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
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.
