Topical formulations for promoting wound healing

Abstract

A topical formulation comprising an active agent, a viscosity increasing agent, an ointment base, an antimicrobial preservative, and an emulsifier wherein the active agent comprises salvigenin and, optionally, asiaticoside.

Description

[0001] This application is a divisional application of an invention patent application with an application date of July 17, 2017, application number 201780009172.7, and invention name “Topical formulation for promoting wound healing”. Technical Field

[0002] This application claims the benefit of the filing date of U.S. Provisional Application No. 62 / 363,284, filed on July 17, 2016, which is incorporated herein by reference in its entirety. Background Art

[0003] Plectranthus amboinicus (formerly Coleus amboinicus Lour., Coleus aromaticus Benth., Coleus aromaticus auct., Plectranthus aromaticus Roxb., Plectranthus aromaticus Benth., and Plectranthus amboinicus (Lour.) Spreng.) is a perennial medicinal plant in the Lamiaceae family (also known as Labiatae) native to southern and eastern Africa. Plectranthus amboinicus is also known as patchouli, Cuban oregano, Indian borage, Indian mint, Mexican mint, Mexican oregano, country borage, and Spanish thyme.

[0004] Centella asiatica (formerly known as Centella asiatica Urban, Centellaasiatica (L.) Urban, Hydrocotyle asiatica L., and Trisanthus cochinchinensis Lour.) is a perennial medicinal plant of the Mackinlayoideae subfamily of the Mackinlayaceae or Apiaceae (also known as the Umbelliferae) family native to Asia, Africa, and South America. Centella asiatica is also known as European water miracle, colora, kola, one molecule, Indian one molecule, swamp one molecule, one yuan grass, Indian ginseng, horse hoof grass, cola bowl, Mo Claw wax autumn snow grass, tiger grass, rattan card grass, or Tono. Centella asiatica extracts generally contain two main compounds: asiaticoside and madecassic acid. Summary of the invention

[0005] The present invention is based, at least in part, on the superior therapeutic effects of a topical formulation comprising a combination of a Plectranthus amboinicus extract and a Centella asiatica extract as described herein, comprising salvigenin and / or asiaticoside, to promote wound healing in diabetic patients, such as those suffering from diabetic foot ulcers (DFUs). For example, the topical formulation successfully enhanced the healing of diabetic foot ulcers, and its efficacy was much higher than that of a positive control group. Topical application of the topical formulation also showed limited systemic distribution of at least the active ingredients (caryophyllein and / or asiaticaside) therein in DFU patients and was found to be safe in patients.

[0006] Thus, provided herein is a topical formulation comprising: (i) an active agent comprising salvigenin in an amount of about 0.0001% to 0.5% (w / w) (e.g., about 0.0001% to 0.1% by weight) and asiaticoside in an amount of about 0.05% to 5% (w / w) (e.g., about 0.05% to 1% by weight); (ii) a viscosity increasing agent in an amount of about 1.0 to 10% (w / w); (iii) an ointment base in an amount of about 5 to 30% (w / w); (iv) an antimicrobial preservative in an amount of about 0.005 to 0.2% (w / w); and (v) an emulsifier in an amount of about 0.5 to 10% (w / w).

[0007] In some embodiments, the active agent is Plectranthus amboinicus (PA) extract, Centella asiatica (CA) extract, or a combination of the PA and CA extracts, which (alone or in combination) comprises carnosine and asiaticoside.

[0008] In one embodiment, the active agent is a combination of Plectranthus amboinicus extract and Centella asiatica extract. The active agent may be about 0.1 to 30% (w / w) and at least comprises trilobate caryophyllacein and asiaticoside. In some cases, the topical formulation may comprise about 0.0001% to 0.5% (w / w), for example, about 0.0001% to 0.1% (w / w) trilobate caryophyllacein. In some cases, the topical formulation may comprise about 0.0001% to 0.1% (w / w) trilobate caryophyllacein and 0.05% to 1% (w / w) asiaticoside. In any topical formulation described herein, the active agent may further comprise cirsimaritin, madecassoside, or a combination thereof.

[0009] In the topical formulation, the Plectranthus amboinicus extract may be about 0.1 to 5% (w / w) or about 0.1 to 1% (w / w). In some embodiments, the Plectranthus amboinicus extract comprises flavonoids, essential oils, phenols, terpenoids, or combinations thereof.

[0010] The Plectranthus amboinicus extract can be prepared by contacting the whole Plectranthus amboinicus or a portion thereof with a solvent having a polarity index of less than 7 (e.g., less than 5) to produce a solution and drying the solution to produce the Plectranthus amboinicus extract. In a specific embodiment, the Plectranthus amboinicus extract is prepared by a method comprising: (i) contacting the whole Plectranthus amboinicus or a portion thereof with an alcohol (e.g., ethanol) to produce a Plectranthus amboinicus crude extract; (ii) contacting the crude extract with a nonionic adsorption resin; (iii) washing the nonionic adsorption resin with a solvent having a polarity index of less than 7 (e.g., less than 5) to produce a solution; and (iv) drying the solution to produce the Plectranthus amboinicus extract.

[0011] Alternatively or additionally, the Centella asiatica extract may comprise asiaticoside, madecassoside, or both. In some embodiments, the Centella asiatica extract is present in the topical formulation at about 0.1 to 20% (w / w) or about 0.5 to 5% (w / w).

[0012] In some embodiments, the ratio between the Plectranthus amboinicus extract and the Centella asiatica extract is about 1:10 to about 10:1, for example, about 1:5 to 5:1.

[0013] The topical formulation may contain about 1.0 to 10% (w / w) of a viscosity increasing agent. Examples of the viscosity increasing agent include, but are not limited to, cetearyl alcohol, cholesterol, stearyl alcohol, chlorocresol, white wax, stearic acid, cetyl alcohol, or a combination thereof.

[0014] The topical formulation may include about 5 to 30% (w / w) (e.g., 10 to 30% by weight) of an ointment base. In some cases, the ointment base may be a cream base. Examples of the ointment base include, but are not limited to, one or more petrolatum compounds (e.g., liquid petrolatum, white petrolatum).

[0015] The antimicrobial preservative in the topical formulation described herein may be about 0.005 to 0.2% (w / w) (e.g., 0.01 to 0.2% by weight). It may include one or more paraben compounds (e.g., methylparaben and / or propylparaben).

[0016] The emulsifier in the topical formulation (e.g. 60 and / or 60) may be about 0.5 to 10% (w / w) (e.g., 0.5 to 6% by weight).

[0017] In some embodiments, the topical formulations described herein may include about 10 to 30% (w / w) of an ointment base, about 0.01 to 0.2% (w / w) of an antimicrobial preservative, and about 0.5 to 6% (w / w) of an emulsifier.

[0018] Any of the topical formulations described herein may further optionally comprise one or more solvents, for example, propylene glycol.

[0019] In one embodiment, the topical formulation described herein comprises a combination of Plectranthus amboinicus extract and Centella asiatica extract in an amount of about 0.1 to 30% (w / w), cetearyl alcohol in an amount of about 1.0 to 10% (w / w) (e.g., about 2 to 8% or about 3 to 7%), a total amount of white petrolatum and liquid petrolatum in an amount of about 5 to 30% (w / w) (e.g., about 5 to 25% or about 5 to 20%), a total amount of methylparaben and propylparaben in an amount of about 0.005 to 0.2% (w / w), and a total amount of sorbitan monostearate and polysorbate 60 in an amount of about 0.5 to 10% (w / w) (e.g., 0.5 to 8% or 0.5 to 6%). Such a topical formulation may also comprise propylene glycol in an amount of about 2 to 20% (w / w) (e.g., 2 to 10% or 4 to 15%).

[0020] In another aspect, the present invention provides a method of wound healing, comprising applying the topical formulation disclosed herein to a wound site of a subject in need thereof. In one embodiment, the topical formulation can be applied at least once a day, for example, twice a day.

[0021] In one embodiment, the subject to be treated may be a human diabetic patient with a diabetic foot ulcer. For example, the human diabetic patient may have type I or type II diabetes. In another embodiment, the subject is a human patient with an open wound, such as an abrasion wound, a cut wound, a laceration wound, a puncture wound, or an avulsion wound. In another embodiment, the subject is a human patient with a chronic wound, which may be a surgical wound, a traumatic wound, a pressure ulcer, a venous ulcer, a diabetic ulcer, or a wound caused by cancer, burns, bedsores, or atopic dermatitis. In some embodiments, the subject is a human patient with acne.

[0022] Also within the scope of the present invention is (i) a topical formulation for promoting wound healing as described herein, and (ii) the use of the Plectranthus amboinicus extract, the Centella asiatica extract, or a combination thereof in the preparation of a medicament for promoting wound healing, wherein the Plectranthus amboinicus extract, the Centella asiatica extract, or a combination thereof is formulated as a topical formulation as described herein.

[0023] The details of one or more specific embodiments of the present invention are described in the following description. Other features and advantages of the present invention will become apparent from the following drawings and detailed description of several specific embodiments, as well as the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is a flow chart showing an exemplary method for preparing Plectranthus amboinicus extract.

[0025] Figure 2 is a flow chart showing an exemplary method for preparing a Centella asiatica extract.

[0026] Figure 3A and Figure 3B The results showed that the extracts of Plectranthus amboinicus (PA) and Centella asiatica (CA) had an effect on the proliferation of endothelial cells determined by MTT assay. Figure 3A ), and on endothelial cell migration as determined by scratch healing assay ( Figure 3B ) in the table below.

[0027] Figure 4A and Figure 4B Contains data showing the effects of Plectranthus amboinicus (PA) extract and Centella asiatica (CA) extract on keratinocyte proliferation as determined by SRB assay ( Figure 4A ), and on keratinocyte migration as determined by scratch healing assay ( Figure 4B ) in the table below.

[0028] Figure 5A and Figure 5B Included are graphs showing the effects of carnosine in promoting wound healing in an in vitro capillary tube formation assay and graphs showing the effects of asiaticaside in promoting keratinocyte migration.

[0029] Figure 6 The graph shows the effects of Plectranthus amboinicus (PA) extract and Centella asiatica (CA) extract on normal wound healing in animal models. DETAILED DESCRIPTION

[0030] Foot ulcers are a common complication associated with diabetes and are caused by the breakdown of skin tissue and exposure of the underlying skin. All people with diabetes are at risk of developing foot ulcers. About 15-20% of people with diabetes will develop a foot ulcer. About 33% of these patients will require amputation.

[0031] Currently available treatments for diabetic foot ulcers (DFUs) include wearing protective gear, such as diabetic shoes, plaster casts, foot braces, compression stockings, and insoles to prevent swelling and callus. Such medical gear can be expensive and often cause inconvenience to patients' daily lives. Alternatively, surgical debridement can be performed to remove the foot ulcer. To date, It is the only drug approved by the U.S. Food and Drug Administration (FDA) for the treatment of DFU. In June 2008, the FDA announced New label changes to indicate exposure to The risk of cancer death increased fivefold in the group.

[0032] The present invention is based at least in part on the development of a topical formulation comprising a combination of Plectranthus amboinicus extract and Centella asiatica extract, which shows significantly higher efficacy in promoting wound healing and low side effects in patients with DFU. The topical formulation is a cream base and comprises at least about 0.0001% to 0.5% (w / w) of trilobatin as an active ingredient. In some specific embodiments, the topical formulation may further comprise about 0.05% to 5% (w / w) of asiaticoside as an additional active ingredient. The topical formulation also comprises about 1.0 to 10% (w / w) of a viscosity increasing agent, about 5 to 30% (w / w) of an ointment base (e.g., a cream base), about 0.005 to 0.2% (w / w) of an antimicrobial preservative; and about 0.5 to 10% (w / w) of an emulsifier. All components can be dissolved in one or more suitable solvents, such as propylene glycol and water.

[0033] The pharmacokinetic study of the topical cream formulation showed its safety in patients with DFU. More importantly, the topical application of the cream formulation resulted in extremely low systemic distribution of at least the two active ingredients, trilobatin and asiaticaside, after topical application to the wound site of the patient, and was found to be safe in patients topically treated with the topical formulation.

[0034] In addition, a Phase III clinical trial was conducted to evaluate the efficacy of a topical formulation of the cream base for the treatment of chronic diabetic foot ulcers. The topical ointment showed significantly higher efficacy in promoting ulcer closure in diabetic patients, especially in those with large ulcers or plantar ulcers.

[0035] Accordingly, provided herein are topical formulations comprising one or both of Plectranthus amboinicus extract and Centella asiatica extract, and methods of using such formulations for promoting wound healing in a subject in need thereof.

[0036] Topical formulations

[0037] The present invention provides a topical formulation for promoting wound healing (e.g., wound healing in diabetic patients), which comprises an active agent, which may be a Plectranthus amboinicus extract comprising carnosine, a Centella asiatica extract comprising asiaticoside, or a combination of the Plectranthus amboinicus extract and the Centella asiatica extract. The topical formulation described herein may comprise carnosine in an amount ranging from about 0.0001% to about 0.5% (w / w), and may optionally comprise asiaticoside, which may be in an amount ranging from about 0.05% to about 5% (w / w). In some specific embodiments, the topical formulation comprises a Plectranthus amboinicus extract, a Centella asiatica extract, or both in an amount ranging from about 0.1-30% (w / w).

[0038] The topical formulation further comprises one or more carriers or excipients, including one or more viscosity increasing agents (e.g., about 1.0 to 10%), one or more ointment bases (e.g., one or more cream bases), which may be about 5 to 30%, one or more antimicrobial preservatives (e.g., about 0.005 to 0.2% by weight), one or more emulsifiers (about 0.5 to 10% by weight), or a combination thereof. These components may be dissolved or dispersed in a suitable solvent.

[0039] (i) Active agent

[0040] The active agent in the topical formulation described herein comprises carnosine, and optionally comprises asiaticoside. In some specific embodiments, the active agent may be a Plectranthus amboinifolius extract comprising carnosine or a Centella asiatica extract comprising asiaticoside, alone, or a combination thereof. The concentration of the active agent in the topical formulation may be about 0.1 to 30% (w / w). For example, the active agent may comprise about 0.1-5%, 0.5-3%, 1-2%, 1-3%, 2-10%, 5-10%, 5-15%, 10-15%, 15-20%, 10-20%, 20-25%, 15-30%, 20-30%, 10-30%, or 25-30% (w / w) of the topical formulation. The concentration of the active agent in the topical formulation described herein refers to the percentage of the total weight of the active agent in the dry form of the total weight of the topical formulation as a whole.

[0041] The topical formulations described herein may include about 0.0001% to 0.5% (w / w) carnosine, for example, about 0.001% to 0.5% (w / w), about 0.001% to 0.3% (w / w), about 0.001% to about 0.2% (w / w), about 0.01% to 0.1% (w / w), about 0.002% to 0.5% (w / w), about 0.002% to 0.4% (w / w), about 0.002% to 0.3% (w / w), about 0.001% to 0.1% (w / w), about 0.005% to 0.01% (w / w), about 0.01% to 0.5% (w / w), about 0.02% to 0.5% (w / w), and about 0.1% to 0.5% (w / w). Alternatively or additionally, the topical formulations described herein may comprise asiaticaside in an amount of about 0.05% to about 5% (w / w), for example, about 0.1-2.5% (w / w), about 0.1-2% (w / w), about 0.1 to 1% (w / w), about 0.25-2.5% (w / w), about 0.5-5% (w / w), about 0.5-2.5% (w / w), or about 0.5-1% (w / w).

[0042] As used herein, the term "about" means strict numerical boundaries for a particular parameter (including upper and lower limits). Those skilled in the art will understand the meaning of "about" associated with a particular context. In some cases, the term "about" refers to a particular value of + / -5% (e.g., + / -3% or + / -2%).

[0043] In one embodiment, the active agent in the topical formulation is a combination of a Plectranthus amboinicus extract comprising trilobatin and a Centella asiatica extract comprising asiaticoside. The ratio between the Plectranthus amboinicus extract and the Centella asiatica extract may be about 1:10 to 10:1. For example, the ratio between the Plectranthus amboinicus extract and the Centella asiatica extract in the topical formulation may be about 1:10 to 1:1, about 1:1 to 10:1, about 1:10 to 1:5, about 1:5 to 1:1, about 10:1 to 5:1, about 5:1 to 1:1, about 1:10 to 1:4, about 1:4 to 1:1, about 10:1 to 4:1, or about 4:1 to 1:1.

[0044] Plectranthus amboinicus extract refers to an extract obtained from the Plectranthus amboinicus plant using one or more suitable solvents. In some cases, the polarity index of at least one solvent used to prepare the extract is less than 7 (e.g., less than 5). See Table 1 below. As used herein, a solvent refers to a substance or mixture of substances that dissolves another substance to form a solution. The Plectranthus amboinicus extract described herein can be prepared using a solvent. The solvent used in each extraction step for preparing the extract described herein (including Plectranthus amboinicus and Centella asiatica extracts) can be a single solvent. Alternatively, it can be a mixture of two or more solvents.

[0045] The Plectranthus amboinicus extract described herein may include terpenoids (e.g., monoterpenes, diterpenes, triterpenes, and / or sesquiterpenes), flavonoids, phenols, essential oils, or combinations thereof. The Plectranthus amboinicus extract may be present in the topical formulation at a concentration of about 0.1-5% (w / w). For example, the topical formulation may include about 0.1-0.5%, 0.5-1%, 1-1.5%, 1.5-2%, 2-2.5%, 2.5-3%, 3-3.5%, 3.5-4%, 4-4.5%, or 4.5-5% (w / w) of the Plectranthus amboinicus extract. Alternatively, the topical formulation may include about 0.1-1%, 0.1-2%, 0.1-3%, 0.1-4%, or 0.1-5% (w / w) of the Plectranthus amboinicus extract. In a particular embodiment, the topical formulation comprises about 0.1-1% or 0.1-0.5% of Plectranthus amboinicus extract.

[0046] The Plectranthus amboinicus extract described herein can be prepared by extracting the whole Plectranthus amboinicus plant or a part thereof with one or more suitable solvents to prepare a solution, and then drying the solution to produce the Plectranthus amboinicus extract. Since the Plectranthus amboinicus extract contains flavonoids, terpenoids (e.g., monoterpenes, diterpenes, triterpenes, and / or sesquiterpenes), phenols, or essential oils as non-polar molecules, at least one extraction solvent may have a relatively low polarity (e.g., having a polarity index of less than 7) to facilitate the dissolution of non-polar molecules. "Extraction" can be performed by directly contacting the Plectranthus amboinicus material with a suitable solvent, or by washing the active ingredients of the Plectranthus amboinicus from the resin to which the active ingredients are attached.

[0047] Table 1 below lists commonly used solvents and their polarity index (Snyder polarity index):

[0048]

[0049]

[0050] In some examples, a solvent with a polarity index of less than 7 can be used to extract active ingredients from Plectranthus amboinicus to produce a Plectranthus amboinicus extract. Such a solvent can be ethyl acetate, methyl acetate, propanol, butanol, or chloroform. Alternatively, the solvent can be a mixture of one or more solvents having different polarity indices. Examples include, but are not limited to, a mixture of ethanol and ethyl acetate, ethyl acetate and butanol, ethanol and propanol, methyl acetate and butanol.

[0051] In some embodiments, the Plectranthus amboinicus extract can be prepared by a method involving the use of a solvent, such as a solvent with a polarity index less than 7 (e.g., < about 6.5, < about 6.0; < about 5.5, < about 5.0, < 4.9, < 4.8, < 4.7, < 4.6, or < 4.5). Examples include, but are not limited to, methanol, ethanol, acetone, ethyl acetate, butanol, dichloromethane, or a combination thereof.

[0052] An exemplary method for preparing a Plectranthus amboinicus extract is provided below. Figure 1 .

[0053] The Plectranthus amboinicus material can be prepared by conventional methods, which can be the whole Plectranthus amboinicus or a part thereof (e.g., leaves). The Plectranthus amboinicus material can be a fresh plant or a part thereof. Alternatively, the Plectranthus amboinicus material can be in a dried form. The Plectranthus amboinicus material can be optionally dried to form a powder, which can be used as the Plectranthus amboinicus material to prepare a Plectranthus amboinicus extract.

[0054] Any of the Plectranthus amboinicus materials described herein may be extracted one or more times with a suitable solvent to produce a crude extract. The solvent used to prepare the crude extract may be a highly polar solvent, for example, with a polarity index greater than 5 and preferably less than 7 (e.g., >5.2; >5.5, >5.8, >6 or higher, preferably less than 7). Examples include, but are not limited to, ethanol, acetone, methanol, water, or a combination thereof. If desired, the crude extract may be concentrated by conventional methods to produce a concentrated crude extract.

[0055] The crude extract may then be contacted with a suitable resin (e.g., a nonionic absorbent resin) under suitable conditions that allow the active components in the crude extract to bind to the resin. Exemplary resins for preparing Plectranthus amboinicus extracts include, but are not limited to: HP20, HP20SS, SP207, Amberlite TM XAD-2, or Amberlite TM XAD-4.

[0056] Thereafter, the resin may be washed one or more times and flushed with a suitable solvent (e.g., a solvent having a polarity index of less than 7) to produce a Plectranthus amboinicus extract, which may then be dried by conventional methods (e.g., freeze drying, spray drying, or concentrated drying) to produce the Plectranthus amboinicus extract which may be in the form of a semi-solid or paste.

[0057] In some embodiments, the resin absorption step can be performed by mixing the crude extract with the resin in a container. In other embodiments, the resin separation step can be performed by setting up a chromatography column.

[0058] The Centella asiatica extract described herein refers to an extract obtained from the whole Centella asiatica plant or a part thereof. The Centella asiatica extract may contain asiaticoside, madecassic acid, or a combination thereof.

[0059] The Centella asiatica extract may be present in the topical formulation at a concentration of about 0.1-20% (w / w). For example, the topical formulation may include about 0.1-0.5%, 0.5-1%, 1-1.5%, 1.5-2%, 2-2.5%, 2.5-3%, 3-3.5%, 3.5-4%, 4-4.5%, 4.5-5%, 5-5.5%, 5.5-6%, 6-6.5%, 6.5-7%, 7-7.5%, 7.5-8%, 8-8.5%, 8.5-9%, 9-9.5%, 9.5-10%, 10-10.5%, 10.5-11%, 11-11.5%, 11.5-12%, 12-12.5%, 12.5-13%, 13-13.5%, 13.5-14%, 14-14.5%, 14.5-15%, 15-15.5%, 15.5-16%, 16-16.5%, 16.5-17%, 17-17.5%, 17.5-18%, 18-18.5%, 18.5-19%, 19-19.5%, or 19.5-20% (w / w) of Centella asiatica extract. Alternatively, the topical formulation may contain about 0.1-5%, 0.1-10%, 0.1-15%, 0.1-20%, 0.5-5%, 0.5-10%, 0.5-15%, or 0.5-20% (w / w) of Centella asiatica extract. In certain examples, the topical formulation can include about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20% (w / w) Centella asiatica extract.

[0060] The Centella asiatica extract can be prepared according to conventional methods, for example, those described in US Pat. Nos. 5,834,437, 6,417,349, 6,475,536, and 6,267,996, and Chinese Patents CN 1313124, CN 1089497, and CN 1194154. The following is an example.

[0061] Centella asiatica material can be prepared by conventional methods. These materials can be fresh Centella asiatica plants or parts thereof, or dried Centella asiatica. The Centella asiatica material can be extracted with a suitable solvent, such as water, ethanol or a mixture thereof, to produce a crude extract. The crude extract, which can be optionally concentrated, can be mixed with a suitable resin or loaded onto a column filled with the resin. After washing one or more times, the resin can be washed with a suitable solvent. The resulting washing liquid can be concentrated to a paste, which can be dried by conventional methods, such as vacuum drying, to produce a powder of the Centella asiatica extract. If necessary, the Centella asiatica powder can be ground through a sieve (e.g., 100 mesh). See also Figure 2 .

[0062] In one example, the extract of Plectranthus amboinicus can be prepared as follows. The aerial parts of Plectranthus amboinicus, including leaves and / or stems (about 1.5 g), can be collected and extracted with a solvent having a polarity of less than 7 (e.g., methanol, ethanol, acetone, ethyl acetate, butanol, dichloromethane, or a combination thereof) and extracted at room temperature for 30 minutes to 6 hours. Alternatively, the extraction method can be carried out at a temperature of about 50 to 80° C. The obtained crude extract can be directly loaded into a non-ionic adsorption resin column and washed with a solvent having a polarity of less than 6 (e.g., ethanol, ethyl acetate, butanol, dichloromethane, hexane, toluene, or a combination thereof). The washed components can be collected and purified by extraction with a solvent having a polarity of less than 6 (e.g., a solvent described herein). The obtained filtrate can be collected to produce a Plectranthus amboinicus extract.

[0063] A Centella asiatica extract can be prepared by the same or similar method as above. The Plectranthus amboinicus extract and / or the Centella asiatica extract can be concentrated using a reduced pressure rotary evaporator.

[0064] (ii) Carriers and excipients

[0065] Topical formulations further comprise one or more carriers and excipients, including viscosity increasing agents, ointment bases (eg, cream bases), antimicrobial preservatives, emulsifiers and / or solvents.

[0066] "Viscosity increasing agents" are agents used to thicken a formulation. Exemplary viscosity increasing agents may include, for example, cetearyl alcohol, cholesterol, stearyl alcohol, chlorinated cresol, white wax, stearic acid, cetyl alcohol, or a combination thereof. The viscosity increasing agent may be present in the topical formulation at a concentration of about 1.0-10% (w / w). For example, the topical formulation may contain about 1-1.5%, 1.5-2%, 2-2.5%, 2.5-3%, 3-3.5%, 3.5-4%, 4-4.5%, 4.5-5%, 5-5.5%, 5.5-6%, 6-6.5%, 6.5-7%, 7-7.5%, 7.5-8%, 8-8.5%, 8.5-9%, 9-9.5%, or 9.5-10% (w / w) of the viscosity increasing agent. Alternatively, the topical formulation may comprise about 1-5%, 2.5-7.5%, or 5-10% (w / w) of the viscosity increasing agent.

[0067] An "ointment base" can be any semisolid formulation or carrier into which an active agent can be incorporated. Exemplary ointment bases include, but are not limited to, oily ointment bases (e.g., white petrolatum or white ointment), absorbent ointment bases (e.g., hydrophilic petrolatum, anhydrous lanolin, Aquabase, TM , as well as ), water / oil emulsion ointment bases (e.g., cold cream, aqueous lanolin, rose water ointment, Hydrocream TM , as well as ), oil / water emulsified ointment bases (e.g., hydrophilic ointments, Dermabase TM , as well as ), and water-soluble ointment bases (e.g., polyethylene glycol (PEG) ointment and Polybase TM). The ointment base may be pharmacologically inert but may entrap water to provide an emollient protective film. In a particular embodiment, the ointment base may be any petrolatum compound (e.g., petrolatum, white petrolatum, white soft paraffin, liquid petrolatum, liquid paraffin). In another particular embodiment, the ointment base is white petrolatum (CAS No. 8009-03-8). The ointment base may be present in the topical formulation at a concentration of about 5-30% (w / w), for example, 10-30% (w / w). For example, the topical formulation may include about 5-25%, 5-20%, 5-15%, 5-15%, 10-15%, 15-20%, 20-25%, or 25-30% (w / w) of the ointment base. In particular, the topical formulation may comprise about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30% (w / w) ointment base.

[0068] In some embodiments, the "ointment base" described herein contains less than 20% water and volatiles, and less than 50% hydrocarbons, waxes, or polyols as carriers.

[0069] In some specific embodiments, the "ointment base" described herein is a "cream base" containing more than 20% water and volatiles and / or usually containing less than 50% hydrocarbons, waxes or polyols as drug carriers. The cream base can be a multiphase preparation containing a lipophilic phase and an aqueous phase. In some cases, the cream base is a lipophilic cream base, which has a lipophilic phase as a continuous phase. Such a cream base usually contains an oil-in-water emulsifier, such as lanolin alcohol, sorbitan esters and monoglycerides. In other cases, the cream base is a hydrophilic cream base, which has an aqueous phase as a continuous phase. Such a cream base usually contains an oil-in-water emulsifier, such as sodium or trihydric phenol soaps, sulfated fatty alcohols, polysorbates and polyoxyethylene fatty acid and fatty alcohol esters, which can be combined with an oil-in-water emulsifier if necessary.

[0070] An "antimicrobial preservative" may be any compound capable of destroying microorganisms, preventing the reproduction or growth of microorganisms, or preventing the pathogenic effects of microorganisms. Exemplary antimicrobial preservatives include, but are not limited to, paraben compounds (esters of p-hydroxybenzoic acid, e.g., p-hydroxybenzoic acid esters, methyl paraben, ethyl paraben, propyl paraben, butyl paraben, heptyl paraben, benzyl paraben, isobutyl paraben, isopropyl paraben, benzyl paraben, or sodium salts thereof), benzalkonium chloride, phenylthorium chloride, benzyl alcohol, boric acid, ciprofloxacin, cetyltrimethylammonium bromide, cetylpyridinium chloride, lohexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethanol, glycerol, hexidineamine, imidourea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and sodium thiomercurate. The antimicrobial preservative can be present in the topical formulation at a concentration of about 0.005-0.2%, such as about 0.01-0.2% (w / w). For example, the topical formulation can contain about 0.005-0.01%, 0.01-0.05%, 0.05-0.1%, 0.1-0.15%, or 0.15-0.2% (w / w) of an antimicrobial preservative. In particular, the topical formulation may comprise about 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, or 0.2% (w / w) antimicrobial preservative.

[0071] An "emulsifier" is a compound or substance that acts as a stabilizer for a mixture of two or more liquids that are normally immiscible (unmixable or incombinable). Exemplary emulsifiers may include, but are not limited to, natural emulsifiers (e.g., gum arabic, agar, alginic acid, sodium alginate, tragacanth, chondroitin, cholesterol, xanthan gum, pectin, gelatin, egg yolk, casein, lanolin, cholesterol, wax, and lecithin), colloidal clays (e.g., bentonite [aluminum silicate] and Veegum [magnesium aluminum silicate]), long chain amino acid derivatives, high molecular weight alcohols (e.g., stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glycerol monostearate, propylene glycol monostearate, and polyvinyl alcohol), carbomers (e.g., carboxypolymethylene, polyacrylic acid, acrylic acid polymers, and carboxyvinyl polymers), carrageenan, cellulose derivatives (e.g., sodium carboxymethylcellulose, powdered cellulose, hydroxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, and methylcellulose), sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan monolaurate [ 20], polyoxyethylene sorbitan[ 60], polyoxyethylene sorbitan monooleate[ 80], Sorbitan monopalmitate[ 40], Sorbitan monostearate[ 60], Sorbitan tristearate[ 65], glycerol monooleate and sorbitan monooleate[ 80]), polyoxyethylene esters (e.g., polyoxyethylene monostearate [ 45], polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, and Solutol), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g., ), polyoxyethylene ethers (e.g., polyoxyethylene lauryl ether [ 30]), and poly(vinyl pyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, Pluronic F 68, poloxamer 188, cetyltrimethylammonium bromide, cetylpyridinium chloride, benzalkonium chloride, sodium octyl thiosuccinate, and / or combinations thereof. The emulsifier may be present in the topical formulation at a concentration of about 0.5-10% (w / w), such as 0.5-6% (w / w). For example, the topical formulation may comprise about 0.5-1%, 1-1.5%, 1.5-2%, 2-2.5%, 2.5-3%, 3-3.5%, 3.5-4%, 4-4.5%, 4.5-5%, 5-5.5%, 5.5-6%, 5-10%, 6-10%, or 8-10% (w / w) of an emulsifier. Specifically, the topical formulation may comprise about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10% (w / w) of an emulsifier.

[0072] The topical formulations of the present invention may also contain one or more solvents (e.g., non-aqueous solvents or water). Exemplary non-aqueous solvents may include, but are not limited to, any known solvents, including propylene glycol, glycols, and mixtures thereof. The non-aqueous solvent may be present in the topical formulation at a concentration of about 2-65% (w / w). For example, the topical formulation may contain about 2-15%, 15-30%, 30-45%, or 45-65% (w / w) of solvent. In some embodiments, the topical formulations of the present invention may also contain water.

[0073] In some embodiments, the topical formulations of the present invention may further include one or more emollients, fragrances or pigments. The topical formulations may also be used in conjunction with a wound dressing (e.g., a bandage with an adhesive, a plaster patch, and the like). (e.g., cyclohexane, n-hexane, n-decane, isooctane, octane, butyl ether, carbon tetrachloride, triethylamine, isopropyl ether, toluene, p-xylene, tert-butyl methyl ether, benzyl ether, methylene chloride, dichloromethane, chloroform, dichloroethane, 1,2-dichloroethane, 1-butanol, isobutyl alcohol, tetrahydrofuran, ethyl acetate, 1-propanol, 2-propanol, methyl acetate, cyclohexanone, methyl ethyl ketone (MEK), nitrobenzene, benzonitrile, 1,4-dichloroethane, Alkane, or di alkyl).

[0074] Ways to promote wound healing

[0075] Any topical formulation described herein can be used to promote wound healing in individuals in need of treatment. The topical formulation can be applied to the wound site following appropriate dosage and treatment regimen. The dosage and dosage regimen of the method described will depend on the nature and condition of the wound being treated, the age and condition of the patient, and any previous or simultaneous treatment. In some cases, the topical formulation can be applied once a week, once every other day, once a day, twice a day, three times a day, or four times a day for a suitable period of time. When the wound recovers, treatment can be terminated. If necessary, treatment can be resumed, for example, if the wound recurs.

[0076] The term "wound" refers to an injury to a living organism's tissue caused by a cut, blow, or other impact (e.g., caused by a medical condition such as a skin disorder), typically an injury in which the skin is severed or broken. A wound may be associated with a medical condition, such as a skin condition. The term "wound healing" refers to the dynamic and complex process of replacing inactivated or missing cellular structures and / or tissue layers. The terms "promotion of wound healing" or "promoting wound healing" refer to inducing an increased level or rate of replacement of inactivated or missing cellular structures and / or tissue layers. For example, promoting wound healing can be indicated by partial or complete ulcer closure or an increase in the rate of healing of an ulcer (including, but not limited to, a more rapid change in ulcer size, area, or severity, a more rapidly closed ulcer, and / or an increase in the percentage change from baseline in ulcer size, area, or severity when compared to a control ulcer treated with a placebo).

[0077] The subject treated with the topical formulation can be a human or non-human mammal. In some embodiments, the subject is a human patient with an open wound, which refers to an injury or damage to a biological tissue (e.g., skin) that disrupts the normal continuity of the biological structure. An open wound may include, but is not limited to, an abrasion, incision, tear, puncture, avulsion, cut, or other similar injury.

[0078] In other embodiments, the subject is a human patient with a chronic wound, which may be an injury or damage to a biological tissue (e.g., skin) that results in a disruption of the normal continuity of the biological structure and does not heal in sequential stages and / or within a predictable amount of time. Chronic wounds may include, but are not limited to, surgical wounds, traumatic wounds, pressure ulcers, venous ulcers, or diabetic ulcers. In other examples, chronic wounds may be associated with a disease or condition, such as cancer, burns, bedsores, skin diseases such as dermatitis odorifera.

[0079] In another example, the subject is a human patient with a foot ulcer associated with diabetes (e.g., type I or type II). Diabetes (also known as diabetes) is a group of metabolic diseases that result in prolonged high blood sugar levels. Diabetes can occur because the pancreas does not produce enough insulin or the body's cells do not respond properly to the insulin produced. The three main types of diabetes are type I (also known as "insulin-dependent diabetes mellitus" (insulin-dependent diabetes mellitus, IDDM) or "juvenile diabetes"; caused by the pancreas not being able to produce enough insulin), type 2 (also known as "non-insulin-dependent diabetes mellitus" (non-insulin-dependent diabetes mellitus, NIDDM) or "adult-onset diabetes"; caused by the cells not being able to respond properly to insulin), and gestational diabetes (observed during pregnancy, high blood sugar levels are observed in the absence of a previous history of diabetes). Many serious complications are observed in diabetic patients, including, but not limited to, chronic wounds, such as diabetic foot ulcers (also known as diabetic ulcers).

[0080] In some embodiments, the subject treated by the methods described herein has a severe wound, such as a wound having an area greater than 2 cm 2 (For example, 3cm 2 , 4cm 2 or 5cm 2 In some instances, the subject suffers from one or more plantar ulcers.

[0081] Wound healing kit

[0082] The present invention also provides a kit for promoting wound healing. Such a kit may include one or more containers containing a topical formulation as described herein, which includes Plectranthus amboinicus extract, Centella asiatica extract, or a combination of Plectranthus amboinicus extract and Centella asiatica extract.

[0083] In some embodiments, the kit may include instructions for use according to any of the methods described herein. The included instructions may include a description of the application of a topical formulation according to any of the methods described herein to promote wound healing. The kit may further include a description of selecting a treatment suitable for the individual based on identifying whether the individual has a wound in need of treatment.

[0084] Instructions for use of a topical formulation generally include information about the dosage, dosing regimen, and route of administration for the intended treatment. The container may be a unit dose, bulk package (e.g., multi-dose package), or subunit dose. The instructions provided in the kit of the invention are typically written instructions on a label or package insert (e.g., paper included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.

[0085] The label or package insert indicates that the composition is used to promote wound healing.Instructions may be provided for practicing any of the methods described herein.

[0086] The kit of the present invention has a suitable package. Suitable packages include, but are not limited to, vials, bottles, cans, soft packages (e.g., sealed polyester films or plastic bags) and the like. At least one active agent in the composition is an active agent selected from the group consisting of Plectranthus amboinicus extract, Centella asiatica extract, and a combination of Plectranthus amboinicus extract and Centella asiatica extract.

[0087] The kit may optionally provide additional components, such as explanatory information. Typically, the kit comprises a container and a label or package insert on or associated with the container. In some embodiments, the invention provides an article comprising the contents of the kit.

[0088] Without further elaboration, it is believed that one skilled in the art can utilize the present invention to its fullest extent based on the above description. Therefore, the following specific embodiments are to be construed as merely illustrative and not limiting in any way the remainder of the present invention. All publications cited herein are incorporated by reference for the purpose or subject matter of reference herein.

[0089] Example 1: Effects of Plectranthus amboinicus extract and Centella asiatica extract on proliferation and migration of endothelial cells (i) MTT assay-amplification

[0090] Plectranthus amboinicus (PA) extract and Centella asiatica (CA) extract were prepared according to the description provided in this article. EA.hy926 endothelial cell line was used in this study. The cells were cultured at 4×10 4 Cells were seeded at a density of 10 cells / well in 96-well plates for one day and then treated with PA or CA extracts in serum-free medium for 70 hours. After treatment, cells were stained with MTT stain for 2 hours.

[0091] like Figure 3A As shown, the PA extract enhanced cell proliferation, while the effect of CA on cell proliferation was not as good as that of the PA extract.

[0092] (ii) Scratch test-migration

[0093] EA.hy926 endothelial cells were cultured at 8×10 4 The cells were seeded at a density of 10 cells / well in 48-well plates for 24 hours. The confluent cell monolayers thus formed were scraped and treated with PA or CA extracts in serum-free medium for 72 hours.

[0094] The results obtained from this study showed that the PA extract significantly enhanced endothelial cell migration, which is essential for wound healing. Cells treated with the CA extract also showed signs of migration, but the results were not as significant as those with the PA extract. Figure 3B .

[0095] Example 2: Effects of Plectranthus amboinicus extract and Centella asiatica extract on keratinocyte proliferation and migration

[0096] (i) SRB analysis - proliferation

[0097] In this study, keratinocyte cell line HaCaT cells were used. The cells were cultured at 4 × 10 4 Cells were seeded at a density of 10 cells / mL in 96-well plates for one day and then treated for 24 hours with the PA or CA extracts described in Example 1. After treatment, the cells were fixed and stained with SRB stain.

[0098] like Figure 4A As shown, both PA and CA increased the cell proliferation of keratinocytes.

[0099] (ii) Scratch test-migration

[0100] HaCaT keratinocytes were cultured at 8 × 10 4 Cells were seeded at a density of 10 cells / well in a 48-well plate for one day. The confluent cell monolayer thus formed was scratched and treated with PA or CA extract for 24 hours. The wound healing area was quantified.

[0101] The results obtained from this study showed that PA extract induced cell migration of keratinocytes in a dose-dependent manner (wound healing effect). CA extract also enhanced the migration of keratinocytes at relatively high concentrations (eg, 30 μg / mL).

[0102] Example 3: Wound healing effects of caryophyllin and asiaticaside

[0103] An in vitro capillary tube formation assay using human umbilical vein cells (HUVEC) was performed to examine the wound healing effect of carnosine, the active agent of the PA extract disclosed herein.

[0104] Briefly, 96-well Nunclon plates were coated with Matrigel (50 μL / well) and incubated at 37°C for 30 minutes to promote gelation. HUVECs were cultured in M200 medium, and cells between passages 2 and 4 were seeded in each Matrigel-coated well (3×10 5 cells). Figure 5A A series of dilutions of the indicated test compounds (including basic fibroblast growth factor (bFGF) and caryophyllin as positive controls) were added to the culture medium to examine their biological effects on tube formation. The cells were incubated at 37°C in 5% CO 2 After 6 hours in a humidified environment, HUVECs were aspirated from the culture medium, fixed, and the complete capillary network was photographed in a designated area at low magnification (x10) and counted with the aid of computer imaging software, ImagePro Plus V.4.5. The test data were expressed as a percentage of complete capillary formation relative to untreated HUVEC control cultures cultured under the same conditions.

[0105] like Figure 5A As shown, carnosine significantly increased tube formation through HUVEC cells.

[0106] In order to study the wound healing effect of Centella asiatica, the following scratch migration assay was performed. HaCaT keratinocytes were cultured at 8×10 4 Cells were seeded at a density of 10 cells / well in a 48-well plate for one day. The resulting confluent cell monolayer was scraped and treated with asiaticoside for 24 hours. The wound healing area was quantified.

[0107] The results of this study showed that asiatica inducing cell migration of keratinocytes (wound healing effect).

[0108] like Figure 5B As shown, asiaticaside significantly promoted HaCaT cell migration, which would contribute to wound healing.

[0109] Example 4: Plectranthus amboinicus extract promotes normal wound healing

[0110] This study was conducted to investigate the effects of various topical formulations of cream bases, including PA extract, on normal wound closure in an animal model. Sprague-Dawley rats were randomly divided into 4 groups. The rats were then anesthetized with pentobarbital and the hair in the surgical area was removed. The skin (full thickness) was excised from the mid-dorsal area of ​​each rat using a surgical scalpel (2 cm x 2 cm).

[0111] The four groups of rats were treated with the following cream formulation, which was prepared according to the method described herein:

[0112] -G1: Cream was used as blank control group,

[0113] -G2: Winsolve-b ointment (from Winsun Pharmaceutical Co., Ltd., Taiwan, China) as a reference control group,

[0114] - G3: Formulation I (1.25% PA extract), and

[0115] - G4: Formulation II (0.25% PA extract).

[0116] The formulation was applied twice daily to each wound site and the wound was covered with gauze. A shield was placed over the neck of Sprague-Dawley rats (n=6; 12 weeks old) to protect the wound from scratching by the animals. For wound closure measurements, wound photographs were taken on days 4, 6, 8, 10, 12, 14, and 16. A ruler was placed next to the wound while photographing. Before analyzing the wound using Image pro (Media cybernetics), the length was standardized using a standard ruler in the image to avoid errors caused by different drawing distances.

[0117] The results obtained from this study showed that both Formulation I and Formulation II showed beneficial effects in promoting wound closure in rats. Figure 6 .

[0118] Table 2. Wound closure effect

[0119] Group CT50 (day)* G1 (cream control group) 10 G2 (reference control group) 9.9 G3 (Formula I) 7.7 G4 (Formula II) 8.3

[0120] *CT50: Time for 50% wound healing (days)

[0121] Example 5: In vivo pharmacokinetic study of a topical cream containing Plectranthus amboinicus extract and Centella asiatica extract

[0122] The pharmacokinetic properties of a topical formulation containing Plectranthus amboinicus and Centella asiatica extracts (1.25% total weight, containing 0.0001% to 0.1% trilobatin and 0.05% to 1% asiatica glycoside by weight) were evaluated in human patients with chronic diabetic foot ulcers (DFU). The PA and CA extracts in the topical formulation were prepared according to the method described herein. The topical formulation is a cream base, further comprising cetearyl alcohol (about 1.0-10% by weight, such as about 3.5-6.5% by weight), white petrolatum and liquid petrolatum 350cps (about 5%-30% by weight in total, such as about 5-15% by weight in total), methylparaben and propylparaben (about 0.005% to 0.2% by weight in total, such as 0.005-0.2% by weight in total), an emulsifier such as 60 and above 60 (about 0.5-10% by weight in total, for example, about 0.5-5% by weight in total), and one or more solvents, such as propylene glycol (about 2-20% by weight, for example, about 4-10% by weight). The extract is formulated in a mixture containing liquid petrolatum, propylene glycol, cetearyl alcohol, white petrolatum, 60. 60. Propylparaben, methylparaben, and water in topical cream-based formulations.

[0123] A total of 12 patients completed the study. Each subject received a dose of the cream formulation on day 1, followed by a blood draw, and then twice daily from day 2 to day 13, with further blood draws scheduled on day 14 after the last dose. The blood samples were analyzed to measure the plasma concentrations of the two active ingredients in the cream, carnosine and asiaticoside, using a validated LC / MS / MS method. Results obtained from day 1 showed that among the 12 patients, 2 patients had detectable plasma concentrations of carnosine ≤ 12.403 pg / mL; and 5 patients had detectable plasma concentrations of asiaticoside ≤ 9.276 ng / mL.

[0124] The results obtained from day 14 showed that 4 of the 12 patients had detectable plasma concentrations of trilobatin ≤ 16.972 pg / mL; and 5 of the 12 patients had detectable plasma concentrations of asiaticaside ≤ 6.154 ng / mL.

[0125] Overall, the results obtained from this study indicate that topical administration of the topical formulation used in this clinical study resulted in limited systemic distribution of carnosine and asiaticoside.

[0126] A total of 6 adverse events (AEs) were reported by 3 patients. All adverse events were considered mild in intensity and were considered "unrelated" or "not very related" to the study treatment by the investigator. After completion, 4 adverse events improved and 2 adverse events did not change. No major adverse events, deaths, or serious adverse events were reported.

[0127] Conclusion: This 14-day study showed that topical application of the topical formulation used in this study resulted in limited systemic distribution of at least the active ingredients trilobatin and asiaticaside and was safe in patients with DFU.

[0128] Example 6: Phase III clinical trial using a topical cream containing Plectranthus amboinicus extract and Centella asiatica extract

[0129] In the interim analysis, a total of 124 patients with DFU were randomized in a multicenter trial to evaluate the wound healing effects of the topical cream formulation described in Example 5 above. The inclusion and exclusion criteria for participation in the trial were as follows. Dressing (ConvaTec, Princeton, NJ, USA) served as a control treatment.

[0130] Selection criteria

[0131] Patient inclusion criteria

[0132] Patients were eligible if they met all of the following criteria:

[0133] 1. Signed written informed consent before the first study assessment;

[0134] 2. Aged 20 and under 80;

[0135] 3. Diabetes (type 1 or type 2) with HbA1c <12.0% measured during the screening period or within three months before randomization;

[0136] 4. The ankle-brachial index of the target limb was at least 0.8 measured during the screening period or within three months before randomization;

[0137] 5. Target ulcer with one of the following characteristics:

[0138] a. Wagner Ulcer Classification System grade 1 or 2;

[0139] b. Not higher than the ankle;

[0140] c. No active infection;

[0141] d. The cross-sectional area after debridement is 1 to 25 cm 2 ;as well as

[0142] e. At least 4 weeks before randomization;

[0143] 6. Those with childbearing potential who have a negative pregnancy test and are not breastfeeding at the time of screening; and

[0144] 7. Able and willing to attend regular visits and comply with study procedures.

[0145] Exclusion criteria

[0146] Patients were excluded from the study if the following circumstances applied:

[0147] 1. Necrosis, pus or sinus tract cannot be removed by debridement surgery;

[0148] 2. Clinical and / or radiological examinations confirmed acute Charcot neuroarthropathy;

[0149] 3. Underwent vascular reconstruction surgery to increase blood flow to the target limb within 4 weeks before randomization;

[0150] 4. Poor nutritional status, defined as albumin <2.5 g / dL;

[0151] 5. AST and / or ALT>3 times the upper limit of normal value;

[0152] 6. Serum creatinine > 2 times the upper limit of normal value;

[0153] 7. Treatment with immunosuppressants or chemotherapeutic agents, radiotherapy or systemic corticosteroids within 4 weeks before randomization;

[0154] 8. Use of any study drug or treatment within 4 weeks before randomization;

[0155] 9. Has a mental health condition (e.g., suicidal thoughts), current or chronic alcohol or drug abuse problem, who is considered a threat to compliance; or

[0156] 10. The investigator determines that the patient is not suitable for this study due to any other reason.

[0157] Discontinuation criteria for individual patients

[0158] Patients can withdraw from the study at any time for any reason, without giving a reason, and without any penalty or prejudice against the patient.

[0159] Additionally, patients will be discontinued from the study if any of the following apply:

[0160] 1. Violations and / or major deviations from the research protocol;

[0161] 2. Poor efficacy (defined as deterioration of Wagner grade to grade 3);

[0162] 3. Safety considerations;

[0163] 4. Loss of follow-up;

[0164] 5. Withdrawal of consent;

[0165] 6. If it is in the best interests of the patient; or

[0166] 7. The sponsor terminates the study.

[0167] Research objectives and results

[0168] The primary objective of this study was to evaluate the effects of a cream-based topical formulation on wound healing in human patients with DFUs.

[0169] The primary variable was the number of target ulcers healed in each group at week 16. The primary efficacy outcome was the comparison of the incidence of complete healing of target ulcers between the two treatment groups at the end of treatment.

[0170] For the purposes of this study, complete healing was defined as complete epithelialization maintained without drainage for at least 2 weeks and confirmed by a blinded assessor.

[0171] Secondary efficacy outcomes were: time to complete ulcer healing (raw healing time as healing time); percent change in ulcer surface area from baseline; percentage of subjects with a 50% reduction in ulcer surface area; and incidence of target ulcer infection.

[0172] Safety outcomes included assessments of the incidence of treatment-emergent adverse events, clinical laboratory values, and vital signs.

[0173] The exploratory endpoint was recurrence of the target ulcer during the follow-up assessment period in subjects who demonstrated complete wound healing at the end of the comparison period.

[0174] result

[0175] The results of the experiments using Example 5 or In the overall FAS and mITT populations, the results shown in Table 3 showed that compared with the topical cream formulation described in Of the patients treated, a higher percentage of those treated with the topical formulation achieved complete healing of their ulcers. This result suggests that at least in terms of complete healing of ulcers, which was the primary endpoint of this study, the topical formulation used in this study is superior to existing products as a treatment for wound healing.

[0176] In addition, the baseline ulcer area was > 5 cm 2 Subgroup analysis was performed in a patient population with plantar ulcers. This type of ulcer is well known in the art to be difficult to heal. The results in Table 3 show that the complete healing rate of patients treated with the topical formulation was higher than that of patients treated with the topical formulation. Complete healing rate among treated patients.

[0177] Table 3: Primary endpoint: complete healing rate

[0178]

[0179] *: Chi-square test

[0180] Therefore, the interim results of this Phase III clinical trial indicate that The control group, in which the tested topical cream formulation (comprising PA and CA extracts) showed excellent therapeutic effect in promoting wound healing in patients with DFUs.

[0181] Specifically, the present invention relates to the following technical solutions:

[0182] 1. A topical formulation comprising:

[0183] (i) an active agent comprising salvigenin in an amount of about 0.0001% to 0.5% (w / w) and asiaticoside in an amount of about 0.05% to 5% (w / w);

[0184] (ii) a viscosity increasing agent in an amount of about 1.0 to 10% (w / w);

[0185] (iii) an ointment base having an amount of about 5 to 30% (w / w);

[0186] (iv) an antimicrobial preservative in an amount of about 0.005 to 0.2% (w / w); and

[0187] (v) an emulsifier, present in an amount of about 0.5 to 10% (w / w).

[0188] 2. A topical formulation as described in technical solution 1, wherein the active agent is a Plectranthus amboinicus extract, a Centella asiatica extract or a combination thereof, and wherein the Plectranthus amboinicus extract and / or the Centella asiatica extract contains trilobatin and / or asiaticoside.

[0189] 3. A topical formulation as described in technical solution 3, wherein the content of the active agent in the topical formulation is about 0.1 to 30% (w / w).

[0190] 4. A topical formulation as described in any one of technical solutions 1 to 3, wherein the topical formulation comprises 0.0001% to 0.1% (w / w) of trilobatin, and / or wherein the topical formulation comprises 0.05% to 1% (w / w) of asiaticaside.

[0191] 5. The topical formulation of any one of technical solutions 1 to 4, wherein the active agent further comprises cirsimaritin, madecassoside or a combination thereof.

[0192] 6. A topical formulation as described in any one of technical solutions 1 to 5, wherein in the topical formulation, the ointment base is about 10 to 30% (w / w), the antimicrobial preservative is about 0.01 to 0.2% (w / w), and the emulsifier is about 0.5 to 6% (w / w).

[0193] 7. A topical formulation as described in any one of technical solutions 1 to 6, wherein the Plectranthus amboinicus extract is prepared by contacting the whole Plectranthus amboinicus or a portion thereof with a solvent having a polarity index of less than 7 to produce a solution and drying the solution to produce the Plectranthus amboinicus extract.

[0194] 8. The topical formulation of any one of technical solutions 1 to 7, wherein the topical formulation comprises the Plectranthus amboinicus extract and the Centella asiatica extract in a weight ratio of about 1:10 to 10:1, preferably about 1:5 to 5:1.

[0195] 9. A topical formulation as described in any one of technical solutions 1 to 8, wherein the viscosity increasing agent is cetostearyl alcohol, cholesterol, stearyl alcohol, chlorinated cresol, white wax, stearic acid, cetyl alcohol, or a combination thereof, the ointment base contains one or more petrolatum compounds, and / or the antimicrobial preservative contains one or more paraben compounds.

[0196] 10. The topical formulation according to any one of technical solutions 1 to 9, wherein the ointment base is a cream base.

[0197] 11. A topical formulation as described in any one of technical solutions 1 to 10, wherein the formulation further comprises one or more solvents.

[0198] 12. A topical formulation as described in technical solution 11, wherein the one or more solvents contain propylene glycol.

[0199] 13. A topical formulation as described in technical solution 1, wherein the topical formulation comprises a combination of the Plectranthus amboinicus extract and the Centella asiatica extract in an amount of about 0.1 to 30% (w / w), a cetearyl alcohol content of about 1.0 to 10% (w / w), a combination of white petrolatum and liquid petrolatum in an amount of about 5 to 30% (w / w), a combination of methylparaben and propylparaben in an amount of about 0.005 to 0.2% (w / w), and a combination of sorbitan monostearate and polysorbate 60 in an amount of about 0.5 to 10% (w / w).

[0200] 14. The topical formulation of claim 13, further comprising propylene glycol in an amount of about 2 to 20% (w / w).

[0201] 15. The topical formulation of any one of technical solutions 1 to 14, which is used to promote wound healing in an individual.

[0202] 16. A topical formulation as described in technical solution 15, wherein the formulation is used for topical application at least once a day, preferably twice a day.

[0203] 17. The topical formulation of claim 15 or 16, wherein the subject is a human diabetic patient suffering from diabetic foot ulcers.

[0204] 18. A topical formulation as described in any one of technical solutions 15 to 17, wherein the individual is a human patient with an open wound, and the open wound is optionally an abrasion wound, a cut wound, a laceration wound, a puncture wound, or an avulsion wound.

[0205] 19. A topical formulation as described in technical solution 15 or 16, wherein the individual is a human patient with a chronic wound, and the chronic wound is optionally a surgical wound, a traumatic wound, a pressure ulcer, a venous ulcer, a diabetic ulcer, or a wound caused by cancer, burns, bedsores, or atopic dermatitis.

[0206] 20. The topical formulation of claim 15 or 16, wherein the individual is a human patient suffering from acne.

[0207] Other specific embodiments

[0208] All features disclosed in this specification may be combined together in any manner. Each feature disclosed in this specification may be replaced by an alternative feature that provides the same, equivalent or similar purpose. Therefore, unless otherwise expressly stated, each feature disclosed is merely an example of a general series of equivalent or similar features.

[0209] From the above description, those skilled in the art can easily determine the basic characteristics of the present invention, and can make various changes and modifications to the present invention without departing from its spirit and scope to adapt it to various uses and conditions. Therefore, other specific embodiments are also within the scope of the patent application.

[0210] Equivalence and Scope

[0211] In the claims, definite articles such as "a," "an," and "the" may mean one or more than one, unless indicated to the contrary or obvious from the context. If one, more than one, or all of the group members are present in, applied to, or otherwise relevant to a given product or process, a claim or description including "or" between one or more members of the group is considered satisfied unless indicated to the contrary or obvious from the context. The invention includes specific embodiments in which exactly one member of the group is present in, applied to, or otherwise relevant to a given product or process. The invention includes specific embodiments in which more than one or all of the group members are present in, applied to, or otherwise relevant to a given product or process.

[0212] In addition, the present invention includes all variations, combinations and arrangements in which one or more limitations, elements, clauses and descriptive terms of one or more of the listed claims are introduced into another claim. For example, any claim attached to another claim may be modified to include one or more limitations found in any other claim attached to the same basic claim. Wherein the elements are presented in a list form, such as in the form of Markush groups, each subgroup of the elements is also disclosed, and any element can be removed from the group. It should be understood that, in general, when the present invention or aspects of the present invention are referred to as including specific components and / or features, some specific embodiments of the present invention or aspects of the present invention are composed of these components and / or features or are substantially composed of these components and / or features. For the purpose of simplification, these specific embodiments are not specifically described in the same words herein. It should also be noted that the words "including" and "containing" are intended to be open and allow the inclusion of additional components or steps. Endpoints are included in the case of a given range. Furthermore, unless otherwise indicated or apparent from the context and understanding of one of ordinary skill in the art, the values ​​expressed as ranges may be within any particular value or sub-range within the stated range in different specific embodiments of the present invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0213] This application is related to various invented patents and published patent applications, all of which are incorporated herein by reference. If there is a conflict between any cited document and the real-time description, the description shall prevail. In addition, any specific embodiment of the present invention that belongs to the prior art may be explicitly excluded from any one or more of the claims. Since these specific embodiments are considered to be known to ordinary technicians in the field, these specific embodiments may be excluded even if the exclusion is not explicitly stated in this article. Any specific embodiment of the present invention may be excluded from any claim for any reason, whether or not it is related to the existence of the prior art.

[0214] Those skilled in the art will recognize or be able to ascertain, using no more than routine experimentation, many equivalents to the specific embodiments described herein. The scope of the embodiments described herein is not intended to be limited to the foregoing description, but is as set forth in the appended claims. Those of ordinary skill in the art will appreciate that various changes and modifications may be made to this specification without departing from the spirit or scope of the invention as defined by the appended claims.

Claims

1. A topical formulation comprising: (i) an active agent comprising salvigenin in an amount of about 0.0001% to 0.5% (w / w) and asiaticoside in an amount of about 0.05% to 5% (w / w); (ii) a viscosity increasing agent in an amount of about 1.0 to 10% (w / w); (iii) an ointment base having an amount of about 5 to 30% (w / w); (iv) an antimicrobial preservative in an amount of about 0.005 to 0.2% (w / w); and (v) an emulsifier, present in an amount of about 0.5 to 10% (w / w).

2. The topical formulation of claim 1, wherein the active agent is Plectranthus amboinicus extract, Centella asiatica extract, or a combination thereof, and wherein the Plectranthus amboinicus extract and / or Centella asiatica extract comprises carnosine and / or asiaticoside.

3. The topical formulation of claim 3, wherein the active agent is present in the topical formulation in an amount of about 0.1 to 30% (w / w).

4. The topical formulation of any one of claims 1 to 3, wherein the topical formulation comprises 0.0001% to 0.1% (w / w) caryophyllin, and / or wherein the topical formulation comprises 0.05% to 1% (w / w) asiaticaside.

5. The topical formulation of any one of claims 1 to 4, wherein the active agent further comprises cirsimaritin, madecassoside, or a combination thereof.

6. The topical formulation of any one of claims 1 to 5, wherein in the topical formulation, the ointment base is about 10 to 30% (w / w), the antimicrobial preservative is about 0.01 to 0.2% (w / w), and the emulsifier is about 0.5 to 6% (w / w).

7. The topical formulation of any one of claims 1 to 6, wherein the Plectranthus amboinicus extract is prepared by contacting the whole or a portion of Plectranthus amboinicus with a solvent having a polarity index of less than 7 to produce a solution and drying the solution to produce the Plectranthus amboinicus extract.

8. The topical formulation of any one of claims 1 to 7, wherein the topical formulation comprises the Plectranthus amboinicus extract and the Centella asiatica extract in a weight ratio of about 1:10 to 10:1, preferably about 1:5 to 5:

1.

9. The topical formulation of any one of claims 1 to 8, wherein the viscosity increasing agent is cetostearyl alcohol, cholesterol, stearyl alcohol, chlorocresol, white wax, stearic acid, cetyl alcohol, or a combination thereof, the ointment base comprises one or more petrolatum compounds, and / or the antimicrobial preservative comprises one or more paraben compounds.

10. The topical formulation of any one of claims 1 to 9, wherein the ointment base is a cream base.

11. The topical formulation of any one of claims 1 to 10, wherein the formulation further comprises one or more solvents.

12. The topical formulation of claim 11, wherein the one or more solvents comprises propylene glycol.

13. The topical formulation of claim 1, wherein the topical formulation comprises a combination of the Plectranthus amboinicus extract and the Centella asiatica extract in an amount of about 0.1 to 30% (w / w), cetearyl alcohol in an amount of about 1.0 to 10% (w / w), a total amount of about 5 to 30% (w / w) of a combination of white petrolatum and liquid petrolatum, a total amount of about 0.005 to 0.2% (w / w) of a combination of methylparaben and propylparaben, and a total amount of about 0.5 to 10% (w / w) of a combination of sorbitan monostearate and polysorbate 60.

14. The topical formulation of claim 13, further comprising propylene glycol in an amount of about 2 to 20% (w / w).

15. A topical formulation as claimed in any one of claims 1 to 14 for use in promoting wound healing in a subject.

16. The topical formulation of claim 15, wherein the formulation is for topical application at least once a day, preferably twice a day.

17. The topical formulation of claim 15 or 16, wherein the subject is a human diabetic patient suffering from a diabetic foot ulcer.

18. The topical formulation of any one of claims 15 to 17, wherein the subject is a human patient having an open wound, optionally an abrasion wound, an incision wound, a laceration wound, a puncture wound, or an avulsion wound.

19. The topical formulation of claim 15 or 16, wherein the subject is a human patient having a chronic wound, optionally a surgical wound, a traumatic wound, a pressure ulcer, a venous ulcer, a diabetic ulcer, or a wound caused by cancer, burns, bedsores, or atopic dermatitis.

20. The topical formulation of claim 15 or 16, wherein the subject is a human patient suffering from acne.

Images