the subjects receiving only sunlight (no MT-1) received almost twice as much total sun exposure to the back as compared with the combination group(Table 6).

Since the group receiving MT-1 in protocol 3 experienced the greatest cumulative drug exposure to date, the question of adverse effects has specialimportance. The most common adverse effect was again facial and upper truncal flushing, which occurred variably. There were 9 instances of flushing in 3of the 5 subjects. As in our prior studies, the onset was within minutes of MT-1 injection, and it typically resolved within 30 to 60 minutes. The mostserious adverse effect was nausea, which was experienced by 2 of the 5 subjects.This began within 40 minutes of the first injection in patient 817. To preventnausea, the next 3 MT-1 injections in this subject were given after 10 mg of prochlorperazine (Compazine; GlaxoSmithKline, Research Triangle Park, NC)had been administered orally. Another subject (patient 816) also experienced nausea after the second dose and received 10 mg of oral prochlorperazine beforethe next 3 doses. Latter MT-1 doses were given with no antiemetics, and there was no significant nausea. The only other reported adverse effect was afternoonfatigue or somnolence, which was reported in 3 subjects. For example, during week 3, one subject described a 2-hour period of fatigue after each injection.In another subject, general fatigue was described throughout the second week of the injections, with some persistence over the weekend (when no MT-1 wasadministered). All of these adverse effects were of mild intensity, and there was no evidence of cumulative toxic effects. Indeed, most adverse effectswere reported during the first 2 weeks of the 4-week regimen, and only 1 instance of flushing and 1 instance of fatigue were reported in the last (fourth) weekof MT-1 dosing.

COMMENT

The primary goal of the present study was to characterize the safety of MT-1 combined with small amounts of UV light. The results show that thesynthetic superpotent melanotropin, MT-1, can be safely combined with smallamounts of UV-B from a solar simulator or with brief exposures to full sunlight.The latter combination produced a marked enhancement of skin tanning, with the most rapid onset seen for sunlight added at the start of MT-1 dosing.We have further shown that MT-1 can be administered for 20 days over 4 weeksat a daily dose of 0.16 mg/kg, without producing cumulative, more intense,or new adverse effects. The trials also confirm that the 0.16-mg/kg dose of MT-1 is superior to the 0.08-mg/kg dose used in the original clinical study8 in terms of the degree of tanning, as well as thenumber of anatomic sites that responded by darkening. For example, in our original study of a 0.08-mg/kg dose in 28 male subjects, significant skindarkening was only observed on the forehead, cheek, and neck.8 In contrast, the results from protocol 2 show that significant darkening canbe achieved at most body sites. In some cases this included the buttocks, wherein MC1R densities are reported to be very low.21 Wewere also able to demonstrate enhancement of pigmentation at sites receiving concomitant UV-B radiation from the solar simulator. However, there was 1female subject in protocol 2 with type IV skin by history who did not respond at any skin site to the 0.16-mg/kg dose of MT-1. This is the first observationof a completely nonresponsive individual, and there is no clear explanation at this time for the total lack of response.

The 50% reduction in the number of sunburn cells seen in protocol 1suggests that MT-1 may be able to reduce the biological consequences of solarradiation. However, there are several caveats. First, this was a small pilot study involving only 8 subjects, and only 4 subjects received MT-1. Second,there was minimal tanning of the neck induced by the 0.08-mg/kg dose of MT-1used. Finally, there was substantial variability between subjects in the numberof sunburn cells produced by 3 MED of UV-B radiation. This variability suggests that large sample sizes will be required to determine whether a reductionof UV-B–induced sunburn cells comprises a reliable surrogate of a solar-protective effect of MT-1. However, a preliminary description of a phase 2 trial withpositive results showing a 50% reduction in sunburn cell development with the 0.16-mg/kg dose of MT-1 in subjects with skin types I and II was recentlyreported by EpiTan Ltd, Melbourne, Australia (http://www.epitan.com.au).

If the goal of sun exposure is simply to obtain a tan, then MT-1 incombination with a minimal amount of sunlight should provide a tan, which reduces the need for substantial solar exposure. This might considerably reduce the damage to skin from solar exposure. The results, therefore, have importantimplications relative to the use of a tanning booth to acquire a tan, whichhas recently been associated with nonmelanoma skin cancer risk.22 Itshould also be noted that the degree of darkening induced by MT-1 in the absence of solar or UV light is rather minimal even after 20 days of administration.It is therefore the synergistic action of sunlight and MT-1 on the duration and intensity of the pigmentary response that is remarkable.

The native hormone, α-MSH, has been reported to have broad anti-inflammatory activities in experimental models of inflammation.10 These effects include inhibition of arthritis in a rat model,11 reduction of endotoxin-induced liver inflammation in a septic shock model,12 andimproved survival in a model of endotoxemia and peritonitis.10 These effects may be mediated by α-MSH–induced inhibition of the synthesisand activity of cytokines such as interleukin 1 in neutrophils.10 Direct effects on neutrophil migration and superoxide dismutase production have alsobeen reported.23 Protocol 2 indirectly addressed the issue of immunologic activity for MT-1 in humans receiving 10 injectionsof 0.16 mg/kg. At the end of the 2-week dosing period, we could not demonstrate any significant changes in the absolute numbers of 17 different white bloodcell subtypes in the peripheral blood of 7 of these subjects. However, the effectiveness of these peripheral blood cells to mount an immunologic reactionwas not evaluated, and therefore we cannot rule out an alteration in immune response induced by MT-1. On the other hand, no infections have been observedin any of the approximately 100 healthy subjects treated with MT-1 to date.8,9,13 Thus, while the question of whether MT-1 induces immunologic alterations in humans is still largelyunanswered, we do know that it does not acutely alter the number of several classes of white blood cells in the peripheral blood. The lack of an immunologiceffect for MT-1 is also consistent with a study in mice wherein the native hormone blocked contact hypersensitivity reactions, but MT-1 did not.24

The other adverse effects of MT-1 seen in this study are similar to those previously reported.8,9,13 Nauseainduced by MT-1 was seen in about 20% of the current subjects and required antiemetic treatment in only 2 subjects. This effect may be mediated by interactionof MT-1 with melanocortin-3 receptors (MC3Rs), which have been found in the gut tissues of animals.25 The fact that thesemild gastrointestinal adverse effects were infrequent and were not cumulative suggests that any activation of MC3R by MT-1 is not dose limiting. The biochemicalpathway for the other MT-1 adverse effects, notably facial flushing and fatigue, are not known. However, the acute flushing reactions in the upper trunk maybe caused by the production of the vasodilatory molecule nitric oxide, which has been observed following MSH binding to MC1R on keratinocytes and melanotyces.26,27 The only other adverse effect, fatigue,was also seen in our prior phase 1 dose-escalation study, wherein it was dose-dependent in severity.13 In our present study, fatigueof a mild nature was noted at some time in about one third of the subjects.Similar to the other toxic effects, fatigue did not recur with each dose andwas not cumulative in intensity when it did recur. Whether these effects aremediated by binding to MC3R and MC4R, which are found in the brain,28 is not known.

Perhaps the most important observation in the 3 clinical studies of MT-1 is the observation of marked tanning synergy with the combination ofUV-B light (protocol 2) or sunlight (protocol 3). The degree of skin darkening measured at both light exposed sites was significantly greater than that achievedwith UV light, sunlight, or drug alone. Indeed, the tanning observed in the sun-exposed back in protocol 3 is the most intense we have ever measured.Furthermore, the combination of MT-1 plus sunlight produced a long-lasting tan at the sun-exposed back sites, which had not begun to return to baselinereflectance values 11 weeks after MT-1 dosing started. This is significantly longer than what we have seen previously using a 2-week course of MT-1, 0.16mg/kg.13 The 4-week course of MT-1 used in protocol 3 also represents the largest cumulative exposure to drug to date.Importantly, we saw no new adverse effects or more intense adverse effects with this doubled exposure to MT-1.

Current studies with MT-1 are being performed in Australia by Epitan Ltd. They are investigating the effect of a MT-1–induced tan on sundamage in a population of individuals at high risk for sun injury, namely, those with a history of actinic keratoses and poorly tanning skin types (typeI-II by Fitzpatrick scale). In addition, genetic phenotyping is planned to evaluate whether the drug is active in individuals with polymorphisms at the MC1R gene locus.29 A 1-monthdepot formulation has also been developed following observations of enhanced efficacy of slowly released drug in animal models.30 Thesestudies will help to define whether MT-1 has potential as a chemoprotectant for human skin cancer.

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Article Information

Correspondence: Robert T. Dorr, PhD, Arizona Cancer Center, 1515 N Campbell Ave, Tucson, AZ 85725-5024 (bdorr@azcc.arizona.edu).

Accepted for publication January 23, 2004.

This work was funded entirely by Program Project Grant P01 CA-27502 from the National Cancer Institute, National Institutes of Health, Bethesda,Md (Dr Alberts).

The studies were conducted under an Investigator-Sponsored Investigational New Drug Application (No. 35121) from the US Food and Drug Administration(submitted May 24, 1990) (Dr Levine). Dr Humphrey is the Manager-Clinical Development at EpiTan Ltd, Melbourne, Australia, and provided detailed critiquesof the manuscript and analysis of the data. These studies were conducted entirely at the Arizona Cancer Center, Tucson, between 1994 and 1996. Statistical analyseswere provided by Mikel Aiken, PhD, who headed the Biometry Core Service of the Arizona Cancer Center at the time the studies were performed.

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