Patent Publication Number: US-2006018851-A1

Title: Methods and compositions for preventing and treating hyperpigmentation of skin

Description:
CROSS-REFERENCE TO RELATED APPLICATION  
      This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application No. 60/583,325 filed Jun. 28, 2004, which provisional application is incorporated herein by reference in its entirety. 
    
    
     BACKGROUND OF THE INVENTION  
      1. Field of the Invention  
      The present invention generally relates to the treatment of dermatological conditions and, more specifically, to the treatment of conditions related to hyperpigmentation of skin, by topical application of a composition comprising a peptide manganese complex.  
      2. Description of the Related Art  
      Melanin is a dark pigment found in the skin of humans that is responsible for the darkening of the skin. Melanin is produced by specialized cells in the skin called melanocytes through a complicated series of chemical and enzymatic reactions, mainly involving the manganese containing enzyme tyrosinase. The produced melanin pigments are packaged in granules called melanosomes. Melanosomes are transferred to the outer layer of the skin where they are responsible for the darkening of the skin, the degree of darkening being associated with skin type, sun exposure, and/or certain dermatological conditions.  
      There are a number of dermatological conditions associated with unwanted or excessive production of melanin. Conditions associated with overproduction of melanin are termed hyperpigmentation. For example, melanosis or melasma is a condition characterized by the development of sharply demarcated blotchy, brown spots usually in a symmetric distribution over the cheeks, forehead, and sometimes on the upper lip and neck. This condition frequently occurs during pregnancy ( melasma gravidarum  or “mask of pregnancy”), and at menopause. Also, this condition is frequently found among those taking oral contraceptives, and is occasionally found among nonpregnant women who are not taking oral contraceptives, and sometimes among men. A pattern of facial hyperpigmentation, similar to that described above, may be associated with a chronic liver disease called chloasma. A common condition associated with aging skin is the development of dark spots sometimes referred to as “liver spots.” Other forms of hyperpigmentation can be caused by UV irradiation, result from a genetic predisposition for the condition, or may come about during the course of wound healing.  
      Manganese is an essential nutrient involved in the formation of bone and in amino acid, cholesterol, and carbohydrate metabolism. Enzymes, which utilize manganese for activity, include arginase, glutamine synthetase, manganese superoxide dismutase, prolidase, and some carbohydrate transferases. The Adequate Intake levels for men and women have been set at 2.3 and 1.8 mg/day, respectively. The enzyme Superoxide Dismutase is one of the most important defenses against oxidative damage in the body. There are two types in humans, namely, the Cu—Zn Superoxide Dismutase (SOD1), which is found mainly in, the cytosol of the cell, and the Mn Superoxide Dismutase (SOD2), which is found in the mitochondria (see Kobayashi et al.,  Acta Dermato - Venereologica  73(1):41-45 (1993)).  
      Small molecular weight complexes of manganese have been shown to possess superoxide dismutase activity. For example, U.S. Pat. Nos. 5,223,538 and 5,227,405 to Fridovich et al. describe water soluble manganese complexes useful to reduce and prevent superoxide radical induced toxicity. In addition, U.S. Pat. No. 5,118,665 to Pickart discloses peptide manganese complexes with superoxide dismutase activity useful for enhancing or restoring the resistance of an animal to oxidative or inflammatory damage.  
      Manganese is also an important component of the enzyme Prolidase. This is a manganese dependent exopeptidase (i.e., a protease which cuts off amino acids from the end of the peptide chain). Prolidase cleaves proline from peptides inside the cell and provides a vital source of proline for new collagen synthesis. The addition of manganese to increase intracellular manganese increases the activity of Prolidase in deficient cells (see Hechtman et al.,  Pediatric Research  24(6):709-712 (1998)). Another manganese requiring enzyme is Arginase. Arginase is an enzyme responsible for the conversion of the amino acid arginine to urea in keratinocytes. The addition of L-arginine and manganese to keratinocyte cultures results in the increase of urea synthesis (see Wohlrab et al.,  Skin Pharmacology and Applied Skin Physiology  15(1):44-54 (2002)).  
      A number of compounds and plant extracts are reported to have activity against hyperpigmentation, including ascorbic acid and derivatives thereof, kojic acid and compounds related thereto, licorice (glycyrrhiza) extract, and bearberry extract. However, while these chemical compounds and extracts are active in the reversal and prevention of hyperpigmentation, they can be irritating to the skin with prolonged use. The only drug approved in the United States for the treatment of hyperpigmentation is hydroquinone.  
      Accordingly, there remains a need in the art for more effective and otherwise improved methods for treating dermatological conditions related to hyperpigmentation by, for example, topically applying compositions, having a desired degree of effectivity, to areas of skin in need thereof. The present invention fulfills this need and provides further related advantages.  
     BRIEF SUMMARY OF THE INVENTION  
      In brief, the present invention is directed to the treatment of dermatological conditions related to hyperpigmentation by topical application of a composition comprising at least one peptide manganese complex to an area of affected skin. It has been surprisingly found that such compositions can be used topically to substantially diminish signs of hyperpigmentation.  
      In one embodiment, the present invention is directed to a method for treating hyperpigmentation of skin, by contacting an area of skin in need thereof with an effective amount of a composition comprising at least one peptide manganese complex. In a further embodiment, the composition further comprises retinol, a retinol derivative, or a mixture thereof. Topical application of an effective amount of such compositions to areas of skin in need thereof results in significant reduction of the hyperpigmentation found on the areas contacted.  
      In other further embodiments, the present invention is directed to methods for such treatment wherein the at least one peptide manganese complex is encapsulated in a liposome, microsponge, polymer matrix or other encapsulation technology adapted to aid in the delivery of the peptide manganese complex to the areas of skin need thereof, or to enhance the stability of the composition. In yet other further embodiments, the at least one peptide manganese complex is formulated in an instrument adapted to deliver the peptide manganese complex via iontophoresis or ultrasound to the areas of affected skin.  
      In yet other further embodiments, the composition further comprises an inert and physiologically-acceptable carrier or diluent, a skin lightening agent, a sunscreen agent, a skin conditioning agent, a skin protectant, an emollient, a humectant, or a mixture thereof in addition to the at least one peptide manganese complex. In other related embodiments, the composition further comprises an active drug substance or an active cosmetic substance.  
      In still other further embodiments, the composition further comprises an emulsifying agent, a surfactant, a thickening agent, an excipient, or a mixture thereof, and/or the composition is in the form of a liquid, cream, gel, fluid cream, lotion, oil, topical serum, emulsion or microemulsion.  
      These and other aspects of this invention will be evident upon reference to the attached drawings and following detailed description of the invention. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       FIG. 1  is a graph showing the amount of melanin secretion as a function of GHK-Mn concentration for B16 melanocytes in culture.  
       FIG. 2  is a photograph of the two culture flasks of melanocytes, one control and the other incubated with GHK-Mn, of Example 5.  
       FIGS. 3A and 3B  are photographs of the control melanocyte culture and the GHK-Mn containing culture of Example 5. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION  
      As noted above, in one embodiment, disclosed is a method for treating hyperpigmentation of skin by topically applying, to areas of skin in need thereof, an effective amount of a composition comprising at least one peptide manganese complex. As used herein the word “treat,” “treating” or “treatment” refers to using the compositions of the present invention either prophylactically to prevent hyperpigmentation, or therapeutically to ameliorate an existing condition characterized by hyperpigmentation. Also, as used herein, the words “patient” and “subject” refer to a warm-blooded mammals, including humans.  
      The compositions utilized may be in any form suitable for topical application, including: a liquid, a cream, a lotion, a gel, a fluid cream, an oil, a topical serum, an emulsion or a microemulsion. Some examples of compositions formulated as cosmetic preparations, useful for cleansing and protecting, in addition to treating, skin are: creams for the face, hands, feet, or the entire body (i.e., day creams, night creams, make-up removal creams, and foundation creams); make-up removal formulations; protective or skin care body milks; skin care lotions, gels, or foams (such as cleansing or disinfecting lotions); bath compositions; deodorant compositions; and aftershave and pre-shave gels or lotions.  
      In more specific embodiments, the at least one peptide manganese complex comprises glycyl-L-histidyl-L-lysine:manganese(II) (“GHK-Mn”), L-alanyl-L-histidyl-L-lysine:manganese(II) (“AHK-Mn”), L-valyl-L-histidyl-L-lysine:manganese(II) (“VHK-Mn”), L-leucyl-L-histidyl-L-lysine:manganese(II) (“LHK-Mn”), L-isoleucyl-L-histidyl-L-lysine:manganese(II) (“IHK-Mn”), L-phenylalanyl-L-histidyl-L-lysine: manganese(II) (“FHK-Mn”), L-prolyl-L-histidyl-L-lysine:manganese(II) (“PHK-Mn”), L-seryl-L-histidyl-L-lysine:manganese(II) (“SHK-Mn”), or L-threonyl-L-histidyl-L-lysine:manganese(II) (“THK-Mn”).  
      As used herein, the abbreviations for the naturally occurring amino acids are:  
                                                          Alanine   Ala   A           Arginine   Arg   R           Asparagine   Asn   N           Aspartic Acid   Asx   D           Cysteine   Cys   B           Glycine   Gly   G           Glutamine   Gln   Q           Glutamic Acid   Glu   E           Histidine   His   H           Isoleucine   Ile   I           Leucine   Leu   L           Lysine   Lys   K           Methionine   Met   M           Phenylalanine   Phe   F           Proline   Pro   P           Serine   Ser   S           Threonine   Thr   T           Tryptophan   Trp   W           Tyrosine   Tyr   Y           Valine   Val   V                      
 
      As used herein, the expression “peptide manganese complex” generally refers to a coordination compound comprising a peptide molecule and a manganese(II) ion non-covalently complexed with the peptide. As is well understood in the art, manganese (II) designates a manganese ion having a valence of 2 (i.e., Mn +2 ). The peptide molecule serves as the complexing agent by donating electrons to the manganese ion to yield the non-covalent complex. The peptide molecule is a chain of two or more amino acid units or amino acid derivative units covalently bonded together via amide linkages, the formation of such linkages being accompanied by the elimination of water.  
      Generally, an amino acid consists of an amino group, a carboxyl group, a hydrogen atom, and an amino acid side-chain moiety—all bonded, in the case of an alpha-amino acid, to a single carbon atom that is referred to as an alpha-carbon. The amino acid units of the present invention may be provided by amino acids other than alpha-amino acids. For example, the amino acids may be beta- or gamma-amino acids, such as the following:  
                 
 
 where X is the amino acid side-chain moiety bonded, along with the amino group and hydrogen, to an alpha-, beta-, or gamma-carbon atom. 
 
      As another example, the amino acids of the present invention include, but are not limited to, naturally occurring alpha-amino acids. Naturally occurring amino acids are those from which the amino acids units of naturally occurring proteins are derived. Some of these amino acids, along with their respective amino acid side chain moieties, are shown below in Table 1. The naturally occurring amino acids shown are all in the L configuration, referring to the optical orientation of the alpha carbon or other carbon atom bearing the amino acid side chain. A peptide molecule of the present invention may also comprise amino acids that are in the D optical configuration, or a mixture of D and L amino acids.  
               TABLE 1                           Naturally Occurring Amino Acid Side-Chain Moieties                             Amino Acid Side Chain Moiety   Amino Acid                       —H   Glycine           —CH 3     Alanine           —CH(CH 3 ) 2     Valine           —CH 2 CH(CH 3 ) 2     Leucine           —CH(CH 3 )CH 2 CH 3     Isoleucine           —(CH 2 ) 4 NH 3   +     Lysine           —(CH 2 ) 3 NHC(NH 2 )NH 2   +     Arginine                                             Histidine                       —CH 2 COO—   Aspartic Acid           —CH 2 CH 2 COO—   Glutamic Acid           —CH 2 CONH 2     Asparagine           —CH 2 CH 2 CONH 2     Glutamine                                             Phenylalanine                                             Tyrosine                                             Tryptophan                       —CH 2 SH   Cysteine           —CH 2 CH 2 SCH 3     Methionine           —CH 2 OH   Serine           —CH(OH)CH 3     Threonine                                             Proline                      
 
 Other naturally occurring amino acids include hydroxyproline and gamma-carboxyglutamate. 
 
      Representative amino acid derivatives include those set forth in Table 2.  
               TABLE 2                       Amino Acid Derivatives                                                                      where X 2  = H or the following moieties:           —(CH 2 ) n CH 3  where n = 1-20           —(CH 2 ) n CH(CH 3 )(CH 2 ) m CH 3  where n, m = 0-20 (when n = 0,           m ≠ 0 or 1 and when n = 1, m ≠ 0)           —(CH 2 ) n NH 2  where n = 1-20 (n ≠ 4)           —(CH 2)   n CONH 2  where n = 3-20           —(CH 2)   n COOH where n = 3-20                                                                                                                                                     —(CH 2 ) n SH where n = 2-20           —(CH 2 ) n S(CH 2 ) m CH 3  where n, m = 1-20 (when n = 2, m ≠ 0)           —(CH 2 ) n CH 2 OH where n = 1-20           —(CH 2 ) n CH(CH 3 )OH where n = 1-20,           and where X 1  = H or the following moieties:           —(CH 2 ) n CH 3  where n = 0-20           —(CH 2 ) n CH(CH 3 )(CH 2 ) m CH 3  where n, m = 0-20.                      
 
      In addition, histidine derivatives of this invention include compounds having the structure:  
                 
 
 where n=1-20, and Y 1  and Y 2  are independently selected from alkyl moieties containing from 1-12 carbon atoms or aryl moieties containing from 6-12 carbon atoms. In certain embodiments, n is 1, Y 2  is methyl, and Y 1  is H (i.e., 3-methyl histidyl) or Y 2  is H and Y 1  is methyl (i.e., 5-methyl histidine). 
 
      Similarly, arginine derivatives of this invention include compounds having the structure:  
                 
 
 where n=1-20 (excluding n=3) 
 
      As used herein, “alkyl” means a straight chain or branched, cyclic or noncyclic, substituted or unsubstituted, saturated or unsaturated aliphatic hydrocarbon containing from 1 to 18 carbon atoms. Representative saturated straight chain alkyls include methyl, ethyl, n-propyl and the like; while saturated branched alkyls include isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, and the like. Representative, saturated cyclic alkyls include cyclopropyl, cyclobutyl, cyclopentyl, —CH 2 cyclohexyl, and the like; while unsaturated cyclic alkyls include cyclopentenyl, cyclohexenyl, and the like. Unsaturated alkyls contain at least one double or triple bond between adjacent carbon atoms (referred to as an “alkenyl” or “alkynyl,” respectively). Representative alkenyls include ethylenyl, 1-butenyl, isobutylenyl, 2-methyl-2-butenyl, and the like; while representative alkynyls include acetylenyl, 2-butynyl, 3-methyl-1-butynyl, and the like.  
      Also, as used herein, “aryl” means an aromatic carbocyclic moiety such as phenyl or naphthyl, and may be substituted or unsubstituted. “Arylalkyl,” as used herein, means an alkyl having at least one alkyl hydrogen atom replaced with a substituted or unsubstituted aryl moiety, such as benzyl (i.e., —CH 2 phenyl, —(CH 2 ) 2 phenyl, —(CH 2 ) 3 phenyl, —CH(phenyl) 2 , and the like).  
      A peptide manganese complex of the present invention may have the formula [R 1 —R 2 —R 3 ]:manganese(II) where R 3  is at least one amino acid or amino acid derivative, as defined above, bonded to R 2  by a peptide bond. Where R 3  is a single amino acid or amino acid derivative, then the peptide of the peptide manganese complex is generally classified as a tripeptide. As another example, a peptide manganese complex of the present invention may have the formula [R 1 —R 2 —R 3 ]:manganese(II) where R 3  is a chemical moiety bonded to the R 2  moiety by an amide bond. The expression “chemical moiety,” as used herein and with reference to R 3 , includes any chemical moiety having an amino group capable of forming an amide bond with the carboxyl terminus of R 2  (i.e., the carboxyl terminus of histidine, arginine, or derivatives thereof).  
      As a more particular example, where R 3  is a chemical moiety bonded to the R 2  moiety by an amide bond, R 3  is —NH 2 , an alkylamino moiety having from 1-20 carbon atoms, or an arylamino moiety having from 6-20 carbon atoms. As used herein, an “alkylamino moiety” encompasses alkyl moieties containing an amino moiety, wherein the alkyl moiety is as defined above, and includes, but is not limited to, octyl amine and propyl amine. Similarly, an “arylamino moiety” encompasses aryl moieties containing an amino moiety, wherein the aryl moiety is as defined above, and includes, but is not limited to, benzylamine and benzyl-(CH 2 ) 1-14 -amine. Further examples of suitable chemical moieties having amino groups capable of forming an amide linkage with the carboxyl terminus of R 2  include polyamines such as spermine and sperimidine.  
      It should be understood that R 3  may include more than one chemical moiety. For example, additional amino acids or amino acid derivatives may be bonded to the above-described peptide manganese complexes comprising tripeptides to yield peptide manganese complexes comprising peptides having four or more amino acids and/or amino acid derivatives. For purposes of illustration, Table 3, shown below, presents various representative examples of peptide manganese complexes of the present invention.  
               TABLE 3                       Representative Peptide-Manganese Complexes                  Examples of [R 1 —R 2 ]:manganese(II)                     glycyl-histidine:manganese   alanyl-histidine:manganese       glycyl-(3-methyl)histidine:manganese   alanyl-(3-methyl)histidine:manganese       glycyl-(5-methyl)histidine:manganese   alanyl-(5-methyl)histidine:manganese       glycyl-arginine:manganese   alanyl-arginine:manganese       (N-methyl)glycine-histidine:manganese   (N-methyl)glycine-arginine:manganese                 Examples of [R 1 —R 2 —R 3 ]:manganese(II)       where R 3  is Chemical Moiety Linked by Amide Bond                     glycyl-histidyl-NH 2 :manganese   glycyl-arginyl-NH 2 :manganese       glycyl-(3-methyl)histidyl-NH 2 :manganese   alanyl-(3-methyl)histidyl-NH 2 :manganese       glycyl-arginyl-NH 2 :manganese   alanyl-arginyl-NH 2 :manganese       (N-methyl)glycine-histidyl-   (N-methyl)glycine-arginyl-       NH 2 :manganese   NH 2 :manganese       glycyl-histidyl-NHoctyl:manganese   glycyl-arginyl-NHoctyl:manganese                 Examples of [R 1 —R 2 —R 3 ]:manganese(II)       where R 3  is Amino Acid or Amino Acid Derivative Linked by Peptide Bond                     glycyl-histidyl-lysine:manganese   glycyl-arginyl-lysine:manganese       glycyl-(3-methyl)histidyl-lysine:manganese   glycyl-(5-methyl)histidyl-lysine:manganese       alanyl-histidyl-lysine:manganese   alanyl-arginyl-lysine:manganese       alanyl-(3-methyl)histidyl-   alanyl-(5-methyl)histidyl-       lysine:manganese   lysine:manganese       glycyl-histidyl-phenylalanine:manganese   glycyl-arginyl-phenylalanine:manganese       glycyl-(3-methyl)histidyl-   glycyl-(5-methyl)histidyl-       phenylalanine:manganese   phenylalanine:manganese       alanyl-histidyl-phenylalanine:manganese   alanyl-arginyl-phenylalanine:manganese       alanyl-(3-methyl)histidyl-   alanyl-(5-methyl)histidyl-       phenylalanine:manganese   phenylalanine:manganese       glycyl-histidyl-lysyl-phenylalanyl-   glycyl-arginyl-lysyl-phenylalanyl-       phenylalanyl:manganese   phenylalanyl:manganese       glycyl-(3-methyl)histidyl-lysyl-   glycyl-(5-methyl)histidyl-lysyl-       phenylalanyl-phenylalanyl:manganese   phenylalanyl-phenylalanyl:manganese       (N-methyl)glycyl-histidyl-   (N-methyl)glycyl-arginyl-       lysine:manganese   lysine:manganese       valyl-histidyl-lysine:manganese   glycyl-histidyl-lysyl-prolyl-phenylalanyl-       prolyl-histidyl-lysine:manganese   proline:manganese       glycyl-D-histidyl-L-lysine:manganese   Leucyl-histidyl-lysine:manganese       seryl-histidyl-lysine:manganese                  
 
      In addition, the expression “peptide manganese complex,” as used herein, encompasses peptide manganese complex derivatives. The expression “peptide manganese complex derivative,” as used herein, refers to a peptide manganese complex where the peptide molecule thereof has: 1) at least one amino acid side chain moiety that is a modification and/or variation of a naturally occurring, amino acid side-chain moiety; and/or 2) at least one of the hydrogens, bonded to an amide linkage nitrogen atom, substituted with a different moiety; and/or 3) the carboxyl group of the carboxyl terminal residue esterified or otherwise modified; and/or 4) at least one hydrogen, bonded to the nitrogen atom of the amino-terminal residue, substituted with a different moiety. Accordingly, the method of the present invention, in another embodiment, employs a composition comprising at least one peptide manganese complex derivative. For example, derivatives of GHK-Mn have the general formula: 
 
[glycyl-histidyl-lysine-R]: manganese(II) 
 
 wherein R may be, for example, an alkyl moiety containing from one to eighteen carbon atoms, an aryl moiety containing from six to twelve carbon atoms, an alkoxy moiety containing from one to twelve carbon atoms, or an aryloxy moiety containing from six to twelve carbon atoms. 
 
      Further examples of the peptide manganese complex and peptide manganese complex derivatives encompassed by the present invention include, but are not limited to, those disclosed and described in the above- and below-cited U.S. Patents that are directed to peptide manganese complexes, as well as those disclosed and described in the published PCT application having the International Publication Number WO 94/03482, which is incorporated herein by reference in its entirety.  
      The synthesis of the above-described peptide manganese complexes is described in detail in the above-referenced patents. For example, the peptides of the peptide manganese complexes disclosed herein may be synthesized by either solution or solid phase techniques known to one skilled in the art of peptide synthesis. The general procedure involves the stepwise addition of protected amino acids to build up the desired peptide sequence. The resulting peptide may then be complexed to manganese (at the desired molar ratio of peptide to manganese) by dissolving the peptide in water, followed by the addition of manganese chloride or another suitable manganese salt and adjusting the pH to greater than 4.0. The peptide manganese complex thus formed may be used as a solution or as a dry powder after, for example, freeze-drying or spray drying.  
      The compositions of the present invention may be prepared from aqueous solutions of peptide manganese complexes. Such aqueous solutions are prepared by methods that are well known to those skilled in the art. For example, an amount of dried peptide manganese complex, suitable for a desired concentration, is readily dissolved in water with mixing and gentle heating. An alternative method is to prepare a solution of the desired peptide, followed by the addition of a manganese salt in the desired molar ratio to yield the desired solution of the peptide manganese complex. Examples of manganese salts that may be used are manganese chloride and manganese acetate. When aqueous solutions of peptide manganese complexes are prepared, the solutions are neutralized, typically with NaOH or HCl.  
      In yet another embodiment of the present invention, the peptide moiety of the at least one peptide manganese complex may be of natural origin. In this embodiment, the peptide is formed by the hydrolysis of naturally occurring proteins, polypeptides, or larger peptides of either plant, microbial, or animal origin. Hydrolysis may be by enzymatic treatment or by acid or base hydrolysis. The manganese complex of this type of peptide manganese complex is formed by addition of a suitable manganese salt to the aqueous solution of the peptide. Alternatively, the peptide manganese complex may be formed during the manufacturing of a formulation by separate additions of the peptide and manganese salt in a suitable solvent.  
      In more specific embodiments, the composition of the present invention comprises at least one peptide manganese complex, where the concentration of the peptide manganese complex, by weight of the composition, ranges from about 0.01% to about 5%, from about 0.025% to about 1%, or from about 0.05% to about 0.5%, respectively. In other more specific embodiments, the molar ratio of peptide to manganese in the peptide manganese complex ranges from about 1:1 to about 3:1 in some embodiments, and from about 1:1 to about 2:1 in other embodiments.  
      In additional embodiments of the method disclosed herein, the composition used comprises at least one peptide manganese complex that is encapsulated in a liposome or microsponge adapted to aid in the delivery of the peptide manganese complex to the area of skin being treated; and, in other embodiments, is formulated in an instrument adapted to deliver the peptide manganese complex via iontophoresis to the area of skin in need of treatment.  
      Exemplary methods of encapsulating pharmaceutical or cosmetic actives are disclosed in the following U.S. Patents:  
      U.S. Pat. No. 6,572,892 to loulalen et al. entitled “Cosmetic or dermopharmaceutical composition in the form of beads and methods for preparing same”, which describes an anhydrous solid composition comprising at least a hydrophobic wax, an oil and talcum, having the form of beads with size ranging from 1 to 10,000 microns.  
      U.S. Pat. No. 6,572,870 to Ribier et al. entitled “Anhydrous cosmetic makeup composition containing a fatty phase”, which describes an anhydrous cosmetic makeup composition containing in addition to a fatty phase formed from oils, fatty bodies and surfactants, and optionally waxes, a vesicular lipidic phase that contains at least one ionic or nonionic amphiphilic lipid and optionally additives.  
      U.S. Pat. No. 6,565,886 to Simonnet et al. entitled “Nanocapsules based on poly(alkylene adipate), process for their preparation and cosmetic or dermatological compositions containing them”, which describes nanocapsules consisting of a lipid center forming or containing a lipophilic active compound, and of a water-insoluble continuous envelope comprising at least one polyester of poly(alkylene adipate) type.  
      U.S. Pat. No. 6,565,873 to Shefer et al. entitled “Biodegradable bioadhesive controlled release system of nano-particles for oral care products”, which describes a controlled release system comprising a nano-particle, having an average particle diameter of from about 0.01 microns to about 10 microns, which comprises a biodegradable solid hydrophobic core and a bioadhesive/mucoadhesive positively charged surface.  
      U.S. Pat. No. 6,548,690 to Mimoun entitled “Porous polymethylsilsesquioxane with adsorbent properties”, which describes a porous polymethylsilsesquioxane useful as an encapsulation matrix.  
      U.S. Pat. No. 6,534,549 to Newton et al. entitled “Controlled release formulations”, which describes a method for producing a controlled release composition in which a film-forming composition comprising a mixture of a substantially water-insoluble film-forming polymer and amylose in a solvent system comprising (1) water and (2) a water-miscible organic solvent, which on its own is capable of dissolving the film-forming polymer, is contacted with an active material and the resulting composition dried.  
      U.S. Pat. No. 6,455,088 to Dasseux entitled “Peptide/lipid complex formation by co-lyophilization”, which describes peptide/lipid vesicles and complexes through the co-lyophilization of peptides.  
      As noted above, in certain embodiments, the composition utilized in the method of the present invention further comprises retinol, a retinol derivative, or a mixture thereof, in addition to a peptide manganese complex. Retinol is also known as vitamin A and has the formula 3,7-dimethyl-9-(2,6,6-trimethyl-1-cyclohexen-1-yl)-2,4,6,8-nonatetraen-1-ol. Other terms that are used for retinol are axerophthol and vitamin A alcohol. In certain specific embodiments of the present invention that use compositions comprising retinol, the isomeric forms of the retinol used are: all-trans-retinol; 1,3-cis-retinol; 3,4-didehydro-retinol; and 9-cis-retinol, respectively. In other specific embodiments of the present invention that use compositions comprising a retinol derivative, the latter is an ester of retinol selected from C 1 -C 30  esters of retinol; C 2 -C 20  esters of retinol; and C 2 , C 3 , and C 16  esters of retinol, respectively. More specifically, the ester of retinol may be retinyl palmitate, retinyl acetate and retinyl propionate. Other retinol derivatives that may be used are retinoic acid and retinyl aldehyde. The concentration of the retinol, retinol derivative, or mixture thereof, ranges from about 0.001% to about 10% in some embodiments; from about 0.01% to about 1% in other embodiments; and from about 0.01% to about 0.5% in yet other embodiments, by weight of the composition.  
      In further embodiments of the methods of the present invention, the compositions used may comprise at least one active agent in addition to the peptide manganese complex. In one such embodiment, the composition is formulated as a pharmaceutical preparation and comprises at least one active drug substance. In another such embodiment, the composition further comprises at least one active agent for rendering the composition suitable as a cosmetic preparation. Active agents, as defined herein, are compounds that provide benefits to the skin and/or provide desirable properties to a composition formulated as a cosmetic preparation. Some examples of active agents, other than drug substances, are sunscreen agents, skin lightening agents, tanning agents, skin conditioning agents, skin protectants, emollients and humectants.  
      Representative sunscreen agents are active ingredients that absorb, reflect, or scatter radiation in the UV range at wavelengths from 290 to 400 nanometers. Specific examples include benzophenone-3 (oxybenzone), benzophenone-4 (sulisobenzone), benzophenone-8 (dioxybenzone), butyl methoxydibenzoylmethane (Avobenzone), DEA-methoxycinnamate (diethanolamine methoxycinnamate), ethyl dihydroxypropyl PABA (ethyl 4-[bis(hydroxypropyl)]aminobenzoate), ethylhexyl dimethyl PABA (Padimate O), ethylhexyl methoxycinnamate (octyl methoxycinnamate), ethylhexyl salicylate (octyl salicylate), homosalate, menthyl anthranilate (Meradimate), octocrylene, PABA (aminobenzoic acid), phenylbenzimidazole sulfonic acid (Ensulizole), TEA-salicylate (trolamine salicylate), titanium dioxide, and zinc oxide. One skilled in the art will appreciate that other sunscreen agents may be used in the compositions and preparations of the present invention.  
      Representative skin lightening agents include, but are not limited to, ascorbic acid and derivatives thereof, kojic acid and derivatives thereof, hydroquinone and derivatives thereof, azelaic acid, various plant extracts such as those from licorice, grape seed and bear berry, and mixtures of any one or more of the foregoing. Those skilled in the art will appreciate that other skin lightening agents may be included in the compositions used for some of the methods of the present invention.  
      Hydroquinone (p-dihydroxybenzene or p-hydroxyphenol) is a phenolic compound having the following structure:  
                 
 
 Derivatives of hydroquinone include other substituted phenolic compounds such as N-acetyl-4-S-cysteaminylphenol (4-S-CAP), Arbutin (hydroquinone-beta-D-glucopyranoside), t-butyl hydroquinone:  
                 
 
 and other alkyl substitutions. Esters of hydroquinone are also possible, such as Hydroquinone mono-methyl ether (p-Hydroxyanisole). 
 
      Kojic acid (5-hydoxy-4-pyran-4-one-2-methyl) is a fungal metabolic product having the following structure:  
                 
 
 Derivatives of kojic acid consist of fatty acid esters such as kojic acid dipalmitate (Hexadecanoic Acid, 4-Oxo-6-[[(1-Oxohexadecyl)Oxy]Methyl]-4H-Pyran-3-yl Ester):  
                 
 
 monopalmitate, iso-palmitate, and the like. 
 
      As noted above, the compositions may further comprise skin conditioning agents. Such agents comprise substances that enhance the appearance of dry or damaged skin, as well as materials that adhere to the skin to reduce flaking, restore suppleness, and generally improve the appearance of skin. Representative examples of skin conditioning agents include: acetyl cysteine, N-acetyl dihydrosphingosine, acrylates/behenyl acrylate/dimethicone acrylate copolymer, adenosine, adenosine cyclic phosphate, adensosine phosphate, adenosine triphosphate, alanine, albumen, algae extract, allantoin and derivatives, aloe barbadensis extracts, aluminum PCA, amyloglucosidase, arbutin, arginine, azulene, bromelain, buttermilk powder, butylene glycol, caffeine, calcium gluconate, capsaicin, carbocysteine, carnosine, beta-carotene, casein, catalase, cephalins, ceramides, chamomilla recutita (matricaria) flower extract, cholecalciferol, cholesteryl esters, coco-betaine, coenzyme A, corn starch modified, crystallins, cycloethoxymethicone, cysteine DNA, cytochrome C, darutoside, dextran sulfate, dimethicone copolyols, dimethylsilanol hyaluronate, DNA, elastin, elastin amino acids, epidermal growth factor, ergocalciferol, ergosterol, ethylhexyl PCA, fibronectin, folic acid, gelatin, gliadin, beta-glucan, glucose, glycine, glycogen, glycolipids, glycoproteins, glycosaminoglycans, glycosphingolipids, horseradish peroxidase, hydrogenated proteins, hydrolyzed proteins, jojoba oil, keratin, keratin amino acids, and kinetin.  
      Other examples of skin conditioning agents are: lactoferrin, lanosterol, lauryl PCA, lecithin, linoleic acid, linolenic acid, lipase, lysine, lysozyme, malt extract, maltodextrin, melanin, methionine, mineral salts, niacin, niacinamide, oat amino acids, oryzanol, palmitoyl hydrolyzed proteins, pancreatin, papain, PEG, pepsin, phospholipids, phytosterols, placental enzymes, placental lipids, pyridoxal 5-phosphate, quercetin, resorcinol acetate, riboflavin, RNA, saccharomyces lysate extract, silk amino acids, sphingolipids, stearamidopropyl betaine, stearyl palmitate, tocopherol, tocopheryl acetate, tocopheryl linoleate, ubiquinone,  vitis vinifera  (grape) seed oil, wheat amino acids, xanthan gum, and zinc gluconate. Skin conditioning agents, other than those listed above, may also be used, as is readily appreciated by those skilled in the art.  
      In other embodiments, the compositions may include a skin protectant, defined herein as a compound that protects injured or exposed skin or mucous membrane surfaces from harmful or irritating external compounds. Representative examples thereof include: algae extract, allantoin, aluminum hydroxide, aluminum sulfate, betaine, camellia sinensis leaf extract, cerebrosides, dimethicone, glucuronolactone, glycerin, kaolin, lanolin, malt extract, mineral oil, petrolatum, potassium gluconate, and talc. Those skilled in the art will readily appreciate that skin protectants, other than those listed above, may be included in the compositions used for the methods of the present invention.  
      An emollient, as the term is used herein, is a cosmetic ingredient that can help skin maintain a soft, smooth, and pliable appearance. Emollients are able to provide these benefits, largely owing to their ability to remain on the skin surface or in the stratum corneum to act as a lubricant and reduce flaking. Some examples of emollients, suitable for embodiments of this invention, are: acetyl arginine, acetylated lanolin, algae extract, apricot kernel oil PEG-6 esters, avocado oil PEG-11 esters, bis-PEG-4 dimethicone, butoxyethyl stearate, C 18 -C 36  acid glycol ester, C 12 -C 13  alkyl lactate, caprylyl glycol, cetyl esters, cetyl laurate, coconut oil PEG-10 esters, di-C 12 -C 13  alkyl tartrate, diethyl sebacate, dihydrocholesteryl butyrate, dimethiconol, dimyristyl tartrate, disteareth-5 lauroyl glutamate, ethyl avocadate, ethylhexyl myristate, glyceryl isostearates, glyceryl oleate, hexyldecyl stearate, hexyl isostearate, hydrogenated palm glycerides, hydrogenated soy glycerides, hydrogenated tallow glycerides, hydroxypropyl bisisostearamide MEA, isostearyl neopentanoate, isostearyl palmitate, isotridecyl isononanoate, laureth-2 acetate, lauryl polyglyceryl-6 cetearyl glycol ether, methyl gluceth-20 benzoate, mineral oil, myreth-3 palmitate, octyidecanol, octyldodecanol, odontella aurita oil, 2-oleamido-1,3 octadecanediol, palm glycerides, PEG avocado glycerides, PEG castor oil, PEG-22/dodecyl glycol copolymer, PEG shorea butter glycerides, phytol, raffinose, stearyl citrate, sunflower seed oil glycerides, and tocopheryl glucoside. Those skilled in the art will readily appreciate that emollients, other than those listed above, may also be used.  
      Humectants are cosmetic ingredients that help maintain moisture levels in skin. Some examples of suitable humectants are: acetyl arginine, algae extract, aloe barbadensis leaf extract, betaine, 2,3-butanediol, chitosan lauroyl glycinate, diglycereth-7 malate, diglycerin, diglycol guanidine succinate, erythritol, fructose, glucose, glycerin, honey, hydrolyzed wheat protein/PEG-20 acetate copolymer, hydroxypropyltrimonium hyaluronate, inositol, lactitol, maltitol, maltose, mannitol, mannose, methoxy PEG, myristamidobutyl guanidine acetate, polyglyceryl sorbitol, potassium PCA, propylene glycol, sodium PCA, sorbitol, sucrose, and urea. Other humectants may be used for yet additional embodiments of this invention, as will be appreciated by those skilled in the art.  
      In addition to the foregoing active agents, the compositions employed in the methods of the present invention may also comprise inert and physiologically acceptable carriers or diluents. Suitable carriers or diluents include, but are not limited to: water, physiological saline, bacteriostatic saline (e.g., saline containing 0.9 mg/ml benzyl alcohol), petrolatum based creams (e.g., USP hydrophilic ointments and similar creams), various types of pharmaceutically acceptable gels, and short chain alcohols and glycols (e.g., ethyl alcohol and propylene glycol).  
      In other further embodiments, the compositions employed may comprise additional ingredients such as fatty alcohols, fatty acids, organic or inorganic bases, preserving agents (such as phenoxyethanol and mixtures of various parabens), wax esters, steroid alcohols, triglyceride esters, phospholipids such as lecithin and cephalin, polyhydric alcohol esters, fatty alcohol ethers, hydrophilic lanolin derivatives, hydrophilic beeswax derivatives, cocoa butter waxes, silicon oils, pH balancers, cellulose derivatives, hydrocarbon oils such as palm oil, coconut oil, and mineral oil, and mixtures thereof.  
      Additional ingredients may be included in the above compositions to vary the texture, viscosity, color and/or appearance thereof, as is appreciated by one of ordinary skill in the art. Accordingly, in a further embodiment, the compositions, in addition to at least one peptide manganese complex, comprise an emulsifying agent, a surfactant, a thickening agent, an excipient or a mixture thereof.  
      More specifically, emulsifiers and surfactants may be included in those compositions used for the present invention that are formulated as emulsions. Either water-in-oil or oil-in-water emulsions may be formulated. Examples of suitable surfactants and emulsifying agents include: nonionic ethoxylated and nonethoxylated surfactants, abietic acid, almond oil PEG, beeswax, butylglucoside caprate, C 18 -C 36  acid glycol ester, C 9 -C 15  alkyl phosphate, caprylic/capric triglyceride PEG-4 esters, ceteareth-7, cetyl alcohol, cetyl phosphate, corn oil PEG esters, DEA-cetyl phosphate, dextrin laurate, dilaureth-7 citrate, dimyristyl phosphate, glycereth-17 cocoate, glyceryl erucate, glyceryl laurate, hydrogenated castor oil PEG esters, isosteareth-11 carboxylic acid, lecithin, lysolecithin, nonoxynol-9, octyldodeceth-20, palm glyceride, PEG diisostearate, PEG stearamine, poloxamines, polyglyceryls, potassium linoleate, PPG&#39;s, raffinose myristate, sodium caproyl lactylate, sodium caprylate, sodium cocoate, sodium isostearate, sodium tocopheryl phosphate, steareths, TEA-C 12 -C 13  pareth-3 sulfate, tri-C 12 -C 15  pareth-6 phosphate, and trideceths. Other surfactants and emulsifiers may be used, as will be appreciated by those skilled in the art.  
      Examples of thickening (i.e., viscosity increasing) agents include, but are not limited to, those agents commonly used in skin care preparations, such as: acrylamides copolymer, agarose, amylopectin, bentonite, calcium alginate, calcium carboxymethyl cellulose, carbomer, carboxymethyl chitin, cellulose gum, dextrin, gelatin, hydrogenated tallow, hydroxytheylcellulose, hydroxypropylcellulose, hydroxpropyl starch, magnesium alginate, methylcellulose, microcrystalline cellulose, pectin, various PEG&#39;s, polyacrylic acid, polymethacrylic acid, polyvinyl alcohol, various PPG&#39;s, sodium acrylates copolymer, sodium carrageenan, xanthan gum, and yeast beta-glucan. Thickening agents other than those listed above may also be used in related embodiments of the present invention.  
      As heretofore noted, the compositions used for the methods of the present invention, being products for topical application to human skin, are, accordingly, formulated as a liquid, cream, gel, fluid cream or milk, lotion, oil, topical serum, emulsion or microemulsion. Also, the above compositions may be further combined with suitable excipients adapted for application to the face and neck. Suitable excipients should have a high affinity for the skin, be well tolerated, stable, and yield a consistency that allows for easy and pleasant utilization.  
      Typically, for a method of the present invention, aside from the content of the composition used, a small amount of the composition (from about 1 ml to about 5 ml) is applied to exposed areas of skin from a suitable container or applicator, and, if necessary, the composition is then spread over and/or rubbed into the skin using the hand, finger, or other suitable device. Each composition disclosed herein is typically packaged in a container that is appropriate in view of the composition&#39;s viscosity and intended use by the consumer. For example, a lotion or fluid cream may be packaged in a bottle, roll-ball applicator, capsule, propellant-driven aerosol device, or a container fitted with a manually operated pump. A cream may simply be stored in a non-deformable bottle, or in a squeeze container, such as a tube or a lidded jar.  
      The following examples are provided for the purpose of illustration, not limitation.  
     EXAMPLES  
      The examples which follow illustrate the preparation, characterization and utility of certain compositions used for exemplary embodiments directed to the methods of the present invention; and illustrate the effectiveness of such methods in treating conditions related to hyperpigmentation of the skin of a human patient.  
     Example 1  
      The composition of a representative moisturizing lotion used for a method of the present invention is shown below.  
                              REPRESENTATIVE MOISTURIZING LOTION                             Ingredients   % w/w                                         Water   73.80%           Glycerin   1.00%           xanthan gum   0.50%           diisopropyl adipate   4.00%           isocetyl stearate   6.00%           octyl palmitate   10.00%           glyceryl stearate   1.00%           cetyl alcohol   1.00%           stearyl alcohol   0.80%           behenyl alcohol   0.50%           palmitic acid   0.25%           stearic acid   0.25%           glycyl-L-histidyl-L-lysine manganese   0.30%           complex           propylene glycol   0.55%           diazolidinyl urea   0.03%           iodopropynyl butylcarbonate   0.02%           total   100.00%                      
 
     Example 2  
      The composition of a representative moisturizing cream used for a method of the present invention is shown below.  
                              REPRESENTATIVE MOISTURIZING CREAM                             Ingredients   % w/w                                         purified water   77.35%           ethylhexyl palmitate   8.00%           Octyldodecanol   2.50%           butyloctyl calicylate   2.00%           Squalane   1.50%           jojoba oil   0.50%           tocopheryl acetate   0.20%           Bisabolol   0.20%           Polyacrylamide   1.50%           laureth-7   0.50%           Glycerin   3.00%           Panthenol   0.60%           Allantion   0.10%           Cyclomethicone   0.50%           Carbomer   0.10%           polysorbate 20   0.20%           L-alanyl-L-histidyl-L-lysine manganese   0.25%           complex           propylene glycol   0.56%           diazolidinyl urea   0.30%           Methylparaben   0.11%           Propylparaben   0.03%           total   100.00%                      
 
     Example 3  
      The composition of a representative oil-in-water emulsion type face cream used for a method of the present invention is shown below.  
                              REPRESENTATIVE OIL-IN-WATER       EMULSION TYPE FACE CREAM                             Ingredients   % w/w                                         purified water   75.20%           Glycerin   4.00%           steareth-100   0.60%           steareth-2   0.35%           xanthan gum   0.35%           isopropyl palmitate   4.00%           Isohexanodecane   1.00%           isostearyl isostearate   1.20%           octyl dodecanol   1.00%           stearic acid   2.50%           cetostearyl alcohol   2.50%           Petrolatum   4.00%           glycyl-L-histidyl-L-lysine manganese   0.10%           complex           Phenoxyethanol   3.00%           Methylparaben   0.11%           Propylparaben   0.03%           Butylparaben   0.02%           Isopropylparaben   0.02%           Isobutylparaben   0.02%           total   100.00%                      
 
     Example 4  
      The composition of a representative moisturizing topical serum used for a method of the present invention is shown below.  
                              REPRESENTATIVE MOISTURIZING SERUM                             Ingredients   % w/w                       Water    75.12%             Methyl Gluceth-20    7.50%            PEG-8    5.00%            Propylene Glycol    4.00%              Bambusa Vulgaris  Extract    2.00%              Pisum Sativum  (Pea) Extract    2.00%            Glucosamine HCl    2.00%            Phenoxyethanol    1.40%            Chlorphenesin    0.30%            Glycerin    0.25%            Methylparaben    0.13%            Benzoic Acid    0.10%            Manganese Tripeptide-1 (GHK-Mn)    0.20%              Camellia Oleifera  (Green Tea) Leaf   &lt;1%           Extract             Vitua Vinifera  (Grape) Seed Extract   &lt;1%           Sodium PCA   &lt;1%           Betaine   &lt;1%           Sorbitol   &lt;1%           Glycine   &lt;1%           Alanine   &lt;1%           Proline   &lt;1%           Serine   &lt;1%           Threonine   &lt;1%           Arginine   &lt;1%           Lysine   &lt;1%           Glutamic Acid   &lt;1%           Tocopheryl Acetate   &lt;1%           Diisopropyl Dimer Dilinoleate   &lt;1%           Diisostearyl Dimer Dilinoleate   &lt;1%           Tocopherol   &lt;1%           Oryzanol   &lt;1%           Ubiquinone   &lt;1%           Retinyl Palmitate   &lt;1%           Tocopheryl Linoleate   &lt;1%           Sodium Carboxymethyl Betaglucan   &lt;1%           Sodium Hyaluronate   &lt;1%           Butylene Glycol   &lt;1%           PEG-12 Dimethicone   &lt;1%           PEG-26 Buteth-26   &lt;1%           PEG-40 Hydrogenated Castor Oil   &lt;1%           Hydroxyethylcellulose   &lt;1%           Triethanolamine   &lt;1%           total    100.00%                        
 
     Example 5  
     Inhibition of Pigment Formation in Cells by a Peptide Manganese Complex  
      Melanin is a nitrogen containing pigmented polymer produced by melanocytes and concentrated in melanosomes within the cells. Depending on the skin type, melanocytes in the skin produce varying ratios of eumalanin, a brown to black melanin, and pheomelanin, a red to yellow melanin. Differences in skin pigmentation are due primarily to the ratio of eumalanin to pheomelanin, amount of melanocyte activity, and environmental factors which stimulate melanin production.  
      The effect of peptide manganese complexes on pigmentation was determined by determining the amount of melanin produced by B16 melanocytes in culture. Various amounts of a representative peptide manganese complex (glycyl-L-histidyl-L-lysine:manganese) were added to the culture medium. After 5 days, the culture media was collected and analyzed for pigment content. In addition, pictures were taken of the culture plates.  
      The results shown in Table 4 and  FIG. 1  show that the addition of GHK-Mn complex (glycyl-L-histidyl-L-lysine:manganese complex) inhibited the formation of melanin pigments in cultured melanocytes in a dose responsive manner.  
               TABLE 4                          Percent Inhibition of Pigmentation in Melanocytes by       glycyl-L-histidyl-L-lysine:manganese complex                         Concentration   Plate I   Plate II               0.1 ug/mL   42%   28%       1.0 ug/ml   68%   35%        10 ug/ml   88%   81%                  
 
      At the highest concentration tested, the formation of pigment was inhibited by more than 80% relative to the control. Furthermore, the inhibition observed at 25 ug/ml of the GHK-Mn was approximately equivalent to that of 200 ug/mi of kojic acid, a known inhibitor of tyrosinase and melanin accumulation (Kim, D. H., Hwang, J-S., Baek, H. S., Ki,, K-J., Lee, B. G., Chang, I., Kang, H. H., and Lee, O. S. Development of 5-[(3-Aminopropyl)phosphinooxyl]-2-(Hydroxymethyl)-4H-pyran-4-one as a Novel Whitening Agent. Chem. Pharm. Bull. 51(2): 113-116 (2003)).  
      Visual examination of cell culture flasks containing the treated and control melanocytes clearly showed the lower amount of pigment in the cells treated with the peptide manganese complex.  FIG. 2  is a photograph of two culture flasks of melanocytes, one control and the other incubated with GHK-Mn. The control culture flask (PBS or phosphate buffered saline) contains a visible amount of secreted melanin that darkens the culture media. On the other hand, the flask incubated with the GHK-Mn contains relatively little melanin.  
      The same effect can be seen in  FIGS. 3A and 3B  in high magnification.  FIG. 3A  is a photograph of the control melanocyte culture, containing signature granules of melanin.  FIG. 3B  is a photograph of the GHK-Mn containing culture, containing much less melanin in the cells. These results clearly demonstrate that the GHK-Mn complex inhibits the accumulation of melanin.  
      All of the above U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referred to in this specification and/or listed in the Application Data Sheet, are incorporated herein by reference, in their entirety.  
      From the foregoing, it will be appreciated that, although specific embodiments of the present invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the present invention is not limited except as by the appended claims.