CN105147533B - Bakuchiol composition for treating postinflammatory hyperpigmentation - Google Patents

Abstract

Methods for treating hyperpigmentation, including treatment of post-inflammatory hyperpigmentation (PIH), are disclosed. The disclosed method comprises administering to a mammal a composition comprising bakuchiol substantially free of furanocoumarins. Compositions comprising bakuchiol and methods for their preparation are also disclosed.

Description

Bakuchiol compositions for treating post-inflammatory hyperpigmentation

References to related applications

This application claims the benefit of PCT Application No. US2011/026594, filed March 1, 2011, and U.S. Provisional Patent Application No. 61/438,890, filed February 2, 2011, pursuant to 35 U.S.C. §119(e).

background

technical field

The present invention generally relates to bakuchiol compositions and their use for the treatment of post-inflammatory hyperpigmentation.

Related technical description

Post-inflammatory hyperpigmentation (PIH) is a rare skin pigmentation disease state associated with increased melanin synthesis and deposition. PIH is also characterized by apoptosis of melanocytes due to oxidative stress and invasion from mediators and cytokines of inflammatory and immune responses. Melanin deposition (i.e., hyperpigmentation) occurs beyond the epidermal level with marked release of melanin into the papillary dermis and capture by large immune cells. These unique histological features of PIH create many difficulties in the treatment of PIH with traditional agents.

Common treatments for PIH focus on preventing further pigment development by using corticosteroids and controlling inflammation with photoprotectants. Chemical peel compounds such as salicylic acid and glycolic acid are also used to promote skin renewal and to remove or reduce hyperpigmentation. Topical retinoids have also been used to treat PIH, but such approaches require up to 40 weeks before significant benefit is observed.

Tyrosinase inhibitors or skin lightening agents such as hydroquinone, azelaic acid, kojic acid and licorice extract have also been used to treat PIH. A significant disadvantage of using conventional skin lighteners or tyrosinase inhibitors is generalized depigmentation of normal skin adjacent to the site of PIH. This effect weakens the color of the background skin and makes the PIH site more prominent. Therefore, these agents must be applied with great care at the site of PIH. Furthermore, tyrosinase inhibitors are only effective against epidermal hyperpigmentation, since the epidermis is where melanin is synthesized by tyrosinase. Since post-inflammatory hyperpigmentation occurs in the deeper layers of the skin (e.g., the papillary dermis), continuous application of hydroquinone drugs over 6 months is required before visible changes in dark marks are observed. Finally, hydroquinone-type skin lighteners or tyrosinase inhibitors are associated with side effects including skin irritation, dryness, teratogenicity, and induction of vitiligo and skin cancer.

Post-inflammatory hyperpigmentation originates from endogenous inflammatory skin disorders such as acne, atopic dermatitis, allergic contact dermatitis, incontinence pigmentosa, lichen planus, lupus erythematosus, morphea. Other causes of PIH include exogenous inflammatory stimuli such as mechanical trauma, ionizing and non-ionizing radiation, burns, laser therapy, and skin infections. Therapeutic agents currently used for the above skin conditions are ineffective in preventing, alleviating, reducing or treating PIH. For example, anti-inflammatory agents such as retinoic acid, COX inhibitors (eg, salicylic acid), nonsteroidal anti-inflammatory drugs (NSAIDs), antimicrobials, or steroids are often used to treat the above skin conditions, but these treatments have proven Not effective for PIH.

Although significant progress has been made in this field, there is still a great need in the field for methods of preventing, alleviating, reducing or treating hyperpigmentation. For example, methods for treating post-inflammatory hyperpigmentation are needed. The present invention fulfills these needs and provides further related advantages.

overview

In general, the present invention relates to methods for preventing, alleviating, reducing or treating hyperpigmentation. Hyperpigmentation can be the result of a disease state derived from an inflammatory skin disease state. For example, one embodiment of the invention is a method for preventing, alleviating, reducing or treating post-inflammatory hyperpigmentation (PIH). This PIH can arise from a number of skin conditions, including acne. The method comprises administering to the mammal an effective amount of a composition comprising bakuchiol and less than 500 ppm total furanocoumarin impurities.

Contrary to other skin lightening agents, the bakuchiol compositions disclosed herein are not tyrosinase inhibitors. Thus, the disclosed compositions depigment particularly at the site of PIH and are useful for treating hyperpigmentation in the deeper layers of the skin (e.g., the papillary dermis). Accordingly, the presently disclosed methods include several advantages over previous methods for treating hyperpigmentation and/or PIH.

Accordingly, one embodiment of the present disclosure is directed to a method for preventing, alleviating, reducing or treating hyperpigmentation caused by a condition derived from an inflammatory skin disorder, the method comprising administering to a mammal an effective amount of A composition of phenol or a pharmaceutically acceptable salt thereof, a pharmaceutically acceptable carrier and a total furanocoumarin impurity of less than 500ppm.

In some embodiments, the disease state is post-inflammatory hyperpigmentation. In other embodiments, the composition comprises less than 100 ppm total furanocoumarin impurities. In other embodiments, the furanocoumarin impurity comprises psoralen, isopsoralen, or a combination thereof. In some embodiments, the composition exhibits no tyrosinase inhibitory activity relative to a kojic acid control.

In other embodiments, bakuchiol is chemically synthesized or isolated from plants. For example, in some embodiments, bakuchiol is isolated from a plant. In other embodiments, the plant is from a plant of the Psoralea genus, such as Psoralea corylifolia L. (Leguminaceae) or Psoraleaglandulosa L. (Papilionaceae).

In other embodiments, bakuchiol is isolated from seeds, stems, bark, branches, tubers, roots, root bark, shoots, rhizomes, flowers or other reproductive organs, leaves or other aerial parts, or combinations thereof .

In some other embodiments, postinflammatory hyperpigmentation (PIH) is derived from acne, atopic dermatitis, allergic contact dermatitis, incontinence pigmentosa, lichen planus, lupus erythematosus, morphea, mechanical trauma, ionization or non-ionizing radiation, burns, laser or drug treatments, skin infections, or a combination thereof. For example, in some aspects, post-inflammatory hyperpigmentation (PIH) results from acne.

In other embodiments, the composition comprises 0.001% to 99.9% by total weight of bakuchiol and a pharmaceutically, dermatologically or cosmetically acceptable carrier. For example, in some aspects, the composition comprises 0.1% to 2.0% bakuchiol by total weight, 1.0% bakuchiol by total weight, or 0.5% bakuchiol by total weight.

In other embodiments, the dermatologically acceptable carrier includes a non-adhesive gauze, bandage, swab, wipe, plaster, mask, or protectant. In some other embodiments, the cosmetically acceptable carrier includes a cleanser or antiseptic.

In some aspects, the composition is formulated for topical administration. For example, in some aspects, the composition further comprises a cream, lotion, ointment, gel, emulsion, liquid, paste, soap, powder, or combinations thereof.

In other embodiments, the composition further comprises an adjuvant, a skin penetration enhancer, or liposomes. In other embodiments, the adjuvants include alpha-hydroxy acids, salicylic acid, linolenic acid, retinoic acid, benzoyl peroxide, sulfacetamide sodium, clindamycin, erythromycin, dapsone, Tetracycline, Deoxyoxytetracycline, Minocycline, Zinc, Estrogen or its Derivatives, Antiandrogens, Sulfur, Steroids, Cortisone, Tazarotene, Curcumin Extract, Gum Arabic Extract, Scutellaria Baicalensis Extract extract, green tea extract, grape seed extract, or combinations thereof.

In some embodiments, the compositions are formulated in capsule form, eg, controlled release capsules. In other embodiments, the composition is administered topically by aerosol, by suppository, intradermally, intramuscularly or intravenously.

In some aspects, the methods prevent hyperpigmentation. In other aspects, the methods alleviate hyperpigmentation. In other aspects, the methods reduce hyperpigmentation. In other aspects, the methods treat hyperpigmentation.

In other embodiments, the hyperpigmentation occurs in the deeper layers of the skin, e.g., in the papillary dermis of the skin. In other embodiments, the method further comprises reducing superoxide anion. In some other embodiments, the method further comprises reducing melanogenesis. In other embodiments, the method further comprises reducing melanocyte proliferation. In other embodiments, the method further comprises preventing melanocyte apoptosis.

In some other embodiments, the mammal is a human. In some other embodiments, the mammal is in need of preventing, alleviating, reducing or treating hyperpigmentation resulting from a condition resulting from an inflammatory skin disorder. For example, a mammal may be in need of treatment for PIH.

In another embodiment, the present disclosure relates to a method for reducing melanogenesis, reducing melanocyte proliferation, or preventing melanocyte apoptosis, the method comprising administering to a mammal an effective amount of A composition of a pharmaceutically acceptable salt with a pharmaceutically acceptable carrier and less than 500 ppm total furanocoumarin impurities. In some other embodiments, the method further comprises reducing superoxide anion.

In other embodiments, the composition comprises less than 100 ppm total furanocoumarin impurities. In other embodiments, the furanocoumarin impurity comprises psoralen, isopsoralen, or a combination thereof. In some embodiments, the composition exhibits no tyrosinase inhibitory activity relative to a kojic acid control.

In other embodiments, bakuchiol is chemically synthesized or isolated from plants. For example, in some embodiments, bakuchiol is isolated from a plant. In some other embodiments, the plant is from a plant of the Psoralea genus, such as Psoralea corylifolia L. (Leguminaceae) or Psoraleaglandulosa L. (Papilionaceae).

In other embodiments, bakuchiol is isolated from seeds, stems, bark, branches, tubers, roots, root bark, shoots, rhizomes, flowers or other reproductive organs, leaves or other aerial parts, or combinations thereof .

In some embodiments, melanogenesis, melanocyte proliferation, or melanocyte apoptosis is a result of post-inflammatory hyperpigmentation (PIH). In some other embodiments, post-inflammatory hyperpigmentation (PIH) is derived from acne, atopic dermatitis, allergic contact dermatitis, incontinence pigmentosa, lichen planus, lupus erythematosus, morphea, mechanical trauma, Ionizing or non-ionizing radiation, burns, laser or drug treatments, skin infections, or a combination thereof. For example, in some aspects, post-inflammatory hyperpigmentation (PIH) results from acne.

In other embodiments, the composition comprises 0.001% to 99.9% by total weight of bakuchiol and a pharmaceutically, dermatologically or cosmetically acceptable carrier. For example, in some aspects, the composition comprises 0.1% to 2.0% bakuchiol by total weight, 1.0% bakuchiol by total weight, or 0.5% bakuchiol by total weight.

In other embodiments, the dermatologically acceptable carrier includes a non-adhesive gauze, bandage, swab, wipe, plaster, mask, or protectant. In some other embodiments, the cosmetically acceptable carrier includes a cleanser or antiseptic.

In some aspects, the composition is formulated for topical administration. For example, in some aspects, the composition further comprises a cream, lotion, ointment, gel, emulsion, liquid, paste, soap, powder, or combinations thereof.

In other embodiments, the composition further comprises an adjuvant, a skin penetration enhancer, or liposomes. In other embodiments, the adjuvants include alpha-hydroxy acids, salicylic acid, linolenic acid, retinoic acid, benzoyl peroxide, sulfacetamide sodium, clindamycin, erythromycin, dapsone, Tetracycline, Deoxyoxytetracycline, Minocycline, Zinc, Estrogen or its Derivatives, Antiandrogens, Sulfur, Steroids, Cortisone, Tazarotene, Curcumin Extract, Gum Arabic Extract, Scutellaria Baicalensis Extract extract, green tea extract, grape seed extract, or combinations thereof.

In some embodiments, the compositions are formulated in capsule form, eg, controlled release capsules. In other embodiments, the composition is administered topically by aerosol, by suppository, intradermally, intramuscularly or intravenously.

In some aspects, the methods prevent hyperpigmentation. In other aspects, the methods alleviate hyperpigmentation. In other aspects, the methods reduce hyperpigmentation. In other aspects, the methods treat hyperpigmentation. In some embodiments, hyperpigmentation occurs in the deeper layers of the skin, e.g., in the papillary dermis of the skin.

In some other embodiments, the method reduces melanogenesis. In other embodiments, the methods reduce melanocyte proliferation. In other embodiments, the methods prevent melanocyte apoptosis.

In some other embodiments, the mammal is a human. In some other embodiments, the mammal is in need of treatment to reduce melanogenesis, reduce melanocyte proliferation, or prevent melanocyte apoptosis.

In other embodiments, the composition further comprises salicylic acid or a pharmaceutically acceptable salt thereof.

Other embodiments of the present disclosure relate to methods of treating inflammatory or non-inflammatory lesions comprising administering to a mammal an effective amount of a compound comprising bakuchiol or a pharmaceutically acceptable salt thereof and salicylic acid or a pharmaceutically acceptable salt thereof. A composition of a salt and a pharmaceutically acceptable carrier. For example, in some embodiments, the lesions comprise inflammatory acne lesions. In other embodiments, the methods treat inflammatory and non-inflammatory lesions.

In some other embodiments of the foregoing, the mammal is a human. In some other embodiments, the mammal is in need of treatment for an inflammatory or non-inflammatory condition.

In other embodiments, the present invention includes compositions comprising bakuchiol, or a pharmaceutically acceptable salt thereof, and salicylic acid, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. In some embodiments, the composition is formulated for topical administration.

These and other aspects of the invention will be apparent upon reference to the following detailed description.

Brief description of the drawings

In the drawings, the same reference numerals designate similar elements. The size and relative positions of elements in the drawings are not necessarily drawn to scale and some of these elements are arbitrarily enlarged and positioned to improve drawing legibility. Furthermore, the particular shapes of elements as drawn are not intended to convey any information about the actual shape of the particular elements, and have only been chosen for ease of identification in the drawings.

Figure 1 depicts the chromatograms of bakuchiol, psoralen, and isopsoralen standards.

Figure 2 shows chromatograms of bakuchiol compositions before and after hydrolysis.

Figure 3 provides data showing the strong antioxidant properties of bakuchiol compositions.

Figure 4 is a graph of the tyrosinase inhibitory activity of bakuchiol compositions and kojic acid.

Figure 5 shows the variation in the severity of PIH in individual test individuals.

Figure 6 represents a graph of the percent change in PIH-infected facial area for individual test individuals.

Figure 7 shows the mean percent change in PIH and PIH severity for the five tested individuals.

Figure 8 depicts the mean grade level reduction in PIH and PIH severity at each visit compared to baseline.

Figure 9 shows photographs of two study participants at different time intervals.

Detailed Description

In the following description, several specific details are set forth in order to provide a thorough understanding of various embodiments. However, it will be understood by those skilled in the art that the present invention may be practiced without these details. In other instances, well-known structures have not been shown or described in detail to avoid unnecessarily obscuring the embodiments. Unless the context requires otherwise, in the following description and claims, the word "comprise" and its variants, such as "comprises" and "comprising", are to be interpreted in an open, inclusive sense, That is, for example "including but not limited to". Furthermore, headings provided herein are for convenience only and do not indicate the scope or meaning of the claimed invention.

In this specification, reference to "one embodiment" or "an embodiment" means that a particular feature, structure or characteristic described with respect to the embodiment is included in at least one embodiment. Thus, appearances of the phrase "in one embodiment" or "in an embodiment" in various places in this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner in one or more embodiments. Furthermore, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. It should also be noted that general usage of the term "or" includes "and/or" unless the content clearly dictates otherwise.

definition

As used herein and unless the context dictates otherwise, the following terms have the meanings set forth below.

As used herein, "bakuchiol" refers to a compound having the formula:

Wherein the benzyl double bond can be cis or trans. As used herein, bakuchiol includes pharmaceutically acceptable salts and tautomers of bakuchiol. Phenolic compounds structurally related to bakuchiol are also included in this definition.

"Bakutrol ^™ " is a composition comprising bakuchiol and may additionally comprise fatty acids extracted from plants of the genus Psoralea.

"UP256" refers to a 0.5% (wt/wt) formulation of bakuchiol.

"Preventing," "prevention," and "prevent" in the context of the disclosed methods all refer to prophylactic methods that arrest or halt the occurrence of a particular disease state, such as PIH.

"Alleviating", "alleviation" and "alleviate" in the context of the disclosed methods all refer to alleviating or alleviating the effects or symptoms of a particular disease state, such as PIH.

"Reducing", "reduction" and "reduce" in the context of the disclosed methods all refer to reducing the effects or symptoms of a particular disease state, such as PIH.

"Treating", "treatment" and "treat" in the context of the disclosed methods all refer to techniques or methods intended to ameliorate the symptoms or reduce or stop the occurrence of a particular disease state such as PIH .

"Impurities" include anything that is undesirable in a bakuchiol composition, usually derived from bakuchiol isolated from natural sources. The term impurity includes, but is not limited to, furanocoumarin compounds including, but not limited to, psoralen, isopsoralen, and other coumarin-type impurities. Impurities also refer to impurities originating from the synthetic methods used to obtain these compositions.

"Treatment" includes treatment and/or prophylaxis. When used, treatment refers to humans as well as other animals.

A "pharmaceutically, cosmetically or therapeutically effective dose or amount" refers to a dosage level sufficient to induce a desired biological or functional result. The result can be alleviation of a disease, a sign, symptom or cause of a skin disease state, or any other desired change in a biological system.

"Placebo" refers to a drug or a therapeutically effective dose or amount administered in place of a drug or a therapeutically effective dose or amount sufficient to induce the desired biological sign, symptom, or cause of a disease that can be alleviated with an inactive substance.

A "host" or "individual" or "patient" is a living individual, human or animal, to whom a composition described herein is administered. Accordingly, the compositions described herein are useful in veterinary as well as human applications and the terms "patient" or "individual" or "recipient" should not be construed in a limiting manner. In the case of veterinary applications, the dosage range can be administered according to the body weight of the animal as described hereinafter.

As noted above, one embodiment of the present disclosure relates to the use of a composition comprising bakuchiol substantially free of furanocoumarin impurities for the prevention, alleviation, reduction or treatment of disease states arising from inflammatory skin disorders Hyperpigmentation. For example, the disclosed methods are useful in the treatment of post-inflammatory hyperpigmentation (PIH). In some embodiments, PIH can be derived from acne. The disclosed methods have been shown to be effective in preventing, alleviating, reducing and treating post-inflammatory pigmentation resulting from skin conditions such as acne, atopic dermatitis, allergic contact dermatitis, incontinence pigmentosa, lichen planus, lupus erythematosus, morphea Hyperpigmentation; and post-inflammatory hyperpigmentation induced by mechanical trauma, ionizing and non-ionizing radiation, burns, lasers, and drug treatments, and by the use of synthetic bakuchiol or psoralea without furanocoumarins Human Clinical Efficacy of ) Extract Bakuchiol Composition in Skin Infections. These and other aspects and various embodiments of the present disclosure will become apparent upon reference to the description that follows.

A. Bakuchiol Compositions

In one embodiment, the present disclosure provides compositions comprising bakuchiol substantially free of impurities, particularly furanocoumarin impurities. This composition is also referred to herein as Bakutrol ^™ . In some embodiments, the organic synthesis from simple compounds or from plants is obtained as demonstrated in the literature (Hongli Chen and Yuanchao Li, Letters in Organic Chemistry, 2008, 5, 467-469). combination. In some embodiments, the bakuchiol composition is isolated from a plant. Plant sources of bakuchiol include plant families including, but not limited to, Luguminosae, Papilionaceae, Lauraceae, and Magnoliaceae, and the plant genera , said plant genera include but are not limited to Psoralea (Psorlea), Sassafras (Sassafras), Magnolia (Magnolia) and Atractylodes (Atractylodes). For example, the bakuchiol composition may be isolated from Psoralea corylifolia L. (Fauceae) or Psoralea glandulosa L. (Papilionaceae). Can be from the whole plant or from one or more individual parts of the plant including, but not limited to, seeds, stems, bark, branches, tubers, roots, root bark, shoots, rhizomes, flowers or other reproductive organs, leaves or other aerial parts Said composition is obtained in or a combination thereof. Methods for isolating bakuchiol from plants may include solvent extraction, supercritical fluid extraction, distillation, physical expression, or combinations thereof.

Bakuchiol, the structure of which is exemplified below, is a phenolic compound having a hydroxyl group in an aromatic ring and an unsaturated hydrocarbon chain. Although represented in trans in the structures below, the benzyl double bond of bakuchiol may also be in cis.

The amount of bakuchiol (i.e., weight percent (w/w%)) in a pure plant extract depends on the extraction method and the degree of purification of the crude extract. In one embodiment, as shown in Table 2, the amount of bakuchiol in the extract is from 13.7% to 29.1%. In other embodiments, the amount of bakuchiol in the extract is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% %. In some embodiments, the amount of bakuchiol in the extract is 100%. In other embodiments, the amount of bakuchiol in the composition is not less than 60%. Examples 6-8 provide examples of extracts containing various amounts of bakuchiol.

Although bakuchiol is a biologically active natural product with great potential for the prevention and treatment of various diseases and conditions, there are many limitations associated with the use of this compound. Some limitations include its low concentration in natural sources and the presence of toxic components co-existing in bakuchiol sources. The impurities present in the bakuchiol composition vary with the bakuchiol source. For example, psoralen, also known as furanocoumarin, is a naturally occurring secondary metabolite in the Psoralea genus (a source of bakuchiol) and is also found in many fruits and vegetables. exists in. Examples of furanocoumarins that are often found co-occurring with bakuchiol include psoralen and isopsoralen.

Numerous health risks are associated with the handling, topical application and ingestion of psoralen-containing plants and the synthesis of psoralen. Psoralens are known to be phototoxic agents that increase skin sensitivity to ultraviolet radiation and promote skin cancer (Epstein (1999) Med. Surg. 18(4):274-284). Psoralen was shown to induce growth inhibition in rats (Diawara et al. (1997) Cancer Lett. 114(1-2):159-160). Gonadal toxicity of crude extracts derived from plants of the genus Psoralea is directly linked to disruption of the hypothalamic-pituitary-gonadal axis (Takizawa et al. (2002) J. Toxicological Sciences 27(2) :97-105). Oral administration of psoralen, bergapton (5-methoxypsoralen), and xanthotoxin (8-methoxypsoralen) in the diet of female rats reduced birth rate, transplantation in a dose-dependent manner Number of sites, puppies, corpus luteum, full and empty uterine weight and circulating estrogen levels (Diawara et al. (1999) J. Biochem. Molecular Toxicology 13(3/4):195-203 ). Psoralen has also been shown to induce mRNAs for the hepatic enzymes CYP1A1 and UGT1A6, suggesting that increased estrogen metabolism by psoralen may explain reproductive toxicity and decreased ovarian follicle function and ovulation observations (Diawara et al. (2003 May - June) Pediatr Pathol Mol Med. 22(3):247-58.). Due to the toxicity of furanocoumarins, it is important to remove psoralen and isopsoralen from bakuchiol compositions intended for the treatment of post-inflammatory hyperpigmentation or other disease states.

Psoralen and isopsoralen comprise about 0.1%-2% dry weight of psoralen seeds and about 1%-20% by weight in solvent or supercritical fluid extracts. Crude extracts from plants of the Psoralea genus can be obtained by solvent extraction or supercritical fluid extraction, distillation, physical expression or a combination of the above extraction methods. Enriched bakuchiol compositions can be obtained by chromatographic separation, solvent layering (Indian Patent Publication #00570/KOL/2005), distillation, recrystallization and other wet chemical and physical methods. Published U.S. Patent Application No. 2006/0251749, which is incorporated herein by reference in its entirety, discloses solvent extraction followed by hydroxylation to decompose the furanocoumarin ring and obtain enriched supplements substantially free of furanocoumarin impurities. Boralol composition (eg, less than 500 ppm or less than 100 ppm furanocoumarin impurity). The disclosed methods include the steps of extracting the compound from the plant source, hydrolyzing the crude extract with heat using an alkaline solution, and purifying by methods including, but not limited to, column chromatography, extraction followed by crystallization, solvent layering, recrystallization, and combinations thereof. Applicants have discovered that such compositions of bakuchiol-enriched psoralea extract substantially free of furanocoumarin impurities are useful for preventing, alleviating, reducing or treating hyperpigmentation. For example, the disclosed bakuchiol compositions are effective in preventing, alleviating, reducing or treating post-inflammatory hyperpigmentation (PIH).

The present disclosure also relates to methods for isolating and purifying crude compositions of bakuchiol and related compounds obtained from natural sources. Methods used to isolate and purify these compositions include extraction of compounds from plant sources, hydrolysis of crude extracts using alkaline solutions, and extraction by methods including, but not limited to, column chromatography, extraction followed by crystallization, solvent layering, recrystallization, and combinations thereof Purification steps. The crude extract purified in this manner is substantially free of furanocoumarin impurities such as psoralen and isopsoralen. Thus, the potential phototoxicity, local irritation, carcinogenicity and reproductive toxicity associated with these compounds are substantially eliminated.

In some embodiments, the disclosed compositions comprise less than 500 ppm, less than 250 ppm, less than 100 ppm, or less than 50 ppm total furanocoumarin impurities. The concentration of furanocoumarin impurities can be determined by any method known to those skilled in the art. For example, in one embodiment, furanocoumarin content can be determined by HPLC.

As described in Example 2, the efficacy of bakuchiol extraction from plant sources was evaluated using six different organic solvent systems under two sets of extraction conditions. The results are set forth in Table 2. Referring to Table 2, it can be seen that bakuchiol can be extracted from Psoralea plants using a number of organic solvents and/or combinations thereof. The amount of bakuchiol in each extract ranged from 13.7% to 29.1% by weight. Other extraction methods include but are not limited to _CO2 supercritical fluid extraction and water distillation. Pressed exudates from fresh plant parts such as seeds can also be used to obtain bakuchiol compositions from natural sources.

The efficacy of purification of crude bakuchiol extract by column chromatography is demonstrated in Example 3 and Table 3. Specifically, eight different types of resins were evaluated for their ability to separate bakuchiol from furanocoumarin impurities. Both the silica gel resin and the CG-161 resin showed satisfactory separation. However, column chromatography separation of crude plant extracts is usually not economically feasible on an industrial scale because it requires expensive equipment and reagents as well as experienced personnel. The very low loading capacity of these samples due to the complexity of the crude plant extracts also makes industrial scale column chromatography difficult.

Example 4 describes an economical process for the separation of bakuchiol from furanocoumarin impurities. The method includes treating a composition comprising a furanocoumarin impurity with a base. As exemplified by Scheme 1 below, to illustrate the use of NaOH, base heating was used to open the lactone rings of the furanocoumarins, thereby converting them to the corresponding carboxylate salts. These salts can then be easily separated from the remainder of the mixture by a variety of methods. The disclosed methods allow for the preparation of bakuchiol compositions that are substantially free (eg, less than 500 ppm) of furanocoumarin impurities. Such highly pure bakuchiol compositions cannot be obtained using standard chromatographic techniques without the disclosed hydrolysis.

Reaction scheme 1. Hydrolysis of furanocoumarins

The base solution may include any base capable of opening the lactone ring, including but not limited to sodium hydroxide, potassium hydroxide, calcium hydroxide, lithium hydroxide, or combinations thereof. Solutions can have varying concentrations and pH values to maximize conversion to acid salts. The reaction mixture can also be heated at different temperatures and pressures to maximize reaction rate, efficiency and yield.

The reaction process was followed by HPLC to ensure complete conversion of the furanocoumarins to their corresponding carboxylates. The HPLC chromatograms of the crude composition before and after hydrolysis are illustrated in FIG. 2 . After completion of the reaction (as determined by HPLC), the reaction solution can be worked up using a variety of methods including, but not limited to, column chromatography, crystallization, solvent layering, precipitation, solvent washing, or combinations thereof. Organic solvents that can be used for solvent separation include, but are not limited to, petroleum ether, ethyl acetate, diethyl ether, hexane, chloroform, propanol, butanol, and methylene chloride, as well as other water-immiscible organic solvents.

The crude extract purified in this manner is substantially free of furanocoumarin impurities such as psoralen and isopsoralen. For example, the purified extract may contain less than 500 ppm, less than 250 ppm, less than 100 ppm, or even less than 50 ppm of furanocoumarin impurities. Furthermore, these highly pure furanocoumarin-free, bakuchiol compositions are light brown or red in color and they are very stable with respect to the color and composition of the active agent, making them particularly suitable for formulation, storage and cosmetic applications.

Also included in the present disclosure are methods for analyzing compositions of bakuchiol that enable the detection and quantification of impurities. In this embodiment, the method for analyzing the bakuchiol composition comprises the step of analyzing said composition by high pressure liquid chromatography (HPLC). Analysis by HPLC enables the quantification of individual components in the mixture and also provides a method for tracking bakuchiol, psoralen, isopsoralen and other natural components in Psoralea plants to Provides guidance on extraction, hydrolysis and purification methods. The method for analyzing the composition of bakuchiol using high pressure liquid chromatography (HPLC) is described in Example 1 (Table 1).

B. Treatment of Hyperpigmentation Using Bakuchiol Compositions

One embodiment of the present disclosure relates to the use of compositions comprising bakuchiol substantially free of furanocoumarin impurities for preventing, alleviating, reducing or treating hyperpigmentation caused by conditions derived from inflammatory skin disorders. For example, the disclosed methods include preventing, alleviating, reducing or treating post-inflammatory hyperpigmentation (PIH). In some embodiments, PIH may arise from acne. The disclosure includes formulation of bakuchiol compositions in typical cosmetic vehicles and also in skin creams, gel lotions and other formulations described in more detail below. As shown in the Examples, the applicants have demonstrated unexpected human clinical efficacy of bakuchiol compositions in the prevention, alleviation, reduction or treatment of post-inflammatory hyperpigmentation (PIH) derived from skin disorders Such as acne, atopic dermatitis, allergic contact dermatitis, incontinence pigmentosa, lichen planus, lupus erythematosus, morphea; and inflammation caused by mechanical trauma, ionizing and non-ionizing radiation, burns, lasers, and drug treatments Post-hyperpigmentation as well as skin infections.

The disclosed methods comprise administering to a mammal (e.g., a human patient) an effective amount of a composition comprising bakuchiol substantially free of furanocoumarin impurities. For example, the composition may contain less than 500 ppm of furanocoumarin impurities. The composition may comprise from about 0.0001% to about 100% bakuchiol. For example, in some embodiments, the composition comprises from about 0.1% to about 2% bakuchiol or from about 0.5% to about 1% bakuchiol. In other examples, the composition comprises about 0.5% or about 1.0% bakuchiol. In some embodiments, the mammal is a human, and in other embodiments, the mammal is in need of preventing, alleviating, reducing or treating hyperpigmentation resulting from a condition resulting from an inflammatory skin disorder, such as the mammal may PIH needs to be treated.

The present disclosure exhibits unexpectedly unique biological properties of synthetic or natural bakuchiol compositions. As shown in Example 5 and Table 4, the Bakutrol composition comprising about 57.35% bakuchiol has unexpectedly high antioxidant capacity, especially against superoxide anion (>69,000 μmole TE/g), against The five main active species had combined ORAC values at >92,000 μmole TE/g.

Superoxide is an anion with the chemical ^formula _O2- . Chronic inflammatory disease states such as acne vulgaris can have markedly increased superoxide anion production from keratinocytes stimulated by Gram-positive anaerobic bacteria such as Propionibacterium acnes (P. acnes) (Grange PA. et al., Plos Pathogens 2009, 5(7) 1-14.). Superoxide is very biotoxic and is dispersed by the immune system to kill invading microorganisms. In phagocytes, superoxide is produced in large quantities by the enzyme NADPH oxidase for an oxygen-dependent killing mechanism of invading pathogens. Superoxide anion and other reactive oxygen species in inflamed skin can also induce melanogenesis, melanocyte proliferation, and melanocyte apoptosis, which are the main pathogenesis of post-inflammatory hyperpigmentation. Accordingly, one embodiment of the present disclosure is to alleviate, reduce or treat disease states arising from inflammatory skin disorders by reducing superoxide using a composition comprising bakuchiol substantially free of furanocoumarin impurities. Way to hyperpigmentation. In one embodiment, the disease state is PIH. In another embodiment, the present disclosure provides methods of reducing melanogenesis or melanocyte proliferation or inhibiting melanocyte apoptosis, eg, by reducing superoxide anion. The method comprises administering to the mammal an effective amount of a composition comprising bakuchiol substantially free of furanocoumarin impurities. In some embodiments, the mammal is a human, and in other embodiments, the mammal is in need of reducing melanogenesis or melanocyte proliferation or inhibiting melanocyte apoptosis.

As demonstrated in Example 6 and Figure 3, a composition comprising 77.02% of bakuchiol substantially free of impurities, particularly furanocoumarin impurities, was shown to have a significant effect on 4-tetrabutylphenol (4-TBP)-induced Protection from oxidative stress. Cytotoxicity against melanocytes from reactive oxygen species generated by 4-TBP was protected by the bakuchiol composition, tested at two concentrations. While not wishing to be bound by said theory, the applicants believe that the unexpected clinical benefits of reducing, alleviating, preventing or treating post-inflammatory hyperpigmentation (PIH) from synthetic or natural bakuchiol compositions derive from Its unique and unexpected ability to neutralize reactive oxygen species, especially superoxide anion, and protect melanocytes from oxidative stress.

In addition to its unexpectedly high antioxidant capacity, the applicants have found that the disclosed bakuchiol composition is not a tyrosinase inhibitor. This is in contrast to other reports disclosing bakuchiol as a skin lightening agent through tyrosinase inhibition (Japanese Patent No. P1107123). This unexpected finding led the applicants to the presently disclosed method for the treatment of PIH, where pigmentation occurs in deep skin layers and where tyrosinase inhibitors are ineffective. The disclosed bakuchiol compositions without tyrosinase inhibition are shown in Example 7 and Figure 4. Both pure bakuchiol (100%) and enriched bakuchiol (77.02%) with no more than 100 ppm of furanocoumarins from natural sources had no tyrosinase inhibitory function at eight different doses .

The safety of compositions comprising bakuchiol was evaluated at concentrations of 86.54% and 77.02% bakuchiol. As shown in Example 9 and Table 6, based on in vitro and human clinical trials, the Bakutrol (UP256) composition exhibited no eye irritation, no skin irritation to normal or chafed skin, no skin contact sensitization, no Phototoxic and not mutagenic. The topical cream of the bakuchiol composition was well tolerated by all humans and in vitro.

As demonstrated in Example 10, containing 77.02% bakuchiol and less than 100 ppm furan tested in a human clinical trial on individuals with post-inflammatory hyperpigmentation (PIH) from mild or moderate acne vulgaris Coumarin is a natural bakuchiol composition (Bakutrol ^™ ) extracted and enriched from the seeds of Psoralea corylifolia. A bakuchiol composition was formulated for topical application at 0.5% bakuchiol. Significant reductions in post-inflammatory hyperpigmentation (PIH) were observed in all five individuals following daily topical application of 0.5% Bakutrol cream. As shown in Figure 5, all five individuals had at least one grade level reduction in PIH severity. Greater than 50% improvement in PIH-affected facial areas was achieved after 8 weeks of continuous topical application of 0.5% Bakutrol cream (Figure 6). Mean percent and absolute grade level improvements for both PIH and its severity are summarized in FIGS. 7 and 8 . Greater than 40% improvement, or greater than one grade level reduction, in both PIH and severity was achieved as early as 4 weeks after use of the bakuchiol composition. As shown in Figure 9, a substantial reduction in PIH on the affected facial skin sites is evident in the photographs of both individuals. Two individuals showed progressive improvement in post-inflammatory hyperpigmentation (PIH) of the skin associated with mild and moderate acne following topical application of Bakutrol® cream.

Table 7 (Example 10) summarizes the clinical outcomes of using a bakuchiol composition without furanocoumarins (i.e., Bakutrol) compared to popular acne treatment products containing antibacterial or anti-inflammatory agents or combinations thereof. The data in Table 7 clearly demonstrates that the bakuchiol composition without furanocoumarins not only improves inflammatory and non-inflammatory lesion counts, but also significantly improves skin post-inflammatory hyperpigmentation. Based on its lack of tyrosinase inhibitory activity, it was unexpected for PIH to benefit from the bakuchiol composition.

Table 8 (Example 11) provides data showing a reduction in PIH grade (i.e., degree of pigmentation). The data clearly show that bakuchiol is more effective than placebo and salicylic acid for the treatment of PIH. Furthermore, the applicants have also found that bakuchiol (or compositions comprising said bakuchiol) are effective for the treatment of inflammatory lesions such as acne lesions. Table 9 (Example 11) demonstrates the effectiveness of bakuchiol for the treatment of inflammatory lesions compared to treatment with placebo or salicylic acid.

In addition to methods involving treatment with a composition comprising bakuchiol, the invention includes embodiments wherein a mammal is treated with a composition comprising bakuchiol and salicylic acid. For example, Applicants have discovered that salicylic acid is effective in the treatment of non-inflammatory lesions, while bakuchiol is effective in the treatment of inflammatory lesions. Accordingly, one embodiment of the invention is directed to a method of treating an inflammatory lesion (e.g., an acne lesion) comprising administering to a mammal an effective amount of a composition comprising bakuchiol or a pharmaceutically acceptable salt thereof. Another embodiment is directed to a method of treating inflammatory and/or non-inflammatory lesions (e.g., acne lesions) comprising administering to a mammal an effective amount of a drug comprising bakuchiol and salicylic acid (or a pharmaceutically acceptable thereof) salt) composition. Other embodiments include treating non-inflammatory lesions by administering to a mammal an effective amount of a composition comprising salicylic acid or a pharmaceutically acceptable salt thereof. In some of the preceding embodiments, the mammal is a human. In other embodiments, the mammal is in need of treatment for inflammatory and/or non-inflammatory lesions such as acne.

In addition to treating lesions, the combination of bakuchiol and salicylic acid is effective in treating any of the aforementioned disease states (e.g., PIH, reducing melanogenesis, reducing melanocyte proliferation, or preventing melanocyte apoptosis, etc.) kind of effective. Accordingly, some embodiments relate to treatment with a composition comprising bakuchiol and salicylic acid. Other embodiments include compositions comprising bakuchiol or a pharmaceutically acceptable salt thereof, salicylic acid or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

The foregoing methods are effective in substantially eliminating inflammatory and/or non-inflammatory lesions. For example, in some embodiments, the methods reduce lesions by about 1% to about 99%, or about 10% to about 90%. In other embodiments, the methods reduce lesions by greater than 50%.

The ratio of bakuchiol to salicylic acid is not particularly limited and can be determined by those of ordinary skill in the art based on desired results. For example, in some embodiments, the weight ratio of bakuchiol to salicylic acid is from about 1:100 to about 100:1. In other embodiments, the weight ratio is about 10:90, about 20:80, about 30:70, about 40:60, about 50:50, about 60:40, about 70:30, about 80:20 to about 10:90. The compositions can be formulated according to any of the formulations described herein.

C. Preparation of Bakuchiol Compositions

The bakuchiol compositions of the present disclosure can be formulated by any method known to those skilled in the art. As shown in Example 8 and Table 5, the compositions of the present disclosure can be formulated as pharmaceutical, cosmetic or dermatological compositions and can contain other components such as pharmaceutically and/or cosmetically acceptable actives, excipients agent, adjuvant, carrier or a combination thereof. Excipients are inert substances that serve as diluents or vehicles for dermatologically and cosmetically acceptable prodrugs and drugs. Examples of such excipients include, but are not limited to, water, buffers, saline, glycerol, hydrated silicon dioxide, propylene glycol, aluminum oxide, carrageenan, cellulose carboxymethyl ether, titanium dioxide, Ringer's solution, Dextrose solution, mannitol, Hank's solution, preservatives and other aqueous physiologically balanced salt solutions. Nonaqueous vehicles, such as fixed oils, sesame oil, ethyl oleate, or triglycerides may also be used. Other useful formulations include suspensions containing viscosifying agents, such as sodium carboxymethylcellulose, sorbitol or dextran.

In Example 8, a composition of the present disclosure was formulated in carbitol, or caprylic triglyceride, or polysorbate-20, or purified water, or a combination of two or more of the foregoing vehicles. Excipients can also contain minor amounts of additives such as EDTA, disodium DDTA, BHA, BHT, diammonium citrate, nordihydroguaiaretic acid, propyl gallate, sodium gluconate, sodium metabisulfite, tert-butyl hydroquinone, SnCl ₂ , H ₂ O ₂ and 2,4,5-trihydroxybutyrophenone, vitamin C, vitamin E, vitamin E acetate, phenonip and other substances that increase isotonicity and chemical stability.

Examples of substances used to adjust the pH of the formulation include sodium hydroxide, sodium carbonate, sodium bicarbonate, pentasodium triphosphate, tetrasodium pyrophosphate, sodium lauryl sulfate, calcium peroxide, phosphate buffer, bicarbonate Saline buffer, tris buffer, histidine, citrate and glycine or mixtures thereof. Examples of flavoring agents include, but are not limited to, thimerosal, m- or o-cresol, formalin, fruit extracts, and benzyl alcohol. Standard formulations can be liquid or solid which can be dissolved in suitable liquids such as suspensions or solutions for administration. Thus, in non-liquid formulations, excipients can include dextrose, human serum albumin, preservatives, etc., to which sterile water or saline can be added prior to administration.

In one embodiment, bakuchiol compositions can be formulated with other active compounds targeting a different mechanism of action for reducing skin pigmentation. Such actives include, but are not limited to, hydroquinone, monobenzyl ether, arbuting, deoxyarbutin, p-methoxyphenol, N-acetyl-4-S-cysteinephenol, koji azelaic acid, glycolic acid, gentisic acid, flavonoids, aloesin, stilbene and stilbene derivatives, licorice extract, bearberry extract, mulberry extract, aloe vera gel, glabridin, Vitamin C derivatives, magnesium ascorbyl phosphate, tetrahexyldecyl ascorbic acid, vitamin E derivatives, tranexamic acid and its derivatives, biomimetic of TGF-B protein, cyanidin, niacinamide, PAR-2 Inhibitors, lectins, glycomimetic proteins, resorcinol and its derivatives, and Nivitol ^™ .

In another embodiment, the composition comprises anti-inflammatory and antibacterial agents that act synergistically with the bakuchiol composition to reduce infection, infection-related inflammation, and accelerate epidermal turnover. Such actives include, but are not limited to, alpha hydroxy acids, salicylic acid, linolenic acid, tretinoin, benzoyl peroxide, sodium sulfacetamide, clindamycin, erythromycin, dapsone, tetracycline, deoxygenated Oxytetracycline, Minocycline, Zinc, Estrogens and Derivatives, Antiandrogens, Sulfur, Steroids, Cortisone, Tazarotene, Curcumin Extract, Gum Arabic Extract, Skullcap Extract, Green Tea Extract and Grape Seed Extract.

In some embodiments, the composition includes an adjuvant or carrier. Generally, an adjuvant is a substance that generally enhances the biological response of a mammal to a particular biologically active agent. Suitable adjuvants include, but are not limited to, Freund's reagent; other bacterial cell wall components; salts of aluminum, calcium, copper, iron, zinc, magnesium, tin; silicon dioxide; microdermabrasion agents, polynucleotides; toxoids ; serum proteins; viral capsid proteins; other bacterial-derived preparations; gamma interferon; block copolymer adjuvants such as Hunter's Titermax adjuvant (Vaxcel. available from Ribi ImmunoChem Research, Inc., Hamilton, Mont.); and saponins and their derivatives, such as Quil A (available from Superfos Biosector A/S, Denmark). Carriers are typically compounds that increase the half-life of the therapeutic composition in the treated recipient. Suitable carriers include, but are not limited to, polymeric controlled release formulations, biodegradable implants, liposomes, nanocapsules, nanoparticles, bacteria, viruses, oils, esters and glycols.

In other examples, the composition is prepared as a controlled release formulation that slowly releases the composition to the recipient. As used herein, a controlled release formulation comprises a bakuchiol composition in a controlled release vehicle. Suitable controlled release vehicles are known to those skilled in the art. Examples of controlled release formulations are biodegradable (i.e., biodigestible) and include capsules.

In one embodiment, a suitable ointment is generally selected from 0.001% to 100% effective, non-toxic amount of UP256 (bakuchiol), 65% to 100% (e.g. , 75% to 96%) white soft paraffin, 0% to 15% liquid paraffin, and 0% to 7% (eg, 3% to 7%) lanolin or its derivatives or synthetic equivalents. In another embodiment, an ointment may comprise a polyethylene-liquid paraffin base.

In one embodiment, a suitable cream consists of an emulsifying system together with the desired concentration of UP256 (bakuchiol) synthetic and/or isolated from a single plant or plants as provided above. The emulsifying system preferably comprises 2% to 10% polyoxyethylene alcohol (for example, a mixture available under the trade mark CetomacrogolTM 1000), 10% to 25% stearyl alcohol, 20% to 60% liquid paraffin and 10% to 65% together with one or more preservatives such as 0.1% to 1% of N,N"-methylenebis[N'-[3-(hydroxymethyl)-2,5-dioxo-4 -imidazolidinyl]urea] (obtained under the name Mimidurea USNF), 0.1% to 1% of an alkyl 4-hydroxybenzoate (such as a mixture available from Nipa Laboratories under the trade name Nipastat), 0.01% to 0.1% Sodium 4-hydroxybenzoate butyrate (commercially available from Nipa Laboratories under the trade name Nipabutylsodium) and 0.1% to 2% phenoxyethanol.

In one embodiment, suitable gels consist of semisolid systems in which the liquid phase is confined within a three-dimensional polymer matrix with a high degree of crosslinking. The liquid phase may contain water together with desired amounts of UP256 (bakuchiol), 0.01% to 20% of water soluble additives such as glycerol, polyethylene glycol or propylene glycol and 0.01% to 10%, preferably 0.5% Up to 2% of thickeners, which may be natural products such as tragacanth, pectin, carrageenan, agar and alginic acid, or synthetic or semi-synthetic compounds such as methylcellulose and polycarboxylates Ethylene (Carbopol); together with one or more preservatives such as 0.1% to 2% methyl 4-hydroxybenzoate (methylparaben) or phenoxyethanol - differential. Another suitable formulation consists of the desired amount of UP256 (bakuchiol), together with 70% to 90% polyethylene glycol (for example, a polyethylene glycol containing 40% polyethylene glycol 3350 and 60% polyethylene glycol 400). Ethylene glycol ointment, prepared according to the United States Formulary (USNF), 5% to 20% water, 0.02% to 0.25% antioxidant (such as butylated hydroxytoluene), and 0.005% to 0.1% chelating agent (such as ethylenediaminetetraacetic acid (EDTA)).

The term soft paraffin as used above includes cream or ointment-like white soft paraffin and yellow soft paraffin. The term lanolin includes both natural wool fat and refined wool fat. Derivatives of lanolin especially include those that have been chemically modified to change their physical or chemical properties and synthetic equivalents of lanolin especially include synthetic or Semi-synthetic compounds and mixtures and may eg be called lanolin substitutes.

A suitable synthetic equivalent of lanolin that may be used is the material available under the trade name Softisan(TM), known as Softisan 649. Softisan 649, available from Dynamit Nobel Aktiengesellschaft, is a glycerol ester of natural vegetable fatty acids, isostearic acid and fatty acids; its properties are described by H.Hermsdorf in Fette, Seifen, Anstrichmittel, Issue No. 84, No.3 (1982), Discussed in pp.3-6.

The other substances mentioned above as ingredients of suitable ointment or cream bases and their properties are discussed in standard reference works, eg the US Pharmacopoeia. Cetomacrogol 1000 has the general formula CH ₃ (CH ₂ ) _m (OCH ₂ CH ₂ ) _n OH, where m can be 15 or 17 and n can be 20-24. Butylated hydroxytoluene is 2,6-di-tert-butyl-p-cresol. Nipastat is a mixture of methyl, ethyl, propyl and butyl 4-hydroxybenzoate.

The compositions disclosed herein can be prepared by conventional techniques of pharmacy. Thus, for example, the above compositions may be conveniently prepared by mixing together soft paraffin, liquid paraffin, if present, and lanolin or derivatives or synthetic equivalents thereof at elevated temperature, such as 60°C to 70°C. The mixture can then be cooled to room temperature and after addition of the hydrated crystalline calcium salt of mupirocin, along with the corticosteroid and any other ingredients, stirred to ensure adequate dispersion.

Finally, bakuchiol has a partition coefficient of log P=6.13. The partition coefficient of a chemical compound provides a thermodynamic measure of its hydrophilicity/lipophilicity balance and thus its potential bioavailability. Having a partition coefficient of 6.13 means that the compound has high cell membrane penetration and bioavailability when formulated in a delivery system. Quantification of skin penetration of the active compound bakuchiol in skin creams in an in vitro test on isolated human skin. Results showed good skin penetration and bioavailability. In some embodiments, the disclosed compositions include a skin penetration enhancer.

D. Administration of bakuchiol compositions

Compositions of the present disclosure can be administered by any method known to those of ordinary skill in the art. For example, the disclosed compositions can be administered orally or topically. Methods of administration include, but are not limited to, enteral (oral), parenteral (intravenous, subcutaneous, and intramuscular) administration, and topical application. In some embodiments, the composition is administered topically.

The bakuchiol composition can be present in the final skin care product for PIH in an amount ranging from 0.001% to 99.9% by weight. In some embodiments, the composition comprises 0.1% to 2% bakuchiol. In other embodiments, the composition comprises 0.5% or 1.0% bakuchiol. In some embodiments, the amount of the bakuchiol composition in the PIH skin cream is between 0.5% and 1%. The methods of the present disclosure comprise orally or topically administering to a mammal a therapeutically effective amount of a composition comprising bakuchiol, which is wholly synthetic or isolated from a natural source (or a combination thereof) and substantially free of impurities, particularly furanol Soybein impurities (eg, less than 500 ppm).

The therapeutic agents of the present disclosure can be administered topically by any suitable method known to those skilled in the art for topical administration of the therapeutic composition. Such methods of administration include, but are not limited to, in the form of an ointment, gel, lotion or cream base or in the form of an emulsion, in the form of a plaster, dressing or mask, non-adherent gauze, bandage, swab or wipe. Such topical application can be administered topically to any affected area using any standard method known for topical administration. Therapeutic compositions can be administered in a variety of unit dosage forms depending on the method of administration. For a particular mode of delivery, therapeutic compositions can be formulated with such excipients as described above. The therapeutic compositions of the present disclosure can be administered to any recipient, preferably to a mammal, and more preferably to a human. The particular mode of administration depends on the disease state being treated.

Regardless of the mode of administration, specific dosages are calculated based on the approximate body weight of the recipient. Further optimization of calculations necessary to determine therapeutically appropriate dosages in relation to each of the formulations described above is routinely performed by those of ordinary skill in the art and is within the purview of their routine performance without undue experimentation, particularly in light of the dosage information and testing disclosed herein. . These doses can be ascertained through the use of established assays for use in conjunction with appropriate dose-response data in determining the dose to use. In some embodiments, the dosage of the composition comprising bakuchiol is 0.001 mg to 200 mg per kilogram of body weight.

The following examples are offered for purposes of illustration and not limitation.

Example

Example 1

Quantification of bakuchiol, psoralen and isopsoralen by HPLC

Quantification of Bakuchiol, Psoralen and Isopsoralen in Extracts, Fractions, Process Materials, Components and Final Formulated Products by High Pressure Liquid Chromatography (HPLC) Using Photodiode Array Detector (HPLC/PDA) amount. The target compound was eluted from a Luna phenyl-hexyl column (250mm x 4.6mm) using a gradient of acetonitrile (ACN) or methanol and water from 36% to 100% CAN in 12 minutes followed by 100% CAN in 3 minutes. The detailed HPLC conditions used are set forth in Table 1. The chromatogram of the HPLC separation is shown in FIG. 1 . Target compounds were identified and quantified based on retention time and UV peak area using commercially pure bakuchiol, psoralen, and isopsoralen as quantitative standards. The retention times of bakuchiol, psoralen and isopsoralen were 18.19 minutes, 7.33 minutes and 7.95 minutes, respectively.

Table 1. HPLC Conditions for Quantification of Bakuchiol, Psoralen, and Isopsoralen

Example 2

General method for extracting bakuchiol from plants of the genus PSORALEA

Method A - Add solvent (100 mL) and psoralen seed powder (10 g) to a flask and shake the mixture on a hand shaker at room temperature for 1 hour. Then, the mixture was passed through a filter and the filtrate was collected. The extraction process was repeated once with fresh solvent, the filtrates were combined, the solvent was removed on a rotary evaporator and the residue was dried under high vacuum.

Method B - Solvent (50 mL) and psoralen seed powder (10 g) were added to a flask, and the mixture was refluxed for 40 min. Then, the solution was filtered and the extraction process was repeated twice with fresh solvent. The filtrates were combined and the solvent was evaporated to obtain a dry extract.

Following the extraction method described above, the sample plant matter was extracted using the following solvents: dichloromethane (DCM), ethyl acetate (EtOAc), acetone, methanol (MeOH), petroleum ether (BP 35-60°C) and petroleum ether (BP 60- 90°C). Extracts and plant matter were then analyzed by HPLC analysis as described in Example 1. The results are set forth in Table 2.

Table 2. Quantification of various psoralen extracts

Example 3

Chromatographic method for purification of bakuchiol extract

Various chromatographic methods were used to purify bakuchiol from the crude solvent extract isolated from the seeds of Bakuchiol using the method described in Example 2. Demonstrate the efficiency of a specific column enrichment method as a means to obtain high purity bakuchiol free of furanocoumarin contaminants, particularly psoralen/isopsoralen contaminants. Briefly, individual empty column shells (1.3 cm internal diameter (ID) and 20 mL volume, from Bio-Rad) were packed with different media and eluted with different solvents in an attempt to separate the furanocoumarin impurities from bakuchiol . Fractions (10 mL each) were collected in tubes and analyzed using a silica gel TLC plate developed with 20% EtOAc/petroleum ether. The target compounds, bakuchiol, psoralen, and isopsoralen, were identified based on their retention times determined using solutions of standard compounds. The results are set forth in Table 3. A number of methods described in Table 3 were used to isolate furanocoumarins and bakuchiol from synthetic and natural sources, however the cost of the method may not be economically feasible for large-scale production.

Table 3. Summary of column chromatography separation of bakuchiol from furanocoumarins in crude extract of psoralen

Example 4

Hydrolysis of an extract isolated from the seeds of psoralen

A hexane extract or a _CO supercritical fluid extract of Bakuchiol seeds containing about 25% bakuchiol was mixed with a 1 M NaOH solution. The solution is heated to a temperature at or above 80°C in the reaction vessel for at least 1 hour. A small portion of the solution was periodically withdrawn from the flask and analyzed by HPLC as described in Example 1. The reaction was stopped and the peaks showing psoralen and isopsoralen completely disappeared after HPLC analysis. Then, the reaction mixture was cooled to room temperature and the aqueous phase was removed. After the solution was washed several times with saturated NaCl solution, the organic layer was extracted with ethyl acetate or other organic solvents. The organic solution is filtered, washed, dried and evaporated to produce a brown-red syrup having a bakuchiol content of not less than 50% and a total of not more than 100 ppm of psoralen and isopsoralen (isopsoralen) .

Example 5

Antioxidant properties of bakuchiol compositions without furanocoumarins

A natural bakuchiol composition containing 57.35% bakuchiol and a total of less than 100 ppm psoralen and isopsoralen (isopsoralen) was evaluated at Brunswick Laboratories, Norton, MA USA (batch No. UP256-0906MP) for the antioxidant capacity of peroxyl radicals, hydroxyl radicals, peroxynitroso, superoxide anions and singlet oxygen. According to published techniques (Ou, B. et al., J Agric and Food Chem (agricultural and food chemistry journal), 2001, 49 (10): 4619-4626; Prior, RL. et al., J Agric and Food Chem (agricultural and Journal of Food Chemistry), 2005, 53:4290-4302) to detect the total oxygen free radical absorbance capacity of the bakuchiol composition. The results are tabulated in Table 4.

Table 4: Antioxidant properties of Bakutrol ^TM (UP256)

Example 6

Evaluation of Antioxidative Protection of Bakuchiol Compositions Against 4-TBP Cytotoxicity

The compound containing 77.02% bakuchiol and Antioxidant properties of natural bakuchiol compositions with a total of less than 100 ppm of psoralen and isopsoralen (isopsoralen). Oxidative stress was determined by examining the production of reactive oxygen species (ROS) using the Image-iT Live Green Reactive Oxygen Species Detection Kit (InVitrogen). In this assay, carboxy-2',7'-dichlorofluorescein diacetate is added to cultured cells for 30 minutes, where it diffuses to melanocytes and undergoes intracellular ester hydrolysis to 2 ',7'-dichlorofluorescein (DCF), which reacts with ROS to produce fluorescent DCF. After 5 days of treatment with 200 μM or 400 μM 4-TBP, ROS production in melanocytes demonstrated a dose-response (i.e., mild to strong, respectively), whereas untreated and DMSO-treated melanocytes showed no ROS production. When the treatment regimen included the test compound, as shown in Figure 3 UP256 (bakuchiol) exhibited strong antioxidant properties.

Example 7

Tyrosinase inhibitory activity of bakuchiol compositions

Two natural bakuchiol compositions comprising 77.02% bakuchiol (100% purity) were tested for tyrosinase inhibitory activity. Both substances contained a total of less than 100 ppm total psoralen and isopsoralen (isopsoralen).

Tyrosinase inhibition assays were performed using the method reported by Jones et al., (2002) Pigment. Cell Res. 15 :335. Using this method, the conversion of L-dopa, the substrate of tyrosinase, to dopachrome is followed by monitoring the absorption at 450 nm. Tyrosinase was prepared in 50 mM potassium phosphate buffer at 2000 U/ml, pH 6.8 (assay buffer) and stored in 1 ml aliquots at -20°C until use. For use in the assay, the stored enzyme solution was thawed and diluted to 200 U/ml with assay buffer. Treatment solutions of 2 mM substrate, L-DOPA, were prepared in assay buffer for each assay. Samples were dissolved in 10% DMSO (0.5ml) and diluted to 5ml with assay buffer. The reaction mixture contained 0.050ml 2mM L-DOPA, 0.050ml 200U/ml mushroom tyrosinase and 0.050ml inhibitor. Adjust the reaction volume to 200 μl with assay buffer. Assays were performed in 96-well Falcon 3097 flat bottom microtiter plates (Beckton Dickinson, NJ). The appearance of dopachrome was detected using a WALLAC 1420 multilabel counter (Turku, Finland). The average rate was determined from the linear enzyme rate as detected by the change in absorbance (ΔA ₄₅₀ ) at 450 nm per minute. The percent inhibition of tyrosinase by the test sample was determined by comparing the absorbance of the sample to the control using formula (1):

(Negative Control Absorption - Sample Absorption)/Negative Control Absorption×100 (1)

As shown in Figure 4, the two bakuchiol compositions exhibited no tyrosinase inhibitory activity, while the positive control (kojic acid) showed dose-response tyrosinase inhibition with an _IC50 value of 63.9 μΜ.

Example 8

Formulation of bakuchiol compositions in the form of cosmetic creams, gels and lotions

Two natural bakuchiol compositions comprising 86.54% bakuchiol and 77.02% bakuchiol were formulated as cosmetic vehicles or complex skin creams, gels or lotions as shown below.

Formulation A

Formulation B

Formulation C

Table 5. Formulation D

Example 9

Evaluation of the Safety Properties of Bakuchiol Compositions

Two formulations containing 86.54% bakuchiol and 77.02% bakuchiol and less than 100ppm total psoralen and isopsoralen (isopsoralen) in a beauty vehicle or complex skin cream natural bakuchiol compositions and test their safety profile in in vitro models or in human clinical trials. As shown in Table 6, the bakuchiol composition exhibited no eye irritation, no skin irritation, no skin sensitization contact sensitization, and no phototoxicity. The composition has a solid safety profile (20% to 100% bakuchiol by weight) and good skin penetration properties at a wide range of concentration levels.

Table 6: Results of Bakutrol ^TM Safety Trial

Example 10

Clinical evaluation of bakuchiol compositions without furanocoumarins

A natural bakuchiol combination extracted and enriched from the seeds of psoralen and containing 77.02% bakuchiol and less than 100 ppm furanocoumarins was formulated in the form of a cosmetic skin cream (Formulation D, Example 8) drug (Bakutrol ^TM ) and tested in human clinical trials. The study was a pilot, open-label human study to evaluate the clinical benefit of Bakutrol ^™ at a concentration of 0.5% after topical application. The study included 5 individuals meeting the exclusion/inclusion criteria to evaluate the effectiveness of a natural bakuchiol composition for improving post-inflammatory hyperpigmentation (PIH). The study duration was 12 weeks. Subjects were instructed to apply Bakutrol ^™ 0.5% Cream twice daily, morning and evening, and return to the site for a total of 9 visits, including the screening visit. Evaluations included investigator's global assessment of skin condition (IGA), and full grade and severity of skin post-inflammatory hyperpigmentation (PIH) and other relevant skin disease states, including erythema, dryness, peeling, oiliness, safety, and tolerance. receptivity evaluation. Individual questionnaires included safety and compliance questions related to irritation, skin comfort, use of other products and sunscreens. Photographs were taken at Baseline, Week 4, Week 8, and Week 12. Change from baseline in PIH severity, change from baseline in PIH grade, and proportion of success according to the IGA scale were recorded and analyzed. The following 6 levels of PIH grades of severity (0=none, 1=mild, 2=mild, 3=moderate, 4=moderately severe, 5=severe) and the total area of the buccal surface infected by PIH were used clinically output analysis.

As shown in Figure 5, all five subjects treated with the topical cream containing Bakutrol ^™ at 0.5% had at least one grade reduction in PIH severity. The percentage improvement of PIH-infected facial areas was greater than 50% after 8 weeks of continuous application (see Figure 6). Mean percent and absolute grade level improvements for both PIH and its severity are summarized in FIGS. 7 and 8 . PIH and severity II were achieved as early as 4 weeks after using a natural bakuchiol composition extracted and enriched from the seeds of psoralen and containing 77.02% bakuchiol and less than 100 ppm furanocoumarins Improvement of greater than 40% or greater than one grade level. Significant reduction of PIH on infected facial skin sites is clearly demonstrated in the photographs of the two individuals shown in FIG. 9 . Two individuals showed progressive improvement in post-inflammatory hyperpigmentation (PIH) of the skin associated with mild and moderate acne following topical application of Bakutrol ^™ cream.

Table 7 summarizes the clinical output using bakuchiol compositions without furanocoumarins compared to prevalent acne treatment products containing antibacterial or anti-inflammatory agents or combinations thereof. The data clearly show that the bakuchiol composition without furanocoumarins not only improved inflammatory and non-inflammatory lesion counts but also significantly improved skin post-inflammatory hyperpigmentation. As demonstrated in Example 7, the PIH benefit from a bakuchiol composition was unexpected based on its lack of tyrosinase inhibition.

Table 7. Summary of Clinical Reports for Bakutrol and OTC Drugs

Example 11

Evaluation of the effect of bakuchiol compositions without furanocoumarins on reducing post-inflammatory hyperpigmentation

The safety and efficacy of bakuchiol (UP256) cream 0.5% (wt/wt) for the treatment of acne-related PIH was evaluated in a double-blind placebo and active control study. The study evaluated bakuchiol cream at a concentration of 0.5%, salicylic acid cream 2%, and placebo cream (vehicle) in an Asian population. Participants were instructed to apply the study cream to the face twice daily (AM/PM). Study participants were given application instructions and provided sunscreen.

The study subjects consisted of male and female individuals greater than 18 years of age and less than 40 years of age and in general good health as determined by medical records. Eighteen subjects were enrolled in the bakuchiol study group, 20 subjects in the salicylic acid (SAL) study group, and 19 subjects in the placebo study group.

Investigators and researchers discuss and agree on a clear definition of PIH as associated with acne or other tissue damage (which does not include freckles (Ephilides), solar lentigines (lentigines) or melisma). The PIH grade is a measure of the severity of hyperpigmentation (higher number = more severe hyperpigmentation). The patient population had PIH grade >3 and acne was mild to moderate grade 2-3, the main factor being PIH related to inflammatory current or post-inflammatory acne.

Main research objectives:

1. PIH IGA Timeframe: Baseline and Weeks 2, 4 and 8

2. PIH% Distribution Timeframe: Baseline and Weeks 2, 4 and 8

PIH efficacy was assessed based on change from baseline (p<0.05) and over other treatment groups (p<0.05) using t-tests or/and analysis of variance.

Secondary Objective: Safety Assessment

Individual assessment questionnaires and tolerability assessments were collected at Baseline and at Weeks 2, 4, and 8. Urine pregnancy tests were collected from females of reproductive potential at baseline and week 8.

data analysis

Collect the following data:

1. Change in PIH severity from baseline;

2. Change in PIH grade from baseline; and

3. Change in Lesion Count from Baseline

result:

Data were analyzed from investigator assessments at each visit and from photographs taken at defined time points. Photographs from Baseline, Week 4 and Week 8 were evaluated by a second investigator group and two separate dermatologists before the studies were pooled to further confirm that the population met the criteria. The data analyzed were based on confirmed assessments, and there were no photographs from the second week, so the data from the second week were not included in the analysis. Table 8 summarizes the data.

Table 8. Changes in PIH Grades by Study Group

The Bakutrol (UP256) group showed a significant change (p<0.05) from baseline in PIH grade (ie, lower PIH grade) at week 8. Furthermore, the Bakutrol group showed a significant change over placebo at week 8 (p<0.05). The data also showed that the bakutrol-treated group was the only group with significant p-values for time and treatment (p=0.0083).

Table 9. Inflammatory acne lesions in all groups

The Bakutrol(UP256) group showed a significant change from baseline at weeks 4 and 8 (p<0.001). The Bakutrol group also showed a significant change over placebo at week 8 (p<0.05). Furthermore, UP256 was the only group (57%) to achieve a greater than 50% reduction in inflammatory lesions at week 8.

There was no significant change in the Bakutrol (UP256) group in the non-inflammatory lesion category (data not shown); however, the salicylic acid treatment group achieved a p-value < 0.05 in the non-inflammatory lesion category at week 4 of treatment.

Table 10: Overview of the Questionnaire and Investigator Safety Assessment

in conclusion:

The results showed that Bakutrol (UP256) cream significantly reduced post-inflammatory hyperpigmentation (PIH) associated with acne after only 4 weeks of topical application. UP256 also significantly reduced inflammatory acne lesions (<0.05) by 57% after 8 weeks.

Bakutrol (UP256) cream had a good safety profile and was well tolerated by study participants. The cosmetic acceptability of the creams was considered to be better than or equal to that of their previous topical over-the-counter (OTC) acne treatments.

Example 12

Treatment of Inflammatory and Noninflammatory Acne Lesions

A composition comprising both bakuchiol and salicylic acid was prepared. Acne patients with both inflammatory, non-inflammatory, or both types of lesions are treated using the composition. The composition was effective in treating inflammatory and non-inflammatory lesions with a p-value < 0.05 at week 4 of treatment. The reduction in lesions is from about 10% to about 90%.

The various embodiments described above can be combined to provide further embodiments. All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent applications mentioned in the specification and/or cited in the Application Data Sheet are hereby incorporated by reference in their entirety. Aspects of the embodiments can be modified, if desired, to employ concepts of the various patents, applications and publications to provide additional embodiments. These and other changes can be made to the embodiments in light of the above detailed description. Generally, in the following claims, the terms used should not be construed as limiting rights to the specific embodiments disclosed in the specification and claims, but rather should be construed to include all possible embodiments along with equivalents to which such claims give the full range of . Accordingly, the claims are not limited by the disclosure.

Claims (13)

1. the composition comprising Bakuchiol or its pharmaceutically acceptable salt prepare for prevent, alleviate, reduce or treat by Purposes in the topical formulations of hyperpigmentation caused by morbid state from inflammatory skin disorders.

2. purposes as described in claim 1, wherein the Bakuchiol is the Bakuchiol of synthesis.

3. purposes as claimed in claim 2, wherein the Bakuchiol of the synthesis is the mixture of stereoisomer.

4. purposes as described in claim 1, wherein the Bakuchiol is from Psoralea, Sassafras, Magnolia or rhizoma atractylodis It is detached in the plant species of category.

5. purposes as described in claim 1 or 4, wherein the Bakuchiol is from entire plant or from seed, stem, tree It is detached in skin, branch, root, root skin, young shoot, flower or other reproductive organs, leaf or other aerial parts or combinations thereof.

6. purposes as claimed in claim 5, wherein the stem includes stem tuber and rhizome.

7. purposes as described in claim 1, wherein the hyperpigmentation from inflammatory skin disorders result from acne, Atopic dermatitis, allergic contact dermatitis, bloch-Siemens syndrome, lichen planus, lupus erythematosus, morphoea, mechanical trauma, electricity From or Non-ionizing radiation, burn, laser or drug therapy, skin infection or combinations thereof.

8. purposes as described in claim 1, wherein the color excessive caused by the morbid state from inflammatory skin disorders It is plain calm for acne spot.

9. purposes as described in claim 1, wherein the composition includes the psoralea corylifolia of 0.001% to 99.9% total weight Phenol and pharmaceutically acceptable, dermatology is acceptable or the acceptable carrier of beauty.

10. purposes as described in claim 1, wherein the composition also includes one kind or mixture of skin whitener, institute It states skin whitener and is selected from quinhydrones, single-benzyl ether, ursin, deoxyarbutin, p methoxy phenol, N- acetyl group -4-S- half Cystamine base phenol, kojic acid, azelaic acid, glycolic, gentianic acid, flavonoids, Aloesin, talan and diphenyl ethylene derivatives, Radix Glycyrrhizae extract, black bearberry extract, mulberry extract, Aloe Vera Gel, glabridin, vitamin C derivatives, vitamin e derivative, Tranexamic acid and its derivative, the biosimulation object of TGF-B protein, centaurcidin, niacinamide, PAR-2 inhibitor, agglutination Element, Neoglycoproteins, resorcinol and its derivative and Nivitol.

11. purposes as claimed in claim 10, wherein the vitamin C derivatives include magnesium ascorbyl phosphate and four hexyls Decyl ascorbic acid.

12. purposes as described in claim 1, wherein the composition also includes one kind or mixture of skin conditioner, institute It states skin conditioner and is selected from 'alpha '-hydroxy acids, salicylic acid, linolenic acid, vitamin A acid, benzoyl peroxide, Guttae Sulfacetamidi Natrici, crin Mycin, erythromycin, dapsone, tetracycline, doxycycline, minocycline, zinc, estrogen and its derivative, antiandrogen, Sulphur, steroids, cortisone, tazarotene, curcumin extract, Arabic gum extract, radix scutellariae extract, Green tea extract and Grape Seed Extract.

13. purposes as described in claim 1, wherein the composition is formulated as in ointment, gel, lotion, emulsifiable paste matrix Emulsion, plaster, dressing or facial mask, nonsticking gauze, bandage, cotton swab or cleaning wiping cloth.