JPH0611710B2 - Pharmaceutical composition containing alpha-MSH analogue - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to pharmaceutical compositions for administering compounds that stimulate the production of melanin in vertebrates by topical administration of certain alpha-MSH analogs.

In vertebrates, their skin color, fur, and characteristics are determined by the number and distribution of cells with certain colors, such as melanocytes, and the number and distribution of melanocytes are genetically under control. . Melanocytes in mammals are localized in the substratum of the epithelium, the dermal-epithelial junction, and within the hair follicles. The synthesis of pigment (melanin) in these melanocytes is controlled by the activity of tyrosinase, an enzyme localized in the organelles of cells, premelanosomes. Upon activation of tyrosinase, eumelanin (brown-black) or phaeomelanin (yellow-red) pigments are deposited within organelles; after complete melanization, premelanosomes are color-dependent Melanosom
e), more particularly known as (eumelanosomes or pheomelanosomes) [see Fitzpatrick, T.
B., Y. Hori, K. Toda, M. Seiji, Jap. J. Derm. 79: 278 (196
9)]. Melanosomes are supplied to the surrounding keratinocytes of the skin or to cells within the growing hair shaft by a method known as the secretion of cytocrine.

Although melanin synthesis and hair patterns are genetically expressed, hair follicle melanogenesis and hair color changes in mammals are associated with alpha-melanotropin (also alpha-melanocyte stimulating hormone, or alpha-MSH). Known as), the formula: Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro.
It may be hormonally controlled by tridecapeptide of -Val-NH _2. This hormone is a mediator of the pituitary gland (pars intermedi
a) and stimulates adenylate cyclase activity, tyrosinase activity, and subsequent production of melanin (see Hadley, ME, CBHoward, VJHruby,
TK Sawye, and YCS Young, Pigment Cell &: 323 (19
80)].

In humans, alpha-MSH is only found in the fetal pituitary gland, not in adults. In adult humans, some level of melanin production is genetically determined and constitutively present. Fluctuating melanin synthesis above this baseline level can result in UV stimuli, such as
It is directly dependent on the sun's rays; exposure to high levels of sun rays initiates an increase in the production of melanin, which eventually darkens the skin. This response may be an evolutionary adaptation that protects a person against the aging and mutagenic properties of UV. Exposure to low levels of UV results in low levels of integumental melanin synthesis, fading of skin color, and a reduction in the blocking effect that allows the skin to absorb greater amounts of radiation. Adults do not synthesize alpha-MSH in the pituitary, but human melanocytes will respond to the hormone (and its racemized preparation).

Although skin hypopigmentation in humans results from local defects in the production of melanin within melanocytes, the etiology for many such disorders of hypopigment remains unknown.

It is estimated that almost 1% of the world's population suffers from some form of hypopigmentation dysfunction. Alpha-MSH and certain analogs of alpha-MSH, when administered subcutaneously,
It is known to cause darkening in amphibian reticulum animals and that alpha-MSH is associated with darkening of the skin in adrenalectomized humans when administered intramuscularly (Lerner, AB, and JSMcGuire, NEJMed.
270: 539-546 (1964)], but these routes of administration are inadequate for repeated administrations necessary to achieve and maintain the desired effect. Prior to the present invention, no suitable means of treating these hypopigmentation disorders was known.

It has now been shown that alpha-MSH and certain analogs of alpha-MSH can be effectively administered transdermally, and that these compounds will reach the active form of melanocytes and stimulate melanin production. It's been found. Thus, according to the present invention, a topical composition comprising an analog of alpha-MSH is applied to impair hypopigmentation dysfunction, e.g. post-inflammatory hypopigmentation, e.g. , Vitiligo,
It is now possible and convenient to achieve idiopathic drop melanin reduction; and normalization of nerve depigmentation.
Furthermore, it is now possible to achieve graying of gray hairs by aging by topical administration of an analogue of alpha-MSH. It is also possible to increase the value of the fur of commercial animals by darkening these analogues via topical administration. Furthermore, it is now possible to achieve darkening of the skin in the total absence of sun or UV radiation.

Therefore, it is an object of the present invention to stimulate the production of melanocytes in adult humans in the total absence of sun or UV light radiation, thus requiring exposure to high levels of UV aging and mutagenic effects. Is to provide a safe method of sunburn avoidance.
Not only is the damaging effect of UV previously required to obtain tannins avoided, but also once tannins are obtained, it protects the skin against subsequent exposure to UV radiation. Will promote.

The present invention provides a pharmaceutical composition for the administration of a compound that stimulates the production of melanin in a vertebrate, in an amount sufficient to cause the production of melanin in said vertebrate,
The following groups, (1) Formula Ac-Ser-Tyr-Ser-M-Glu-His-D-Phe-Arg-Trp-Gly-Lys-Pro
-Val-NH _{2 wherein} M is selected from the group consisting of Met, Nle and Cys, (2) Formula R ₁ -WD-Phe-YZR ₂ (wherein R ₁ is Ac-Gly, Ac-Met-Glu, Ac-Nle-Glu and Ac-
Selected from the group consisting of Tyr-Glu, W selected from the group consisting of His and D-His, Y selected from the group consisting of Arg and D-Arg, Z selected from the group consisting of Trp and D-Trp And
R ₂ is an alpha-MSH analogue having NH ₂ , Gly-NH ₂ and Gly-Lys-NH ₂ (wherein this analogue is A
c-Nle-Glu-His-D-Phe-Arg-Trp-Gly-Lys-NH ₂ ), and (3) the following [Nle ⁴ , D-Phe ⁷ ] -alpha-MSH [Nle ⁴ , D-Phe ⁷ ] -alpha-MSH _4-10 [Nle ⁴ ,, D-Phe ⁷ ,, D-Trp ⁹ ] -alpha-MSH _4-11 [Nle ⁴ , D-Phe ⁷ ] -alpha-MSH _4-9 There is provided a composition comprising an alpha-MSH analog selected from the group consisting of: a compound selected from: and a pharmaceutically acceptable carrier for topical administration.

These compounds can be synthesized according to the procedures set forth in US Pat. No. 4,457,864 and US Pat. No. 4,485,039, or according to the well known methods used in preparing synthetic alpha-MSH. These compounds have an alpha character in one or more of the following properties:
-Better than MSH: potency as measured by in vivo and in vitro frog and / or lizard assays; duration of in vivo action in such assays; and / or resistance to blood enzyme degradation by serum enzymes.

The compounds useful in the present invention can be administered transdermally or via other epithelia. Several other routes of administration can also be used, such as intraocular, intranasal, oral and intravenous. These compounds are formulated into suitable compositions according to the intended means of administration, according to methods and procedures well known in the art. For example, a compound suitable for use in the invention may be formulated or formulated into a preparation, eg, a preparation, eg, depending on the desired method for administration of these ingredients to an individual using a variety of conventional bases. It can be a cream, ointment, gel, lotion or spray. In the preparation of these preparations, the compositions can also be mixed with customary thickeners, softeners, surface-active agents, pigments, perfumes, preservatives, fillers and emulsifiers, all of which are It is well known and commonly used in the preparation of formulations for transdermal or other administration. Typically these non-active ingredients will make up a larger portion of the final preparation.
Preferably, the composition is configured to allow for slow or timed release delivery.

A further understanding of the invention can be obtained from the following non-limiting examples. In these examples, all temperatures and temperature ranges are from the Celsius system, and ambient or room temperature is about 20 ° C. Percentages or (%) are weight percentages and moles are gram moles.

Example 1 Preparation of Compounds All compounds shown in Examples 1 to 3 were prepared according to Sawyer et al., PN.
S.77: 5754-5758 (1980), or Sawyer et al., PNSUS.
A.79: 1751-1755 (1982), or Sawyer et al., J. Med. Chem.
25: 1022-1027 (1982) and synthesized and purified by solid phase synthesis.

Briefly summarized, each compound is first prepared p- methylbenzhydrylamine resin was continuously coupling a desired amino acid as its N ^alpha -Boc derivative thereto. The groups on the reactive side chain of each trifunctional amino acid were protected by the incorporation of appropriate protecting groups. After all amino acid residues were attached to the resin, the amino terminus of the peptide-resin was acetylated. Following acetylation, the protected peptide was cleaved from the resin and all protecting groups were removed. For cyclic disulfide compounds, the sulfhydryl form was oxidized to the cyclic disulfide compound by oxidation of ferricyanide. The crude compound was purified by ion exchange chromatography on silica gel using the appropriate solvent. The value of optical rotation is Perkin-Elmer
(Perkin-Elmer) 241 MC Polarimeter measured at mercury-green line (546 nm).

Alpha-M used for comparison purposes in Examples 1-3
SH, Yang et al., Int. J. Pept. Protein Res. 15: 130-138 (1
980). [Nle ⁴ ] -alpha-MSH was also used for comparative analysis and was purchased from Penninsula Laboratories (San Carlos, Calif.) Or Hruby et al., J. Med. Chem. 23. : 1432-1437 (1980).

Example 2 The biological effects on in vivo and in vitro melanocyte dispersion were demonstrated by frog (Rana pipiens) and lizard (Anolis caroli).
niensis). [See Hadley et al., Scie.
nce213: 1025-1027 (1981)); similar test is performed by Shimume, E.
A skin assay as described in ndrochrinology, 54: 553-560 (1954) was used for duration of ability to stimulate melanocyte dispersion in vitro. The in vitro results are listed in Table I.

The resistance of the compounds according to the invention to the degradation of serum proteolytic enzymes is determined in Corning flasks containing Ham's F-10 medium containing 10% horse serum and 2% fetal calf serum. It was verified by incubating the compounds at 37 ° C under aseptic conditions. In this procedure, samples of medium containing peptide (10 nmol) were taken at time zero and 24, 48 and 72 hours. Samples were immediately frozen and assayed for biological activity using an in vitro frog skin bioassay. Using this protocol, resistance to degradation of serum proteolytic enzymes was clearly demonstrated.

The salient properties of the compounds of the invention are also due to existing diagnostics,
They will be useful as alternatives to alpha-MSH and [Nle ² ] -alpha-MSH in therapeutic and basic research programs. In the area of diagnostic procedures, the compounds of the invention, in particular those which have been radioiodinated or bound to gamma-ray emitters, are associated with melanoma cells on the basis of their association with melanotropin receptors in melanoma. Exceptionally well suited for looking and / or differentially characterizing. It is known that the serum stability of the compounds of the present invention makes them a prime candidate in the proposed selective drug delivery system, where the target tissues have high concentrations of melanotropin receptors. The relatively high potency and prolonged activity of the compounds of the invention in phenomena associated with color change stimulate hormones and their synthetic analogs,
It is expected to be repeated with respect to other biological effects previously recognized for naturally occurring melanocytes.

Examples 3 and 4 show topical or subcutaneous alpha-MSH, [Nl, for hair follicle melamine development in mice.
e ⁴ , D-Phe ⁷ ] -alpha-MSH, [Nle ⁴ , D-Phe ⁷ ] -alpha-MSH ^4-10 and [Nle ⁴ , D-Phe ⁷ ] -alpha-MSH ^4-11
Example 3 Subcutaneous Injection Alpha-MSH and [Nle ⁴ , D-Phe ⁷ ] -alpha-MSH were synthesized. Jackson Laboratories
atories) (Bar Harbor, Maine), mice (C57BL / 6JA ^y ) originally obtained were kept in our laboratory with continuous access to food and water.

Alpha-MSH is (C
57BL / 6JA ^y ) yellow mice were shown to stimulate hair follicle melanogenesis. Alpha-MSH, when injected in mice to stimulate melanogenesis, produced the analog [Nle ⁴ , D
-Phe ^7] - compared to alpha -MSH may require 10,000 times higher concentration was found. A single injection (0.05 ml) of the analogue (10 ^-6 mol) caused a shift from the production of pheomelanin to the synthesis of eumelanin in the hair roots after 24 hours, and eumelanin development was 96 hours after a single injection. Consecutive. The color of the animal's entire coat is [Nle ⁴ , D-Phe ⁷ ]
-It was found to be altered by daily 14-day injections of alpha-MSH. Melanotropin induces melamine development only during hair growth, and as long as hair stays in the anagen (proliferation) phase of the hair cycle,
Melanosomes are continuously integrated into the cells of the hair shaft.
The synthesis of eumelanin in wide-growing hair only results in the formation of patterns that show specific areas on the animal during the anagen phase of the hormone injection.

Example 4 Topical Administration Each of alpha-MSH and [Nle ⁴ , D-Phe ⁷ ] -alpha-MSH was dissolved in a vehicle consisting of propylene glycol (26 wt% PEG400 and 74 wt% PEG33509. Postnatal 48. The hairy area (2-3 cm ² ) on the back of the back of the day mouse was swollen; in this age mouse, the hair follicles in the back of the back were telogen (tel) of the hair cycle.
ogen) (rest) period. Peeling of hairs from resting follicles stimulates the growth of new hairs, and by 7 days these hairs begin to grow from the surface of the skin. Mouse cardboard collars were fitted to prevent ointment spread to the study and untreated areas of the skin and then individually caged. 〔Nl
An ointment containing e ⁴ , D-Phe ⁷ ] -alpha-MSH (0.5 ml, 10 ⁻⁶ mol) was applied daily to the puffy area as new hair grew. Emerging hair samples were applied before topical application of melanotropin and every 24 hours thereafter for 7 days.

Microscopic examination revealed eumelanin in the hair root 24 after application of the analog. Continuous daily application of the analogue
It resulted in continuous synthesis of eumelanin and transferred the melanosomes to cells that moved into the shaft of the hair. Hair follicle melanogenesis was not limited to hair roots at the treated site, but was observed microscopically in hair roots taken from untreated areas of animals in which hair growth was progressing. Eumelanin was not visible in the hair taken from control mice. Electron microscopy is [Nl
e ^4, D-Phe ^7] - confirmed the presence of Maranosomu in Eumeranin in melanocytes of the hair follicles of mice treated with alpha-MSH. Only pheomelanosomes were present in mice treated with control ointment containing no melanotropin. Alpha-MSH was unable to induce the development of eumelanin even when applied to the skin of mice for as long as 14 days at the same concentration as the analogue (10 ^-8 molar or less). Alpha-MSH applied at higher concentrations (10 ^-5 to 10 ^-7 molar) did indeed cause local and systemic follicular melanogenesis.

These examples demonstrate the transdermal delivery of both the natural hormone alpha-MSH and the analog [Nle ⁴ , D-Phe ⁷ ] -alpha-MSH. The action of the analogues was determined by the peptide as determined in several bioassays, alpha-M.
It may be related to the fact that it is about 10 to 1000 times more potent than SH. In addition, the analogue is a rapidly inactivating alpha-M
Unlike SH, it is resistant to enzyme inactivation. Both male and female mice responded similarly to melanotropins. Transdermal delivery of analogs occurred equally well when applied to the shaved area of skin or when applied to the base of the hair. As these results demonstrate, the transfer of melanotropins proceeded through an intact area of skin. Mimicking melanogenesis is [Nl
The transdermal transfer of the peptide was probably much more rapid, although it could be seen 24 hours after the application of e ⁴ , D-Phe ⁷ ] -alpha-MSH. Because activation of the activity of tyrosinase is a genomic event involving both transcriptional and translational processes known to occur only many hours after contact with melanotropin.

Example 5 Alpha-MSH or [Nle ⁴ , DP in propylene glycol
he ⁷ ] -Alpha-MSH and 1-dodecylazacycloheptan-2-one, a synthetic polar surfactant known to enhance transdermal penetration of other compounds Example 4 was repeated. Reference, eg St
oughton, RB, Arch.Dematol.118: 479 (1982) ； Stougho
nRB and WOMcClure, Drug Devel.Indust.Pharm.9
(4): 725 (1983); (Azone). The vehicle was solid at room temperature, but when applied to the shaved skin of mice, it immediately softened and spread easily.

In this example, the azone concentration was 1.8% and the final concentration of MSH was 0.001% (1 mg / 100g cream base) or 0.003%.

Example 6 Mice Mice with some pigmentation on the skin were treated with the same cream as in Example 5. The treated area remained unchanged, but the tail darkened significantly.

The tails of treated animals were darkened with or without direct topical treatment. Histological examination revealed increased pigment in areas of non-follicle melanocytes. These results led to the following conclusions: (1) [Nle ⁴ , D-Phe ⁷ ] -alpha-MSH have systemic effects after topical administration, and (2) non-hair follicle melanocytes have these effects. It is sensitive to the melanogenic action of melanotropins.

Example 7 The Ames microbiological assay was used for these tests. These tests were performed with and without the microsomal enzyme / NADPH generation system. Briefly, this test measures the number of backmutations that occur in a particular histidine-dependent strain of Salmonella typhimurium. Positive strains (mutations) are scored as the number of revertants. The revertants are capable of forming colonies in histidine-deficient growth medium after exposure to mutagens.

This particular test involved three melanotropins
T: Alpha-MSH, [Nle^Four, D-Phe⁷] -Alpha-MSH,
And [Nle^Four, D-Phe⁷] -Alpha-MSH_4-10-NH₂. Everything
All substances are sterile 0.9% sodium chloride (preservative
10) in^-2,Ten^-3And 10^-FourDissolves in molarity
did. The TA-98 strain of S. typhimurium was used, and the experiment
S-9 rat liver microsomal enzyme and NADPH generation system
Was performed with and without exposure to. This enzyme's power
Kuter metabolizes some compounds into mutagenized species
Can be activated. In addition to melanotropins
, Doxorubicin (Adriamycin )
Were also tested at the same time. Doxorubicin
in) is highly mutagenic in this assay.
Known to be. It is an antibiotic added to DNA
It Thus, to act it as a positive control
included. The following table shows the results of these tests. Return collision
However, for the actual evaluation of mutants, specific test compound
One sex was blind.

As the results of these preliminary experiments suggest, both natural and synthetic melanotropins are mutagenic in vitro. Adriamycin was significantly mutagenic and this was increased after exposure of S-9 microsomes to the enzyme. Thus, bacterial assays and metabolic activation systems appear to work well, and mutagenicity with melanotropins does not appear to be an artifact.

Example 8 Toxicity To 6 rats, [Nle ⁴ , D-Phe ⁷ ] -alpha-MSH (0.2 ml of a 10 ⁻⁴ molar solution of peptide; 0.0 daily for 5 consecutive days).
2 mg) was injected. Four female and two male rats were clearly not adversely affected by this treatment. On the last day of treatment, animals were sacrificed and serum samples were obtained. Blood glucose analysis showed that the levels of glucose were not different between control and experimental animals. If melanotropins stimulate the adrenal gland, it would be expected that blood glucose levels would change (as in Cushing's disease in humans).

Mice injected with melanotropin at the same unusually high concentration of hormone were clearly unaffected.
Their behavior did not appear to change during the observation time.

Additional work was done with 0.25 ml of 10 ^-3 mol [Nle ⁴ , D-Phe ⁷ ].
-Performed in male CD-2 injected alpha-MSH ip. Core temperature was monitored by a rectal probe connected to a digital heat transducer. There was no invariant change in core temperature, with core temperatures averaging 35.2 ° C until 24 hours post injection. The level of activity is
It was also normal in these animals. 24 after administration
At time, mice were killed by rapid cervical dislocation and blood was drawn for analysis of liver and kidney chemicals and for characterization of hematological indices. These studies showed no changes in liver or kidney function. Serum levels of electrolytes were also normal. Analysis of whole blood showed no hematological toxicity, and white blood cells, red blood cells and platelets were at normal levels. The properties of red blood cells (size, level of hemoglobin) were normal. Body weight did not change in any of the animals in this short time study.

Example 9 Model of human cadaver and excised skin for transdermal delivery of melanotropins Human cadaver and mastectomy, facelift (faca li)
ft) using excised skin, such as alpha-MSH
[Nle ⁴ 4H, D-Phe ^7] of the analogue - to determine the in vitro penetration of alpha-MSH. Studies have shown that in vitro transdermal delivery in these types of skin accurately predicts the in vivo case.

Subcutaneous tissues are removed from these skin samples and they are placed on a specially designed permeation cell [LGA] for 24 hours at 38 ° C. Alpha-M in PFG
An analog of SH is applied to the epithelial surface. The skin surface of the sample is immersed in saline. The amount of analog of alpha-MSH that goes across the skin into saline is quantified by a frog skin bioassay.

The data from these experiments support the following conclusions: 1) Mouse skin is well penetrated by the analogs and is relevant for in vivo studies; 2) Full thickness abdominal chest human skin. (Epithelium + dermis)
Is not completely penetrated. This is an important finding for topical administration (ie the drug can remain localized in the skin).

3) The full-thickness skin of the human scalp is penetrated by the analogue (probably associated with the presence of multiple hair follicles).

4) Human slit skin sample (0.5mm and 0.7m
m) is permeated by the analogs, suggesting that the stratum corneum of the skin is not the major barrier to the analogs and therefore the analogs can reach melanocytes in the epithelium.

Thus, although preferred embodiments of the invention have been illustrated and described, the invention is susceptible to variations and modifications and is therefore not limited to the precise terminology set forth, but adapting the invention to various applications and conditions. It is to be understood that such changes and modifications can be made in order.
Accordingly, such changes and modifications are properly encompassed within the full scope of equivalents and, therefore, within the scope of the following claims.

Thus, the present invention and methods of making and using the same have been described in such complete, clear, concise, and precise terms so that one of ordinary skill in the art would understand the present invention.
And it could be made and used.

 ─────────────────────────────────────────────────── ─── Continued Front Page (72) Inventor Dol, Robert United States, Arizona 85749, Toson, South Avenida Kona Rare 1130 (72) Inventor Levin, Norman United States, Arizona 85718, Toson, No -Sukamino Arco 6202 (56) Reference European publication 146113 (EP, A) Journal of Chromatography, Vol. 314 (1984), P. 313-321 Helvetica Chimica Acta, Vol. 58, Fasc. 6, No. 168 (1975) P. 1528-1535

Claims (8)

[Claims] 1. A pharmaceutical composition for the administration of a compound that stimulates the production of melanin in a vertebrate, the group of the following: (1) which is in an amount sufficient to cause the production of melanin in the vertebrate. Formula Ac-Ser-Tyr-Ser-M-Glu-His-D-Phe-Arg-Trp-Gly-Lys-Pro
-Val-NH _{2 wherein} M is selected from the group consisting of Met, Nle and Cys, (2) Formula R ₁ -WD-Phe-YZR ₂ (wherein R ₁ is Ac-Gly, Ac-Met-Glu, Ac-Nle-Glu and Ac-
Selected from the group consisting of Tyr-Glu, W selected from the group consisting of His and D-His, Y selected from the group consisting of Arg and D-Arg, Z selected from the group consisting of Trp and D-Trp And
R ₂ is an alpha-MSH analogue having NH ₂ , Gly-NH ₂ and Gly-Lys-NH ₂ (wherein this analogue is A
c-Nle-Glu-His-D-Phe-Arg-Trp-Gly-Lys-NH ₂ ), and (3) the following [Nle ⁴ , D-Phe ⁷ ] -alpha-MSH [Nle ⁴ , D-Phe ⁷ ] -alpha-MSH _4-10 [Nle ⁴ ,, D-Phe ⁷ ,, D-Trp ⁹ ] -alpha-MSH _4-11 [Nle ⁴ , D-Phe ⁷ ] -alpha-MSH _4-9 A composition comprising an alpha-MSH analog selected from the group consisting of: a compound selected from: and a pharmaceutically acceptable carrier for topical administration. 2. The compound is [Nle ⁴ , D-Phe ⁷ ] -alpha-MSH.
The composition of claim 1 which is _4-10 . 3. The composition according to claim 1, wherein the compound is [Nle ⁴ , D-Phe ⁷ , D-Trp ⁹ ] -alpha-MSH _4-11 . 4. The compound is [Nle ⁴ , D-Phe ⁷ ] -alpha-MSH.
The composition of claim 1 which is _4-9 . 5. The compound is Ac-Ser-Tyr-Ser-Cys-Glu-His-DP.
Claim 1 which is he-Arg-Trp-Gly-Lys-Pro-Val-NH ₂ .
The composition according to the item. 6. A composition according to claim 1 wherein the compound is the alpha-MSH analogue of (2). 7. A composition according to claim 1 wherein the compound is the alpha-MSH analog of (2) wherein R ₁ is Ac-Nle-Glu. 8. The compound is [Nle ⁴ , D-Phe ⁷ ] -alpha-MSH.
The composition of claim 1 which is