HK1165741B - Compositions and methods for treating hyperpigmentation - Google Patents

Description

Compositions and methods for treating hyperpigmentation

Related patent application

Priority of U.S. provisional patent application 61/142,094 (the contents of which are incorporated herein by reference in their entirety) filed 12/31/2008 is claimed in this application in accordance with 35U.S.C. § 119.

Technical Field

The present invention relates to the treatment of hyperpigmentation (hyperpigmentation) and other undesirable pigmentation in the skin. The present invention provides compositions and methods for improved delivery of therapeutic agents for treating hyperpigmentation and other undesirable pigmentation in the skin.

Background

Melanin is a generic term for a class of compounds found in animals, plants and protists. In humans, melanin is formed in melanosomes, which are cellular structures found in cells called melanocytes, which are located in the lowest layers of the epidermis, basal layer, and basal cells. Melanin is transported to keratinocytes (cornecocytes) in the stratum corneum of the skin via keratinocytes of the epidermis, and imparts a brown pigment to the stratum corneum of the skin. Melanin is responsible for human skin pigmentation due to the presence of melanin in the stratum corneum.

Melanin is capable of absorbing ultraviolet radiation and thus plays an important role in protecting the human body, particularly the skin, from damage and potentially carcinogenic effects of sunlight and other environmental sources of ultraviolet radiation. When exposed to ultraviolet radiation, the human body naturally increases the production of melanin in the exposed skin area as a defense mechanism. The increased production of melanin leads to darkening of the exposed skin, a phenomenon commonly referred to as tanning. In some cultures, darkened skin associated with tanning is considered desirable and aesthetically pleasing.

However, increased melanin production is not always considered desirable. For example, in some cultures, light-colored skin is considered more aesthetically pleasing than tanned skin. In addition, certain skin diseases can result in the uneven production of melanin in the skin, resulting in the appearance of uneven skin pigmentation. For example, hyperpigmentation disorders are characterized by local darkening of skin color caused by locally high levels of melanin (see, e.g., Voet D., Voet J.G., PrattCW. fundamentals of biochemistry. New York: Von Hoffmann Press, 2001: 657). Hyperpigmentation can be caused by: the melanin production by existing melanocytes is increased, or the proliferation of activated melanocytes.

Hyperpigmentation and other conditions of uneven skin pigmentation are generally considered undesirable and unsightly. For example, the occurrence of acne, rash, scratch or injury to the skin can cause post-inflammatory hyperpigmentation characterized by the presence of unwanted dark spots on the face or other parts of the body. Chloasma (melasma) is a condition associated with hormonal changes caused by pregnancy, the administration of contraceptives, or menopausal changes, which is often masked by pigments deposited on the surface of the epidermis or deeper in the dermis. Lentigines (also known as liver plaques) are dark discolorations caused by sun damage, which typically occur in older individuals. Freckles (ephelides), more commonly referred to as freckles (freckles), are small spots common in young people with a white and clean skin that is prone to sunburn when exposed to sunlight (CayeKA and feldmann st. hyperbipication: a review of common options.j. drugs department.2004; 3: 668-) -678).

Skin discoloration associated with hyperpigmentation or tanning can generally be reduced by the use of the bleaching agent hydroquinone. Hydroquinone has been approved by the U.S. Food and Drug Administration (FDA) for gradual fading of dark discoloration in skin and is available in the form of Over The Counter (OTC) skin bleaching formulations (up to 2% concentration) and prescription formulations (3-4% concentration). However, in recent years, studies have been conducted to challenge the safety of hydroquinone. These studies, which are currently being reviewed by the FDA, provide evidence of toxicity, carcinogenicity in animals and indicate the development of exogenous brown yellow disease in humans. The FDA may prohibit the use of hydroquinone in the treatment of hyperpigmentation.

In view of the potential safety hazards of using hydroquinone, it would be desirable to develop compositions and methods for lightening skin tone caused by hyperpigmentation or tanning that do not include hydroquinone. However, the skin lightening agents identified to date, which may be alternatives to hydroquinone, tend to have low efficacy or undesirable side effects, such as toxicity or skin irritation. For example, although kojic acid has been found to be useful as a skin lightening agent, conventional topical kojic acid formulations for the treatment of hyperpigmentation have certain disadvantages. Since kojic acid does not readily penetrate human skin, conventional kojic acid preparations contain a relatively high concentration of kojic acid to provide sufficient transdermal flux to achieve a skin lightening effect. However, kojic acid is known to be an irritant at high concentrations, which has the potential to sensitize and cause contact dermatitis. Furthermore, in conventional topical formulations comprising kojic acid, the high concentrations of kojic acid necessary to achieve a skin lightening effect are potentially serious health risks, and several studies have shown that high doses of kojic acid may be mutagens and/or promote tumor formation. Because of these disadvantages, some countries have partially banned the use of currently available formulations of kojic acid for reducing skin pigmentation associated with excess melanin.

Therefore, there is a need for new, safer formulations for treating hyperpigmentation.

Summary of The Invention

The present invention provides compositions and methods for reducing the intensity of melanin-related pigmentation of the skin. The compositions and methods of the present invention may be used to treat any condition associated with increased melanin production, including hyperpigmentation and tanning.

One embodiment of the present invention provides a topical composition for treating hyperpigmentation. The compositions comprise a skin lightening agent (skin-lightening agent) and a positively charged carrier present in an amount sufficient to enhance transdermal transport of the skin lightening agent. In a preferred embodiment, the skin lightening agent is kojic acid or a derivative of kojic acid.

Another embodiment of the present invention provides a method for reducing skin pigmentation. The method includes identifying an area of skin to be treated and applying a composition to reduce pigmentation of the skin in the selected area. The compositions comprise a skin lightening agent and a positively charged carrier molecule present in an amount to enhance transdermal transport of the skin lightening agent. In a preferred embodiment, the skin lightening agent comprises kojic acid or a derivative of kojic acid.

Another aspect of the invention provides a kit for reducing skin pigmentation. The kit comprises a skin lightening agent and a positively charged carrier present in an amount to enhance transdermal transport of the skin lightening agent. The skin lightening agent and the positively charged carrier may be stored separately as components of a kit and combined immediately prior to use, or may be pre-mixed.

Detailed Description

The present invention provides compositions for reducing unwanted pigmentation in skin, for example associated with hyperpigmentation or unwanted skin darkening (e.g., from tanning). In a preferred embodiment, the composition according to the invention comprises a skin lightening agent and a delivery molecule capable of enhancing dermal penetration of the skin lightening agent upon topical application. The present invention also provides methods of reducing skin discoloration associated with hyperpigmentation by topically applying a skin lightening agent and a delivery molecule capable of enhancing dermal penetration of the skin lightening agent.

The skin lightening agent to which the present invention relates is not particularly limited, and includes synthetic and naturally occurring compounds capable of reducing discoloration associated with excess melanin. Non-limiting examples of skin lightening agents to which the present invention relates include kojic acid, azelaic acid, ascorbic acid, tretinoin (retinol), topical glucocorticoids, linoleic acid, niacinamide, 4-tert-butylcatechol, tranexamic acid and licorice extract. The invention also relates to combinations of skin lightening agents.

In a preferred embodiment, the skin lightening agent is kojic acid or a derivative of kojic acid. Kojic acid (C)₆H₆O₄(ii) a 5-hydroxy-2- (hydroxymethyl) -4-pyrone) has the following chemical structure:

and may be obtained from a class of fungi known in japan as koji (aspergillus oryzae). Kojic acid blocks melanin formation by inhibiting the activity of tyrosinase, an enzyme that catalyzes in vivo chemical reactions associated with melanin formation. Kojic acid acts as a skin lightening agent by preventing the synthesis of melanin. In certain preferred embodiments of the present invention, the skin lightening agent is a kojic acid derivative. As used herein, the term "kojic acid derivative" refers to kojic acid that has undergone one or more chemical or functional changes, but which still has the ability to lighten skin discoloration caused by undesirably high levels of melanin. Kojic acid derivatives having the ability to lighten skin discoloration have been previously reported (see, e.g., U.S. patents 5,486,624; 5,523,421; 5,824,327 and 5,968,487; the contents of which are incorporated herein by reference in their entirety). Non-limiting examples of kojic acid derivatives to which the present invention relates include 2- (2-hydroxybenzoyl) oxymethyl-5-hydroxy-4H-pyran-4-one, 2- (3-hydroxybenzoyl) oxymethyl-5-hydroxy-4H-pyran-4-one, 2- (4-hydroxybenzoyl) oxymethyl-5-hydroxy-4H-pyran-4-one, 2- (2, 3-dihydroxybenzoyl) oxymethyl-5-hydroxy-4H-pyran-4-one, and 2- (3, 4-dihydroxybenzoyl) oxymethyl-5-hydroxy-4H-pyran-4-one.

One aspect of the present invention is the recognition that kojic acid cannot easily reach the desired skin-related structures to inhibit melanin production. Without wishing to be bound by theory, it is believed that delivery of kojic acid to relevant skin-related structures is difficult, which results in the use of high concentrations of kojic acid in certain kojic acid formulations. Thus, preferred embodiments of the present invention provide positively charged carrier molecules capable of enhancing the transdermal flux of kojic acid and/or directing kojic acid to associated skin-related structures. Delivery occurs without covalent modification of the skin lightening agent.

By "positively charged" is meant that the carrier has a positive charge under at least some solution phase conditions, more preferably at least some physiologically compatible conditions. More specifically, "positively charged" as used herein means that the group of interest comprises a functional group (functional) that is charged under all pH conditions, such as a quaternary amine, or comprises a functional group that can acquire a positive charge under certain solution phase conditions, such as in the case of a primary amine, a change in pH. More preferably, "positively charged" as used herein refers to a group that, under physiologically compatible conditions, behaves in conjunction with an anion. It will be apparent to those skilled in the art that the polymer having multiple positively charged moieties need not be a homopolymer. Other examples of positively charged moieties are well known in the art and can be readily used, as will be apparent to those skilled in the art.

Typically, a positively charged carrier comprises a positively charged backbone, which is typically a chain of atoms, and groups in the chain carry a positive charge at physiological pH, or the groups carrying a positive charge are attached to side chains extending from the backbone. Preferably, the positively charged backbone does not have a defined enzymatic or therapeutic biological activity per se. The linear backbone is a hydrocarbon backbone, which in some embodiments is interrupted by heteroatoms selected from nitrogen, oxygen, sulfur, silicon, and phosphorus. Most of the backbone atoms are typically carbon. The main chain is usually a polymer of a repeating unit (e.g., amino acid, poly (oxyethylene), poly (allylamine), polyalkyleneimine (polyalkyleneimine), or the like), but may be a heteropolymer. In one set of embodiments, the positively charged backbone is polypropyleneamine (polypropylenamine), in which a number of amine nitrogen atoms are present as positively charged ammonium groups (tetra-substituted). In another embodiment, the positively charged backbone is a non-peptidic polymer, which may be a heteropolymer or homopolymer, such as a polyalkyleneimine, for example polyethyleneimine or polypropyleneimine, having a molecular weight of about 100 to about 2,500,000D, preferably about 250 to about 1,800,000D, most preferably about 1000 to about 1,400,000D. In another set of embodiments, the backbone has attached thereto a plurality of side chain moieties that include positively charged groups (e.g., ammonium, pyridyl, phosphonium, sulfonium, guanidinium, or amidinium groups). In this group of embodiments, the pendant moieties may be spaced along the backbone, with the spacing being uniform or variable. In addition, the length of the side chains may be the same or different. For example, in one set of embodiments, the side chain may be a straight or branched hydrocarbon chain having from one to twenty carbon atoms and terminating distally (away from the backbone) with one of the positively charged groups described above. In all aspects of the invention, the biologically active agent and the carrier are associated by non-covalent interactions, non-limiting examples of which include ionic interactions, hydrogen bonding, van der waals forces, or combinations thereof.

In one set of embodiments, the positively charged backbone is a polypeptide having a plurality of positively charged side chain groups (e.g., lysine, arginine, ornithine, homoarginine, etc.). Preferably, the polypeptide has a molecular weight of about 100 to about 1,500,000D, more preferably about 250 to about 1,200,000D, most preferably about 1000 to about 1,000,000D. It will be appreciated by those skilled in the art that when amino acids are used in this part of the invention, the side chains may have a D-or L-form (R or S configuration) at the center of attachment. In certain preferred embodiments, the polypeptide has a molecular weight of about 500 to about 5000D, more preferably about 1000 to about 4000D, more preferably 2000 to about 3000D. In other embodiments, the polypeptide has a molecular weight of at least about 10,000.

In another embodiment, the backbone moiety is polylysine, and the efficiency group is attached to the polylysine as described herein. Polylysine may have a molecular weight of from about 100 to about 1,500,000D, more preferably from about 250 to about 1,200,000D, and most preferably from about 1000 to about 3000D. It may also be any commercially available (Sigma Chemical Company, st. louis, mo., USA) polylysine, such as, for example, polylysine with a MW > 70,000D, polylysine with a MW of 70,000 to 150,000D, polylysine with a MW of 150,000 to 300,000D and polylysine with a MW > 300,000D. The selection of a suitable polylysine depends on the other components of the composition, and will be sufficient to provide a total net positive charge to the composition and a length preferably one to four times the combined length of the negatively charged components.

Alternatively, the backbone may be a polypeptide analog, such as a peptoid. See, e.g., Kessler, angelw.chem.int.ed.engl.32: 543 (1993); zuckermann et al, Chemtracts- -Macromol. chem.4: 80 (1992); and Simon et al, proc.nat' l.acad.sci.usa 89: 9367(1992)). Briefly, a peptoid is a polyglycine in which the side chains are attached to the backbone nitrogen atoms instead of the α -carbon atoms. As noted above, a portion of the side chains typically terminate in positively charged groups to provide a positively charged backbone component. The synthesis of peptoids is described, for example, in U.S. patent 5,877,278, which is incorporated herein by reference in its entirety. As the term is used herein, positively charged backbones having a peptoid backbone structure are considered "non-peptidic" in that they do not consist of amino acids having side chains naturally occurring at the α -carbon position.

A variety of other backbones can be used, for example, steric or electronic mimetics of polypeptides in which the amide bond of the peptide is replaced by a surrogate such as an ester bond, thioamide (- -CSNH- -), retro thioamide (- -NHCS- -), aminomethylene (- -NHCH)₂- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -₂NH- -) group, keto-methylene (- -COCH)₂- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -₂RCH₂- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -₂RNH- -), reverse peptide (- -NHCO- -), trans olefin (- -CR ═ CH- -), fluoroolefin (- -CF ═ CH- -), dimethylene (- -CH- -)₂CH₂- - - - - -, thioether (- -CH)₂S- -), hydroxyethylidene (- -CH (OH) CH₂- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -₂O- -), tetrazole (CN)₄) Sulfonamido (- -SO)₂NH- -), methylenesulfonamido (- -CHRSO)₂NH- -), reverse sulfonamido (- -NHSO)₂- -) and backbones with malonate and/or gem-diaminoalkyl subunits, e.g., as discussed by Fletcher et al ((1998) chem. rev.98: 763), which is incorporated herein by reference.

In each of the backbones provided above, a pendant group may be attached, the pendant group carrying a positively charged group. For example, a sulfonamido-linked backbone (- -SO)₂NH- -and- -NHSO₂- -) may have a side chain group attached to the nitrogen atom. Similarly, hydroxyethylidene (- -CH (OH) CH₂- - - -) linkages may have a side chain group attached to a hydroxyl substituent. One skilled in the art can readily employ other chemical linkages to provide positively charged side chain groups using standard synthetic methods.

In one embodiment of the invention, only positively charged carriers are necessary for transdermal delivery of skin lightening agents, said carriers having positively charged efficiency groups. In certain embodiments, as described above, the positively charged backbone is a polypeptide having a plurality of positively charged side chain groups (e.g., lysine, arginine, ornithine, homoarginine, etc.). In another embodiment, the positively charged carrier comprises a non-peptidic polymer, such as a polyalkyleneimine having a plurality of positively charged side chain groups having a molecular weight of about 100 to 1,500,000D. The polyalkyleneimines include polyethyleneimine and polypropyleneimine.

In a preferred embodiment, the positively charged carrier comprises a positively charged backbone having a plurality of attached efficiency groups. As used herein, an "efficiency group" is any agent that has the effect of promoting transfer of a positively charged backbone through a tissue or cell membrane. Non-limiting examples of efficiency groups include- (gly)_n1-(arg)_n2HIV-TAT or fragments thereof, or a protein transduction domain of Antennapedia (Antennapedia), or fragments thereof, wherein subscript n1 is an integer from 0 to 20, more preferably from 0 to 8, more preferably from 2 to 5, and subscript n2 is individually an odd integer from about 5 to about 25, more preferably from about 7 to about 17, most preferably from about 7 to about 13. Further preferred are embodiments wherein the HIV-TAT fragment has the formula (gly)_p-RGRDDRRQRRR-(gly)_q、(gly)_p-YGRKKRRQRRR-(gly)_qOr (gly)_p-RKKRRQRRR-(gly)_qWherein subscripts p and q are each independently integers of 0 to 20 and the fragments are linked to the backbone via the C-terminus or N-terminus of the fragment. Preferred HIV-TAT fragments are those wherein subscripts p and q are each independently an integer from 0 to 8, more preferably from 2 to 5.In another preferred embodiment, the efficiency group is the antennapedia (Antp) Protein Transduction Domain (PTD), or a fragment thereof that retains activity. These are known in the art, see, for example, Console et al, j.biol.chem.278: 35109(2003), and a non-limiting example of an Antp PTD to which the present invention relates is SGRQIKIWFQNRRMKWKKC. Preferred positively charged carriers include at least about 0.05% (percent of total carrier weight), preferably from about 0.05 to about 45 weight%, and most preferably from about 0.1 to about 30 weight% of the efficiency groups. For having the formula- (gly)_n1-(arg)_n2Most preferably in an amount of from about 0.1 to about 25%. Preferred positively charged efficiency groups include, for example, -Gly-Gly-Gly-arg-arg-arg-arg-arg-arg (-Gly)₃Arg₇) HIV-TAT or fragments thereof, and antennapedia protein PTD or fragments thereof.

In other embodiments of the invention, the positively charged carrier is a relatively short polylysine or Polyethyleneimine (PEI) backbone (which may be linear or branched) and which has positively charged efficiency groups. A non-limiting example of such a vector is the amino acid sequence RKKRRQRRRG- (K)₁₅-GRKKRRQRRR. In preferred embodiments, such carriers are used to minimize uncontrolled aggregation of the skin lightening agent in the backbone and the therapeutic composition, which results in a significant reduction in transport efficiency. In some embodiments, when the carrier is a relatively short linear polylysine or PEI backbone, the backbone has a molecular weight of less than 75,000D, more preferably less than 30,000D, and most preferably less than 25,000D. For example, in certain embodiments, the carrier is a relatively short, branched polylysine or PEI backbone, having a molecular weight of less than 60,000, more preferably less than 55,000D, and most preferably less than 50,000D.

The compositions of the invention are preferably applied to the skin of a subject or patient (i.e., a human or other mammal in need of specific treatment) in the form of a product. The term "need" is intended to include pharmaceutical or health related needs, as well as cosmetic and subjective needs, such as altering or improving the appearance of facial tissue. Typically, the compositions are prepared by mixing the skin lightening agent with a positively charged carrier, and optionally with one or more additional pharmaceutically acceptable carriers or excipients. In their simplest form, they may comprise a simple aqueous pharmaceutically acceptable carrier or diluent, such as buffered saline (e.g. phosphate buffered saline). However, the compositions may contain other ingredients typically found in topical pharmaceutical or cosmeceutical compositions, including dermatologically or pharmaceutically acceptable carriers, vehicles, or vehicles (i.e., carriers, vehicles, or vehicles that are compatible with the tissue to which they are to be applied). As used herein, the term "dermatologically or pharmaceutically acceptable" means that the compositions or components thereof are suitable for use in contact with such tissues or in patients, and generally do not have undue toxicity, incompatibility, instability, allergic response, and the like. The compositions of the invention may comprise, as the case may be, any ingredient conventionally used in the fields concerned, in particular in cosmetics and dermatology. The composition may also comprise a certain amount of small anions, preferably polyvalent anions, such as phosphate, aspartate or citrate.

Depending on their form, the compositions of the invention may include solutions, emulsions (including microemulsions), suspensions, creams, lotions, gels, powders or other generally solid or liquid compositions for application to the skin and other tissues with which the compositions may be used. The compositions may contain, in addition to skin lightening agents and carriers, other ingredients commonly used in such products, such as antimicrobial agents, moisturizers and hydrating agents, penetrants, preservatives, emulsifiers, natural or synthetic oils, solvents, surfactants, cleansers, lubricants, antioxidants, fragrances, fillers, thickeners, waxes, deodorants, dyes, colorants, powders, and optionally including anesthetics, anti-itch additives, botanical extracts, conditioners, lightening agents, glitter, moisturizers, micas, minerals, polyphenols, silicones or derivatives thereof, sunscreens, vitamins, and botanicals (phytomedicinals).

In a particularly preferred embodiment, the composition comprisesGelling agents and/or viscosity modifiers. These agents are typically added to increase the viscosity of the composition, thereby making application of the composition easier and more accurate. In addition, these agents help to prevent the aqueous skin lightening agent/carrier solution from drying out, which tends to cause a reduction in the activity of the skin lightening agent. Particularly preferred agents are those that are uncharged and do not interfere with the skin lightening agent activity or the efficiency of the toxin carrier complex to penetrate the skin. The gelling agent may be certain cellulose-based gelling agents, such as hydroxypropyl cellulose (HPC). In some embodiments, the skin lightening agent/carrier complex is formulated in a composition with 2-4% HPC. Alternatively, the viscosity of the solution containing the skin lightening agent/carrier complex may be altered by the addition of polyethylene glycol (PEG). In other embodiments, the skin lightening agent/carrier solution is admixed with a pre-mixed adhesive (e.g., a skin lightening agent/carrier solutionHumectant) combination.

The invention also relates to a kit comprising one or more skin lightening agents and a positively charged carrier according to the invention. The one or more skin lightening agents and the positively charged carrier may be pre-mixed or may be present in the kit as separate components that are mixed prior to administration. The kit may include a device for delivering one or more skin lightening agents and a positively charged carrier. Non-limiting examples of such devices include skin patches and conventional applicators.

It is understood that the following examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are incorporated by reference in their entirety for all purposes.

Examples

Example 1

In vitro assay-tyrosinase inhibition assay

This example reports a comparative study in which the transdermal flux of kojic acid formulations (with and without the exemplary positively charged carrier molecule according to the invention) through porcine skin was measured. As detailed below, the results show that the transdermal flux of kojic acid with exemplary positively charged molecules of the present invention is more than twice or more greater than that observed for the same kojic acid formulation that does not contain a positively charged carrier molecule.

The measurements reported in this example make use of the following facts: the presence of kojic acid inhibits the ability of tyrosinase to oxidize phenols, such as tyrosine. By monitoring the extent to which the tyrosinase activity is reduced by kojic acid penetrating the skin of pigs, the transdermal flux of kojic acid corresponding to various kojic acid formulations can be measured. In these studies, high levels of tyrosinase inhibition indicate high levels of transdermal flux of kojic acid through the porcine skin. The inhibition reaction was monitored using optical photometry, since the enzymatic reaction between tyrosine and tyrosinase was accompanied by a color change, which can be monitored by measuring the optical density at 475nm and is directly proportional to the concentration of tyrosinase.

For the studies reported in this example, all reagents were obtained from Sigma-Aldrich, st. A1.8 mM L-tyrosinase solution and a 1.25U/. mu.L mushroom tyrosinase solution were prepared using 67mM potassium phosphate buffer (pH 6.8). Polyaspartic acid flux buffer (concentration: 77ng/ml) was prepared in PBS with 1% BSA. Prepared with 0.9% NaCl at a concentration of 10. mu.g/. mu.L having the formula RKKRRQRRRG- (K)₁₅A positively charged carrier molecule of-GRKKRRQRRR (hereinafter "RTP 004"). RTP004 was synthesized using tBoc and/or Fmoc solid phase chemistry. A0.625 mM kojic acid solution (molecular weight 142g/mol) was prepared in potassium phosphate buffer and polyaspartic acid flow buffer. IC of kojic acid₅₀The value was calculated to be 30. mu.M (4.26. mu.g/mL). Thus, more than 2000 times concentrated kojic acid solution (i.e., about 4260. mu.g of kojic acid in 200. mu.l buffer) was used in the flow experimentsAcid) to account for the dilution of the buffer that occurs during the collection of kojic acid across porcine skin, as described below.

Validation of kinetic assays in flow buffer

Before using the kinetic assay to measure the transdermal flux of the kojic acid formulation through porcine skin, the kinetic assay was validated to confirm the sensitivity of the assay used to detect inhibition of tyrosinase by kojic acid. Validation of the assay included measuring the extent of inhibition of tyrosinase activity by kojic acid and the dose dependence of inhibition of tyrosinase activity by kojic acid at various time points and serial dilutions. More specifically, 140. mu.L of 0.625mM kojic acid, 35. mu.L of 1.8mM L-tyrosine, 25. mu.L of 1.25U/. mu.L mushroom tyrosinase were loaded to a 96-well plate and incubated at 37 ℃. Kinetic measurements were performed at 1,2, 3 and 4 hour time points for 30 minutes, with the Optical Density (OD) at 475nm read and the results collected at 1 minute intervals (spectra max M5, Molecular Devices, Sunnyvale, CA). Some wells of the 96-well plate were control wells to which flow buffer was added without any tyrosinase or L-tyrosine. To calibrate the amount of kojic acid present in each reaction well, the measured amount of kojic acid was incorporated into the flow buffer stream of the control well per hour. These values were used to provide a positive control to assess the amount of kojic acid in reaction wells containing a mixture of tyrosinase, tyrosine and kojic acid.

Franz compartment assay for transdermal flux

The transdermal flux of kojic acid was measured for the following three formulations: (1) a formulation containing 4mg kojic acid in 200 μ L flow buffer but no positively charged carrier molecules; (2) a formulation comprising 4mg kojic acid and 12 micrograms RTP004 in 200 μ L flow buffer; and (3) a control formulation containing 200 μ L of flow buffer but no kojic acid or RTP 004.

The transdermal flux of kojic acid associated with the three formulations was measured using a Franz compartment (PermeGear, Bethlehem, Pa.; Isco Retriever IV, Lincoln, NE). In short, the Franz compartment is a device that allows measuring the flux of a compound through a membrane (here porcine skin). The various formulations to be studied were placed on one side of the pig skin and allowed to diffuse through to the other side (for 4 hours). The solution that passed through the pig skin (i.e., the flow-through solution) entered a continuously circulating stream of 0.9% NaCl buffer, which was finally collected and analyzed in a kinetic assay.

More specifically, the series of cells (in-line cells) were mounted to the Franz compartment and the circulation vessel was filled with 0.9% NaCl. Porcine skin (0.45mm thickness) was loaded into the tandem cells and 200 μ L of the three formulations were added to each cell. The Franz compartment was run at 8 μ Ι/min (min) for 4 hours with changing compartments (shuttle) once per hour (total 480 μ Ι _ per sample) for a total of 5 samples per group. Three different flow samples were tested in a total volume of 200 μ L/cell (N5/sample). Note that the samples were mixed and incubated for 5 minutes at room temperature before loading into each individual Franz cell.

Flow-through solutions obtained from each of the three formulations were collected and subjected to kinetic measurements. In the kinetic assay, 35. mu.L of 1.8mM L-tyrosine, 25. mu.L of 1.25U/. mu.L mushroom tyrosinase and 140. mu.L sample were loaded into each well of a 96-well plate. The sample solution is a collected flow-through solution, or a solution obtained by subjecting a flow-through solution to a serial series of two-fold dilutions. Kojic acid plus peptide delivery was measured by the percentage of the amount of loading that appeared in the flow through solution. For the positive control, kojic acid was not added.

The results show that the formulation containing kojic acid and RTP004 exhibited greater transmembrane flux of kojic acid than the formulation containing kojic acid but no RTP 004. More specifically, the percentage of kojic acid passing through the pig skin was determined to be 12.28% (for formulations containing kojic acid and RTP 004), and 5.62% (for formulations containing kojic acid only), relative to the amount of added kojic acid. As expected, no kojic acid flux was observed for the control formulation.

Thus, this example shows that transdermal flux of kojic acid can be enhanced by using positively charged carrier molecules of the present invention. The results indicate that the topical compositions of the present invention can allow the same transdermal flux of skin lightening agent (e.g., kojic acid) to be achieved with lower concentrations of the skin lightening agent in the topical composition. Thus, the composition of the present invention may help to mitigate the deleterious effects caused by a high local concentration of kojic acid.

Claims (58)

1. A topical composition comprising:

kojic acid, and

a positively charged carrier molecule comprising a positively charged backbone and a plurality of efficiency groups attached to the backbone;

wherein the positively charged backbone is a polyamino acid and the efficiency group is an amino acid sequence selected from the group consisting of: (gly)_p-RKKRRQRRR-(gly)_qWherein subscripts p and q are each independently an integer from 0 to 20;

wherein the kojic acid and the carrier molecule are non-covalently bound;

wherein the kojic acid is present in an amount sufficient to cause skin lightening; and

wherein the positively charged carrier molecule is present in an amount sufficient to enhance transdermal transport of kojic acid.

2. The topical composition according to claim 1, wherein the polyamino acid is selected from polylysine, polyarginine, polyhistidine and polyornithine.

3. Use of a topical composition comprising kojic acid and a positively charged carrier molecule in the manufacture of a medicament for reducing pigmentation in skin, wherein the positively charged carrier molecule comprises a positively charged backbone and a plurality of efficiency groups attached to the backbone;

wherein the positively charged backbone is a polyamino acid and the efficiency group is an amino acid sequence selected from the group consisting of: (gly)_p-RKKRRQRRR-(gly)_qWherein subscripts p and q are each independently an integer from 0 to 20;

wherein the kojic acid and the positively charged carrier molecule are non-covalently bound; and

wherein the positively charged carrier molecule is present in the topical composition in an amount sufficient to enhance transdermal transport of kojic acid.

4. Use according to claim 3, wherein the polyamino acid is selected from polylysine, polyarginine, polyhistidine and polyornithine.

5. A topical composition according to claim 1, wherein the positively charged carrier molecule is present in the topical composition in a sufficient amount such that the transdermal flux of kojic acid is more than twice the transdermal flux of kojic acid for an otherwise identical reference composition that does not contain a positively charged carrier molecule.

6. Use according to claim 3, wherein the positively charged carrier molecule is present in the topical composition in a sufficient amount such that the transdermal flux of kojic acid is more than twice the transdermal flux of kojic acid for an otherwise identical reference composition not comprising a positively charged carrier molecule.

7. The topical composition according to claim 1, wherein subscripts p and q are each independently an integer from 0 to 8.

8. The topical composition according to claim 1, wherein subscripts p and q are each independently an integer from 2 to 5.

9. Use according to claim 3, wherein the subscripts p and q are each independently an integer from 0 to 8.

10. Use according to claim 3, wherein the subscripts p and q are each independently an integer of from 2 to 5.

11. The topical composition according to claim 1, wherein the composition reduces melanin production or melanin-related pigmentation of the skin.

12. The topical composition according to claim 1, wherein the composition treats a condition associated with increased melanin production.

13. The topical composition according to claim 12, wherein the condition is hyperpigmentation, tanning, or a combination thereof.

14. The topical composition according to claim 1, further comprising a dermatologically, pharmaceutically, cosmetically, or pharmaceutically acceptable carrier.

15. The topical composition according to claim 1, wherein the composition is in the form of a solution, emulsion, suspension, cream, lotion, gel or powder.

16. The topical composition according to claim 15, wherein the composition is in the form of a microemulsion.

17. The topical composition according to claim 15 or 16, further comprising a gelling agent, a viscosity modifier, or a combination thereof.

18. The topical composition according to claim 17, wherein the gelling agent is cellulose-based.

19. The topical composition according to claim 17, wherein the gelling agent is hydroxypropyl cellulose.

20. The topical composition according to claim 19, wherein the hydroxypropyl cellulose is present in the composition in an amount of 2-4% of the total composition.

21. The topical composition according to claim 17, wherein the viscosity modifier is a humectant.

22. The topical composition according to claim 17, wherein the viscosity modifier is polyethylene glycol.

23. The topical composition according to claim 15 or 16, further comprising an additive selected from the group consisting of: antimicrobial agents, moisturizers, hydrating agents, penetrants, preservatives, natural oils, synthetic oils, solvents, surfactants, cleansers, lubricants, antioxidants, fragrances, fillers, thickeners, waxes, odor eliminators, dyes, colorants, powders, or combinations thereof.

24. The topical composition according to claim 23, wherein the surfactant is an emulsifier.

25. The topical composition according to claim 15 or 16, further comprising an additive selected from the group consisting of: anesthetics, botanical extracts, conditioners, glitters, minerals, polyphenols, silicones, sunscreens, botanicals, or combinations thereof.

26. The topical composition according to claim 25, wherein the conditioning agent is at least one conditioning agent selected from the group consisting of anti-itch additives, lightening agents, moisturizers, and vitamins; or wherein the mineral is mica.

27. Use according to claim 3, wherein the medicament treats a condition associated with increased melanin production.

28. The use according to claim 27, wherein the condition is hyperpigmentation, tanning, or a combination thereof.

29. The topical composition according to claim 2, wherein the polyamino acid is polylysine.

30. A topical composition according to claim 1 wherein the positively charged backbone has a molecular weight of from 100 to 1,500,000D.

31. A topical composition according to claim 1 wherein the positively charged backbone has a molecular weight of from 250 to 1,200,000D.

32. A topical composition according to claim 1 wherein the positively charged backbone has a molecular weight of from 1000 to 1,000,000D.

33. The topical composition according to claim 29, wherein the polylysine has a molecular weight of 100 to 1,500,000D.

34. The topical composition according to claim 29, wherein the polylysine has a molecular weight of 250 to 1,200,000D.

35. The topical composition according to claim 29, wherein the polylysine has a molecular weight of 1000 to 1,000,000D.

36. The topical composition according to claim 29, wherein the polylysine has a molecular weight of 1000 to 3000D.

37. The topical composition according to claim 29, wherein the polylysine has a molecular weight of greater than 70,000D.

38. The topical composition according to claim 29, wherein the polylysine has a molecular weight of 70,000 to 150,000D.

39. The topical composition according to claim 29, wherein the polylysine has a molecular weight of 150,000 to 300,000D.

40. The topical composition according to claim 29, wherein the polylysine has a molecular weight of greater than 300,000D.

41. Use according to claim 4, wherein the polyamino acid is polylysine.

42. Use according to claim 4, wherein the positively charged backbone has a molecular weight of from 100 to 1,500,000D.

43. Use according to claim 4, wherein the positively charged backbone has a molecular weight of 250 to 1,200,000D.

44. Use according to claim 4, wherein the positively charged backbone has a molecular weight of 1000 to 1,000,000D.

45. The use according to claim 41, wherein the polylysine has a molecular weight of from 100 to 1,500,000D.

46. The use according to claim 41, wherein the polylysine has a molecular weight of from 250 to 1,200,000D.

47. The use according to claim 41, wherein the polylysine has a molecular weight of from 1000 to 1,000,000D.

48. Use according to claim 41 wherein the polylysine has a molecular weight of from 1000 to 3000D.

49. The use according to claim 41, wherein the polylysine has a molecular weight of greater than 70,000D.

50. The use according to claim 41, wherein the polylysine has a molecular weight of from 70,000 to 150,000D.

51. The use according to claim 41, wherein the polylysine has a molecular weight of 150,000 to 300,000D.

52. The use according to claim 41, wherein the polylysine has a molecular weight of greater than 300,000D.

53. The topical composition according to claim 1, wherein the polyamino acid is (Lys)₁₅。

54. The topical composition according to claim 1, wherein the efficiency group is an amino acid sequence selected from the group consisting of: RKKRRQRRRG and GRKKRRQRRR.

55. The topical composition according to claim 1, wherein the positively charged carrier molecule has the formula: RKKRRQRRRG- (K)₁₅-GRKKRRQRRR。

56. Use according to claim 3, wherein the polyamino acid is (Lys)₁₅。

57. Use according to claim 3, wherein the efficiency group is an amino acid sequence selected from the group consisting of: RKKRRQRRRG and GRKKRRQRRR.

58. Use according to claim 3, wherein the positively charged carrier molecule has the formula: RKKRRQRRRG- (K)₁₅-GRKKRRQRRR。