Evaluation of melanotan-II a superpotent cyclic melanotropic peptide in a pilot phase-1 clinical study

Dorr RT, Lines R, Levine N, Brooks C, et al.

The University of Arizona, Tucson, USA

Summary

A pilot phase I study was conducted with a cyclic heptapeptide analog of a-melanocyte stimulating hormone (a-MSH). The lactam-bridged molecule, called Melanotan-Il (MT-II), has the structure Ac-N1e4- 0-NH2 (MT-II) and has superpotent melanotropic activity in vitro. A single-blind, alternating day (saline or MT-II, placebo-controlled trial was conducted in 3 normal male volunteers at the starting dose of 0.01 mg/kg of MT-II. Subcutaneous injections of the peptide MT-II or saline were given daily (Monday-Friday) for 2 consecutive weeks. Two subjects were escalated by 0.005 mg/kg increments to 0.03 mg/kg and one to 0.025 mg/kg. The 0.03 mg/kg dose produced Grade II somnolence and fatigue in one of two subjects (WHO standards). Mild nausea, not requiring antiemetic treatment, was reported at most MT-II dose levels. A stretching and yawning complex appeared to correlate with the onset of spontaneous, penile erections which were intermittently experienced for 1-5 hours after MT-II dosing, depending on the MT-II dose. Two subjects had increased pigmentation in the face, upper body and buttock, as measured by quatitative reflectance and by visual perception 1 week after MT-II dosing ended. These results demonstrate that MT-II has tanning activity in humans given only 5 low doses every other day by subcutaneous injection. The recommended single MT-II dose for future Phase I studies is 0.025 mg/kg/day.

Alpha-melanocyte stimulating hormone (a-MSH) is a linear, tridecapeptide which is synthesized and secreted by the pars intermedia of lower vertebrate pituitary glands (1). It has structural homology with the 4-10 region of lI- and a-MSH as well as with ACTH. However, a-MSH lacks corticotropic activity and instead, is used to control integumental coloration via binding to melanocortin (MC) receptors on the plasma membranes of melanocytes in the skin of some animals (2). Numerous linear peptide analogs of native cz-MSH have been synthesized to enhance melanotropic potency (3). In frog or lizard skin bioassays two substitutions in amino acids appear substantially increase melanotropic potency; a norleucine (Nie) substitution for methionine at the 4-position, and racemization to -phenyla1anine at the 7-position (3). The resulting molecule, [Nle,4-Phe7]a-MSH has both enhanced melanotropic potency (26 to 100-fold) and increased resistance to degradation by normal plasma enzymes (4). This analog, called Melanotan-I (MT-1) has been shown to be nonmutagenic and non-toxic in mice (5), non-toxic in pigs (unpublished study) and nonteratogenic in pregnant rats (6). A preliminary clinical trial of MT-I in normal males showed that tanning could be achieved in the absence of sunlight exposure, and there were only minor side effects, consisting of transient facial flushing and rarely, gastrointestinal upset (7).

A second series of cyclic, conformationally constrained melanotropins containing a disulfide bridge has been synthesized (8). Subsequent cyclic analogs which contained a lactam bridge rather than a disulfide, possessed even greater melanotropic potency in frog skin and especially in lizard skin bioassays (9,10). One cyclic analog molecule in this series had enhanced potency which was quite prolonged in vitro (11). This heptapeptide contains the conserved MC receptor (4-10) binding region of a-MSH, along with the Nie4 and .-Phe7 substitutions with a lactam bridge between aspartic acid (Asp) and lysine (Lys).

This compound, called Melanotan-II(MT-II), was non-toxic in rodents (unpublished data) and was shown to have a relatively long half life of 1.5 hours in rats given a 0.3 mg/kg I.V. dose (12). In a light-colored, adult male German shepherd dog, one mg of MT-II given daily for 3 weeks induced a profound and complete coat color change from a yellow-tan to black color (13). This was maximal 2 months after starting MT-II and the dog’s coat color gradually returned to normal over the ensuing 2 months. These results suggested that MT-II should be safe for testing and might have potent tanning activity in humans. The current trial reports a pilot Phase I study of MT-II in 3 normal male volunteers.

Dose Escalation and Side Effects

Four dose escalations were required to define the maximally-tolerated dose of subcutaneously administered MT-II on an “every-other-day” schedule. Table II summarizes the side effects at each dose level. At doses up to 0.02 mg/kg, side effects were minimal and transient. They consisted of stretching and yawning for a few hours after dosing, mild GI cramping or nausea (without a decrease in appetite), and facial flushing. At a dose of 0.025 mg/kg (3 escalations from baseline), spontaneous, nonpainful penile erections were reported in all 3 subjects. These involved partial tumescence and were intermittently experienced between 1 and 5 hours post-dosing. At the fourth dose level of 0.025 mg, the stretching/ yawning complex and penile erections were reported by ail 3 subjects. Mild fatigue was also reported in one subject at this dose level and the duration of nausea was longer (6 hours) in one subject.

Because a penile erection was obtained after the first dose in subject 1, a second 0.01 mg/kg dose was administered instead of escalating to 0.015 mg/kg. Thus, this subject did not receive the highest dose of 0.03 mg/kg. The maximal 0.03 mg/kg dose was associated with prolonged fatigue (> 1/2 waking hours) in one of 2 treated subjects. There was no nausea nor a change in appetite at this dose level. Both subjects also reported frequent stretching and yawning for up to 10 hours after dosing. None of the 3 subjects reported nausea, stretching and yawning, or penile erections on the alternating days of saline injection. Because of the prolonged (Grade 2) somnolence and fatigue experienced by one subject at the 0.03 mg/kg dose, the next-lower dose of 0.025 mg/kg was determined to be the maximally-tolerated dose for a single subcutaneous injection of MT-II. With 6 months of follow-up, none of the subjects have reported any adverse symptoms related to this MT-II administration.

Discussion

This pilot trial shows that MT -II can increase human skin pigmentation at cumulative doses much lower than with the linear, 13-amino acid homolog, Nle4-Phe7-a-MSH (MT-I) (7). The cumulative dose of MT-1 which produced a similar degree of tanning was 0.8 mg/kg (ten SC doses of 0.08 mg/kg, each) (7). This compares with a cumulative MT-II dose of 0.10 mg/kg (and only 5 active injections) in the current study. MT-IIwas also more potent in terms of side effects, particularly nausea, which was seen in a mild form at all dose levels in one or more subjects. However, Grade 2 fatigue and somnolence were the overall acute dose-limiting toxicities of MT-II. Whether these effects would be increased with more frequent drug administration, is not known. These effects have not been reported with MT-I or with natural a-MSH, although formal Phase I dose-escalation studies with these melanotropins have not been performed. The alternate day administration schedule for MT-II was used both to observe for toxicities over a longer time period after dosing, and also to estimate the background incidence of “side effects” such as penile erections on the day of saline injection. There were no prolonged side effects of MT-II, and no side effects were reported on the alternating saline treatment days. To prevent moderate toxicity, further Phase I/H studies with MT-II should limit single SC doses to 0.025 mg/kg.

Alternatively, Melanotan-I may be the preferred agent for tanning since significant pigmentation can be obtained with daily SC dosing and with no moderate toxicities (7). Nonetheless, tanning effects with MT-II were produced in both subjects with Type HI skin (tans gradually and uniformly, burns moderately), but not in the one subject with Type il skin (burns easily, tans minimally). As in the prior MT-I study, the tanning effect seen with MT-II was most prominent on the face, and particularly, the forehead. This is probably explained by the greater number of melanocytes in this anatomic area (19).

These results complement prior studies showing that humans can respond with increased pigmentation to exogenously-administered native a-MSH and ACTH (20,21), or l3-MSH (21). The subcutaneous route of injection of MT-II was selected due to the poor bioavailability of only 4.6% for an oral MT-II dose administered to rats (22). The penile erection/stretching and yawning syndrome seen in this study has also been observed in rats given either ACTH or MSH directly into the central nervous sytem (23). However, this report is the first confirmation of the effect in humans given parenteral melanotropins outside the CNS. These observations suggest that MT-II may interact with recently-identified melanocortin-3 (MC-3) receptors which are not found in melanocytes but are uniquely expressed in the brain (cortex, thalamus, hippocampus and hypothalamus), the placenta and in the gut (24). This differs significantly from the expression of melanocortin-1 (MC-1) receptors for a-MSH which are primarily found in melanocytes, and melanocortin-2 (MC-2) receptors for ACTH which are only found in the adrenal gland (25). Thus, MT-II may produce the broader activities seen in this pilot trial by interacting with MC-1 receptors in melanocytes to produce tanning, and with MC-2 receptors in the brain to produce erections, and at high doses, with MC-3 receptors in the gut to produce fatigue and nausea. This is compatible with the current findings that MT-II is a cyclic MSH peptide which possesses tanning and erectogenic activities at low subcutaneous doses.