Melanocortin receptor antagonists and modulations of melanocortin receptor activity

The clinical outcome of disseminated melanoma is grim. Small molecular weight antagonists (preferably about seven amino acid residues) specific for MCR on melanoma cells are provided for the therapy of melanoma as well as in other conditions where modulation of MCR is of clinical significance. A particularly preferred antagonist is p-anisoyl-[D-Arg.sup.6,9, D-Lys.sup.11, D-Leu.sup.12 ] dynorphin A(6-12)-NH.sub.2, which is an excellent antagonist of the MCR-1 receptor.

FIELD OF THE INVENTION
 The present invention relates to pharmaceutical compositions containing
 melanocortin receptor antagonists, and more particularly to methods for
 administering melanocortin receptor antagonists so as to modulate
 melanocortin receptor activity.
 This invention was made with government support under Grant No. DA00091,
 awarded by the National Institutes of Health. The government has certain
 rights in this invention.
 BACKGROUND OF THE INVENTION
 Melanocytes are embryonically derived from the neural crest. These cells
 migrate to the skin during fetal development, sit on the basal lamina of
 the epidermis and interdigitate with basal cells via dendrites. Melanin is
 produced in the Golgi apparatus of the cell and this pigment is packaged
 (melanosomes) and delivered to keratinocytes and the hair follicle. In
 some cold-blooded vertebrates (frogs, fish, and reptiles), the cells
 synthesize melanosomes but do not pass it on to other cells. The
 melanosomes can move back and forth from the nucleus to the peripheral
 processes (dendrites) and this mechanism of dispersion and aggregation
 gives these animals the ability to change skin color from dark to light or
 vice versa. In cold-blooded vertebrates melanocytes are called
 melanophores.
 .alpha.-, .beta.-, and .gamma.-Melanocyte-Stimulating-Hormone (MSH) and
 adrenocorticotropin (ACTH) are melanocortin receptor (MCR) peptide
 agonists derived (in humans) from post-translational processing of
 pro-opiomelanocortin (POMC). MCR are seven-transmembrane domain G-protein
 coupled receptors first discovered in 1992. Five subtypes have been cloned
 and named MCR-1 to MCR-5. There is consensus that MCR-1 exists on normal
 and neoplastic melanocytes and activation of these receptors results in
 increased melanogenesis (via G-protein stimulation, cAMP accumulation and
 tyrosinase activation). The MCR-4 receptor is implicated in body weight
 regulation. For example, inventors Gu et al. describe using melanocortin-4
 receptor as a target to treat body weight disorders by modulating the
 activity of that receptor, WO 97/47316, published Dec. 18, 1997.
 Melanoma is a tumor originating from unrestrained proliferation of
 melanocytes, which are pigment cells residing mainly in the epidermis.
 This tumor has an annual incidence in the United States of about 35,000
 cases, with a mortality of approximately 7300 deaths (for 1997). The
 incidence of melanoma has been increasing significantly (with a 300
 percent increase in the past 40 years). Currently, the lifetime risk of
 melanoma in the U.S. is approximately one percent. But in some countries
 such as Australia and New Zealand the lifetime risk is as high as 1/15 or
 6.7%.
 The reason for the increased incidence of this disease is uncertain but may
 stem from greater recreational sun exposure, especially early in life, and
 a just released study suggests that sunscreen does not protect against
 skin cancer, including melanoma. Individuals most susceptible to
 development of melanoma are those with fair complexions, red or blond
 hair, blue eyes, and freckles and who are poor tanners and easy
 sunburners. Other factors associated with increased risk include family
 history of melanoma (approximately one in ten melanoma patients have a
 family member with melanoma) and the presence or excess of atypical moles.
 Malignant melanoma is usually first detected as a change in size or shape
 of a pigmented area of the skin and confirmed by histological examination
 of the biopsy specimen. The five-year survival for localized disease
 (clinical stages I and II) is about 85 percent. For clinical stage III
 (clinically palpable nodes that contain tumor cells), a five-year survival
 of about 50 percent is noted when only one node is involved and about 15
 to 20 percent when four or more nodes are involved. Five-year survival for
 clinical disseminated disease (stage IV) is less than five percent.
 Fortunately, the majority of melanomas are diagnosed in clinical stages I
 and II and melanomas less than 0.76 mm thick are usually cured by surgical
 removal (five-year survival rates range from 96 to 99 percent). On the
 other hand, metastatic melanoma to organs such as brain, liver, and lung,
 is associated with survival of less than one year.
 Current treatment of the disseminated disease is usually palliative to
 improve the quality of life. Surgical excision of a single metastasis to
 the lung or to accessible brain sites can be associated with prolonged
 survival. Radiation therapy can provide local relief for recurrent tumors
 or metastatic sites. Patients who have advanced regional disease isolated
 to a limb may benefit from localized intra-arterial limb perfusion with
 chemotherapeutic agents. However, chemotherapy has a response rate of only
 20 to 25 percent and rarely induces complete remission. The lack of
 response to traditional cancer treatments has led to many trials using
 agents such as retinoids, high-dosage chemotherapy with autologous bone
 marrow trans-plantation, antipigmentary agents, and antibodies conjugated
 to isotopes, drugs, and toxins. More recently, immunotherapy with
 interleukin-2 and .alpha.-interferon has been used. Marginally improved
 response rates have resulted from these experimental methods.
 Wei and Thomas discovered that a number of dynorphin A peptides had the
 unusual property of preventing the increased vascular permeability of
 small blood vessels that occurs after tissue injury. This
 anti-inflammatory property of dynorphin A peptides and certain des-Tyr
 dynorphin A compounds and analogs is described in U.S. Pat. No. 5,482,930,
 issued Jan. 9, 1996.
 Some MCR antagonists have been found in the agouti proteins (named after
 the South American rodent), which are 131 (mouse)/132 (human) amino acid
 proteins elaborated by hair follicle melanocytes and by brain tissue. The
 elaboration of agouti-signaling protein (ASP) determines the hair color of
 fur-coated animals. Agouti also antagonizes MCR-2 to MCR-5, but analyses
 of the binding coefficients do not indicate the clearcut characteristics
 of competitive antagonism which is found for MCR-1. (Yang et al., Mol.
 Endocrinol., 11, pp. 274-280, 1997.) Siegrist et al. recently showed that
 ASP binds to the MCR-1 with an almost identical affinity to that of
 .alpha.-MSH, and that it had antiproliferative action with a half-maximal
 effective concentration of 13 nM. Agouti protein was also found to induce
 MC down-regulation. They also showed that ASP will inhibit the
 .alpha.-MSH-stimulated growth of B16-F1 mouse melanoma cells in culture.
 Siegrist et al., J. Recept. Signal. Transduct. Res., 17, pp. 75-98 (1997);
 Siegrist et al., Biochem. Biophys. Res. Commun., 218, pp. 171-175 (1996).
 However, an agouti-related protein (ARP) which was recently cloned from
 non-epidermal tissues had little effect on MCR-1 at concentrations up to
 100 nM, although it did cause a dose dependent inhibition for MCR-3 and
 MCR-4. Ollmann et al., Science, 278, pp. 135-138 (1997).
 SUMMARY OF THE INVENTION
 It is an object of the present invention to provide antagonists to
 mammalian MCR receptors, particularly in the form of small or relatively
 small peptides.
 The present invention provides a method for modulating the activity of a
 melanocortin receptor in which a melanocortin receptor antagonist is
 administered so as to be therapeutically useful in reducing the activity
 of melanocortin receptors. Thus, the present invention provides that small
 MCR antagonists may be used for the therapy of melanoma as well as in
 other conditions where modulation of MCR may be of clinical significance.
 One example of such uses to modulate is in the case of cancerous
 melanocytes (melanoma) in which MCR agonists stimulate growth and
 metastasis. Administration of an antagonist is useful in inhibiting the
 melanocytes. Another example is where the antagonist administered
 modulates the activity of MCR-4 receptors, so that modulation is useful in
 treating body weight disorders.
 Thus, in an aspect of the present invention, a method of treating melanoma
 is provided in which a subject in need of treatment is administered a
 melanocortin receptor antagonist. The method inhibits melanocortin
 receptors on melanocytes when the melanocytes have administered thereto a
 melanocortin receptor antagonist having about seven amino acid residues
 and which is in an amount effective at concentrations of less than 250 nM
 so as to block the actions of .alpha.-melanocyte stimulating hormone.
 Antagonists of the invention include peptides selected from:
 (a) Xaa-Arg-Xaa-Arg-Pro-Xaa-Xaa (SEQ ID NO:1), such as dynorphin A(6-12),
 where Xaa.sup.6 is Arg or D-Arg, Ala or D-Ala, Xaa.sup.8 is Ile or Ala,
 Xaa.sup.11 is Lys or D-Lys, and Xaa.sup.12 is amidated Leu, D-Leu, or Ala.
 The Arg in the sixth and/or ninth position is preferably in the D-Arg
 stereoconfiguration, which substantially increases potency. Additional,
 particularly preferred modifications are where the peptides have an
 acylated amino terminus, or an anisoylated (methoxybenzoyl) N-terminus,
 and have an amidated carboxyl terminus (e.g. amidated Leu);
 (b) a mystixin having the sequence T.sub.N -A.sub.1 -A.sub.2 -A.sub.3
 -A.sub.4 -A.sub.5 -A.sub.6 -T.sub.C, where T.sub.N is an amino terminal
 portion having a molecular weight less than about 600 daltons and is
 selected to convey resistance against enzymatic degradation. A preferred
 T.sub.N, for example, is N-acetylated Tyr(Me) or methoxybenzoyl; A.sub.1
 is D- or L-arginine and D-lysine; A.sub.2 is lysine or arginine; A.sub.3
 is leucine or isoleucine; A.sub.4 is leucine, isoleucine, methionine, or
 valine; A.sub.5 is methoxybenzoyl-ethyl-Gly, methoxybenzoylmethyl-D-Ala,
 Tyr(Me), Trp, Tyr, Leu, Lys, Arg, 4' substituted Phe (4'F, 4'I, 4'Cl,
 4'NO.sub.2), D-His, D-Lys, D-Arg, D-Leu, D-Pro, or D-Trp; A.sub.6 is
 isoleucine; with the proviso that not all of the A.sub.1 -A.sub.6 are in
 the L-configuration; and T.sub.C is isoleucineamide, D-leucineamide,
 D-valineamide; or
 (c) is a compound having the sequence Arg-Tyr-Tyr-Arg-Trp-Lys (SEQ ID NO:4)
 or Arg-Try-Try-Arg-D-Trp-Lys which may include the modifications as
 described in (a).
 In another aspect of the present invention, a method of modulation,
 particularly in treating body weight disorders, comprising administering
 an antagonist selective for the MCR-4 receptor. Antagonists of the
 invention for the MCR-4 receptors in accordance with the invention include
 an agouti-related protein fragment (83-132), which is preferably amidated.
 Although therapeutic uses are contemplated, particularly because there have
 been so few known antagonists of melanocortin receptors known to date, the
 antagonists herein described will also be useful in drug screening assays.
 Aspects of the invention will be illustrated through inhibition of
 melanocyte cells in which a substance decreases the activity of a
 melanocortin receptor agonist (i.e. .alpha.-melanocyte stimulating
 hormone, or .alpha.-MSH) on frog melanophores or on mammalian cells
 transfected with melanocortin receptors. The inhibition constant
 (K.sub.i), given in units of concentration, which is defined as the
 concentration of the antagonist which doubles the amount of agonist
 required to produce the same degree of effect, is computed; or the
 inhibitory concentration of the antagonist (IC.sub.50), which reduces by
 50% the activity of a fixed concentration of agonist (usually 200 to 600
 picoM of .alpha.-MSH), is obtained. Particularly preferred antagonists for
 therapeutic utility have value of K.sub.i or IC.sub.50 of less than 250
 nM.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
 Hormone receptors are increasingly of interest as pharmaceutical targets,
 and the use of hormone-receptor antagonists for therapy is
 well-established in the treatment of breast and prostate cancers. Primary
 examples are tamoxifen and flutamine, which are antagonists of the
 steroidal estrogen and androgen receptors, respectively (Goodman and
 Gilman, Pharmacological Basis of Therapeutics, 1997).
 We have discovered certain peptides that are antagonists against mammalian
 melanocortin receptors and which are useful in modulating the activity of
 these receptors. Thus, two of us have recently discovered that some
 dynorphin A peptides antagonize the human MCR-1, MCR-3, and MCR-4
 receptors (Quillan and Sadee, Pharm. Research, 14, pp. 713-719, 1997).
 Thus, dynorphin A(1-13) -NH.sub.2 was found to be the most potent of a
 series with an IC.sub.50 of 110 to 145 nM (against 600 pM .alpha.-MSH).
 Further shortening of the carboxyl terminal end was found to cause
 reduction in antagonist potency. Analogs of [des-Tyr] dynorphin A were
 also tested. Dynorphin (2-17) was found to have an IC.sub.50 value of 190
 nM (against 600 pM .alpha.-MSH). Dynorphin A(6-17) was also tested for
 antagonist activity, but was found to be inactive. Antagonists useful in
 the subject invention further include dynorphin A(6-12), mystixins, an
 agouti-related protein (ARP) fragment, and some ligands for an orphan
 opioid receptor. These will now be more fully described.
 Dynorphin A Fragments
 We have surprisingly discovered that dynorphin A(6-12) and some variations
 thereof are particularly excellent antagonists of the MCR-1 receptor.
 Dynorphin fragments of the present invention, which include dynorphin
 A(6-12), have the general structure shown by Formula 1 (SEQ. ID NO:1):
 FORMULA 1
 Xaa-Arg-Xaa-Arg-Pro-Xaa-Xaa
 6 7 8 9 10 11 12
 where Xaa.sup.6 is Arg or D-Arg, Ala or D-Ala, Xaa.sup.8 is Ile or Ala,
 Xaa.sup.11 is Lys or D-Lys, and Xaa.sup.12 is amidated Leu, D-Leu, or Ala.
 The Arg in the sixth and/or ninth position is preferably in the D-Arg
 stereoconfiguration, which substantially increases potency. Additional,
 particularly preferred modifications are where the peptides have an
 acylated amino terminus, or an anisoylated N-terminus, and have an
 amidated carboxyl terminus (e.g. amidated Leu). Of the various
 modifications or substituents possible with peptides in accordance with
 the present invention, the most potent modification appears to be the
 attachment of an anisoyl (methoxybenzoyl) group to the N-terminus. The
 position of the methoxy group on the benzene ring does not appear to be a
 critical determinant of activity as para, meta, and ortho anisoyl-
 substituted analogs exhibited similar potencies. The p-nitrobenzoyl analog
 was as active as the anisoyl analogs. Other alternative modifications,
 such as p-methylpenylacetyl, p-toylacetyl, phenoxyacetyl, and
 p-aminobenzoyl derivatives, are possible.
 In the case of dynorphin A(6-12), Xaa is arginine at position 6, isoleucine
 at position 8, lysine at position 11, and leucine at position 12. A
 particularly preferred peptide of the invention with good potency is
 sometimes referred to as "SQW-5," which is p-anisoyl-[D-Arg.sup.6,9,
 D-Lys.sup.11, D-Leu.sup.12 ] dynorphin A(6-12)-NH.sub.2.
 FIG. 1 graphically illustrates the activity a dynorphin A(6-12)-amide
 analog, SQW-5, which antagonizes the stimulatory effects of .alpha.-MSH on
 melanin dispersion in Xenopus frog melanophores. The presentation of data
 is in the form of a Schild Plot wherein the goal is to obtain a inhibitory
 constant (K.sub.i) which estimates the antagonist potency of the test
 compound. At varying doses of the antagonist, the dose-ratio for the
 amount .alpha.-MSH to produce darkening of the melanophores is shifted. A
 regression analysis yields the K.sub.i value at the intercept on the
 abscissa. The K.sub.i value is defined as the concentration of antagonist,
 in this case 12 nM of SQW-5, which is sufficient to require a doubling of
 the concentration of .alpha.-MSH required to produce an equivalent effect.
 The slope of the regression line is near unity, which is indicative of
 reversible competitive antagonism.
 Although less potent than the particularly preferred dynorphin A(6-12)
 analog, dynorphin A(1-13)-amide is another antagonist useful in practicing
 the subject invention.
 Mystixins
 Mystixins contain the sequence
 -Arg-Lys-Leu-(Leu/Met)-X-Ile-(Leu/D-Leu)-NH.sub.2 where X may be an
 anisolyated glutamic derivative (A*) or other aromatic residues. U.S. Pat.
 No. 5,480,869, issued Jan. 2, 1996, inventors Wei and Thomas describes
 mystixins that have anti-inflammatory properties and are useful in
 inhibiting vascular leakage in injured tissue. These mystixins have now
 been found to be antagonists against mammalian MCR-1 receptors.
 Thus, useful mystixins have the sequence T.sub.N -A.sub.1 -A.sub.2 -A.sub.3
 -A.sub.4 -A.sub.5 -A.sub.6 -T.sub.C, where T.sub.N is an amino terminal
 portion having a molecular weight less than about 600 daltons and is
 selected to convey resistance against enzymatic degradation. A preferred
 TN, for example, is N-acetylated Tyr(Me) or methoxybenzoyl; A.sub.1 is D-
 or L-arginine and D-lysine; A.sub.2 is lysine or arginine; A.sub.3 is
 leucine or isoleucine; A.sub.4 is leucine, isoleucine, methionine, or
 valine; A.sub.5 is methoxybenzoyl-ethyl-Gly, methoxybenzoylmethyl-D-Ala,
 Tyr(Me), Trp, Tyr, Leu, Lys, Arg, 4' substituted Phe (4' F, 4' I, 4' Cl,
 4'NO.sub.2), D-His, D-Lys, D-Arg, D-Leu, D-Pro, or D-Trp; A.sub.6 is
 isoleucine; with the proviso that not all of the A.sub.1 -A.sub.6 are in
 the L-configuration; and Tc is isoleucineamide, D-leucine-amide,
 D-valineamide.
 Some other of the melanocortin receptor antagonists of the present
 invention have also previously been shown to have anti-inflammatory
 properties. It is not known whether the previously known anti-inflammatory
 properties are related to antagonism on melanocortin receptors. Indeed, as
 will be hereinafter discussed, in some instances peptides useful in
 accordance with the present invention as melanocortin receptor antagonists
 do not have significant anti-inflammatory properties.
 Agouti-Related Protein (ARP) Fragment
 In practicing the present invention, we have found that the amidated
 carboxyl-terminal ARP fragment (83-132)-NH.sub.2 is potently active with
 an inhibitory equilibrium disassociation constant (K.sub.i) of 0.7 nM for
 the frog melanophore receptor. The sequence of this fragment (SEQ. ID
 NO:2) is:
EQU SSRRCVRL HESCLGQQVP CCDPCATCYC RFFNAFCYCR KLGTAMNPCS RT
 Human ARP and human ASP are two proteins found in tissues which have the
 property of antagonizing melanocortin receptors. Currently, these proteins
 are made by recombinant methods but the structural features of the
 proteins which determine MCR antagonism are not understood.
 As will be further described, the peptide fragments of human ARP consisting
 of the amidated form of (83-132) were synthesized on an Applied
 Biosystem's 433A automated peptide synthesizer (Foster City, Calif.) using
 para-methyl-benzylhydrylamine resin as the solid support and standard
 solid-phase F-moc methods. N-terminal F-moc amino acids were deprotected
 with 20% piperidine in dimethylformamide and coupled with
 o-benzotriazole-N,N,N',N'-tetramethyl-uronium hexa-fluorophosphate in
 1-hydroxybenzotriazole. Peptide was cleaved from resin with hydrogen
 fluoride, washed with diethyl ether, extracted with water/acetonitrile,
 and cyclized at pH 8.5. Preparatory HPLC separations of the extracts
 yielded a peptide which was 99% pure by analytical HPLC. MALDI-TOF mass
 spectra of the linear fragments gave molecular weights which corresponded
 to theoretical values. As yet, the precise arrangement of disulfide
 bridges within ARP and its C-fragment, which contain 11 and 10 Cys,
 respectively, are undetermined.
 In FIG. 2, the activity of ARP-(83-132)-NH.sub.2 as an antagonist of
 .alpha.-MSH (0.1 nM) MC in frog melanophores is shown. The EC.sub.50 of
 .alpha.-MSH alone is 0.03 nM. In the presence of ARP fragment, 30 nM, the
 EC.sub.50 of .alpha.-MSH is increased to 19 nM. This parallel shift is
 indicative of competitive binding for the MC receptor site.
 A Schild plot regression analysis of ARP (83-132)-NH.sub.2 illustrates the
 potency of this peptide fragment. The K.sub.i value of 0.21 nM is less
 than that of dynorphin A peptides and mystixin peptides, but comparable to
 the intact protein.
 Ligands for an Orphan Opioid Receptor
 An orphan opioid receptor (ORL1) was recently identified and its endogenous
 ligand characterized and called nociceptin/orphanin FQ.
 Nociceptin/orphanin FQ is a 17-amino acid residue peptide with structural
 homology to dynorphin A(1-17). C. T. Dooley, C. Spaeth, L. Toll, and R. A.
 Houghten presented an abstract at the 27th Meeting of the International
 Narcotics Research Conference, abstract TU13, Long Beach, Calif. 1996;
 cited in detail in Trends in Pharmacological Sciences, 18, pp. 293-300
 (August 1997), (article by G. Henderson and A. T. McKnight, "The Orphan
 Opioid Receptor and its Endogenous Ligand--Nociceptin/Orphanin FQ") in
 which they used combinatorial libraries of hexapeptides to screen for and
 to find ligands for the ORL1 receptor. A ligand was identified,
 Ac-Arg-Tyr-Tyr-Arg-Trp-Lys-NH.sub.2, SEQ. ID NO:3, which exhibited
 affinity and agonist activity at the ORL1 receptor.
 We have now found that this ligand, as well as several analogs prepared
 with modifications to the N-terminus thereof, exhibits activity as a
 melanocortin receptor antagonists. One of these ligands has a potency
 similar to dynorphin A(1-13)-NH.sub.2. These data are shown in Example 4.
 EXPERIMENTAL
 A standard procedure for evaluating MCR antagonists is to use cells which
 bear such receptors and are activated by .alpha.-MSH, and to examine the
 shift in concentration-response curves to .alpha.-MSH in the presence of
 the antagonists. Examples of such methods are given in Quillan and Sadee
 (Pharm. Res., 14, pp. 713-719, 1997) and Ollmann et al. (Science, 278, pp.
 135-138, 1997).
 Pure cultures of melanophores derived from Xenopus laevis embryos have been
 used to study MCR. Xenopus melanophore cell cultures are maintained in 5
 parts Leibovitz's L-15 medium (Gibco), 2 parts FBS, 3 parts
 deionized-distilled H.sub.2 O, and phosphate-buffered saline. Cultured
 melanophore cells retain their ability to aggregate (lightened state) or
 disperse (darkened state) cytoplasmic pigment vesicles in response to
 hormonal stimulation, melatonin causes aggregation whereas .alpha.-MSH,
 vasotocin, histamine, norepinephrine, and serotonin cause dispersion.
 Concentration-response curves were measured in melanophores by quantifying
 the change in absorbance at 630 nm through a monolayer of cells using a
 BT2000 Microplate Reader (FisherBiotech, Pittsburgh, Pa.) after treatment
 with test compounds, and curves were fit using a logistic equation.
 Initial absorbance (A.sub.i) was measured upon addition of test compounds
 after 90 minutes pretreatment with carrier alone (7 parts L-15 medium in 3
 parts distilled-deionized H.sub.2 O containing 0.5% (w/w) bovine serum
 albumin), or carrier plus 1 nM melatonin, to initiate tests from either
 the dispersed state (high absorbance) or an aggregated state (low
 absorbance), respectively.
 A second method for studying MCR antagonists is to use mammalian cells
 stably transfected with the cDNA for various human MCR. These transfected
 cells manifest an increased CAMP response to .alpha.-MSH stimulation and
 agents which antagonize MCR will block this stimulating effect of
 .alpha.-MSH. The inhibitory effects of antagonists can be quantified by
 the change in the effective concentration (EC.sub.50) of .alpha.-MSH i the
 presence of antagonist, or by computation of K.sub.i value by a Schild
 regression analysis.
 EXAMPLE 1
 Dyn A(6-12) Analogs Demonstrate MCR Antagonist Activity by Blocking
 .alpha.-MSH Activation of Xenopus MC Receptors
 Dynorphin A peptides have no direct stimulatory effect on MC receptors
 endogenous to xenopus melanophores but inhibited activation of these
 receptors by .alpha.-MSH. To compare the relative potency of various
 dynorphin A(6-12) analogs, IC.sub.50 measurements were made against a
 fixed concentration (600 pM) of .alpha.-MSH. Melanophores were used
 because dynorphin peptides were of comparable potency at both frog and
 human MC receptors.
 A graphical illustration for the determination of the antagonist potency,
 as measured by Ki, is shown for the analog SQW-5 (FIG. 1). The
 melanophores in the presence of 600 pM of .alpha.-MSH have an absorbance
 200% greater than the initial value (A.sub.i). Increasing concentrations
 of SQW-5 block the .alpha.-MSH effect and the increased light passing
 through the aggregated melanosomes is measured by the decrease in
 absorbance. The logistic fit of the data gives the IC.sub.50 value which
 is the statistically most reliable point for measurement of potency.
 None of the dynorphin A(6-12) analogs tested caused stimulation (darkening)
 of melanophores, but most were active as antagonists against .alpha.-MSH
 with IC.sub.50 values ranging from 17 to 1600 nM. The results are shown in
 Table 1. The more potent analogs (SQW-1, 4, 5, 6) with IC.sub.50 of 17 to
 42 nM were active as anti-edema agents, but the correlation of anti-edema
 to MCR antagonist potencies was weak. For example, the good MCR antagonist
 potency of SQW-6, 8, and 13 was not matched by a corresponding enhanced
 potency in suppression of edema. In analogs singly substituted with
 alanine, reduction of the number of side-chain carbons in residue 11
 significantly decreased MCR antagonist potency. Substitution of the L-Arg
 residues by the D-Arg isomer retained the MCR antagonist activity and the
 anti-edema potency. These analogs have not been previously described.
 To further quantify the activity of candidate prototypes, dose-response
 curves were measured in the presence of several concentrations of
 antagonists and Schild plots were used to estimate equilibrium
 dissociation K.sub.d values for SQW-1 and SQW-5. This analysis yielded
 K.sub.i values of 27 and 12 nm, respectively, which are significantly
 lower than values of 71 and 79 nM found for dynorphin A(1-13)-NH.sub.2 and
 dynorphin A(2-13), respectively. The Schild regression slopes for SQW-1
 and SQW-5 are almost unity which indicate a mechanism of competitive
 inhibition at the MCR for the two analogs.
 Dynorphin A(2-17) lowers blood pressure of anesthetized rats. It was
 therefore of interest to determine if a prototype dynorphin A(6-12)
 analog, with a lower molecular weight, would exhibit similar hypotensive
 activity. The dose-effect curves of dynorphin A(2-17) and
 p-anisoyl-[D-Leu.sup.12 ] dynorphin A(6-12)-NH.sub.2 on the mean arterial
 pressure of pentobarbital-anesthetized rats were compared. Analysis of the
 integrated hypotensive activities, measured by the area-under-curve, for
 the two peptide, indicated that, on a molar basis, the two peptides had
 similar hypotensive activities. The potencies for anti-edema and
 hypotensive actions were not directly correlated as dynorphin A(2-17) was
 at least twice more active than p-anisoyl-[D-Leu.sup.12 ] dynorphin
 A(6-12)-NH.sub.2 as an anti-edema agent. The anti-edema activities were
 detectable at doses which did not produce significant hypotension.
 The selectivity of the MC receptor antagonism by dynorphin A(6-12) analogs
 was examined. Under the same experimental conditions, the dispersion of
 melanin initiated by norepinephrine, serotonin, and vasotocin were not
 inhibited by SQW-1. Peptides which inhibit heat-induced edema such as
 human CRH, urocortin, sauvagine, xenopsin, and a neurotensin(8-13) analog,
 D-Arg-Arg-Pro-Trp-Ile-Leu, were relatively inactive in the frog MCR assay
 with IC.sub.50 values of &gt;10 .mu.M.
 TABLE 1
 Melanocortin receptor antagonist activities of anisoyl-dynorphin A(6-12)
 --NH.sub.2 analogs

IC.sub.50 nM
 in Frog
 Melano-
 phore
 Analog Assay
 1 Ac-Arg-Tyr-Tyr-Arg-Trp-Lys-NH.sub.2 1050 .+-.
 170
 2 Ac-Arg-Tyr-Tyr-Arg-D-Trp-Lys-NH.sub.2 867 .+-.
 85
 3 p-Anisoyl-Arg-Tyr-Tyr-Arg-D-Trp-Lys-NH.sub.2 175 .+-.
 27
 All three peptides exhibited at least some activity as melanocortin
 receptor antagonists, with analog (3) having the best potency. Dynorphin
 A(1-13)-NH.sub.2, which was also tested, had an IC.sub.50 of 145.+-.14 nM.
 The peptide antagonists of the invention may be administered in combination
 with a pharmaceutically acceptable carrier, such as isotonic saline,
 phosphate buffer solution or the like. Topical administration is feasible
 for those preferred peptides that are relatively small.
 Compositions and excipients useful for the administration of small peptides
 through the nasal mucosa are, for example, described by U.K. patent
 application no. 8719248, published Feb. 24, 1988, applicant Sandoz
 Limited. Topical compositions with enhanced penetration through intact
 skin preferably include a potentiator, many of which are described by U.S.
 Pat. No. 4,091,090, issued May 23, 1978, inventor Sipos.
 The peptides form pharmaceutically acceptable salts and can be administered
 in salt form. Examples of suitable acids for the formation of
 pharmaceutically acceptable salts are hydrochloric, sulfuric, phosphoric,
 acetic, benzoic, citric, malonic, salicylic, malic, fumaric, succinic,
 tartaric, lactic, gluconic, ascorbic, maleic, fumaric, succinic, tartaric,
 lactic, gluconic, ascorbic, maleic, benzenesulfonic, methane- and
 ethanesulfonic, hydroxymethane- and hydroxyethane-sulfonic. Salts may also
 be formed with suitable organic pharmaceutically acceptable base addition
 salts. These organic bases form a class whose limits are readily
 understood by those skilled in the art. Merely for purposes of
 illustration, the class may be said to include mono-, di-, and
 trialkylamines, such as methylamine, dimethylamine, and triethylamine;
 mono-, di-, or trihydroxyalkylamines such as mono-, di-, and
 triethanolamine; amino acids such as arginine and lysine; guanidine;
 N-methylglucosamine; N-methylglucamine; L-glutamine; N-methylpiperazine;
 morpholine; ethylenediamine; N-benzylphenethylamine; tris
 (hydroxymethyl)aminomethane; and the like. (See for example,
 "Pharmaceutical Salts," J. Pharm. Sci., 66:1, pp. 1-19, 1977.)
 For many applications, the peptide will preferably be formulated so as to
 enhance bioavailability. For example, U.S. Pat. No. 5,514,670, issued May
 7, 1996, describes emulsions with a plurality of submicron particles, a
 bioactive peptide, and an aqueous continuous phase so as to enhance oral
 bioavailability of the peptide. Controlled release liposomal liquid
 pharmaceutical formulations for injection or oral administration are
 described in U.S. Pat. No. 4,016,100, and other liposomal/peptide
 compositions are described by U.S. Pat. No. 4,348,384. These three,
 just-noted patents are incorporated herein by reference.
 EXAMPLE 5
 Protein Synthesis
 The protein fragment of human ARP consisting of residue (83-132) in an
 amidated form was synthesized on an Applied Biosystem's 433A automated
 peptide synthesizer (Foster City, Calif.) using hydroxymethyl or
 paramethyl benzylhydrylamine resin as the solid support and standard
 solid-phase F-moc methods. N-terminal F-moc amino acids were deprotected
 with piperidine in dimethylformamide (20% v/v) and coupled using
 o-benzotriazole-N,N,N',N'-tetramethyl-uronium hexafluorophosphate in
 1-hydroxybenzotriazole (1:1 molar equivalents, in a 0.5 molar excess). The
 protein fragment was cleaved from resin with hydrogen fluoride containing
 anisole (13% v/v) and dimethyl sulfide (1.7% v/v) for 45 minutes at
 0.degree.C., washed with ethyl ether, and extracted with
 water/acetonitrile (50% v/v). The ARP-(83-132)-NH.sub.2 fragment was
 cyclized in an acetonitrile/NH.sub.4 HCO.sub.4 solution adjusted to pH 8.5
 with ammonium hydroxide for 72 hours, and extracted with Bio-Rex 70 cation
 exchange resin for 24 hours. The Bio-Rex resin was then rinsed with
 H.sub.2 O, cyclized protein eluted from the resin with 50% glacial acetic
 acid in H.sub.2 O (v/v), and analyzed by Pauly spray. Major fractions were
 purified by HPLC using a C-18 column over a standard 100% H.sub.2 O (0.1%
 trifluoracetic acid) to 100% acetonitrile (0.1% trifluoracetic acid)
 gradient, rotoevaporated, and lyophilized. Samples were then analyzed
 using matrix assisted laser desorption ionization-time of flight
 (MALDI-TOF) and electrospray mass spectrometry.
 Testing Synthetic ARP Fragments for Functional Antimelanotropic Activity
 Synthetic human ARP fragments were assayed for antagonist activity using
 Xenopus laevis dermal melanophore cell preparations as described by
 Quillan et al., Proc. Natl. Acad. Sci. USA, 92, pp. 2894-2898 (1995), and
 Quillan and Sadee, Pharm. Res., 14, pp. 713-719 (1997). Cultured
 melanophores can translocate cytoplasmic pigment vesicles in response to
 hormonal stimulation by a .alpha.-MSH, norepinephrine, vasotocin,
 pituitary adenylylcyclase activating peptide (AP), and serotonin. These
 compounds cause pigment dispersion and cell darkening. Pigment aggregation
 and cell lightening can be stimulated by melatonin. Melanophore responses
 were determined by measuring absorbance (OD) ratios taken through a
 monolayer of cells using a FisherBiotec BT2000 Microplate Reader
 (Pittsburgh, Pa.). Absorbance was measured before and after a 90 minute
 exposure to test peptides and then expressed as percent of initial
 absorbance. (Measurements were taken after 60 minutes pretreatment with 10
 nM melatonin to initiate readings from an aggregated state.)
 Protein Synthesis and Mass Spectra Analysis
 Preparatory HPLC separations of synthetic extracts yielded protein
 fragments which were 99% pure by analytical HPLC. MALDI-TOF mass
 spectrometric analysis of the linear ARP fragments gave a molecular mass
 of 2984.5 daltons for (25-51) and 3282.5 daltons for (54-82), both of
 which correspond to expected theoretical values. The molecular mass of
 cyclized ARP-(83-132)-NH.sub.2 as determined by MALDI-TOF and electrospray
 mass spectra was 5678.04 (SE 0.1%) and 5677.0 (SE 0.01%) daltons,
 respectively. These values are consistent with the calculated theoretical
 mass of 5676.7 daltons for protein containing a full complement of five
 disulfide bonds.
 Antagonist Activity of the ARP Fragment
 Synthetic ARP-(83-132)-NH.sub.2 produced a dose-dependent suppression of
 .alpha.-MSH-induced dispersion of pigment in melanophore cells. The
 inhibitory equilibrium dissociation consent (K.sub.i) of human
 ARP(83-132)-NH.sub.2 as determined by Schild regression analysis, was 0.7
 (SE 0.4) nM, which is comparable to K.sub.4 values reported for ASP and
 ARP purified by recombinant means. Each of the three protein fragments had
 no "agonist like" activity in melanophore cells; when applied to
 melanophores in the absence of .alpha.-MSH they did not cause pigment
 dispersion in the presence of 10 nM melatonin. The specificity of ARP for
 MC receptor-mediated response was tested by examining ARP(83-132)-NH.sub.2
 for interaction with other ligands known to induce pigment dispersion.
 ARP(83-132)-NH.sub.2 (30 nM) had no effect on concentration-response
 curves evoked with norepinephrine, serotonin, AP-27, and Arg.sup.8
 -vasotocin, indicating that antagonism is selectively directed towards
 responses initialized by .alpha.-MSH stimulation of MC receptors.
 These results demonstrate that a synthetic fragment of human ARP based on
 the amino acid sequence (83-132) can potently antagonize MC receptor
 function in Xenopus melanophores.
 Electrospray mass spectrometry is a very accurate method of determining
 protein molecular mass and purity, but proteins containing disulfide bonds
 can produce multiply charged ions and fragment ions. This problem is
 accentuated with ARP(83-132)-NH.sub.2 because of its multiple disulfide
 sites. Although the major peak in the electrospray mass spectrum was
 5677.0 daltons, it contained a number of minor peaks not amenable to easy
 interpretation. None of the peaks, however, were consistent with protein
 containing less than five disulfide bonds; this was expected since
 cyclization reactions usually proceed to completion. To help better
 resolve the origin of the minor peaks we retested ARP(83-132)-NH.sub.2
 using MALDI-TOF mass spectrometry, a procedure less prone to
 fragmentation, and found only one major peak around 5678.04, which roughly
 corresponded to the expected theoretical mass of 5676.7 daltons. Although
 less precise, MALDI-TOF confirmed that minor peaks seen with the
 electrospray procedure were likely due to fragmentation. Thus, an
 ARP(83-132)-NH.sub.2 structure containing a full complement of disulfide
 bonds appears to account for the biological activity observed in Xenopus
 melanophores.
 It is to be understood that while the invention has been described above in
 conjunction with preferred specific embodiments, the description and
 examples are intended to illustrate and not limit the scope of the
 invention, which is defined by the scope of the appended claims.