Alpha-melanocyte stimulating hormone analogues: the perils and the promise.
2009

Introduction
A recent report in the New England Journal of Medicine highlighted the potential use of an [alpha]-melanocyte-stimulating hormone ([alpha]-MSH) analogue called afamelanotide (Nle4-D-Phe7-[alpha]-melanoctye stimulating hormone), in the treatment of patients with erythropoietic protoporphyria (EPP). If successful, this would represent an exciting new therapeutic option for this chronic skin disorder.

While the use of an [alpha]-MSH analogue as an EPP therapeutic is a recent development, [alpha]-MSH analogues have a long history in the field of dermatology. Initially, [alpha]-MSH analogues (Melanotan II) were examined for their potential use as safe tanning agents. Building upon this initial use, the ability to increase the melanin content of skin is now being applied to the treatment of other diseases.

This review will examine the history of [alpha]-MSH and some of the [alpha]-MSH analogues, discuss possible applications for these molecules in medicine, and finally discuss some potential side effects.

Discussion
Alpha-melanocyte-stimulating hormone is the primary hormone of pigmentation. In addition to its release from the pars intermedia of the pituitary gland, [alpha]-MSH is also produced by keratinocytes and other cells in human skin upon exposure to UV radiation (UVR). Alpha-MSH works by binding the melacortin-1-receptor on melanocytes to induce the synthesis of two forms of melanin and augment DNA repair in melanoctyes.

Eumelanin, a brownish-black pigment, is more common in dark-complected individuals who rarely sunburn, while pheomelanin, a reddish-sulfur containing pigment, is more common in light-complected individuals who sunburn easily. Eumelanin is preferentially produced when melanogenesis is initiated, either by [alpha]-MSH or one of the MSH analogues.

Physiologically, [alpha]-MSH is released at a baseline level and is induced in response to UVR damage. UV-induced [alpha]-MSH release from keratinocytes acts in a paracrine manner to stimulate both the synthesis of melanin and the transport of melanosomes from melanocytes to keratinocytes. Once in keratinocytes, melanosomes congregate above the nucleus, serving as protection against further insult. Because this entire process necessitates an initial UV-stimulus, aside from inducing [alpha]-MSH secretion, UV radiation has the potential to cause DNA damage.

Therefore, teleologically it would be useful to increase one's baseline melanin levels to prevent DNA damage before the initial UV insult. It is on this premise that the induction of melanogenesis and melanin transfer via a non-UVR mechanism, such as treatment with MSH analogues, may protect the skin from damage before it occurs.

Since it was discovered that [alpha]-and [beta]-MSH play a role in determining the skin color of humans, "it is clear that systemically administered [alpha]-melanocyte stimulating hormone is an effective agent for darkening the skin of man" researchers have been studying the use of synthetic analogues in the production of artificial tanning.

A group at the University of Arizona became interested in determining the structural features of [alpha]-MSH that are necessary for its biologic activity. It was determined that the N- and C-terminal portions of [alpha] MSH were not essential for its bioactivity, and so, with a few modifications, the central portion of the protein was synthesized as a potent analogue 10- to 1000-times more active than natural MSH (MT-II).

This initial analogue was termed Melanotan I (Nle, (4) DPhe (7)), now called afamelanotide, and has been utilized in Australia for the production of a therapeutic tan and is the focus of numerous clinical trials. Afamelanotide requires the patient or physician to inject the medicine subcutaneously, although it is currently being tested in an inplant delivery system. A shorter version of Melanotan I, dubbed Melanotan II, was also produced. Like Melanotan I, this analogue had potent activity against the melanocortin receptor; however, it also produced immediate penile erections in men, and was not pursued for dermatologic applications. Another group from Cincinnati has also developed MSH analogues, Ac-His-D-Phe-Arg-Trp-NH2, n-Pentadecanoyl- and 4-Phenylbutyryl-His-D-Phe-Arg-Trp-NH2, which they believe may be administered topically.

Early studies noted that afamelanotide alone was capable of inducing an increase in melanin, however, it was also noted that a synergistic increase in melanization occurred when the skin was exposed to UV light. This meant that when given afamelanotide, the patient required a lower dose of UV light to achieve the same level of tanning. This conclusion had both medical and cosmetic implications since it suggested that afamelanotide would provide a fuller, darker tan with less sunlight exposure; thus, the theoretical risk of skin cancer is reduced. While it might be an obvious assumption that an analogue to MSH, which is secreted by cells for the purpose of increasing melanin content, would exclusively induce hyperpigmentation of skin, the effects of these analogues might extend beyond skin pigmentation. Initial clinical studies on afamelanotide were directed at establishing any cutaneous and extracutaneous side effects.

Preclinical studies found that afamelanotide was well tolerated in rodents causing no lethality, weight gain, or serum chemistry changes; and these observations held for human trials as well. In addition, rodent studies found no effect on fetal development. During phase 1 trials in patients with Fitzpatrick scale III-IV skin, the maximal effective dose of afamelanotide was found to be 0.16 mg/kg/day when delivered over a two-week period daily (Monday through Friday). At higher doses, tanning was not substantially increased however mild side effects such as GI effects and lethargy became more pronounced. Tanning effects of afamelanotide were long lasting extending 5-6 months. In 2006, Barnetson et al. reported that afamelanotide augmented melanin production and provided photo protection in people who meet criteria for Fitzpatrick type I and II (white, fair skin that usually or always burn). (This placebo-controlled study also increased the duration of treatment time to three monthly 10-day courses of 0.16 mg/kg/day without noting any additional deleterious effects. The results were extremely promising. Authors found an increase in melanin density at each of eight skin sites in the [alpha]-MSH treated subjects while noting a decrease in controls. Additionally, there was a decrease in luminance (signifying an increase in darkening) in the treated groups and an increase in luminance in the controls. Researchers also compared skin biopsies stained for melanin at day 0 (baseline) and day 90. They found that in patients with high minimal erythemal dose (MED) skin, and thus higher baseline melanin, there was no significant difference in melanin content; however, in patients with low MED, and low baseline melanin, skin melanin content increased by 61%. There was also a statistically significant decrease in the number of sunburn cells in the treated group as compared to controls (50% versus 26% reduction).

Based on years of epidemiological evidence that increased melanin confers a protective benefit from sunlight induced skin cancer, the authors concluded that increased melanin may accentuate any protective effects against UV damage and convert "melano-compromised Caucasians" into melanocompetent individuals. (This observation suggested that MSH analogues might have the additional utility of making skin less sunlight sensitive, and therefore could represent a novel therapy for certain light-sensitive or UV-induced skin diseases. Disease candidates include patients with erythropoietic protoporphyria (EPP), polymorphous light eruption (PMLE), solar urticaria, and actinic keratoses (AK) and squamous cell carcinoma (SCC) in transplant recipients.

EPP is an autosomal recessive disorder characterized by the accumulation of protoporphyrin in tissues, including the skin. Patients with this disorder report extreme degrees of photosensitivity, described as the sense that their skin is actually burning. There is currently no "cure" for this disease, and patients are advised to avoid direct sunlight. With such limited treatments, patient quality of life is dramatically affected and additional therapies are clearly needed. In a study by Harms et al., the authors report on the preliminary responses of five patients with EPP enrolled in an open-label phase 2 afamelanotide study. In this trial, patients received sustained release subcutaneous resorbable implants of 20 mg afamelanotide administered twice daily for 60 days. The primary endpoint was tolerance to standardized xenon-light irradiation measured on the dorsum of the hand with secondary endpoints including]
The acquired condition known as polymorphous light eruption (PMLE) is the most common form of idiopathic photodermatoses, affecting approximately 10-20% of people in Western Europe and the U.S. Patients present with recurrent, cyclical reactions to sunlight, which often begin in the spring and resolve by autumn. Severity can range from erythematous papules, vesicles and plaques to erythema multiforme. These non-scarring, often pruritic lesions are most commonly found on the chest, arms, legs and face. While the etiology remains largely unknown, and is likely multifactorial, it is believed to be a delayed-type hypersensitivity reaction in response to a cutaneous antigen induced by UVR. The disease process tends to vary among patients from mild, virtually insignificant disease to extremely severe disease, with up to 40% of patients with PMLE reporting symptoms of emotional distress related to their condition, including depression, anxiety and anger. Currently, the best treatment available for these patients is the use of sunscreens or other photoprotective devices aimed at decreasing UV exposure. Treatments such as anti-malarials, beta-carotene and PUVA have been tried but with minimal benefit. In severe cases, corticosteriods can be beneficial in reducing symptoms, but at the expense of a multitude of side effects. In August of 2006, phase 2 trials were conducted on the use of afamelanotide in patients with PMLE. Results of the trial indicated that patients using Melanotan required less systemic corticosteroids and had fewer eruptions. Based on this data, in 2007 Clinuvel began phase 3 trials in the U.K. to study the efficacy of afamelanotide as a therapeutic agent for patients with PMLE. Up to 200 patients were planned for participation in this multi-centered, double bind, placebo-controlled study.

Solar urticaria is a rare form of photodermatosis characterized by the formation of wheals, with associated burning, pruritus and erythema, upon exposure to sun. The symptoms develop in minutes to hours following sun exposure, and in some cases may be life-threatening. It is hypothesized that solar urticaria is mediated by a light-induced serum antigen, which produces an IgE-mediated photosensitivity reaction. Various treatment modalities have been utilized for patients with this condition, including antihistamines, H2 receptor antagonists and antimalarials. However, the success of these treatments is largely based on the characteristics and severity of patient symptoms, and ultimately, the best treatment approach for these patients is avoidance of sunlight and use of photoprotective clothing and sunscreens. Currently, phase 2 trials are underway to study the efficacy of afamelanotide as a potential treatment for this disease.*

Actinic keratoses (AK) are precancerous lesions of the skin that are induced by DNA damage from UV radiation. Histologically these lesions are characterized by atypical keratinocytes confined to the epidermis. If left untreated, approximately 2-5% of these lesions can develop into squamous cell carcinoma. AKs are very common, with prevalence rates of 11-26% based on studies conducted in the U.S. and Australia. Interestingly, there is an alarming prevalence of these lesions in patients who have received solid organ transplants, likely as a result of the immunosuppressive medications that must be taken to prevent organ rejection. Prevalence estimates of the development of some form of skin cancer range from 35-70% within 20 years of transplant. The lesions appear as red, flat or scaly, and tend to occur on previously sun-damaged skin. Due to their malignant potential, close surveillance is indicated. Avoidance of sun exposure and the use of sunscreens reduce the development of AKs. However, once lesions are present, the size and number of lesions determines treatment. For non-hyperkeratotic lesions, liquid nitrogen is the treatment of choice, while larger, thicker lesions are treated with more destructive procedures. Numerous pharmacologic agents have been studied in the treatment of this condition, including 5-flurouracil, diclofenac, imiquimod, topical retinoids and photodynamic therapy. In November of 2007, phase 2 trials were initiated to study the efficacy of afamelanotide in reducing the prevalence of AK and SCC in organ transplant patients. The primary end-point for this double-blind, placebo controlled study was the number of AKs noted on the head, back of hand and forearm during a 24-month student course. Secondary endpoints included the number of SCCs on these same regions.

To date, the vast majority of reported side effects for [alpha]-MSH analogues have been benign and self-limited, including, nausea, peripheral vasodilation, fatigue, vomiting, Gl distress and headache. But the main clinical concern regarding the use of [alpha]-MSH analogs has always been the possibility of inducing melanoma, and multiple laboratories have attempted to address this issue. [alpha]-MSH analogs did not increase the ability of human melanoma cells to grow in soft agar, a measurement of tumor formation, and in some instances the analogs even inhibited tumor formation. Similar results were reported by another laboratory using a melanoma cell line. Further work was done to examine whether [alpha]-MSH analogs increased the ability of melanoma cells to grow in DBA/2J mice, and these results suggested that analogs had no effect. And finally, [alpha]-MSH analogues were found to have no effect on the growth of human melanocytes in SCID mice, suggesting that [alpha]-MSH analogs are not capable of inducing malignant transformation of human melanocytes in vivo.* Therefore, these early studies concluded that there was a low risk of developing melanoma while using [alpha]-MSH analogues.

More recently, case reports have been published which report an increased incidence of new or changing dysplastic nevi. In one such report by Cardones et al., a 40-year-old white male bodybuilder with a past history of melanoma and atypical nevi presented with new, irregular and rapidly growing moles. The patient had started taking Melanotan II six weeks prior to his presentation at the clinic in order to tan for a bodybuilding competition. Fellow body builders had recommended this product to the patient, and he purchased the drug over the Internet. He felt this method of tanning would be safer for him, due to his past history of melanoma, because it allowed him to avoid UV radiation. He reported the development of new nevi along with bizarre changes in preexisting nevi about three weeks prior to presentation in the clinic. On examination, the patient was noted to have new or changed lesions scattered on his trunk, back and extremities. The lesions were noted to have peripheral dots and "pseudopod-like" extensions on the periphery suggesting active growth. Of note, there was no recurrence of pigment at his previous melanoma excision site. Alarmingly, however, one of his new lesions on upper back had clinical and dermoscopic findings suspicious for melanoma. On histopathology, the lesion was reported as "a compound nevus with moderate-to-severe architectural and cytologic atypia extending to the margins." Histopathology also noted pigment laden keratinocytes and occasional pagetoid melanocytes in the epidermis. This area was again excised, and on reexcision no residual nevus was noted. Upon withdrawal of the drug, there was a disappearance of any clinical or dermascopic evidence of rapid growth along with a progressive lightening of the nevi over the subsequent nine months.

In a recent letter to the British Medical Journal by Langan et al., the authors discussed two case reports of changes in moles linked to unlicensed use of Melanotan I and II. The patients had several features in common, namely their Fitzpatrick type I/II skin and their self-reported use of UVR tanning beds. The first patient, a 42-year-old female, presented with two growing and darkening nevi on the sole of her foot. Histopathology was reported as atypical acral nevi, later reported as benign. The second patient, a 30-year-old female, presented with darkening of several moles on her back, that were found to be severely dysplastic nevi. This letter supports the findings of the Cardones case report, and both the Langan and Cardones reports serve as a warning to physicians to suspect the use of MSH analogues in patients presenting with new skin darkening and rapid changes in nevi.

For patients with diseases such as EPP and PMLE, where effective therapies are limited, [alpha]-MSH analogs may provide needed relief from these chronic skin disorders. In addition, the ability to tan with lower amounts of sun exposure may contribute to a decrease in the incidence of non-melanoma skin cancer among individuals who might otherwise expose themselves to excessive UV radiation. But even with these clear potential benefits, it is important to proceed with trepidation when considering [alpha]-MSH analogs as a therapeutic. Although initial data seemed to suggest that [alpha]-MSH analogues were safe with regards to melanocyte growth and tumor progression, recent case reports have suggested that the relationship between [alpha]-MSH analogs and melanocytes in humans may be more complicated than previously thought. Only through continued clinical trials will we better understand both the perils and the promise of [alpha]-MSH analogs.