Shikonin, aryl naphthyl lignans and pharmaceutical compositions containing the same for inhibiting pro-inflammatory gene expression

Abstract

The present invention relates to one or more aryl naphthoyl lignans, in particular shikonin A, and pharmaceutical compositions comprising the same. Shikonin A was isolated from a preparation of the root of comfrey (complexa) and was found to reduce inflammation by interfering with pro-inflammatory gene expression. In particular, it inhibited interleukin 1β (IL-1β)-induced E-selectin expression in HUVEC cells.

Description

Technical Field

[0001] The present invention relates to one or more aryl naphthoyl lignans, referred to herein as shikonin or shikonin compounds, and pharmaceutical compositions comprising them. Exemplary embodiments of the disclosed compounds can be isolated from the root of shikonin (Symphytumofficinale L.). Certain embodiments of shikonin compounds and pharmaceutical compositions comprising them reduce inflammation by interfering with pro-inflammatory gene expression. Background Art

[0002] Consumers are looking for effective and natural ways to relieve pain and inhibit inflammation. Commonly known as comfrey (Boraginaceae), comfrey is a perennial herb common in Europe, Asia and North America (C. Staiger, Phytother Res 26, 1441-1448, 2012; A. Trifan et al., Food Chem Toxicol 112, 178-187, 2018). Comfrey has been used in folk medicine to treat diarrhea, bronchitis, tuberculosis, ulcers and hemorrhoids. Now, clinical trials have demonstrated the efficacy and safety of its topical preparations. Like other species belonging to the Boraginaceae family, comfrey contains hepatotoxic and carcinogenic pyrrolizidine alkaloids (PAs). Therefore, PA-depleted extracts are used in over-the-counter topical medications to reduce inflammation and for treating broken bones, tendon injuries, painful joints, ankle deformities, and muscles (I. Sowa et al., Nat Prod Res 32, 605-609, 2018, M. Adams et al., J. Ethnopharmacol. 121 (2009) 343-359). In fact, liquid hydroalcoholic extracts of Lithospermum officinale roots have been clinically shown to be effective in treating acute upper and lower back pain, arthritis, knee arthritis and blunt trauma (B. Grube et al., Phytomedicine 14, 2-10, 2007; R. Koll et al., Phytomedicine 11, 470-477, 2004; HGPredel et al., Phytomedicine 12, 707-714, 2005, SK Patwardhan et al. Int. J. Pharm. Pharm. Sci., Vol. 21, No. 13, 2020, p. 4671).

[0003] It is reported that the main components of Lithospermum officinale root include allantoin, polyphenols (such as rosmarinic acid, salvianolic acid, ellagic acid and caffeic acid), and rich mucilages and polysaccharides composed of fructose and glucose units (B. Grabias and L. Swiatek, Pharm. Pharmacol. Lett 8, 81-83, 1998; I. Sowa et al., Nat Prod Res 32, 605-609, 2018; C. Staiger Phytother Res 26, 1441-1448, 2012; A. Trifan et al., Food Chem Toxicol 112, 178-187, 2018). In addition, many oleanolic acid and disaccharide chain triterpenoid saponins have been produced from the roots of Lithospermum officinale (VU Ahmad et al., Phytochemistry 32, 1003-1006, 1993; FV Mohammad et al., Phytochemistry 40, 213-218, 1995). Recently, it was reported that the phenolic substances from the hydroalcoholic extract of the root of Polygonum aviculare contain salvianolic acid isomers (A. Trifan et al., Food Chem Toxicol 112, 178-187, 2018). Lithospermum officinale roots also contain hepatotoxic pyrrolizidine alkaloids (PAs) with a 1,2-unsaturated sedumine structure, including sedumine, sedumine, their acetyl and N-oxide derivatives, and polysedumine (RA Coulombe, Adv. Food Nutr. Res. 45, 61-99, 2003; F. Liu et al., Talanta 80, 916-923, 2009; A. Trifan et al., Food Chem Toxicol 112, 178-187, 2018).

[0004] Currently, medicinal products from Lithospermum officinale roots commercialized on the European market contain only extracts from pyrrolizidine-depleted plant material (WO2017 / 068175A1; WO2018 / 224518A1) or are obtained from special cultivars that do not synthesize pyrrolizidine alkaloids (A. Trifan et al., Food Chem Toxicol 112, 178-187, 2018).

[0005] Although the anti-inflammatory and analgesic properties of comfrey have been demonstrated in clinical studies, the bioactive compounds associated with these therapeutic activities have not yet been identified. It is therefore an object of the present invention to provide a better understanding of the chemical composition of comfrey root and to gain an initial understanding of the contribution of its specific metabolites to the anti-inflammatory activity. Summary of the invention

[0006] The present invention relates to one or more aryl naphthyl lignans, wherein the aryl naphthyl lignans have a general structure as shown in the following formula I.

[0007]

[0008] Wherein each R1, R2, R3 and R4 is independently selected from hydrogen, hydroxyl, methyl, methoxy, ethoxy, propoxy, butoxy or pentyloxy; and wherein R5 is selected from hydroxyl, methoxy, ethoxy, propoxy, butoxy, pentyloxy, feruloyl, caffeoyl, coumaroyl, isoprenyloxy or isopentenyloxy residues. Specifically, the present invention relates to an aromatic naphthoyl lignan, named comfreyn A, wherein R1, R2, R3 and R4 are each hydroxyl, and R5 is ethoxy, as shown in formula II

[0009]

[0010] The present invention also relates to a pharmaceutical composition comprising a safe and effective amount of at least one of the one or more aryl naphthyl lignans disclosed herein and a pharmaceutically acceptable carrier. In particular, the pharmaceutically acceptable carrier can be a carrier for topical administration. In addition or alternatively, the pharmaceutically acceptable carrier can be a carrier for internal administration.

[0011] The present invention also relates to a safe and effective amount of one or more aryl naphtholignans as disclosed herein and pharmaceutical compositions comprising them for use in reducing and / or treating inflammation, preferably for treating broken bones, tendon injuries, painful joints and muscles, acute upper and lower back pain, knee arthritis, blunt trauma, for reducing swelling in joint deformities, for improving joint mobility in osteoarthritic joints and / or combinations thereof. Specifically, the reduction and / or treatment of inflammation is achieved by interfering (blocking or reducing) pro-inflammatory gene expression.

[0012] The present invention also relates to a pharmaceutical composition comprising one or more aryl naphthoyl lignans called shikonins, and in particular shikonin A, as active agents in a pharmaceutical carrier, wherein the pharmaceutical formulation may also include a penetration enhancer or any other functional or non-functional excipient. In a preferred embodiment, the pharmaceutical composition comprises shikonin A in an amount of 3 μg / g composition to 5 μg / g composition and additional optional active caffeic acid ethyl ester in an amount of 5 μg / g composition to 7.5 μg / g composition, wherein the pharmaceutical carrier comprises at least functional and non-functional topical excipients.

[0013] The present invention also relates to a method of reducing and / or treating inflammation by topically and / or internally administering a safe and effective amount of one or more aryl naphthoquinones known as shikonins, and particularly shikonin A, as disclosed herein, or a safe and effective amount of one or more shikonins, and particularly shikonin A, in combination with a safe and effective amount of caffeic acid ethyl ester.

[0014] In one embodiment, the method is disclosed for

[0015] Treat broken bones, tendon injuries, painful joints and muscles, acute upper and lower back pain, knee arthritis, and blunt trauma;

[0016] Reduce swelling in deformed joints;

[0017] Improving joint mobility in osteoarthritic joints; and / or combinations thereof,

[0018] Inflammation is reduced and / or treated by topical and / or internal administration of a safe and effective amount of one or more aryl naphthyl lignans known as shikonins, particularly shikonin A, or a safe and effective amount of one or more shikonins, particularly shikonin A, in combination with a safe and effective amount of caffeic acid ethyl ester.

[0019] In another embodiment, a method for inhibiting and / or treating inflammation by interfering with (blocking or reducing) pro-inflammatory gene expression is provided by topical and / or internal administration of a safe and effective amount of one or more shikonins, particularly shikonin A, or a safe and effective amount of one or more shikonins, particularly shikonin A in combination with a safe and effective amount of caffeic acid ethyl ester and / or other additional active agents as disclosed herein.

[0020] The present invention also relates to a kit comprising a safe and effective amount and instructions for use of a pharmaceutical composition as disclosed herein, wherein the pharmaceutical composition comprises one or more aromatic naphthoxylins as disclosed herein, particularly shikonin A, or one or more aromatic naphthoxylins as disclosed herein, particularly shikonin A plus caffeic acid ethyl ester. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 LC-MS curve of hydroalcoholic liquid extract of Polygonum aviculare root in negative ion mode is shown;

[0022] Figure 2 The structures of the major compounds isolated from the roots of Polygonum aviculare are shown;

[0023] Figure 3 Shown is a list of secondary metabolites identified from the roots of Syngonium wilfordii by LC-ESI-HR-MS and LC-ESI-HR-MS / MS;

[0024] Figure 4 Shown in Figure 2 The high-resolution MS spectrum of shikonin A, compound 15, is shown in FIG.

[0025] Figure 5 Shown as Figure 2 The key HMBC (→) and ROESY of shikonin A shown in compound 15 Relevance. DETAILED DESCRIPTION

[0026] Consumers are looking for effective and natural ways to relieve pain and inhibit and reduce inflammation. As disclosed herein, the aryl naphthoyl lignan shikonin A is isolated from the root of Polygonum multiflorum and is shown to inhibit the expression of E-select mRNA in endothelial cells after 90 min of stimulation with 5 ng / mL IL-1β. Thus, a reduction in inflammatory responses is achieved. Therefore, the present invention relates to one or more shikonins, pharmaceutical compositions comprising them, and their medical uses in the treatment of inflammatory conditions and diseases, particularly those associated with pro-inflammatory gene expression.

[0027] While the specification concludes with claims particularly pointing out and distinctly claiming the invention, it is believed that the present invention will be better understood from the following description of embodiments taken in conjunction with the accompanying drawings, wherein like reference numerals refer to like elements.

[0028] The unit "cm" used herein refers to centimeters; the unit "mm" used herein refers to millimeters; the unit "μm" or "micrometer" used herein refers to micrometers; the unit "nm" used herein refers to nanometers. The unit "g" used herein refers to grams; the unit "mg" used herein refers to milligrams. The units "mL" or "ml" used herein refer to milliliters; the units "μL" or "μl" used herein refer to microliters. The unit "min" used herein refers to minutes, and the unit "sec" used herein refers to seconds. The unit "mM" used herein refers to millimoles / liter; the unit "μM" used herein refers to micromole / liter. Unless otherwise indicated, as used herein, the terms "wt%" and "w / w%" mean percentages by weight, and generally all percentages and ratios used hereinafter are by weight of the total topical composition.

[0029] Unless otherwise specified, all percentages, ratios, levels and concentrations referred to herein for ingredients are based on the actual amount of the ingredient and do not include solvents, fillers or other materials that may be used in combination with such ingredients in commercially available products.

[0030] Unless otherwise indicated, all measurements mentioned herein were made at about 22°C (ie, room temperature).

[0031] As used herein, the word "about" means + / - 10%.

[0032] As used herein, the word “or” when used as a conjunction of two or more elements is meant to include the elements individually or in combination; for example, X or Y means X or Y or both.

[0033] As used herein, the articles "a" and "an" are understood to refer to one or more materials being claimed or described, for example, "an active substance" or "a solvent."

[0034] As used herein, the word "comprising" and variations thereof are intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other similar items that may also be useful in the materials, oral compositions, devices, and methods of the invention. The term includes "consisting of" and "consisting essentially of."

[0035] As used herein, the word "include" and variations thereof are intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other similar items that may also be useful in the materials, topical compositions, devices, and methods of the present invention.

[0036] As used herein, the words "preferred", "preferred" and their variations refer to embodiments of the present invention that can provide specific benefits under specific circumstances. However, other embodiments may also be preferred under the same or other circumstances. In addition, the description of one or more preferred embodiments does not mean that other embodiments are not applicable, and is not intended to exclude other embodiments from the scope of the present invention.

[0037] "Actives," "active agents," and "other ingredients" useful herein may be categorized or described herein according to their cosmetic and / or therapeutic benefits or their postulated modes of action or operation. However, it should be understood that in some cases, actives and other ingredients useful herein may provide more than one cosmetic and / or therapeutic benefit, or function or operate via more than one mode of action. Therefore, the categorization herein is for convenience only and is not intended to limit the ingredients to the specifically specified functions or activities listed.

[0038] As used herein, "safe and effective amount" means that within the scope of sound medical judgment, the amount of a component is high enough to significantly (definitely) change the condition to be treated or affect the desired result, but low enough to avoid serious side effects (at a reasonable benefit / risk ratio). The safe and effective amount of a component depends on the specific condition to be treated, the age and physiological state of the patient to be treated, the severity of the condition, the treatment period, the nature of the co-treatment, the specific form used, and the specific carrier used for the component.

[0039] As used herein, the terms "administering," "administering," and "administering" refer to any method within sound medical practice for delivering a composition to a subject in a manner that provides a therapeutic effect.

[0040] As used herein, "drug" refers to a drug such as a medicament, including prescription drugs, over-the-counter drugs, over-the-counter drugs, and combinations thereof. In some examples, the drug may be a supplement.

[0041] Shikonin

[0042] The present invention relates to one or more aryl naphthyl lignans, hereinafter named shikonin. The chemical structure of the one or more shikonin as claimed is shown in Formula I.

[0043]

[0044] wherein each R1, R2, R3 and R4 is independently selected from hydrogen, hydroxyl, methyl, methoxy, ethoxy, propoxy, butoxy or pentyloxy; and wherein R5 is selected from hydroxyl, methoxy, ethoxy, propoxy, butoxy, pentyloxy, feruloyl, caffeoyl, coumaroyl, isoprenyloxy or isopentenyloxy residues. The one or more shikonins as claimed are established as aryl naphthoyl lignans with an unusual delta-lactone ring, corresponding to a coumarin backbone, and are naturally occurring compounds, since there are natural synthetic routes for all the side groups claimed for R1 to R5.

[0045] In a preferred embodiment, the present invention relates to an aryl naphthoyl lignan, hereinafter named shikonin A, wherein each of R1, R2, R3 and R4 is a hydroxyl group, and wherein R5 is an ethoxy group. The chemical structure is shown in Formula II

[0046]

[0047] and corresponds to Figure 2 of compound 15.

[0048] In addition or alternatively, the present invention relates to the therapeutic activity of one or more shikonins as claimed herein, in particular to the therapeutic activity of shikonin A. Shikonin A was shown to reduce inflammation by interfering with proinflammatory gene expression, such as inhibiting E-selectin expression in human venous endothelial cells (HUVEC) stimulated by IL-1β (Table 2). E-selectin is a NF-κB-dependent target gene, and IL-1β is a well-described proinflammatory mediator that plays an important role in the regulation of immune and inflammatory responses, particularly those involving aseptic injuries such as trauma and blunt trauma. In one embodiment, using one or more shikonins as disclosed herein, in particular using shikonin A, E-selectin activation is inhibited by at least 30%, preferably at least 40%, more preferably at least 45%, more preferably at least 48%, and even more preferably at least 50%. In addition or alternatively, the local effective concentration of one or more shikonins as disclosed herein, in particular the local effective concentration of shikonin A that inhibits E-selectin activation in vitro is less than 70 μM, preferably less than 60 μM, and more preferably about 50 μM.

[0049] The present invention also relates to the use of the anti-inflammatory activity of one or more shikonins as disclosed herein, in particular the use of the anti-inflammatory activity of shikonin A to inhibit and / or treat inflammation via interference with pro-inflammatory gene expression. Without wishing to be bound by theory, the anti-inflammatory activity of one or more shikonins as disclosed herein, in particular the anti-inflammatory activity of shikonin A based on inhibition of pro-inflammatory gene expression, can be applied to the location where it is needed by applying one or more shikonins, in particular by applying a safe and effective amount, preferably in an amount providing a local concentration of about 30 μM to about 65 μM, more preferably in an amount providing a local concentration of about 40 μM to about 60 μM, more preferably in an amount providing a local concentration of about 45 μM to about 55 μM, even more preferably in an amount providing a local concentration of 48 μM to 52 μM shikonin A for the treatment of broken bones, tendon injuries, painful joints and muscles, acute upper and lower back pain, knee arthritis, blunt trauma, for reducing swelling of joint deformities, for improving joint mobility in osteoarthritic joints, and / or combinations thereof.

[0050] In a preferred embodiment, a suitable composition comprises shikonin A in an amount of 1 μg / g to 10 μg / g of the composition, particularly in an amount of 2 μg / g to 7.5 μg / g of the composition, and even more particularly in an amount of 3 μg / g to 5 μg / g of the composition, wherein a suitable single dose is 3 g to 5 g of the composition and administration is recommended at least three times a day.

[0051] Additional active agents

[0052] In addition or alternatively and in accordance with the present invention as disclosed herein, one or more shikonin active agents, in particular shikonin A, may be combined with additional active agents. Suitable additional active agents according to the present invention are preferably analgesics, active agents with anti-inflammatory activity, activity enhancers or combinations thereof. In a preferred embodiment, the anti-inflammatory activity of the additional active agent that interferes with the expression of the pro-inflammatory gene as disclosed herein is inhibited by at least 30%, preferably at least 40%, more preferably at least 45%, more preferably at least 48%, even more preferably at least 50%. In addition or alternatively, the effective concentration of the additional active agent that inhibits the expression of the pro-inflammatory gene is less than 70 μM, preferably less than 60 μM, more preferably less than 50 μM, more preferably less than 40 μM, more preferably less than 30 μM and even more preferably less than 25 μM, as shown by in vitro experiments.

[0053] Non-limiting examples of chemical analgesics may include APAP, ibuprofen, ketoprofen, diclofenac, naproxen, aspirin, and combinations thereof. In one example, about 0.5% to about 3.5% of analgesics by weight of the composition may be used as an additional active agent, in another example, about 1% to about 3% of analgesics by weight of the composition may be used, and in another example, about 1.5% to about 2% of analgesics may be used. For example, a dose may contain 325 mg to 500 mg APAP, 200 mg ibuprofen, 200 mg naproxen, or a combination thereof.

[0054] In a preferred embodiment, the additional active agent is a natural analgesic, in particular a natural analgesic extracted from comfrey, more particularly from comfrey root. Without wishing to be bound by theory, it is believed that the combination of more than one active agent, in particular more than one active agent extracted from comfrey root, can increase anti-inflammatory activity. In a preferred embodiment, one or more shikonin, in particular shikonin A, is combined with one or more additional active agents selected from the following items: allantoin, p-hydroxybenzoic acid glucoside, 5-hydroxymethyl-2-furfural, protocatechuic acid, protocatechuic aldehyde, p-hydroxybenzoic acid, caffeic acid, globoidnan B, malashinic acid, 3-carboxy-6,7-dihydroxy-1-(3',4'-dihydroxyphenyl)-naphthalene, (+)-vanillin, rosmarinic acid, hydroxycaffeic acid, globoidnan A, caffeic acid ethyl ester, ternifoliuslignan D, linolenic acid, linoleic acid and hydroxypalmitic acid. In particular, shikonin A is combined with one or more additional active agents selected from the group consisting of globoidnan B, malaxinic acid, globoidnan A, caffeic acid ethyl ester, trifoliol D, 3-carboxy-6,7-dihydroxy-1-(3',4'-dihydroxyphenyl)-naphthalene and chrysanthemum indole.

[0055] In a preferred embodiment, one or more shikonins, in particular shikonin A, are combined with one or more additional active agents, the additional active agent comprising or consisting of: caffeic acid ethyl ester, malashinic acid and globoidnan A or a combination thereof. More preferably, shikonin A is combined with caffeic acid ethyl ester. In one embodiment, caffeic acid ethyl ester is used to inhibit the expression of pro-inflammatory genes by at least 30%, preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 65% and even more preferably 70%. In addition or alternatively, the effective concentration of caffeic acid ethyl ester for inhibiting the expression of pro-inflammatory genes is less than 80 μM, preferably less than 70 μM, more preferably less than 65 μM, as shown in in vitro experiments.

[0056] Caffeic acid ethyl ester can be used as an active agent in a pharmaceutical composition as disclosed herein in a safe and effective amount. Without wishing to be bound by theory, the anti-inflammatory activity of caffeic acid ethyl ester is achieved via inhibition of pro-inflammatory gene expression, and therefore, the present invention relates to inhibiting and / or treating inflammation by applying a safe and effective amount, preferably an amount that provides a local concentration of about 50 μM to about 80 μM caffeic acid ethyl ester at a location where it is needed, more preferably an amount that provides a local concentration of about 55 μM to about 75 μM caffeic acid ethyl ester at a location where it is needed, more preferably an amount that provides a local concentration of about 60 μM to about 70 μM caffeic acid ethyl ester at a location where it is needed, and even more preferably an amount that provides a local concentration of 62 μM to 66 μM caffeic acid ethyl ester at a location where it is needed, preferably for treating broken bones, tendon injuries, painful joints and muscles, acute upper and lower back pain, knee arthritis, blunt injuries, for reducing swelling of joint deformities, for improving joint mobility in osteoarthritic joints, and / or combinations thereof.

[0057] In a preferred embodiment, a suitable composition comprises caffeic acid ethyl ester in an amount of 1 μg / g to 15 μg / g of the composition, particularly in an amount of 2.5 μg / g to 10 μg / g of the composition, and even more particularly in an amount of 5 μg / g to 7.5 μg / g of the composition, wherein a suitable single dose is 3 g to 5 g of the composition and administration is recommended at least three times a day.

[0058] Pharmaceutical composition

[0059] The present invention relates to a pharmaceutical composition comprising one or more aryl naphthyl lignans as disclosed herein, in particular a safe and effective amount of shikonin A as disclosed herein and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" includes one or more compatible solid or semisolid or liquid excipients or diluents suitable for topical or internal administration. As used herein, "compatible" means that the components of the pharmaceutical composition can be mixed but will not interact in a manner that significantly reduces the stability and / or efficacy of the composition.

[0060] The selection of pharmaceutical excipients, carriers or diluents is selected according to the expected route of administration and standard pharmaceutical practice. In some respects, examples of such suitable excipients for various compositions as described herein can be found in "Handbook of Pharmaceutical Excipients" (2nd edition, 1994) edited by A Wade and PJ Weller. In some respects, acceptable carriers are described in, for example, "Remington's Pharmaceutical Sciences" (Mack Publishing Co., A. R. Gennaro edits, 1985). In addition to excipients, carriers or diluents, such compositions may also include additional ingredients. Such additional ingredients include, but are not limited to, any suitable binding agent, lubricant, coating agent, solubilizing agent, emulsifying agent, surfactant, preservative, dye, flavoring agent, stabilizer, buffer and / or suspending agent. In addition, such formulations may also include penetration enhancers or any other functional or non-functional excipients.

[0061] Topical compositions

[0062] The pharmaceutical compositions disclosed herein can be used for topical administration, in particular for topical administration to the skin, that is, "topical composition" as used herein refers to a product that is not used for the purpose of systemic administration of a therapeutic agent during general use, but is applied to the skin and remains on the skin for a sufficient time to achieve the intended purpose, such as inhibiting inflammation, relieving pain, etc.

[0063] Depending on the type and amount of the ingredients, the desired consistency of the topical formulation and the expected mode of administration, the carrier material may include such as pharmacologically acceptable oils, water and / or conventionally used topical formulations, such as carrier materials for such supplementary drug adjuvants in conventional matrices for ointments, creams and gels. In some embodiments, it may be suggested that a structure forming agent, a thickener or a gel forming agent be incorporated into the composition. Suitable agents are particularly highly dispersed silicic acid (e.g., commercial product "Aerosil"), bentonite, modified montmorillonite such as montmorillonite alkylammonium salts (e.g., commercial product "Bentone") (wherein the alkyl group may contain 1 to 20 carbon atoms) (e.g., dimethyl-dialkylammonium salts (wherein the alkyl group contains 16 to 18 carbon atoms)), organic structure forming agents, thickeners and suspending agents, such as xanthan gum, carrageenan, 1860 alcohol and / or modified castor oil products. In addition or alternatively, in addition to excipients, carriers or diluents, such compositions may also include additional ingredients. Such additional ingredients may include, but are not limited to, any suitable binders, lubricants, coatings, solubilizers, solvents, emulsifiers, lipids, polymers, gelling agents, surfactants, penetration enhancers, pH adjusters, buffers, preservatives, dyes, flavorings, stabilizers, buffers and / or suspending agents or fragrances or chelating agents.

[0064] Methods of applying the active ingredient, either alone or as a combination component in a topical formulation as disclosed herein, will generally involve application to an area affected by inflammation and / or degeneration, or an area expected to be affected by inflammation and / or degeneration, or to an area affected by pain, swelling, stiffness, and / or lack of mobility associated with the inflammation and / or degeneration, or expected to be affected by it. In a certain embodiment, the active ingredient is applied to an area surrounding the affected site or an area expected to be affected. For example, the active ingredient may be applied to an area all around the affected area or an area expected to be affected by inflammation and / or degeneration, either alone or as a combination component in a topical formulation of the present invention. Topical formulations will include, but are not limited to, creams, gels, ointments, or lotions, and may be applied to the body surface and gently rubbed in. Types of inflammatory and / or degenerative disorders suitable for the methods and uses of the present invention include, but are not limited to, rheumatoid arthritis, septic arthritis, gout, pseudogout, juvenile arthritis, Still's disease, cervical spondylosis, lumbar spondylosis, frozen shoulder, heel spurs, psoriatic arthritis, ankylosing spondylitis, and osteoarthritis.

[0065] In a certain embodiment of the invention, the subject for administration is a human. However, the method of prophylactic or therapeutic treatment according to the invention may also be applicable to non-human subjects susceptible to inflammatory and / or degenerative disorders, including but not limited to horses, dogs, cats, rabbits and / or cows.

[0066] The dosage regimen will depend on a number of readily determinable factors, such as the degree of inflammation and / or degeneration or pain, swelling, stiffness and / or lack of mobility associated with the inflammation and / or degeneration, and the responsiveness of the inflammation, degeneration, pain, swelling, stiffness and / or lack of mobility to treatment, but will generally be one or more doses per day, with the course of treatment lasting from several days to several months, or until a cure is achieved or the degree of inflammation, degeneration, pain, swelling, stiffness and / or lack of mobility experienced by the subject is significantly reduced. The optimal dosage, method of administration and repetition rate can be readily determined by one of ordinary skill. In a certain embodiment, it is contemplated that the topical formulation will be applied once to five times per day.

[0067] As used herein, "an amount of a composition sufficient to provide an effect" refers to an amount of a topical composition applied to a patient, used or worn by a patient, or instructs a patient to apply, use or wear an amount of a topical composition greater than about 1 g, greater than about 2 g, greater than about 3 g, preferably about 4 g, particularly about 1, 2, 3, 3.5 or 4 g to about 10, 8, 6, 5 or 4.5 g per application, or any other narrower numerical range that falls within such a wider numerical range, such a narrower numerical range seems to be explicitly written herein. Treatment can be applied as needed and will depend on the desired effect, the severity of any condition being treated, the health status and age of the user and similar considerations. It is generally recommended to apply three times a day. In one embodiment, one or more active substances, preferably one or more shikonin active substances, particularly shikonin A can be used in an amount of 1 μg / g composition to 10 μg / g composition, preferably in an amount of 2 μg / g composition to 7.5 μg / g composition, and more preferably in an amount of 3 μg / g composition to 5 μg / g composition.

[0068] The topical formulations according to the present invention can be prepared in any conventional manner, but are not limited to known pharmaceutical practices. Topical formulations as disclosed herein can be aqueous (i.e., containing water), or they can be non-aqueous and optionally used in combination with a closed covering layer so that the moisture evaporated from the body surface after being applied to the body surface and thereafter is retained in the formulation. In addition, the formulation can be directly applied with or without the need to apply a bandage or pressure pad. The composition of the present invention may also include, for example; a wash-free composition, wherein as used herein, "wash-free" refers to a composition intended to be applied to and allowed to remain on keratinous tissue. These wash-free compositions are distinguished from compositions that are applied to the skin and subsequently (within a few minutes or less) removed by washing, rinsing, wiping, etc. Wash-free compositions do not include wash-off applications, such as shampoos, facial cleansers, hand soaps, body washes, or body washes.

[0069] Thus, the topical compositions as disclosed herein are "dermatologically acceptable," which means that the compositions or components are suitable for use in contact with human skin tissue without undue toxicity, incompatibility, instability, allergic response, etc. The topical pharmaceutical compositions as disclosed herein may, for example, be in the form of an ointment, salves, paste, viscous liquid, lotion, gel, cream, mousse, foam or any other adhesive product, or combinations thereof.

[0070] The topical composition may be a single phase composition or may be a combination of two or more separate compositions, wherein the two or more separate compositions may be contained in physically separate compartments of a dispenser and dispensed side by side. As used herein, the term "dispenser" refers to any pump, tube or container suitable for dispensing a topical composition.

[0071] The topical composition can also be incorporated into a delivery vehicle for direct application or attachment to the skin surface. As used herein, the term "delivery vehicle" includes materials or appliances for keeping topical compositions close to the skin surface, but is not limited to those mentioned. The delivery vehicle can be, for example, a strip or a polymer film or a backing film or a patch. These delivery vehicles can be rectangular, arched, curved, semicircular, have rounded corners, have a slit cut into it, have a notch cut into it, bend into a three-dimensional shape or a combination thereof. They can be solid, semi-solid, textured, plastic, flexible, deformable, permanently deformable or a combination thereof. The strip can be, for example, made of a plastic sheet comprising polyethylene, or a wax sheet or a polymer carrier. These sizes are adjusted according to the zone to be treated.

[0072] In addition or alternatively, the topical formulations of the present invention can also be applied through the skin or mucosal tissue using conventional skin patches, wherein the active agent is contained in a laminated structure used as a delivery device to be fixed to the body surface. In such structures, the topical formulation is contained in a layer or "reservoir" below the upper backing layer. The laminated structure may contain a single reservoir, or it may contain multiple reservoirs. In one embodiment, the reservoir includes a polymer matrix of a pharmaceutically acceptable adhesive material, which is used to fix the system to the skin during the delivery of the topical formulation. Typically, the adhesive material is a pressure-sensitive adhesive (PSA) suitable for long-term skin contact and should be physically and chemically compatible with the topical formulation. Examples of suitable adhesive materials include, but are not limited to: polyethylene; polysiloxane; polyisobutylene; polyacrylate; polyacrylamide; polyurethane; plasticized ethylene-vinyl acetate copolymer; and viscous rubbers, such as polyisobutylene, polybutadiene, polystyrene-isoprene copolymer, polystyrene-butadiene copolymer and chloroprene rubber (polychloroprene). A preferred adhesive is polyisobutylene. The backing layer plays the role of the main structural element of the transdermal system, and provides flexibility and preferably closure for the device. The material for the backing layer should be inert and can not absorb any one or more active agents contained in the topical formulation contained in the device. The backing is preferably composed of a flexible elastomeric material, which is used as a protective covering to prevent the loss of medicine and / or vehicle via the upper surface of the patch, and will preferably give the system a certain degree of closure, so that the body surface area covered by the patch becomes hydrated during use. The material for the backing layer should allow the device to follow the contour of the skin and be comfortably worn on the skin area, such as at the joints or other bending points that are usually subjected to mechanical strain, due to the difference in the flexibility or resilience of the skin and the device, the device is rarely or may not be separated from the skin. As mentioned above, the material used as the backing layer is closed or permeable, although the closed backing is preferred, and is usually derived from synthetic polymers (for example, polyester, polyethylene, polypropylene, polyurethane, polyvinylidene chloride and polyetheramide), natural polymers (for example, cellulosic materials) or macroporous woven and nonwoven materials.

[0073] Additionally or alternatively, the reservoir containing the topical formulation and the skin contact adhesive are present as separate and distinct layers, with the adhesive underlying the reservoir, wherein the reservoir may be a polymer matrix as described above. Alternatively, the reservoir may consist of a liquid or semisolid topical formulation contained in a closed compartment or "pouch", or it may be a hydrogel reservoir, or may take some other form.

[0074] Additional layers such as intermediate fabric layers and / or rate controlling membranes may also be present in any of these delivery systems. Fabric layers can be used to facilitate the manufacture of the device, while rate controlling membranes can be used to control the rate at which the topical formulation permeates out of the device.

[0075] Rate controlling membranes, if present, will be included in the system on the skin side of one or more reservoirs. The material used to form such membranes is generally selected to limit the flux of one or more components contained in the topical formulation. Representative materials that can be used to form rate controlling membranes include polyolefins such as polyethylene and polypropylene, polyamides, polyesters, ethylene-ethyl acrylate copolymers, ethylene-vinyl acetate copolymers, ethylene-vinyl methyl acetate copolymers, ethylene-vinyl ethyl acetate copolymers, ethylene-vinyl acetate propyl copolymers, polyisoprene, polyacrylonitrile, ethylene-propylene copolymers, etc.

[0076] Typically, the area of ​​the lower surface of the transdermal device (i.e., the skin contact area) is in the range of about 0.25 cm2 to 200 cm2, preferably 1 cm2 to 25 cm2, more preferably 2 cm2 to 10 cm2. Of course, this area will vary with the size of the area to be treated.

[0077] Delivery systems as disclosed herein can use conventional coating known in the art and lamination technology manufacturing.For example, adhesive matrix system can be prepared by casting the fluid mixture of adhesive and topical formulation of the present invention on the backing layer, and then laminating release liner.Similarly, adhesive mixture can be cast on the release liner, and then laminating backing layer.Alternatively, reservoir can be prepared in the absence of topical formulation, and then loaded by "immersing" in the topical formulation.Usually, these patches are made by solvent evaporation, film casting, melt extrusion, film lamination, die-cutting etc.

[0078] The topical formulation will be incorporated into the device usually during the patch manufacturing period rather than after the preparation device. The delivery system may also include an adhesive cover layer, which is also used as the backing of the delivery system, for better fixation of the patch on the body surface. The size of this cover layer is such that it extends beyond the reservoir so that the adhesive on the cover layer contacts the body surface. The cover layer is useful because the adhesive / reservoir may lose its adhesion due to hydration a few hours after application. By mixing this type of adhesive cover layer, the delivery system remains in place within the required time period.

[0079] Topical carrier

[0080] Topical formulations according to the present invention may take the form of compositions suitable for topical application to body surfaces and may include, for example, creams, lotions, solutions (e.g., liquid sprays), gels, films, ointments, pastes, plasters, paints, bioadhesives, and the like.

[0081] Ointment

[0082] As is well known in the art, ointments are semisolid preparations generally based on vaseline or other petroleum derivatives. As will be appreciated by those skilled in the art, the specific ointment base used is an ointment base that will provide the best delivery of an active agent, and it can also provide other desired properties, such as emollients, etc. As with other carriers or vehicles, ointment bases are generally inert, stable, non-irritating, and non-sensitizing. Ointment bases can be divided into four categories: oleaginous bases; emulsifiable bases; emulsion bases; and water-soluble bases. Oleaginous ointment bases include, for example, vegetable oils, fats obtained from animals, and semisolid hydrocarbons obtained from petroleum. Emulsifiable ointment bases, also referred to as absorbent ointment bases, contain little or no water, and include, for example, hydroxy tristearate sulfate, anhydrous lanolin, and hydrophilic petrolatum. Emulsion ointment base is water-in-oil (w / o) emulsion or oil-in-water (o / w) emulsion, and comprises for example cetyl alcohol, glyceryl monostearate, lanolin, stearic acid and polyethylene glycol of different molecular weight or their combination.Other examples of suitable alkyl include but are not limited to hard, soft or liquid paraffin, glycerine, beeswax, metal soap, glue, oil (such as almond oil, corn oil, peanut oil, castor oil or olive oil) of natural origin, lanolin or its derivative, fatty acid (such as stearic acid or oleic acid) or their combination.All the above-mentioned components that contribute to the method for preparing ointment are not limited to their synthetic properties, and can be natural or natural origin or natural derivative.

[0083] Cream

[0084] Creams, also well known in the art, are viscous liquids or semisolid oil-in-water emulsions. Cream bases can be natural or synthetic washable and contain an oil phase, an emulsifier, and an aqueous phase. The oil phase, also referred to as the "internal" phase, can include petrolatum and fatty alcohols, such as cetyl or stearyl alcohol. The aqueous phase typically (although not necessarily) exceeds the oil phase in volume and typically contains a wetting agent. The emulsifier in the cream formulation is typically a nonionic, anionic, cationic or amphoteric surfactant or a combination thereof. In addition, novel delivery technologies incorporated into the cream base may also be included therein. All of the above ingredients that contribute to the method for preparing the cream are not limited to their synthetic properties and may be natural or naturally derived or naturally derived.

[0085] gel

[0086] Gel or emulsion Gel formulations are generally semisolid, suspension-type systems. Single-phase gels generally contain organic macromolecules substantially uniformly distributed throughout a carrier liquid, which is generally aqueous but may also contain alcohol and optionally oil. Exemplary "organic macromolecules" (i.e., gelling agents) are cross-linked acrylic acid polymers, such as the "carbomer" polymer family (e.g., the Carbopol TM Commercially available carboxypolyolefins). Also exemplified are hydrophilic polymers such as polyethylene oxide, polyoxyethylene-polyoxypropylene copolymers, polyvinyl alcohol, and polyvinyl pyrrolidone; cellulosic polymers such as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methylcellulose, carboxymethyl cellulose, hydroxypropyl methylcellulose phthalate, and methylcellulose; gums such as carrageenan, tragacanth, and xanthan gum; sodium alginate; and gelatin. Examples of suitable gel forming agents include carboxypolyethylene derivatives such as carbomer and pectin. In one embodiment of the invention, the gelling agent is xanthan gum and / or carrageenan.

[0087] In one embodiment, the thickener or gelling agent may be present in the composition, typically in an amount of about 0.2% to 5.0% w / w%. In another embodiment, the thickener or gelling agent may be present in the composition, typically in an amount of about 0.1% to 1.0% w / w%. In another embodiment of the invention, a topical gel formulation may include a gelling agent (e.g., xanthan gum) in an amount of up to 3.0% w / w.

[0088] In order to prepare a uniform gel, a diluent such as an alcohol may be added, or the gelling agent may be dispersed by grinding, mechanical mixing or stirring, or a combination thereof. In addition, the gel or emulsion gel formulation may contain other adjuvants commonly used in formulation types such as preservatives, antioxidants, colorants, fragrances, stabilizers, etc.

[0089] Hydrogels are macromolecular networks that absorb water and therefore swell but are insoluble in water. That is, the hydrogel contains hydrophilic functional groups that provide water absorption, but the hydrogel is composed of cross-linked polymers that produce water insolubility. Typically, the hydrogel is then composed of a cross-linked hydrophilic polymer, such as polyurethane, polyvinyl alcohol, polyacrylic acid, polyoxyethylene, polyvinyl pyrrolidone, poly(hydroxyethyl methacrylate) (poly(HEMA)) or a copolymer or mixture thereof. A particularly preferred hydrophilic polymer is a copolymer of HEMA and polyvinyl pyrrolidone.

[0090] All of the above ingredients that aid in the process of preparing the gel or emulsion gel are not limited in their synthetic nature and may be natural or naturally derived or naturally occurring.

[0091] lotion

[0092] Lotion is a preparation applied to the skin surface without friction, and is a liquid or semi-liquid preparation, wherein solid particles (including active agent) are present in water or alcohol matrix. Lotion is a solid suspension, and for purposes of the present invention, can comprise a liquid oily emulsion of oil-in-water type. Lotion can be used for processing large body areas, because more fluid compositions are easily applied. It is usually desirable that the insoluble material in the lotion is subdivided. Therefore, lotion will usually contain suspending agent to produce better dispersion, and can be used for locating and keeping active agent in contact with the skin, such as methylcellulose, sodium carboxymethylcellulose etc. Lotion can also include an agent that accelerates the drying and cooling of solution on the skin, such as alcohol or acetone. They can also include a wetting agent, such as glycerine, or an oil, such as castor oil or peanut oil.

[0093] The topical formulations of the present invention can also be prepared with liposomes and / or micelles. Liposomes are microscopic vesicles with a lipid wall comprising a lipid bilayer and can be used as a delivery system herein. Typically, liposome formulations are used for poorly soluble or insoluble agents. Liposomal formulations for use in the present invention can include cationic (positively charged), anionic (negatively charged) and neutral formulations. Cationic liposomes are readily available. For example, N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes can be obtained under the trade name Lipofectin TM Obtain. Similarly, anionic and neutral liposomes are also readily available, or can be easily prepared using readily available materials. Such materials include phosphatidylcholine, cholesterol, phosphatidylethanolamine, dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylglycerol (DOPG), dioleoylphosphatidylethanolamine (DOPE), etc. These materials can also be mixed with DOTMA in appropriate ratios. Methods for preparing liposomes using these materials are well known in the art.

[0094] Micelles in aqueous solution are known in the art to be composed of surfactant molecules, which are arranged so that their polar head groups form an outer spherical shell, and the hydrophobic hydrocarbon chain is oriented toward the center of the sphere to form a core. Micelles are formed in an aqueous solution containing a sufficiently high concentration of surfactant so that micelles are naturally produced. Surfactants that can be used to form micelles include but are not limited to potassium laurate, sodium octane sulfonate, sodium decane sulfonate, sodium dodecane sulfonate, sodium lauryl sulfate, sodium docusate, decyl trimethyl ammonium bromide, dodecyl trimethyl ammonium bromide, tetradecyl trimethyl ammonium bromide, tetradecyl trimethyl ammonium chloride, dodecyl ammonium chloride, polyoxyethylene 8 dodecyl ether, polyoxyethylene 12 dodecyl ether, nonoxynol 10 and nonoxynol 30. Micellar formulations can be used in combination with the present invention by being incorporated into a reservoir of a local delivery system or into a topical formulation to be applied to the body surface. Various additives known to those skilled in the art may also be included in the topical formulation of the present invention.

[0095] Additionally or alternatively, solvents, including relatively small amounts of alcohols, may be used to dissolve certain formulation components. Suitable solvents include glycerol, SD 40 alcohol, lecithin, poloxamer, Miglyol TM and methylpyrrolidone or a combination thereof. Other suitable solvents include glycerin, isopropyl alcohol, hexylene glycol and propylene glycol. Menthol may also be added to the topical formulations of the present invention. In one embodiment, the surfactant may be used in the topical formulations according to the present invention, for example, in an amount of about 0.5% to about 5.0% w / w. Suitable surfactants may include, but are not limited to, sodium lauryl sulfate, benzalkonium chloride, cetylpyridinium chloride, sodium laurate, cetyltrimethylammonium bromide, poloxamer (231, 182, 184), Tween (20, 40, 60, 80) and Span TM and their combinations.

[0096] The formulation may also contain irritation-reducing additives to minimize or eliminate the possibility of skin irritation or skin damage caused by the pharmacologically active matrix or other components of the composition. Suitable irritation-reducing additives include, for example: α-tocopherol; monoamine oxidase inhibitors, particularly phenyl alcohols, such as 2-phenyl-1-ethanol; glycerol; ascorbic acid and ascorbate; ionophores, such as amphiphilic amines; ammonium chloride; N-acetylcysteine; cis-urocanic acid; capsaicin.

[0097] In another embodiment of the present invention, the delivery system may include nanoparticles. The nanoparticles may be dispersed in a microemulsion in the formulation of the present invention, or as part of a vehicle for such formulations, for delivering active agents through the skin. Without wishing to be bound by theory, such nanoparticles will easily penetrate the skin and may be retained in a specific layer of the skin. In one embodiment, the nanoparticles have a core consisting of an iron ore that is strongly attracted to a magnet. In addition or alternatively, the nanoparticles include magnetite, which in one embodiment refers to a molecule with the general formula Fe3O4. The nanoparticles may also include chemically equivalent magnetite, such as (FeM)OFe2O3, wherein in one embodiment, M may be Zn, Co, Ni, Mn, Cr, Au or Ag. The nanoparticles used in the delivery system and the method according to the present invention may, for example, be in the size range of 1nm-100nm. The polymer may also be conjugated to the nanoparticles by a range of methods well known to those skilled in the art. The active agent may in turn be conjugated to the polymer, to the nanoparticles, or may be directly fixed to the nanoparticles as a function of the physicochemical properties of the compound. The selection of polymer used can be a function of the particles used. In one embodiment, polymer includes polyacrylic acid, polystyrene sulfonic acid, polyethylene sulfonic acid, polyethylene oxide, polypropylene oxide, polyvinyl alcohol, photopolymerizable macromonomer, biodegradable polymer such as but not limited to PLGA, or a combination thereof. In another embodiment, polymer includes surfactant, polyethylene glycol, lignin sulfonate, polyacrylamide or biopolymer. In another embodiment, biopolymer includes polypeptide, cellulose and its derivatives (such as hydroxyethyl cellulose and carboxymethyl cellulose), alginate, chitosan, lipid, dextran, starch, gellan gum or other polysaccharides, or a combination thereof.

[0098] The topical formulation of the present invention may also include other compositions that promote the delivery of active ingredients through the skin. For example, nanoparticles can be used to prepare active agents into macromolecular assemblies, such as WO2006 / 113668, WO2006 / 129127 and US60 / 671,892 (the entire contents of which are incorporated herein by reference) disclosed. Without wishing to be bound by theory, the macromolecular assemblies of this type usually simulate natural lipoproteins, and therefore it is said that the hydrophobic active agents are dissolved to promote their delivery (e.g., transdermal delivery) by penetrating the intercorneal lipid layer and hydrophilic pores of the skin. Without wishing to be bound by theory, the macromolecular assemblies can be captured in the stratum corneum of the skin, and therefore act as a reservoir of active agents, so that continuous release can be achieved in the deeper layers of the skin, and thus different therapeutic properties are provided. This can in turn enhance the efficacy of the active agent, reduce the required number of applications and the amount of the active agent, and therefore can be more convenient for patients.

[0099] These and other types and configurations of topically applied delivery systems can be used alone or in combination to facilitate the delivery of active agents included in the formulations of the present invention, as will be understood by those skilled in the art of, for example, topical drug delivery. Thus, it should be understood that the delivery system can include any single embodiment or combination of embodiments listed herein.

[0100] Any suitable emollient or skin conditioner may be optionally included in the topical formulation of the present invention. Suitable emollients include, but are not limited to, cholesterol, glycerol, glyceryl monostearate, isopropyl myristate, isopropyl palmitate, 186-hexadecyl alcohol, lanolin alcohol, and mixtures thereof. Optionally, polydimethylsiloxane, mineral oil, or white soft paraffin may also be incorporated into the formulation in relatively small amounts to serve as skin conditioners. Emollients or skin conditioners may be present in topical formulations of the present invention in an amount of about 0.5% to about 50% w / w%. For example, in gel formulations of the present invention, emollients or skin conditioners may be present in an amount of up to about 50% w / w. In another embodiment, cream or lotion formulations of the present invention may include an emollient or skin conditioner in an amount of about 0.5% to about 15% w / w.

[0101] The formulations of the present invention may optionally include a buffer or neutralizer. Examples of suitable buffers include, but are not limited to, citric acid, lactic acid, oleic acid, sodium phosphate, water, triethanolamine, sodium citrate, hydrochloric acid, and the like. The buffer may be present in the composition in any suitable buffering effective amount. The gel formulation will typically contain a base, such as sodium hydroxide, triethanolamine, and the like. The gel formulations of the present invention will also typically include a volatile solvent, such as ethanol, isopropanol, and the like.

[0102] Other agents, including antimicrobial agents, may also be added to the topical formulations of the present invention to prevent spoilage (i.e., inhibit the growth of microorganisms such as yeast and mold) during storage. Suitable antimicrobial agents are generally selected from the group consisting of methyl and propyl esters of parahydroxybenzoic acid (i.e., methylparaben and propylparaben), sodium benzoate, sodium hydroxymethylglycinate, sorbic acid, imidurea, potassium iodide, and combinations thereof. This list is not limited to synthetic materials, but may also be natural or naturally derived or from natural sources.

[0103] The topical formulations of the present invention may optionally include a preservative or antioxidant component that is not limited to natural or synthetic sources. Examples of such preservatives and antioxidants include, but are not limited to, alkyl alcohols, benzyl alcohol, butylated hydroxyanisole, butylated hydroxytoluene (methylparaben and propylparaben), butylparaben, disodium edetate, citric acid, grape seed fruit extract, sesamol, etc. The preservative and / or antioxidant may be present in the formulation of the present invention in an amount of about 0.05% to about 5.0% w / w. In another embodiment, the preservative and / or antioxidant may be present in the formulation of the present invention in an amount of about 0.25% to about 0.5% w / w.

[0104] In certain embodiments, the topical formulations of the present invention may also include other active ingredients, including but not limited to eucalyptus oil (e.g., eucalyptus globulus oil, eucalyptus serrulata oil, eucalyptus camaldulensis), cineole or other terpenes derived from eucalyptus oil, boswellic acid, glycerin, algae, potassium iodate, algin, lavender, 1,8-cineole, linalool, linalool acetate, nut kernel extract, lecithin, licorice extract (e.g., licorice root extract), glabridin, glycyrrhizic acid, monoammonium glycyrrhizinate, 10-β-glycyrrhetinic acid, ellagic acid, turmeric, curcumin, tetrahydrocurcumin (THC), or any combination thereof. Without wishing to be bound by theory, other active ingredients may contribute at least in part to the efficacy of the formulations of the present invention by acting directly or indirectly on physiological or pathophysiological pathways.

[0105] Other matrix carriers in topical compositions as disclosed herein include matrix carrier materials. Suitable matrix carrier materials are non-toxic oils, non-toxic edible oils, saturated or unsaturated fatty alcohols, aliphatic hydrocarbons, long chain triglycerides, fatty esters, and mixtures thereof. In addition or alternatively, the hydrophobic phase may also include silicones, polysiloxanes, and mixtures thereof.

[0106] Suitable aliphatic hydrocarbons can comprise approximately 10, 12, 14 or 16 to approximately 16, 18, 20, 22, 24, 26, 28, 30, 36, 40 carbon atoms, such as decane, 2-ethyldecane, tetradecane, isotetradecane, hexadecane, eicosane and their mixture. Long-chain triglycerides comprise vegetable oil, fish oil, animal fat, hydrogenated vegetable oil, partially hydrogenated vegetable oil, semi-synthetic triglyceride, synthetic triglyceride and their mixture. The fractionation, refining or purification of these types of oils can also be used. Specific examples of suitable long-chain triglyceride-containing oils for use in the compositions of the present invention include almond oil; babassu oil; borage oil; black currant seed oil; canola oil; castor oil; coconut oil; corn oil; cottonseed oil; emu oil; evening primrose oil; linseed oil; grapeseed oil; peanut oil; mustard oil; olive oil; palm oil; palm kernel oil; peanut oil; rapeseed oil; safflower oil; sesame oil; cod liver oil; soybean oil; sunflower oil; hydrogenated castor oil; hydrogenated coconut oil; hydrogenated palm oil; hydrogenated soybean oil; hydrogenated vegetable oil; a mixture of hydrogenated cottonseed oil and hydrogenated castor oil; partially hydrogenated soybean oil; a mixture of partially hydrogenated soybean oil and partially hydrogenated cottonseed oil; triolein; trilinolein; trilinolein; omega-3-polyunsaturated fatty acid triglycerides containing oils; and mixtures thereof. The oil containing long-chain triglycerides can be specifically selected from corn oil, olive oil, palm oil, peanut oil, safflower oil, sesame oil, soybean oil, castor oil, linseed oil, rapeseed oil, rice bran oil, coconut oil, hydrogenated castor oil; partially hydrogenated soybean oil; triolein; trilinolein; omega 3-polyunsaturated fatty acid triglycerides containing oil; and mixtures thereof.

[0107] Suitable saturated or unsaturated fatty alcohols have from about 6 to about 20 carbon atoms, cetearyl alcohol, lauryl alcohol, and mixtures thereof For example, Lipowax (cetearyl alcohol and ceteareth-20) is supplied and manufactured by Lipo Chemical.

[0108] Suitable mineral oils are petrolatum and mixtures thereof.Examples of petrolatum include Snow White Pet-C from Calumet Specialty Products (Indianapoli, IN), G-2191 from Sonneborn (Parsippany, NJ), G-2218 from Sonneborn, G-1958 from Sonneborn, and mixtures thereof.

[0109] General information on silicone fluids, gums and resins, and the manufacture of silicones can be found in Encyclopedia of Polymer Science and Engineering, Vol. 15, Second Edition, pages 204-308, John Wiley & Sons Inc., 1989 and Chemistry and Technology of Silicones, Walter Noll, Academic Press Inc (Harcourt Brue Javanovich, Publishers, New York), 1968, pages 282-287 and 409-426.

[0110] Surfactants

[0111] The types of surfactants that can be used as emulsifiers for the compositions disclosed herein include nonionic emulsifiers, anionic emulsifiers, cationic emulsifiers, amphoteric emulsifiers, synthetic emulsifiers, and mixtures thereof. Many suitable nonionic and amphoteric surfactants are disclosed by U.S. Pat. No. 3,988,433; U.S. Pat. No. 4,051,234, and many suitable nonionic surfactants are disclosed by U.S. Pat. No. 3,959,458. Other emulsifiers that may optionally be used herein include natural emulsifiers such as acacia, gelatin, lecithin, and cholesterol; finely divided solids such as colloidal clays, bentonites, colloidal magnesium aluminum silicate (magnesium aluminum silicate; and synthetic emulsifiers such as salts of fatty acids, sulfates such as sorbitan trioleate, sorbitan tristearate, sucrose distearate, propylene glycol monostearate, glyceryl monostearate, propylene glycol monolaurate, sorbitan monostearate, sorbitan monolaurate, polyoxyethylene-4-lauryl ether, sodium lauryl sulfate, sulfonates such as dioctyl sodium sulfosuccinate, glycerides, polyoxyethylene glycol esters and ethers, diethylene glycol monostearate, PEG 200 distearate and sorbitan fatty acid esters such as sorbitan monopalmitate, and polyoxyethylene derivatives thereof, polyoxyethylene glycol esters such as monostearate, polysorbate 80 (ethoxylated sorbitan monooleate) (provided by Spectrum, etc.), and mixtures thereof.

[0112] water

[0113] The personal care composition may also contain water, preferably less than about 10% water, less than about 8% water, less than about 5%, less than about 3% water, all percentages being by weight of the composition. Alternatively, the personal composition may contain from about 0.5% to about 10% water, alternatively from about 0.75% to about 8%, alternatively from about 1% to about 5% water, all percentages being by weight of the composition.

[0114] Preservatives, buffers, dyes, and flavoring agents may also be included in some embodiments of the compositions.

[0115] Examples of preservatives include, but are not limited to, sodium benzoate, esters of benzoic acid, sorbic acid and p-hydroxybenzoic acid, p-hydroxybenzoic acid esters, particularly propyl p-hydroxybenzoate, ethyl p-hydroxybenzoate, methyl p-hydroxybenzoate, benzalkonium chloride, ethylenediaminetetraacetic acid (EDTA), benzyl alcohol, potassium sorbate, or mixtures thereof. The composition may contain from about 0.01% to about 1% preservative, from about 0.05% to about 0.5% preservative, from about 0.07% to about 0.3% preservative, and preferably from about 0.08% to about 0.15% preservative.

[0116] Examples of buffers include, but are not limited to, acetic acid, sodium acetate, citric acid, sodium citrate, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium carbonate, sodium bicarbonate, succinic acid, sodium succinate, potassium dihydrogen phosphate, and phosphoric acid, and help to establish and maintain a constant pH value. The amount of buffer is determined and based on the pH requirements to be met and can be easily adjusted by a skilled person.

[0117] The present composition may also include any dye or color for increasing the optical acceptance of the consumer to be treated. Non-limiting examples of color may include red, green, amber, orange, yellow, blue, pink, violet, cyan and combinations thereof. In one example, the composition is transparent. The composition may also include a dye providing a color. Non-limiting examples of dyes that may be used in the present invention include FD&C Blue #1, FD&C Blue #2, D&C Blue #4, D&C Blue #9, FD&C Green #3, D&C Green #5, D&C Green #6, D&C Green #8, D&C Orange #4, D&C Orange #5, D&C Orange #10, D&C Orange #11, FD&C Red #3, FD&C Red #4, D&C Red #6, D&C Red #7, D&C Red #17, D&C Red #21 , D&C Red #22, D&C Red #27, D&C Red #28, D&C Red #30, D&C Red #31, D&C Red #33, D&C Red #34, D&C Red #36, D&C Red #39, FD&C Red #40, D&C Violet #2, FD&C Yellow #5, FD&C Yellow #6, D&C Yellow #7, Ext. D&C Yellow #7, D&C Yellow #8, D&C Yellow #10, D&C Yellow #11, and combinations thereof. The composition can include from about 0.001% to about 0.1% of the dye, from about 0.002% to about 0.05% of the dye, or from about 0.003% to about 0.01% of the dye.

[0118] Compositions as disclosed herein may also include fragrance materials acceptable to consumers. In one example, fragrance materials suitable for use herein may include melaleuca oil, anise oil, geranium oil, lemon oil, spearmint oil, eucalyptus oil, myrtle oil, oregano oil, pine oil, camphor oil, thyme oil, cedarwood oil, cedar leaf oil, nutmeg, and mixtures thereof. Additionally or alternatively, fragrance materials may include chemical components of essential oils, such as vanillin, ethyl vanillin, musk, methyl dihydrojasmonate, anethole, catechol, camphor, camphene, ferulic acid, farnesol, hinokitiol, tropolone, menthol, levomenthol, methyl salicylate, carveol, terpineol, verbenone, ratanhia root extract, caryophyllene oxide, citronellal, curcumin, nerolidol, rosin, thymol, or mixtures thereof. These materials may be combined and used at levels and ratios known to those skilled in the art of fragrance materials for use in personal care compositions.

[0119] Internal administration

[0120] Alternatively, one or more shikonin active substances, particularly shikonin A and / or pharmaceutical compositions comprising the same can also be administered internally via systemic pharmaceutical compositions. As used herein, "systemic pharmaceutical compositions" refer to products for the purpose of systemic administration of therapeutic agents during general use, which are administered orally, parenterally, using suppositories, or other internal and systemic administrations in dosage forms and doses sufficient to achieve the intended purpose (e.g., inhibiting inflammation, relieving pain, etc.). The composition may include a variety of dosage forms for internal administration. Non-limiting examples of dosage forms may include liquid drugs, particles suspended in liquid formulations, solids in gelatin or foam, or solid dosages in the form of tablets, capsules, powders, granules, spheres, microspheres, nanospheres, beads or pellets, and combinations thereof.

[0121] Orally administrable dosage forms can be swallowed immediately, slowly dissolved in the mouth, or chewed. In some aspects, the composition can be formulated as a powder, tablet, capsule, enteric-coated dosage form (e.g., for delivery to the ileum / colon), or pill that can be administered to a subject by any suitable route. The lyophilized formulation can be mixed with saline or other solutions prior to administration.

[0122] In some aspects, the pharmaceutical compositions disclosed herein can be enteric-coated. Enteric coatings can protect the active substances of the composition, for example, from gastric acid and provide delivery to the ileum and / or upper colon region. Non-limiting examples of enteric coatings can include pH-sensitive polymers (e.g. FS30D), methyl acrylate-methacrylic acid copolymer, cellulose acetate succinate, hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate succinate (e.g., hypromellose acetate succinate), polyvinyl acetate phthalate (PVAP), methyl methacrylate-methacrylic acid copolymer, shellac, cellulose acetate trimellitate, sodium alginate, zein, other polymers, fatty acids, waxes, shellac, plastics, and plant fibers. In some aspects, the enteric coating is made of a pH-sensitive polymer (e.g., using The composition within the dissolving coating can be formulated similarly to the topical composition as disclosed herein, with the proviso that only carrier materials intended and registered for internal administration, such as vegetable oils (e.g., peanut oil) or emulsifiers accepted for nutritional use, such as lecithin, are used.

[0123] In addition or alternatively, the pharmaceutical compositions disclosed herein may also be provided in the form of tablets. Compositions for tablets are known to the skilled person and typically comprise fillers such as starch, glucose, mannitol and / or sorbitol; binders such as microcrystalline cellulose, starch, starch paste, cellulose ethers and / or gelatin, disintegrants, lubricants and / or optional coatings. The active agent may be dried and mixed directly with the tablet material.

[0124] As used herein, "dose" refers to a volume of medicine, such as a liquid or solid medicine, which contains the amount of active pharmaceutical ingredient suitable for administration on a single occasion according to reliable medical practice. The dose can be taken orally. In one example, the dose can be about 30 mL, about 25 mL in another example, about 20 mL in another example, about 15 mL in another example, and about 10 mL in another example. In another example, the dose of liquid medicine can be about 10 mL to about 75 mL, about 15 mL to about 50 mL in another example, about 25 mL to about 40 mL in another example, and about 28 mL to about 35 mL in another example. In another example, the dose can be half a tablet or capsule, one tablet or capsule in another example, 1.5 tablets or capsules in another embodiment, and 2 tablets or capsules in another embodiment. In another example, the dosage of the solid medication can be about 1 mg to about 100 mg, about 5 mg to about 50 mg in another example, about 10 mg to about 40 mg in another example, about 15 mg to about 30 mg in another example, and about 50 mg to about 90 mg in another example, about 55 mg to about 80 mg in another example, and about 60 mg to about 75 mg in another example. Given a dose size, the concentration of the active ingredient can be adjusted to provide an appropriate dose of the active substance. In one example, the dose is intended to be taken every 4 hours, every 6 hours in another example, every 8 hours in another example, and every 12 hours in another example.

[0125] The active agents disclosed herein, including but not limited to one or more shikonin (especially shikonin A), the combination of shikonin A and caffeic acid ethyl ester, and other active substances mentioned herein and pharmaceutical compositions containing the active substances can be particularly used to treat pain, inflammation, swelling, limited movement, contusion, strain, sprain, arthritis, back pain, muscle discomfort and / or joint discomfort. Without wishing to be bound by theory, the therapeutic effect is achieved, in particular, through the NF-κB pathway, by inhibiting E-selectin transcription.

[0126] Therefore, the present invention also relates to a method for inhibiting and / or treating inflammation by topically and / or internally administering a safe and effective amount of one or more shikonins, particularly shikonin A, or a safe and effective amount of shikonin A in combination with a safe and effective amount of caffeic acid ethyl ester and / or other additional active agents as disclosed herein.

[0127] The present invention also relates to a method for inhibiting and / or treating inflammation by at least partially blocking the activation of the NF-κB pathway, for example, after activation with interleukin 1β (IL-1β), by topically and / or internally administering a safe and effective amount of one or more shikonins, particularly shikonin A, or a safe and effective amount of shikonin A in combination with a safe and effective amount of caffeic acid ethyl ester and / or other additional active agents as disclosed herein.

[0128] The pharmaceutical composition may be administered topically and / or may be administered internally.Topical administration requires that the pharmaceutical composition be administered in a sufficient amount to achieve an effect to the area in need of treatment, wherein the composition remains there for a sufficient period of time to achieve an effect.

[0129] The present invention also relates to a kit comprising a safe and effective amount and instructions for use of a pharmaceutical composition as disclosed herein, the pharmaceutical composition comprising one or more aryl naphthoquinones, particularly shikonin A, or shikonin A plus caffeic acid ethyl ester. The instructions for use will describe some exemplary application scenarios as described herein, but the optimal amount, duration and frequency of application will depend on the desired effect, the severity of any condition to be treated, the health and age of the user, and similar considerations.

[0130] A further preferred embodiment is based on the dependent patent claims. Features of the composition claims can be correspondingly combined with the use claims herein and vice versa, in any desired combination. The invention will be described in more detail below.

[0131] Example

[0132] General Experimental Procedure

[0133] IR measurements were performed on a Bruker IFS-48 spectrometer. UV spectra were obtained on a Beckman DU 670 spectrometer. NMR experiments were obtained at 300 K in methanol-d4 (99.95%, Sigma-Aldrich) on a Bruker DRX-600 spectrometer (Bruker BioSpin GmBH, Rheinstetten, Germany) equipped with a Bruker 5 mm TCI CryoProbe. Data processing was performed with Topspin 3.2 software. ROESY spectra were obtained with tmix=400ms. Size exclusion chromatography was performed on Sephadex LH-20 (GE Healthcare, Sigma Aldrich, Milan, Italy).

[0134] Compound separation

[0135] A PA-depleted hydroalcoholic (20% EtOH w / w) liquid extract of Lithospermum officinale root (DER 1:2) was obtained from Merck KGaA & Co Werk Spittal, Austria. To remove the mucilage, the ethanol content of 500 g of the liquid extract was evaporated and the aqueous phase was partitioned with ethyl acetate (1:1) to obtain 50 g of ethyl acetate extract. 3 g of the ethyl acetate extract was fractionated on a Sephadex LH-20 (GE Healthcare, Sigma Aldrich, Milan, Italy) column (100×5 cm) using methanol as the mobile phase to obtain 80 fractions (8 mL) that were monitored by LC-ESI-HR-MS analysis.

[0136] LC-ESI / LTQ Orbitrap / MS analysis

[0137] LC-MS of mucilage and PA-depleted extract of Lithospermum officinale root showing inhibition of interleukin-1β-induced E-selectin expression in HUVEC cells at a concentration of 20 μg / mL were analyzed by LC-ESI / HR / MS using the "data-dependent scan" mode (where the precursor ion corresponding to the most intense peak is selected). n The procedure was performed as follows:

[0138] The secondary metabolite profile of the hydroalcoholic extract of Lithospermum officinale root was obtained by using an HPLC method coupled to a hybrid mass spectrometer that combines a linear trap quadrupole (LTQ) and an orbitrap mass analyzer. All experiments were performed using a Thermoscientific liquid chromatography system consisting of a quaternary Accela 600 pump and an Accela autosampler connected to a linear trap-orbitrap hybrid mass spectrometer with electrospray ionization (ESI) (LTQ-Orbitrap XL, Thermo Fisher Scientific, Bremen, Germany). Separation was performed on a Kinetex EVO C18 5μm (150mm×2.10mm) column (Phenomenex Aschaffenburg, Germany) using a flow rate of 0.2mL / min and a mobile phase consisting of a combination of A (0.1% formic acid / water, v / v) and B (0.1% formic acid / acetonitrile, v / v). A linear gradient program was used, starting isocratically with 5% B in 5 min, then from 5% to 23% B in 5 min, held at 23% B for 5 min, from 23% to 55% B in 7 min, from 55% to 95% B in 5 min, held at 95% B for 5 min. The mass spectrometer was operated in negative ion mode. The ESI source parameters were as follows: capillary voltage -48 V; tube mirror voltage -176.47; capillary temperature 280 ° C; sheath and auxiliary gas flows (N2), 15 (arbitrary units) and 5 (arbitrary units), purge gas 0 spray voltage 5 kV. The mass range was 120 m / z to 1600 m / z with a resolution of 30000. For fragmentation studies, data-dependent scans were performed and the precursor ions corresponding to the most intense peaks in the LC-MS analysis were selected. Xcalibur software version 2.1 was used for instrument control, data acquisition, and data analysis.

[0139] Preliminary metabolite profiles showed that the metabolites corresponded to different structural classes ( Figure 2 ) of the 20 main compounds ( Figure 1 ). Some of these metabolites were identified by comparing their accurate masses, characteristic fragmentation patterns and retention times with those of authentic standards. Based on the above method, the presence of allantoin (1), protocatechuic acid (4), protocatechuic aldehyde (5), p-hydroxybenzoic acid (6), caffeic acid (7) and its ethyl ester derivative (16), rosmarinic acid (12) and the fatty acids linolenic acid (18), linoleic acid (19) and hydroxypalmitic acid (20) were identified. The remaining compounds (2, 3, 8-11, 13-15 and 17) could not be assigned unambiguously by HR-ESI-MS because their accurate masses did not match any compounds reported by comfrey in the literature or because their preliminary NMR data did not support compounds reported in comfrey with the same molecular weight.

[0140] Isolation procedure of shikonin A (compound 15)

[0141] In order to fully characterize compound 2-3,8-11 and 13-17, first remove the syrup fraction of extract by distributing liquid extract with ethyl acetate.Then, organic phase is subjected to size exclusion chromatography on Sephadex Lh-20 post, and further purified by semi-preparative HPLC-UV.In fact, the dry fraction obtained is diluted in acetonitrile (concentration is 10mg dry residue / 100 μL), and semi-preparative HPLC UV separation is carried out by using Phenomenex C18 Synergi-Hydro-RP (250mm×10mm, 10 μm) post on Agilent1260Infinity system (Agilent Technologies, Palo Alto, CA, USA) equipped with binary pump (G-1312C) and UV detector (G-1314B).Mobile phase is composed of solvent A (H2O+0.1% formic acid) and solvent B (CH3CN+0.1% formic acid).Analysis is carried out at room temperature without constant temperature. Peaks collected at HPLC-UV were evaporated to dryness in vacuo and also freeze-dried prior to NMR analysis.The compound structures were fully characterized by extensive 1D- and 2D-NMR experiments as well as MS / MS analysis.

[0142] Fractions 23-26 (10 mg) corresponded to compound 12, ie, rosmarinic acid.

[0143] Fractions 40-42 (10 mg) corresponded to compound 14, which was identified as globoidnan A (C 26 H 20 O 10 ).

[0144] Fractions 19-22 (51.6 mg) were purified using a linear gradient program at a flow rate of 2 mL / min at a wavelength of 254 nm (0% to 20% B in 15 min; 20% to 40% B in 10 min; 40% to 60% B in 10 min; 60% to 100% B in 11 min, then 6 min at 100% B to give compounds 2 (1.2 mg, Rt=10.3 min), 3 (1.3 mg, Rt=13.5 min), 6 (1.5 mg, Rt=11.2 min, p-hydroxybenzoic acid) and 9 (2.2 mg, Rt=27.3). NMR analysis confirmed that compound 2 was p-hydroxybenzoic acid glucoside and compound 3 was 5-hydroxymethyl-2-furfural, respectively. Compound 9 was purified at m / z 367.1384 [MH] -The main peak is shown at 18 H 24 O 8 Its MS / MS spectrum is at m / z 205.09 [MH-162] - The fragment ions are shown at , indicating the presence of a hexose unit. The NMR data of 9 allowed the deduction of its structure as the glycosylated and prenylated phenolic acid malaxinic acid, which is described for the first time in comfrey herein.

[0145] Fractions 27-39 (42.3 mg), fractions 43-54 (7.1 mg) and fractions 55-80 (22.2 mg) were purified under the same elution conditions at a flow rate of 2 mL / min and at a wavelength of 280 nm: (holding at 5% B for 0 min-5 min, from 5% to 20% B in 15 min; from 20% to 50% B in 20 min, from 50% to 100% B in 10 min, and holding at 100% B for 5 min).

[0146] Under these conditions, compounds 4 (1.1 mg protocatechuic acid, Rt = 22.60 min), 5 (1.2 mg protocatechuic aldehyde, Rt = 27.9 min), 7 (1.5 mg caffeic acid, Rt = 29.4 min), 8 (1.3 mg, Rt = 31.7 min), 11 (1.6 mg, Rt = 34.3), 13 (1.7 mg, Rt = 35.1 min) and 10 (4.4 mg, Rt = 35.3) were isolated from fractions 27-39.

[0147] The HR-ESI-MS spectra of 8, 11 and 14 supported the molecular formulas of C 27 H 22 O 12 , C 36 H 30 O 16 and C 26 H 20 O 10 The analysis of their NMR and MS / MS data allowed the identification of compound 14 as globoidnan A and compound 8 as globoidnan B, respectively. Compound 11 was determined to be (+)-caryophyllene, where these lignans are described for the first time in the root of polychaete. The molecular formula of compound 13 was determined to be C 9 H 10 O 5, and the structural elucidation provided by NMR analysis allowed the identification of compound 13 as hydroxycaffeic acid. Although the MS / MS fragmentation pattern suggested a structural framework similar to that of globoidnan A of compound 10, the interpretation of the NMR data confirmed this hypothesis, determining the structure of 10 as 3-carboxy-6,7-dihydroxy-1-(3',4'-dihydroxyphenyl)-naphthalene.

[0148] Compound 15 (1.6 mg Rt = 41.10) was isolated from fractions 43-54.

[0149] Compound 16 (1.2 mg, Rt = 42.2) was identified as caffeic acid ethyl ester, and 17 (1.0 mg, Rt = 43.3) was isolated from fractions 55-80. Although the MS / MS fragmentation pattern indicated a structural framework similar to that of globoidnan A of compound 17, interpretation of the NMR data determined the structure of compound 17 to be the ethyl ester derivative of compound 10, trifoliolignan D.

[0150] In addition, 3 g of the raw material was extracted with 50 mL of methanol to obtain a methanol extract in order to confirm the natural occurrence of ethyl esters 15-17 in Lithospermum officinale by LC-MS analysis. This showed that compounds 15, 16 and 17 were not artifacts due to the use of ethanol as the extraction solvent, but were naturally present.

[0151] Identification of compound 15; named as shikonin A :

[0152] Shikonin A (Compound 15) appears as an amorphous yellow solid. HR-ESI-MS spectrum of Compound 15 (m / z 381.0601 [MH] - , C 20 H 13 O 8 The calculated value, 381.0610) supports C 20 H 14 O 8 The molecular formula ( Figure 4 The ESI / MS / MS spectrum of compound 15 was at m / z 353.02 ([MH-28] - ) showed a strong ion at m / z 353.02, indicating the presence of an ethyl group. The MS of the ion peak at m / z 309.10 ([MH-44] - ) and 265.10([MH-44-44] - ) corresponds to two CO 2Loss of molecule. 1H and 13C NMR (methanol-d4, 600 MHz and 150 MHz) data are shown in Table 1 below; ESI-HR-MS m / z 381.0601 [MH]-(calculated for C20H13O8, 381.0610).

[0153] Table 1: 13C (150 MHz) and 1H NMR (600 MHz) data of compound 15 .

[0154]

[0155]

[0156] For compound 15 13 Analysis of C and HSQC NMR data indicated a highly unsaturated molecule with 16 aromatic carbons, while its 1H NMR spectrum showed five aromatic singlets at δ8.30 (H-8), 8.02 (H-6'), 7.67 (H-4), 7.34 (H-5), and 6.91 (H-3'). In addition, the 1H NMR of compound 15 showed characteristic signals of ethoxy groups at δ4.43 (2H, q, J = 7.0 Hz) and 1.41 (3H, t, J = 7.0 Hz). In addition, detailed analysis of the HMBC spectrum confirmed the presence of two major fragments ( Figure 5 ). Specifically, key long-range correlations between aromatic proton H-4 and carbon resonances at δ 172.2 (C-9), 130.5 (C-3), 113.4 (C-2), 133.7 (C-4a), 112.4 (C-5) and 124.5 (C-8a), H-5 and carbon resonances at δ 133.7, 151.3 (C-6), 150.3 (C-7) and 124.5, and H-8 and carbon resonances at δ 136.1 (C-1), 151.3 and 133.7 confirmed the presence of 1,2-disubstituted 6,7-dihydroxy-naphthalene-3-carboxylic acids. Based on the HMBC correlations from the protons H-3' and H-6' to the carbon resonances at δ 111.5 (C-1'), 143.8 (C-2'), 149.6 (C-4') and 147.1 (C-5'), the structure of the second fragment was determined to be a 1-substituted benzene-2,4,5-triol. Subsequently, the connectivity between the two fragments was deduced from the HMBC correlations from H-6' to C-1. In addition, the HMBC correlation of the equivalent methylene at δ 4.43 (H-1") with the carbonyl at δ 172.2 connected the ethoxy residue to the naphthalene portion. Examination of the structure of this portion revealed that it contained 13 of the required 14 unsaturations, and only one of the two carbonyl groups was assigned. Therefore, the phenolic ring and the 6,7-dihydroxy-naphthalene-3-carboxylic acid residue must be connected by forming a lactone ring to satisfy the unsaturation index and molecular formula. HMBC experiments were performed with a long-range delay range of 2.75 Hz to 10 Hz. For 5 Hzn The HMBC spectrum optimized by J(C,H) coupling, corresponding to a delay time of Δ2 = 100 ms, shows a four-bond long-range correlation and a stronger double-bond long-range correlation. Finally, the structure of compound 15 was assembled as follows. The carbon resonance at δ 162.5 (C-10) and H-4 were 4 J CH The HMBC correlation allowed the deduction of the attachment of the carbonyl group to C-2, while the ROE correlation between H-6′ and H-8 together with the attachment of H-6′ to C-2 4 J CH HMBC correlation indicated the esterification site at C-2 ( Figure 3 ). Therefore, shikonin A (15) was identified as an aryl naphthoyl lignan with an unusual δ-lactone ring (corresponding to the coumarin skeleton).

[0157] Pharmaceutical Activity of Shikonin A

[0158] J. Seigner et al. (Frontiers in Pharmacology 10.3389 / fphar.2019.00289) recently reported that at a starting concentration of 20 μg / ml, comfrey extract inhibited interleukin-1β (IL-1β)-induced E-selectin mRNA levels by about 70%. E-selectin is an NF-κB-dependent target gene, and IL-1β is a well-described proinflammatory mediator that plays an important role in regulating immune and inflammatory responses, particularly those involving sterile injuries such as trauma and blunt trauma. The tested extracts were shown to contain allantoin (1), protocatechuic acid (5), p-hydroxybenzoic acid (6), caffeic acid (7), rosmarinic acid (12), and globoidnan A (14). Therefore, the anti-inflammatory activity of individual compounds 1, 5, 12, and 14 was tested using IL-1 stimulated HUVEC cells, and the fold change of mRNA expression of E-selectin was determined by real-time PCR analysis. These were compared with the inhibitory activities of the new aryl naphthoyl lignans shikonin A (15) and globoidnan B (8), malaxinic acid (9) and caffeic acid ethyl ester (16), the latter three being identified for the first time in comfrey as disclosed herein.

[0159] Therefore, as described in M. Hoeth et al., Guidance Molecules in Human Endothelial Cells, Plos One 7, 2012, primary HUVEC cells were isolated from the umbilical cord and maintained in M199 medium (Lonza) supplemented with 20% FCS (Sigma), 2mM L-glutamine (Sigma), penicillin (100 units / mL), streptomycin (100 mg / mL), 5 units / mL heparin and 25 mg / mL ECGS (Promocell) until the 5th passage. After 90 min of stimulation with 5ng / mL IL-1β (R&D Systems), HUVEC cells were pre-incubated with various concentrations of purified compounds (1, 5, 8, 9, 12, 14, 15 and 16) for 30 minutes. The cells were then analyzed for changes in E-selectin (SELE) mRNA expression. Therefore, total RNA was isolated using the PeqGold total RNA isolation kit (VWR International) according to the manufacturer's instructions. 1 μg RNA was reverse transcribed using random hexamers (Fermentas) and murine leukemia virus reverse transcriptase (Thermo Scientific Fisher). Primers were designed using the software "Primer3". The following primer sequences were used for qPCR: 3-phosphoglyceraldehyde dehydrogenase (GAPDH) forward, 5′-AGAAGGCTGGGGCTCATTT-3′ and reverse, 5′-CTAAGCAGTTGGTGGTGCAG-3′; E-selectin (SELE) forward, 5′-CCTGTGAAGCTCCCACTGA-3′ and reverse, 5′-GGCTTTTGGTAGCTTCCATCT-3′. Real-time PCR was performed using a StepOnePlus instrument (Applied Biosystems) with SsoAdvanced Universal SYBR Green Supermix (BioRad), and relative mRNA expression was normalized to GAPDH. The fold change of mRNA expression was calculated according to the 2-ΔΔCT method. The results are expressed as the average induction fold of the average Ct value of triplicates and are shown in Table 2:

[0160] Table 2: Inhibitory effects of 1, 5, 8, 9, 12, 14, 15 and 16 on IL-1β-induced E-selectin expression .

[0161]

[0162] The effective concentration (EC) was based on at least three independent experiments.

[0163] Surprisingly, allantoin (compound 1) and rosmarinic acid (compound 12) were shown not to show any inhibitory activity on E-selectin expression. A small effect of 35% inhibition could be achieved with globoidnan A (compound 14) at an effective concentration of 40 μM, where the previously known globoidnan B (compound 8) did not show any inhibitory activity. Shikonin A (compound 15) was found to be more effective in E-selectin inhibition, inhibiting 50% at an EC of 50 μM. Caffeic acid ethyl ester (compound 16) represents the most active inhibitor, inhibiting E-selectin expression by 70% at a concentration of 64 μM. The known protocatechuic acid (compound 5) as well as the newly identified malashinic acid (compound 9) also showed an inhibitory effect greater than 50%, but only at higher effective concentrations. Compared to the latter two compounds, shikonin A (15) and caffeic acid ethyl ester (16) were effective at half the concentration.

[0164] Embodiment composition

[0165] In the following academic examples, compositions are specified, which are intended to be exemplary only and should not limit the scope of the invention.

[0166]

[0167]

[0168]

[0169] The dimensions and values ​​disclosed herein are not to be understood as being strictly limited to the exact numerical values ​​cited. Instead, unless otherwise indicated, each such dimension is intended to represent the stated value and a functionally equivalent range around that value. For example, a dimension disclosed as "40 mm" is intended to mean "about 40 mm".

[0170] Unless expressly excluded or otherwise limited, each document cited herein, including any cross-referenced or related patent or patent application and any patent application or patent to which this application claims priority or the benefit of, is hereby incorporated by reference in its entirety. The citation of any document is not an admission that it is prior art to any of the present invention disclosed or claimed herein, or an admission that it, by itself or in combination with any one or more of the references, proposes, suggests, or discloses any such invention. In addition, to the extent that any meaning or definition of a term in this invention conflicts with any meaning or definition of the same term in a document incorporated by reference, the meaning or definition assigned to that term in this invention shall govern.

[0171] Although specific embodiments of the present invention have been illustrated and described, it will be apparent to those skilled in the art that various other changes and modifications may be made without departing from the spirit and scope of the present invention. Therefore, it is intended that all such changes and modifications within the scope of the present invention be covered in the appended claims. <110> The Procter & Gamble Company <120> Shikonin and arylnaphthoyl lignans inhibit pro-inflammatory gene expression And pharmaceutical compositions containing them <130> CM05167FQ <160> 4 <170> PatentIn Version 3.5 <210> 1 <211> 19 <212> DNA <213> Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) <400> 1 agaaggctgg ggctcattt 19 <210> 2 <211> 20 <212> DNA <213> Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) <400> 2 ctaagcagtt ggtggtgcag 20 <210> 3 <211> 19 <212> DNA <213> E-selectin (SELE) <400> 3 cctgtgaagc tcccactga 19 <210> 4 <211> twenty one <212> DNA <213> E-selectin (SELE) <400> 4 ggcttttggt agcttccatc t 21

Claims

1. One or more aryl naphthyl lignans, wherein the aryl naphthyl lignans have the general structure shown in Formula I wherein each of R1, R2, R3 and R4 is independently hydroxyl; and Wherein R5 is selected from methoxy, ethoxy, propoxy, butoxy or pentoxy.

2. The aryl naphthyl lignan according to claim 1, wherein R1, R2, R3 and R4 are each a hydroxyl group, and R5 is an ethoxy group.

3. A pharmaceutical composition comprising a) an active agent, the active agent comprising one or more aryl naphtholignans according to any one of claims 1 or 2; and b) a pharmaceutically acceptable carrier.

4. The pharmaceutical composition according to claim 3, further comprising an additional active agent.

5. The pharmaceutical composition according to claim 4, wherein the additional active agent is an analgesic, an active agent having anti-inflammatory activity, an activity enhancer or a combination thereof.

6. The pharmaceutical composition according to claim 5, wherein the additional active agent is a natural analgesic.

7. The pharmaceutical composition according to claim 6, wherein the additional active agent is a natural analgesic extracted from comfrey.

8. The pharmaceutical composition of claim 4, wherein the additional active agent comprises caffeic acid ethyl ester, malaxinic acid, globoidnan A, protocatechuic aldehyde, polysaccharide root extract, or a combination thereof.

9. The pharmaceutical composition of claim 8, wherein the additional active agent comprises caffeic acid ethyl ester, malaxinic acid, globoidnan A, or a combination thereof.

10. The pharmaceutical composition according to any one of claims 4 to 9, wherein the additional active agent further comprises allantoin, p-hydroxybenzoic acid glucoside, 5-hydroxymethyl-2-furfural, protocatechuic acid, p-hydroxybenzoic acid, caffeic acid, globoidnanB, 3-carboxy-6,7-dihydroxy-1-(3',4'-dihydroxyphenyl)-naphthalene, (+)-caryophyllene, rosmarinic acid, α-hydroxyhydrocaffeic acid, trifoliol D, linolenic acid, linoleic acid, hydroxypalmitic acid or a combination thereof.

11. The pharmaceutical composition according to claim 10, wherein the additional active agent further comprises globoidnan B, trifoliol D, 3-carboxy-6,7-dihydroxy-1-(3',4'-dihydroxyphenyl)-naphthalene, chrysanthellate and / or a combination thereof.

12. The pharmaceutical composition according to any one of claims 3 to 9, wherein the composition comprises a pharmaceutically safe and effective amount of the one or more aryl naphthol lignan active agents.

13. The pharmaceutical composition of claim 12, wherein the composition comprises the one or more arylnaphthol active agents in an amount to provide a local concentration of 30 μM to 65 μM active agent at the site in need thereof.

14. The pharmaceutical composition of claim 13, wherein the composition comprises the one or more arylnaphthol active agents in an amount to provide a local concentration of 40 μM to 60 μM active agent at the site in need thereof.

15. The pharmaceutical composition of claim 14, wherein the composition comprises the one or more arylnaphthol active agents in an amount to provide a local concentration of 45 μM to 55 μM active agent at the site in need thereof.

16. The pharmaceutical composition of claim 15, wherein the composition comprises the one or more arylnaphthol active agents in an amount to provide a local concentration of 48 μM to 52 μM active agent at the site in need thereof.

17. The pharmaceutical composition of claim 12, wherein the composition comprises the one or more arylnaphthol active agents in an amount of 1 μg / g to 10 μg / g of the composition.

18. The pharmaceutical composition of claim 17, wherein the composition comprises the one or more arylnaphthol active agents in an amount of 2 μg / g composition to 7.5 μg / g composition.

19. The pharmaceutical composition of claim 18, wherein the composition comprises the one or more arylnaphthol active agents in an amount of 3 μg / g to 5 μg / g of the composition.

20. The pharmaceutical composition according to any one of claims 8 to 9, wherein the composition comprises a safe and effective amount of caffeic acid ethyl ester.

21. The pharmaceutical composition of claim 20, wherein the composition comprises caffeic acid ethyl ester in an amount to provide a local concentration of 50 μM to 80 μM caffeic acid ethyl ester at a location in need thereof.

22. The pharmaceutical composition of claim 21, wherein the composition comprises caffeic acid ethyl ester in an amount to provide a local concentration of 55 μM to 75 μM caffeic acid ethyl ester at a location in need thereof.

23. The pharmaceutical composition of claim 22, wherein the composition comprises caffeic acid ethyl ester in an amount to provide a local concentration of 60 μM to 70 μM caffeic acid ethyl ester at the location where it is needed.

24. The pharmaceutical composition of claim 23, wherein the composition comprises caffeic acid ethyl ester in an amount to provide a local concentration of 62 μM to 66 μM caffeic acid ethyl ester at a location in need thereof.

25. The pharmaceutical composition of claim 20, wherein the composition comprises the caffeic acid ethyl ester in an amount of more than 1 μg / g composition to 15 μg / g composition.

26. The pharmaceutical composition according to claim 25, wherein the composition comprises the caffeic acid ethyl ester in an amount of 2.5 μg / g composition to 10 μg / g composition.

27. The pharmaceutical composition according to claim 26, wherein the composition comprises the caffeic acid ethyl ester in an amount of 5 μg / g composition to 7.5 μg / g composition.

28. The pharmaceutical composition of any one of claims 3 to 9, wherein the composition inhibits E-selectin transcription by at least 30% after activation with interleukin 1 β.

29. The pharmaceutical composition of claim 28, wherein the composition inhibits E-selectin transcription by at least 40% upon activation with interleukin 1 β.

30. The pharmaceutical composition of claim 29, wherein the composition inhibits E-selectin transcription by at least 45% upon activation with interleukin 1[beta].

31. The pharmaceutical composition of claim 30, wherein the composition inhibits E-selectin transcription by at least 48% upon activation with interleukin 1 β.

32. The pharmaceutical composition of claim 31, wherein the composition inhibits E-selectin transcription by at least 50% upon activation with interleukin 1 β.

33. A pharmaceutical composition according to any one of claims 3 to 9, wherein the composition is a topical composition or a composition for internal administration.

34. The pharmaceutical composition according to claim 33, wherein the topical composition is in the form of an ointment, a viscous liquid, a mousse or any other adhesive product applied to a strip or a dissolvable strip or a combination thereof.

35. The pharmaceutical composition of claim 33, wherein the topical composition is in the form of an ointment, paste, gel or cream.

36. The pharmaceutical composition of claim 33, wherein the topical composition is in the form of a lotion or paint.

37. The pharmaceutical composition of claim 33, wherein the topical composition is in the form of a foam.

38. The pharmaceutical composition according to claim 33, wherein the composition for internal administration is in the form of a capsule, tablet, lozenge, syrup or a combination thereof.

39. The pharmaceutical composition according to claim 33, wherein the composition for internal administration is in the form of a chewable tablet.

40. The pharmaceutical composition according to claim 33, wherein the composition for internal administration is in the form of chewing gum.

41. The pharmaceutical composition of claim 33, wherein the composition for internal administration is in the form of a liquid.

42. The pharmaceutical composition according to claim 33, wherein the composition for internal administration is in the form of a mousse.

43. The pharmaceutical composition of claim 33, wherein the pharmaceutically acceptable carrier is a water-in-oil emulsion comprising at least one carrier matrix material, a surfactant, and water.

44. The pharmaceutical composition of claim 43, wherein the carrier matrix material is selected from non-toxic oils.

45. The pharmaceutical composition of claim 44, wherein the carrier matrix material is selected from non-toxic edible oils.

46. ​​The pharmaceutical composition of claim 43, wherein the carrier matrix material is selected from the group consisting of saturated or unsaturated fatty alcohols, aliphatic hydrocarbons, fatty esters, and mixtures thereof.

47. The pharmaceutical composition of claim 46, wherein the carrier matrix material is selected from long chain triglycerides.

48. The pharmaceutical composition of claim 43, wherein the carrier matrix material is peanut oil.

49. The pharmaceutical composition of any one of claims 3 to 9, wherein the composition further comprises at least one of a preservative, a dye, a fragrance, a stabilizer, a buffer, or a combination thereof.

50. A pharmaceutical composition according to any one of claims 3 to 9, wherein the composition comprises at least the aromatic naphthoxylin shikonin A, caffeic acid ethyl ester, malayxinic acid and globoidnan A according to claim 3 in a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier comprises at least one non-toxic edible oil, at least one emulsifier, at least one buffer, water and a preservative.

51. The pharmaceutical composition of claim 50, wherein the composition comprises shikonin A in an amount to provide a local concentration of 50 μM shikonin A at a location in need thereof and caffeic acid ethyl ester in an amount to provide a local concentration of 65 μM caffeic acid ethyl ester at a location in need thereof.

52. The pharmaceutical composition according to claim 50, wherein the composition comprises shikonin A in an amount of 3 to 5 μg / g of the composition and caffeic acid ethyl ester in an amount of 5 to 7.5 μg / g of the composition in a pharmaceutical carrier.

53. The pharmaceutical composition of claim 52, wherein the pharmaceutical carrier comprises at least peanut oil, at least one emulsifier, at least one buffer, water, a fragrance, and a preservative.

54. One or more arylnaphtholignans according to any one of claims 1 or 2 and a pharmaceutical composition according to any one of claims 3 to 9 for use in inhibiting and / or treating inflammation.

55. One or more arylnaphtholignans according to any one of claims 1 or 2 and a pharmaceutical composition according to any one of claims 3 to 9 for use in the treatment of broken bones, tendon injuries, painful joints and muscles, acute upper and lower back pain, knee arthrosis, blunt trauma, for reducing swelling in joint deformities, for improving joint mobility in osteoarthritic joints and / or combinations thereof.

56. Use of the aryl naphthyl lignan shikonin A according to claim 2 as a marker substance for quantitative analysis of active substances in products using lithospermum erythrorhizon and / or extracted from lithospermum erythrorhizon.

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