BR112019000820B1 - TOPIC FORMULATION - Google Patents

Abstract

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

Description

CROSS REFERENCE TO RELATED PATENT APPLICATIONS

[001] This patent application claims the benefit of the filing date of US Provisional Patent Application No. 62/363,284, filed on July 17, 2016, the entire contents of which are incorporated herein by reference.

BASICS OF THE INVENTION

[002] Plectranthus amboinicus (also known previously or alternatively as Coleus amboinicus Lour., Coleus aromaticus Benth., Coleus aromaticus auct., Plectranthus aromaticus Roxb., Plectranthus aromaticus Benth. and Plectranthus amboinicus (Lour.) Spreng.) is a medicinal herb perennial of 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, national borage and Spanish thyme.

[003] Centella asiatica (also known previously or alternatively as Centella asiatica Urbana, Centella asiatica (L.) Urbana, Hydrocotyle asiatica L. and Trisanthus cochinchinensis Lour.) is a perennial medicinal plant of the family Mackinlayaceae or subfamily Mackinlayoideae of the family Apiaceae (also known as Umbelliferae) native to Asia, Africa and South America. Centella asiatica is also known as European water-marvel, gotu kola, Kola, pennywort, Indian pennywort, swamp pennywort, pennyweed, Indian ginseng, Horse-hoof grass, Pegaga, Mandookaparni, tiger herb, Spadeleaf or Tono. Centella asiatica extracts generally comprise two main compounds: asiaticoside and madecassic acid.

SUMMARY OF THE INVENTION

[004] The present description is based, at least in part, on the superior therapeutic effects of a topical formulation comprising a combination of an extract of Plectranthus amboinicus and an extract of Centella asiatica as described herein, which comprises salvigenin and/or asiaticoside, in promotion of wound healing, for example in diabetic patients with diabetic foot ulcers. For example, the topical formulation effectively improved foot ulcer healing in diabetic patients and the efficacy was much greater than Aquacel®, a positive control. Local application of the topical formulation also showed limited systemic distribution of at least its active ingredients (salvigenin and/or asiaticoside) in patients with DFU and was considered safe in patients.

[005] Thus, a topical formulation is provided comprising: (i) an active agent comprising salvigenin in an amount of about 0.0001% to 0.5% (w/w) (for example, about 0.0001% to 0.1% by weight) and asiaticoside in an amount of about 0.05% to 5% (w/w) (for example, 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 emulsifying agent in an amount of about 0.5 to 10% (w/w).

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

[007] 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 comprises at least salvigenin and asiaticoside. In some cases, the topical formulation may comprise salvigenin ranging from about 0.0001% to 0.5% (w/w), for example, about 0.0001% to 0.1% (w/w). In some cases, the topical formulation may comprise salvigenin ranging from about 0.0001% to 0.1% (w/w) and asiaticoside ranging from 0.05% to 1% (w/w). In any of the formulations described herein, the active agent therein may additionally comprise cirsimaritin, madecassoside or a combination thereof.

[008] Plectranthus amboinicus extract can be about 0.1 to 5% (w/w) or about 0.1 to 1% (w/w) in the topical formulation. In some embodiments, the Plectranthus amboinicus extract comprises flavonoids, essential oils, phenolics, terpenoids, or a combination thereof.

[009] Plectranthus amboinicus extract can be prepared by placing a whole Plectranthus amboinicus or a part thereof in contact with a solvent having a polarity index less than 7 (for example, less than 5) to produce a solution and dry the solution to produce Plectranthus amboinicus extract. In a specific embodiment, the Plectranthus amboinicus extract is prepared by a process comprising: (i) placing a whole Plectranthus amboinicus or a part thereof in contact with an alcohol (e.g., ethanol) to produce a crude Plectranthus amboinicus extract; (ii) placing the crude extract in contact with a non-ionic absorbent resin; (iii) eluting the non-ionic absorbent resin with a solvent having a polarity index less than 7 (e.g., less than 5) to produce a solution; and (iv) drying the solution to produce the Plectranthus amboinicus extract.

[0010] Alternatively or in addition, the Centella asiatica extract may comprise asiaticoside, madecassoside or both. In some embodiments, the Centella asiatica extract is about 0.1 to 20% (w/w) or about 0.5 to 5% (w/w) in the topical formulation.

[0011] 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.

[0012] The topical formulation may comprise the viscosity increasing agent at about 1.0 to 10% (w/w). Examples of the viscosity increasing agent include, but are not limited to, cetostearyl alcohol, cholesterol, stearyl alcohol, chlorocresol, white wax, stearic acid, cetyl alcohol or a combination thereof.

[0013] The topical formulation may comprise the ointment base at about 5 to 30% (w/w) (for example, 10 to 30% by weight). In some cases, the ointment base may have 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).

[0014] The antimicrobial preservative in the topical formulation described here can be about 0.005 to 0.2% (w/w) (e.g., 0.01 to 0.2% by weight). It may comprise one or more paraben compounds (e.g., methylparaben and/or propylparaben).

[0015] The emulsifying agent (e.g., Span® 60 and/or Tween® 60) in the topical formulation can be about 0.5 to 10% (w/w) (e.g., 0.5 to 6% by weight ).

[0016] In some examples, the topical formulation described herein may comprise the ointment base at about 10 to 30% (w/w), the antimicrobial preservative at about 0.01 to 0.2% (w/w) and the emulsifying agent at about 0.5 to 6% (w/w).

[0017] Any of the topical formulations described herein optionally may additionally comprise one or more solvents, for example, propylene glycol.

[0018] In one example, 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), cetostearyl alcohol in an amount of about 1.0 to 10% (w/w) (e.g. about 2 to 8% or about 3 to 7%), a combination of white petrolatum and liquid petrolatum in a total amount of about 5 to 30 % (w/w) (e.g., about 5 to 25% or about 5 to 20%), a combination of methylparaben and propylparaben in a total amount of about 0.005 to 0.2% (w/w), and a combination of sorbitan monostearate and polysorbate 60 in a total 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 further comprise propylene glycol in an amount of about 2 to 20% (w/w) (e.g., 2 to 10% or 4 to 15%).

[0019] In another aspect, the present description provides a method for wound healing, comprising administering the topical formulation described herein to a wound area of an individual in need thereof. In one embodiment, the topical formulation may be administered at least once daily, e.g., twice daily.

[0020] In one example, the individual to be treated may be a human diabetic patient suffering from diabetic foot ulcers. For example, the human diabetic patient may have type I or type II diabetes. In another example, the subject is a human patient with an open wound, e.g., abrasion, incision, laceration, puncture, or avulsion. In another example, 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 a carcinoma, burn, atopic dermatitis. , bedsore. In some examples, the subject is a human acne patient.

[0021] Also within the scope of the present description are (i) a topical formulation as described herein for use in promoting wound healing and (ii) uses of Plectranthus amboinicus extract, Centella asiatica extract or a combination thereof in the manufacture of a medicament for use in promoting wound healing, wherein Plectranthus amboinicus extract, Centella asiatica extract or combination thereof is formulated into a topical formulation as described herein.

[0022] Details of one or more embodiments of the invention are presented in the following description. Other particularities or advantages of the present invention will be evident in the following drawings and in the detailed descriptions of the various embodiments and also in the attached claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is a flowchart showing an exemplary process for preparing extracts from Plectranthus amboinicus.

[0024] Figure 2 is a flowchart showing an exemplary process for preparing Centella asiatica extract.

[0025] Figures 3A and 3B include graphs showing the effect of Plectranthus amboinicus extract (PA) and Centella asiatica extract (CA) on endothelial cell proliferation as determined in an MTT assay (Figure 3A) and on endothelial cell migration. endothelial cell as determined in a scratch assay (Figure 3B).

[0026] Figures 4A and 4B include graphs showing the effect of Plectranthus amboinicus extract (PA) and Centella asiatica extract (CA) on keratinocyte cell proliferation as determined in an SRB assay (Figure 4A) and migration of the keratinocyte cell as determined in a scratch assay (Figure 4B).

[0027] Figures 5A and 5B include a graph showing the effect of salvigenin in promoting wound healing in an in vitro capillary tube formation assay and a graph showing the effect of asiaticoside in promoting keratinocyte migration.

[0028] Figure 6 is a graph showing the effect of Plectranthus amboinicus extract (PA) and Centella asiatica extract (CA) on normal wound healing in an animal model.

DETAILED DESCRIPTION OF THE INVENTION

[0029] Foot ulcers are a common complication associated with diabetes as a result of skin tissue breaking down and exposing the layers beneath it. All diabetics are at risk of developing foot ulcers. About 15 to 20% of diabetics develop foot ulcers. Among them, approximately 33% of patients require amputation.

[0030] Currently available treatments for diabetic foot ulcers (DFUs) include wear-protective instruments such as diabetic shoes, molds, supports, compression wraps, and shoe inserts to prevent corns and calluses. Such medical instruments can be expensive and often cause an inconvenience in patients' daily lives. Alternatively, surgical procedures may be performed to remove foot ulcers with debridement. To date, Regranex® is the only medication approved by the United States Food and Drug Administration (FDA) to treat DFU. In June 2008, the FDA announced a new label change for Regranex® to indicate that there was a fivefold increased risk of cancer mortality in the group exposed to Regranex®.

[0031] The present description is based, at least in part, on the development of a topical formulation comprising a combination of an extract of Plectranthus amboinicus and an extract of Centella asiatica, which showed significantly greater efficacy in promoting wound healing and fewer side effects. collaterals in patients with DFU. This topical formulation is cream-based and comprises at least about 0.0001% to 0.5% (w/w) salvigenin as an active ingredient. In some embodiments, the topical formulation may further comprise about 0.05% to 5% (w/w) asiaticoside as an additional active ingredient. The topical formulation further comprises about 1.0 to 10% (w/w) viscosity increasing agent(s), about 5 to 30% (w/w) ointment base(s) (e.g., cream base), about 0.005 to 0.2% (w/w) antimicrobial preservative(s); and about 0.5 to 10% (w/w) emulsifying agent(s). All components can be dissolved in one or more suitable solvents, such as propylene glycol and water.

[0032] Pharmacokinetic studies of the aforementioned topical cream formulation showed its safety in patients with DFU. Above all, topical administration of the cream formulation led to very low systemic distribution of at least the two active ingredients, salvigenin and asiaticoside, after local application to the patients' wound zones and was considered safe in patients treated with the topical formulation. locally.

[0033] Additionally, a phase III clinical trial was conducted to evaluate the effectiveness of the cream-based topical formulation to treat chronic diabetic foot ulcers. Unexpectedly, the topical ointment showed significantly greater efficacy in promoting ulcer closure in diabetic patients, particularly those with large ulcers or plantar ulcers, compared to a control product, Aquacel®.

[0034] Thus, provided herein are topical formulations comprising one or both Plectranthus amboinicus extract and Centella asiatica extract and methods of using them to promote wound healing in an individual in need of treatment.

Topical formulations

[0035] The present description herein provides a topical formulation for promoting wound healing (for example, the healing of wounds in diabetic patients), comprising an active agent, which may be an extract of Plectranthus amboinicus comprising salvigenin, an extract of Centella asiatica which comprises asiaticoside or a combination of Plectranthus amboinicus extract and Centella asiatica extract. The topical formulation described herein may comprise salvigenin in an amount ranging from about 0.0001% to about 0.5% (w/w) and optionally asiaticoside, which may be in an amount ranging from about 0.05% (w/w). % to around 5% (w/w). In some embodiments, the topical formulation comprises Plectranthus amboinicus extract, Centella asiatica extract, or both in an amount of about 0.1 to 30% (w/w).

[0036] 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., a or more cream base) which may range from 5 to 30%, one or more antimicrobial preservatives (e.g., about 0.005 to 0.2% by weight), one or more emulsifying agents (about 0.5 to 10% by weight) or a combination thereof. These components can be dissolved or made available in a suitable solvent.

(i) Active agents

[0037] The active agent in the topical formulation described herein comprises salvigenin and optionally asiaticoside. In some embodiments, the active agent may be either an extract of Plectranthus amboinicus comprising salvigenin or an extract of Centella asiatica comprising asiaticoside alone or a combination thereof. The concentration of the active agent in the topical formulation can vary from about 0.1 to 30% (w/w). For example, the active agent may comprise about 0.1 to 5%, 0.5 to 3%, 1 to 2%, 1 to 3%, 2 to 10%, 5 to 10%, 5 to 15%, 10 at 15%, 15 to 20%, 10 to 20%, 20 to 25%, 15 to 30%, 20 to 30%, 10 to 30% or 25 to 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 dry form of the total weight of the topical formulation as a whole.

[0038] The topical formulation described herein may comprise about 0.0001% to 0.5% (w/w) salvigenin, for example, about 0.001% to 0.5% (w/w), about 0.001 % to 0.3% (w/w), about 0.001% to 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) /p), about 0.005% to 0.01% (w/w), about 0.01% to 0.5% (w/w), about 0.02% to 0.5% (w/ p) and around 0.1% to 0.5% (w/w). Alternatively or in addition, the topical formulation described herein may comprise asiaticoside in an amount ranging from about 0.05% to about 5% (w/w), for example, about 0.1 to 2.5%. (w/w), about 0.1 to 2% (w/w), about 0.1 to 1% (w/w), about 0.25 to 2.5% (w/w), about 0.5 to 5% (w/w), about 0.5 to 2.5% (w/w) or about 0.5 to 1% (w/w).

[0039] As used herein, the term “about” means a strict numerical limit for the specified parameters (including both upper and lower limits). One skilled in the art understands the meaning of “about” in association with a specific context. In some cases, the term “about” refers to a particular value +/- 5% (e.g., +/- 3% or +/- 2%).

[0040] In one embodiment, the active agent in the topical formulation is a combination of Plectranthus amboinicus extract comprising salvigenin and Centella asiatica extract comprising asiaticoside. The ratio of Plectranthus amboinicus extract to Centella asiatica extract can be about 1:10 to 10:1. For example, the ratio of Plectranthus amboinicus extract to 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.

[0041] A Plectranthus amboinicus extract refers to an extract obtained from the Plectranthus amboinicus plant using one or more suitable solvents. In some cases, at least one of the solvents used to prepare the extract has a polarity index 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 to form a solution. An extract of Plectranthus amboinicus as described herein can be prepared using a solvent. The solvent used in each extraction step to prepare the extracts described herein (including the extract of both Plectranthus amboinicus and Centella asiatica) may be a single solvent. Alternatively, it may be a mixture of two or more solvents.

[0042] The Plectranthus amboinicus extract described here may comprise terpenoids (e.g., monoterpenoids, diterpenoids, triterpenoids and/or sesquiterpenoids), flavonoids, phenolics, essential oils or a combination thereof. Plectranthus amboinicus extract may be present in the topical formulation at a concentration of about 0.1 to 5% (w/w). For example, the topical formulation may comprise about 0.1 to 0.5%, 0.5 to 1%, 1 to 1.5%, 1.5 to 2%, 2 to 2.5%, 2.5 at 3%, 3 to 3.5%, 3.5 to 4%, 4 to 4.5% or 4.5 to 5% (w/w) of Plectranthus amboinicus extract. Alternatively, the topical formulation may comprise about 0.1 to 1%, 0.1 to 2%, 0.1 to 3%, 0.1 to 4% or 0.1 to 5% percent (w/w) of Plectranthus amboinicus extract. In a specific embodiment, the topical formulation comprises about 0.1 to 1% or 0.1 to 0.5% of Plectranthus amboinicus extract.

[0043] The Plectranthus amboinicus extract described herein can be prepared by extracting a whole Plectranthus amboinicus plant or a part thereof with one or more suitable solvents to produce a solution and then drying the solution to produce the Plectranthus amboinicus extract. Since Plectranthus amboinicus extract comprises flavonoids, terpenoids (e.g. monoterpenoids, diterpenoids, triterpenoids and/or sesquiterpenoids), phenolics or essential oils, which are non-polar molecules, at least one of the extraction solvents may have a relatively polarity. low (e.g., having a polarity index less than 7) to facilitate dissolution of nonpolar molecules. “Extraction” can be carried out either by placing the Plectranthus amboinicus material directly in contact with a suitable solvent or by eluting the active components of Plectranthus amboinicus from the resins in which the active components are bound.

[0044] The following table 1 lists commonly used solvents and their polarity indices (Snyder polarity index): Table 1. Exemplary solvents and their polarity indices

[0045] In some examples, a solvent with a polarity index less than 7 can be used to extract the active components of Plectranthus amboinicus to produce Plectranthus amboinicus extract. A solvent like this can be ethyl acetate, methyl acetate, propanol, butanol or chloroform. Alternatively, the solvent may be a mixture of one or more solvents with 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.

[0046] In some embodiments, Plectranthus amboinicus extract can be prepared by a process that involves 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.

[0047] An exemplary process for preparing the Plectranthus amboinicus extract is provided below. See also Figure 1.

[0048] Plectranthus amboinicus materials, which may be a whole Plectranthus amboinicus or a part thereof (e.g., leaves), may be prepared by routine methodology. The Plectranthus amboinicus material may be a fresh plant or a part thereof. Alternatively, the Plectranthus amboinicus material may be in dried form. Plectranthus amboinicus can optionally be dried to form powders, which can be used as Plectranthus amboinicus material to prepare Plectranthus amboinicus extract.

[0049] Any of the Plectranthus amboinicus materials described herein can be extracted, one or more times, by a suitable solvent to produce a crude extract. The solvent for use in preparing the crude extract may be a high polarity solvent, for example, with a polarity index above 5 and preferably below 7 (e.g., >5.2; >5.5, >5, 8, >6 or above and preferably below 7). Examples include, but are not limited to, ethanol, acetone, methanol, water or a combination thereof. If necessary, the crude extract can be concentrated by a conventional method to produce a concentrated crude extract.

[0050] The crude extract can then be brought into contact with a suitable resin (for example, a non-ionic absorbent resin) under suitable conditions that allow the binding of active components in the crude extract to the resin. Exemplary resins for use in preparing Plectranthus amboinicus extract include, but are not limited to, DIAION® HP20, DIAION® HP20SS, Sepabeads® SP207, Amberlite™ XAD-2 or Amberlite™ XAD-4.

[0051] Subsequently, the resin can be washed one or more times and eluted with a suitable solvent, for example, a solvent with a polarity index less than 7, to produce an extract of Plectranthus amboinicus, which can then be dried by a conventional method (e.g., freeze-drying, spray drying or concentration drying) to produce dried Plectranthus amboinicus extract, which may be in semisolid or paste form.

[0052] In some embodiments, the resin absorption step can be carried out by mixing the crude extract with the resin in a container. In other embodiments, the resin separation step may be performed by a chromatography column configuration.

[0053] Centella asiatica extract as described herein refers to an extract obtained from total Centella asiatica plants or a part thereof. The Centella asiatica extract may comprise asiaticoside, madecassic acid or a combination thereof.

[0054] Centella asiatica extract may be present in the topical formation at a concentration of about 0.1 to 20% (w/w). For example, the topical formulation may comprise about 0.1 to 0.5%, 0.5 to 1%, 1 to 1.5%, 1.5 to 2%, 2 to 2.5%, 2.5 to 3%, 3 to 3.5%, 3.5 to 4%, 4 to 4.5%, 4.5 to 5%, 5 to 5.5%, 5.5 to 6%, 6-6, 5%, 6.5 to 7%, 7 to 7.5%, 7.5 to 8%, 8 to 8.5%, 8.5 to 9%, 9 to 9.5%, 9.5 to 10 %, 10 to 10.5%, 10.5 to 11%, 11 to 11.5%, 11.5 to 12%, 12 to 12.5%, 12.5 to 13%, 13 to 13.5% , 13.5 to 14%, 14 to 14.5%, 14.5 to 15%, 15 to 15.5%, 15.5 to 16%, 16 to 16.5%, 16.5 to 17%, 17 to 17.5%, 17.5 to 18%, 18 to 18.5%, 18.5 to 19%, 19 to 19.5% or 19.5 to 20% (w/w) of Centella extract Asian. Alternatively, the topical formulation may comprise about 0.1 to 5%, 0.1 to 10%, 0.1 to 15%, 0.1 to 20%, 0.5 to 5%, 0.5 to 10% , 0.5 to 15% or 0.5 to 20% (w/w) of Centella asiatica extract. In specific examples, the topical formulation may comprise 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 percent (w/w) of Centella asiatica extract.

[0055] Centella asiatica extract can be prepared following a conventional method, for example, those described in US patents Nos. 5,834,437, 6,417,349, 6,475,536 and 6,267,996, CN 1313124, CN 1089497 and CN 1194154. Below is an example.

[0056] Centella asiatica materials can be prepared through routine practice. Such materials may be the fresh Centella asiatica plant or a part thereof or dried Centella asiatica. Centella asiatica materials can be extracted with a suitable solvent, such as water, ethanol or a mixture thereof, to produce a crude extract. The crude extract, which may optionally be concentrated, may either be mixed with a suitable resin or loaded onto a column packed with the resin. After being washed one or more times, the resin can be eluted with a suitable solvent. The resulting eluent can be concentrated to form a paste, which can be dried by a conventional method, for example, vacuum dried, to produce Centella asiatica extract powders. When necessary, Centella asiatica powder can be crushed through a mesh (for example, a No. 100 mesh). See also Figure 2.

[0057] In one example, an extract of Plectranthus amboinicus can be prepared as follows. A surface portion of Plectranthus amboinicus (about 1.5 g), including leaves and/or stems, can be collected and extracted with a solvent having a polarity less than 7 (e.g., methanol, ethanol, acetone, ethyl acetate , butanol, dichloromethane or a combination thereof) at room temperature for 30 min to 6 hours. Alternatively, this extraction process can be carried out at a temperature of about 50 to 80°C. The resulting crude extract can be directly loaded onto a nonionic absorbent resin column and eluted by a solvent having a polarity of less than 6 (e.g., ethanol, ethyl acetate, butanol, dichloromethane, hexane, toluene, or a combination thereof). . Eluted components can be collected and purified by extraction with a solvent with a polarity less than 6 (e.g., those described herein). The resulting filtrate can be collected to produce a PA extract.

[0058] An extract of Centella asiatica can be prepared by the same or similar process as previously described. The PA extract and/or CA extract can be concentrated using a rotary evaporator that reduces pressure.

(ii) Carriers and excipients

[0059] The topical formulation additionally comprises one or more carriers and excipients, including viscosity increasing agents, ointment bases (e.g., cream bases), antimicrobial preservatives, emulsifying agents and/or solvents.

[0060] A “viscosity increasing agent” is an agent that is used to thicken a formulation. Exemplary viscosity increasing agents may include, for example, cetostearyl alcohol, cholesterol, stearyl alcohol, chlorocresol, white wax, stearic acid, cetyl alcohol or a combination thereof. The viscosity increasing agent may be present in the topical formation at a concentration of about 1.0 to 10% (w/w). For example, the topical formulation may comprise about 1 to 1.5%, 1.5 to 2%, 2 to 2.5%, 2.5 to 3%, 3 to 3.5%, 3.5 to 4 %, 4 to 4.5%, 4.5 to 5%, 5 to 5.5%, 5.5 to 6%, 6 to 6.5%, 6.5 to 7%, 7 to 7.5% , 7.5 to 8%, 8 to 8.5%, 8.5 to 9%, 9 to 9.5% or 9.5 to 10% (w/w) of the viscosity increasing agent. Alternatively, the topical formulation may comprise about 1 to 5%, 2.5 to 7.5% or 5 to 10% (w/w) of the viscosity increasing agent.

[0061] An “ointment base” can be any semisolid preparation or vehicle 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), ointment absorption bases (e.g., hydrophilic petrolatum, anhydrous lanolin, Aquabase™, Aquaphor® and Polisorb® ), ointment water/oil emulsion bases (e.g., cold cream, hydrous lanolin, rose water ointment, Hydrocream™, Eucerin® and Nivea®), ointment oil/water emulsion bases (e.g., hydrophilic ointments, Dermabase™, Velvachol® and Unibase®) and water-miscible ointment bases (e.g. polyethylene glycol (PEG) ointment and Polibase™). Ointment bases may be pharmacologically inert, but they can trap water to provide an emollient protective film. In a specific embodiment, the ointment base may be any petrolatum compound (e.g., petrolatum, white petrolatum, white soft paraffin, liquid petrolatum, liquid paraffin). In a further specific embodiment, the ointment base is white petrolatum (CAS number 8009-03-8). The ointment base may be present in the topical formation at a concentration of about 5 to 30% (w/w), for example 10 to 30% (w/w). For example, the topical formulation may comprise about 5 to 25%, 5 to 20%, 5 to 15%, 5 to 15%, 10 to 15%, 15 to 20%, 20 to 25%, or 25 to 30% ( p/w) of the ointment base. Specifically, 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 percent (w/w) of the ointment base.

[0062] In some embodiments, the “ointment base” described herein contains less than 20% water and volatiles and more than 50% hydrocarbons, waxes or polyols as the carrier.

[0063] In some embodiments, the “ointment base” described herein is a “cream base”, which contains more than 20% water and volatiles and/or typically contains less than 50% hydrocarbons, waxes or polyols such as vehicle for drug substance. The cream base may 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 the continuous phase. A cream base like this typically contains water-in-oil emulsifying agents such as cotton spirits, sorbitan esters and monoglycerides. In other cases, the cream base is a hydrophilic cream base, which has an aqueous phase as the continuous phase. A cream base such as this typically contains oil-in-water emulsifying agents, such as sodium or trolamine soaps, sulfated fatty alcohols, polysorbates, and polyoxyl fatty acid and fatty alcohol esters, which may be in combination with water-in-water emulsifying agents. in-oil if necessary.

[0064] An “antimicrobial preservative” can be any compound capable of destroying microbes, preventing the multiplication or growth of microbes, or preventing the pathogenic action of microbes. Exemplary antimicrobial preservatives include, but are not limited to, a paraben compound (an ester of parahydroxybenzoic acid; e.g., paraben, methylparaben, ethylparaben, propylparaben, butylparaben, heptylparaben, benzylparaben, isobutylparaben, isopropylparaben, benzylparaben, or salts thereof. sodium), benzalkonium chloride, benzethonium chloride, benzyl alcohol, boric acid, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol , phenylmercury nitrate, propylene glycol and thimerosal. The antimicrobial preservative may be present in the topical formation at a concentration of about 0.005 to 0.2%, for example, about 0.01 to 0.2% (w/w). For example, the topical formulation may comprise about 0.005 to 0.01%, 0.01 to 0.05%, 0.05 to 0.1%, 0.1 to 0.15% or 0.15 to 0. 2% (w/w) antimicrobial preservative. Specifically, 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 percent ( w/w) of the antimicrobial preservative.

[0065] An “emulsifying agent” is a compound or substance that acts as a stabilizer for a mixture of two or more liquids that are normally immiscible (which cannot be mixed or combined). Exemplary emulsifying agents may include, but are not limited to, natural emulsifying agents (e.g., acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, fat cotton, 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, glyceryl monostearate, propylene glycol monostearate and poly(vinyl alcohol)), carbomers (e.g. polymethylene carboxy, poly(acrylic acid), acrylic acid polymer and carboxyvinyl polymer), carrageenan, cellulosic derivatives (e.g., sodium carboxymethylcellulose, powdered cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose and methylcellulose), sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan monolaurate [Tween® 20], polyoxyethylene sorbitan [Tween® 60], polyoxyethylene sorbitan monooleate [Tween® 80], sorbitan monopalmitate [Span® 40], sorbitan monostearate [Span® 60], sorbitan triestearate [Span® 65], glyceryl monooleate and sorbitan monooleate [Span® 80]), polyoxyethylene esters (e.g., polyoxyethylene monostearate [Myrj® 45], polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate and Solutol), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g., Cremophor®), polyoxyethylene ethers (e.g., polyoxyethylene lauryl ether [Brij® 30]) and poly(vinyl-pyrrolidone), monolaurate diethylene glycol, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, Pluronic F 68, Poloxamer 188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride and docusate sodium and/or combinations thereof. The emulsifying agent may be present in the topical formation at a concentration of about 0.5 to 10% (w/w), for example, 0.5 to 6% (w/w). For example, the topical formulation may comprise about 0.5 to 1%, 1 to 1.5%, 1.5 to 2%, 2 to 2.5%, 2.5 to 3%, 3 to 3.5% %, 3.5 to 4%, 4 to 4.5%, 4.5 to 5%, 5 to 5.5%, 5.5 to 6%, 5 to 10%, 6 to 10% or 8 to 10 % (w/w) of emulsifying agent. 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 percent (w/w) of the emulsifying agent.

[0066] The topical formulation of the invention may additionally contain one or more solvents (for example, solvents other than water or water). Exemplary non-water solvents may include, but are not limited to, any known solvent including propylene glycol, glycol and mixtures thereof. Solvent other than water may be present in the topical formation at a concentration of about 2 to 65% (w/w). For example, the topical formulation may comprise about 2 to 15%, 15 to 30%, 30 to 45% or 45 to 65% (w/w) of the solvent. In some embodiments, the topical formulation of the invention may also contain water.

[0067] In some embodiments, the topical formulation of the invention may additionally comprise one or more emollients, fragrances or pigments. The topical formula may also be used in conjunction with a wound dressing (e.g., adhesive bandage, plaster patch, and the like). (e.g., cyclohexane, n-hexane, n-decane, i-octane, octane, butyl ether, carbon tetrachloride, triethylamine, i-propyl ether, toluene, p-xylene, t-butyl methyl ether, benzene, benzyl ether, dichloromethane, methylene chloride, chloroform, dichloroethane, ethylene dichloride, 1-butanol, i-butyl alcohol, tetrahydrofuran, ethyl acetate, 1-propanol, 2-propanol, methyl acetate, cyclohexanone, methyl ethyl ketone (MEK), nitrobenzene, benzonitrile, 1,4a-dioxane or p-dioxane)

Methods for promoting wound healing

[0068] Any of the topical formulation described herein can be used to promote wound healing in an individual in need of treatment. The topical formulation can be applied to a wound area after an appropriate dosage and treatment regimen. The dosage and administration regimen for the described method will depend on the nature and condition of the wound treated, the age and condition of the patient, and any previous or current therapy. In some cases, the topical formulation may 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 an appropriate period of time. Treatment can be completed when the wound is healed. When necessary, treatment can be restarted, for example if a wound comes back.

[0069] The term “wound” refers to an injury to living tissue caused by a cut, blow, or other impact (e.g., caused by a medical condition such as a skin disorder), typically one in which the skin is cut or destroyed. The wound may be associated with a medical condition, for example, a skin disorder. The term “wound healing” indicates the dynamic and complex process of replacing lifeless or missing cellular structures and/or tissue layers. The term “promoting wound healing” or “promoting wound healing” indicates the induction of a greater level or rate of replacement for lifeless or missing cellular structures and/or tissue layers. As an example, promotion of wound healing may be indicated by partial or complete closure of the ulcer or an increase in the rate of healing of an ulcer (including, but not limited to, more rapid changes in the size, area, or severity of the ulcer). , faster ulcer closure, and/or an increase in the percentage of change from baseline in ulcer size, area, or severity compared to a control ulcer treated with a placebo).

[0070] The individual to be treated by the topical formulation can be a human or a non-human mammal. In some embodiments, the subject is a human patient with an open wound, which refers to an injury or damage to living tissue (e.g., skin) that causes a disruption in the normal continuity of biological structures. An open wound may include, but is not limited to, an abrasion, incision, laceration, puncture, avulsion, cut or other similar injuries.

[0071] In other embodiments, the subject is a human patient with a chronic wound, which may be injury or damage to living tissue (e.g., skin) that causes a disruption in the normal continuity of biological structures and does not heal in an orderly manner. of steps and/or in a predictable amount of time. A chronic wound may include, but is not limited to: a surgical wound, a traumatic wound, a pressure ulcer, a venous ulcer, or a diabetic ulcer. In other examples, a chronic wound may be associated with a disease or disorder, for example, a carcinoma, burn, bedsore, a skin disorder such as atopic dermatitis.

[0072] In one example, the subject is a human patient with a foot ulcer associated with diabetes (e.g., type I or type II). Diabetes mellitus (also known as diabetes) is a group of metabolic diseases that result in high blood sugar levels over a prolonged period. Diabetes can result from the pancreas not producing enough insulin or the body's cells not responding adequately to the insulin produced. The three main types of diabetes mellitus are Type I (also known as “insulin-dependent diabetes mellitus” (IDDM) or “juvenile diabetes”; it results from the failure of the pancreas to produce enough insulin), Type 2 (also known as “diabetes mellitus non-insulin dependent” (NIDDM) or “adult-onset diabetes”; results from the failure of cells to respond adequately to insulin) and gestational diabetes (seen during pregnancy when high blood sugar levels are observed in the absence of a prior 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 foot ulcers).

[0073] In some embodiments, the individual being treated by the methods described here suffers from a severe wound, for example, having an ulcer with an area greater than 2 cm2 (e.g., 3 cm2, 4cm2 or 5cm2). In some examples, the individual suffers from one or more plantar ulcers.

Kits for use in promoting wound healing

[0074] The present description also provides kits for use in promoting wound healing. Such kits may include one or more containers comprising a topical formulation as described herein, which comprises a Plectranthus amboinicus extract, a Centella asiatica extract, or a combination of the Plectranthus amboinicus extract and the Centella asiatica extract.

[0075] In some embodiments, the kit may comprise instructions for use in accordance with any of the methods described herein. The included instructions may comprise a description of administering the topical formulation to promote wound healing according to any of the methods described herein. The kit may further comprise a description of selecting a suitable individual for treatment based on identifying whether the individual has wounds in need of treatment.

[0076] Instructions regarding the use of a topical formulation generally include information regarding dosage, dose schedule and route of administration for the intended treatment. Containers may be unit doses, bulk packages (e.g., multi-dose packages) or subunit doses. Instructions applied to invention kits are typically instructions written on a label or packaging insert (e.g., a sheet of paper included in the kit), but machine-readable instructions (e.g., instructions loaded onto a storage disk). magnetic or optical) are also acceptable.

[0077] The label or packaging insert indicates that the composition is used to promote wound healing. Instructions may be provided for carrying out any of the methods described herein.

[0078] The kits of this invention are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Milar or plastic bags) and the like. At least one active agent in the composition is an active agent selected from the group consisting of an extract of Plectranthus amboinicus, an extract of Centella asiatica and a combination of an extract of Plectranthus amboinicus and an extract of Centella asiatica.

[0079] The kits may optionally provide additional components, such as interpretive information. Typically, the kit comprises a container and a label or packaging insert(s) on or associated with the container. In some embodiments, the invention provides articles of manufacture comprising the contents of the kits described above.

[0080] Without further elaboration, it is believed that one skilled in the art can, based on the previous description, utilize the present invention to its fullest extent. The following specific embodiments, therefore, should be considered merely illustrative and not limiting the remainder of the description in any way. All publications cited herein are incorporated by reference for the purposes or subject matter referenced herein.

EXAMPLE 1: Effects of Plectranthus amboinicus extract and Centella asiatica extract on endothelial cell proliferation and migration (i) MTT Assay - Proliferation

[0081] The Plectranthus amboinicus extract (PA) and the Centella asiatica extract (CA) were prepared following the descriptions provided here. The EA.hy926 endothelial cell line was used for this study. Cells were plated at a density of 4 x 104 cells/well in a 96-well plate for one day and then treated with PA or CA extract in serum-free medium for 70 hours. After treatment, cells were stained with MTT dye for 2 hours.

[0082] As shown in Figure 3A, PA extract improved cell proliferation, while the effect of CA on cell proliferation was not as good as that for PA extract.

(ii) Scratch test - Migration

[0083] EA.hy926 endothelial cells were plated at a density of 8 x 104 cells/well in a 48-well plate for 24 hours. The confluent cell monolayer thus formed was scratched and treated with the PA or CA extract in a serum-free medium for 72 hours.

[0084] The results obtained in this study show that PA extract significantly improved the migration of endothelial cells, which is essential for wound healing. Cells treated with the CA extract also showed signs of migration, but the result is not as significant as that with the PA extract. Figure 3B.

EXAMPLE 2: Effects of Plectranassim amboinicus extract and Centella asiatica extract on keratinocyte proliferation and migration (i) SRB Assay - Proliferation

[0085] The keratinocyte cell line, HaCaT cells, was used in this study. Cells were plated at a density of 4 x 104 cells/mL in the 96-well plate for one day and then were treated with the PA or CA extract described in Example 1 above for 24 hours. After treatment, cells were fixed and stained with SRB dye.

[0086] As shown in Figure 4A, both PA and CA increase cell proliferation of keratinocyte cells.

(ii) Scratch test - Migration

[0087] HaCaT keratinocyte cells were plated at a density of 8 x 104 cells/well in a 48-well plate for one day. The confluent cell monolayer thus formed was scratched and treated with the PA or CA extract for 24 hours. The area of wound healing was quantified.

[0088] The results obtained in this study demonstrated that PA extract induced cell migration (a wound healing effect) in keratinocytes in a dose-dependent manner. CA extract also improved keratinocyte cell migration at a relatively high concentration (e.g., 30 μg/mL).

EXAMPLE 3: Effect of salvigenin and asiaticoside on wound healing

[0089] An in vitro capillary tube formation assay using human umbilical vein cells (HUVECs) was performed to examine the effect of salvigenin, an active agent of the PA extract described herein, on wound healing.

[0090] Briefly, Nunclon 96-well plates were coated with Matrigel (50 μL/well) and incubated at 37°C for 30 min to promote gelation. HUVECs were grown in M200 medium and cells between passages 2 and 4 were seeded (at 3 x 105 cells in 100 μL of medium) into each of the Matrigel-coated wells. A series of dilutions of test compounds, as shown in Figure 5A (including basic fibroblast growth factor (bFGF) as a positive control and salvigenin), were added into the culture media to examine their biological effects on tube formation. After a 6-hour incubation at 37°C in a humified 5% CO2 atmosphere, HUVECs were aspirated from the media, fixed, and complete capillary tube networks in a given area of a low-magnification field (x10) were photographed. and counted with the help of computing imaging software, ImagePro Plus V.4,5. Test data were expressed as the percentage of complete capillary tube formation relative to untreated HUVEC control cultures incubated under the same conditions.

[0091] As shown in Figure 5A, salvigenin significantly increased tube formation by HUVEC cells.

[0092] To investigate the effect of asiaticoside on wound healing, a migration scratch test was performed as follows. HaCaT keratinocyte cells were plated at a density of 8 x 104 cells/well in a 48-well plate per day. The confluent cell monolayer thus formed was scratched and treated with asiaticoside for 24 hours. The wound healing area was quantified.

[0093] The results obtained in this study demonstrated that asiaticoside induced cell migration (an effect of wound healing) in keratinocytes.

[0094] As shown in Figure 5B, asiaticoside significantly promoted HaCaT cell migration, which would contribute to wound healing.

EXAMPLE 4: Plectranassim amboinicus extracts promoted normal wound healing

[0095] This study was carried out to investigate the effect of various cream-based topical formulations including PA extract on normal wound closure in an animal model.

[0096] Sprague-Dawley rats were randomly assigned into 4 groups. The rats were then anesthetized with pentobarbital and hair was removed in the surgical area. The skins on the mid-dorsal areas in each rat were excised (full thickness) using a surgical blade (2 cm x 2 cm).

[0097] Rats from the four groups were treated with the following cream formulations, which were prepared according to the methods described here: - G1: cream as a blank control, - G2: Winsolve-b ointment (from Yungshin Pharma, Co. LTD, Taiwan) as a reference control, - G3: Formulation I (1.25% PA extract) and - G4: Formulation II (0.25% PA extract).

[0098] The formulations were applied to each wound zone twice a day and the wounds were covered with gauze. Caps were used on the necks of Sprague-Dawley rats (n = 6; 12 weeks old) to prevent the wounds from receiving animal scratches. To measure wound closure, wound photos were taken on day 4, 6, 8, 10, 12, 14, and 16. When taking photos, a standard ruler was placed next to the wounds. Before analyzing the wounds with Image pro (Media cybernetics), the length was standardized with the standard ruler in the photo to avoid errors caused by different photographic distances.

[0099] The results obtained in this study showed that Formulations I and II showed promising effects in promoting wound closure in rats. Table 2 and Figure 6. Table 2. Effects on wound closure * CT50: time (days) for 50% wound closure

EXAMPLE 5: In vivo pharmacokinetic study on a topical cream comprising an extract of Plectranassim amboinicus and an extract of Centella asiatica

[00100] The pharmacokinetic properties of a topical formulation containing extracts of Plectranassim amboinicus and Centella asiatica (1.25% in total, containing 0.0001% to 0.1% salvigenin by weight and 0.05% to 1% of asiaticoside by weight) were evaluated in human patients with chronic diabetic foot ulcers (DFU). PA and CA extracts in the topical formulation were prepared according to the methods described here. The topical formulation is cream-based and additionally comprises cetostearyl alcohol (about 1.0 to 10% by weight, e.g., about 3.5 to 6.5% by weight), white petrolatum and liquid petrolatum 350 cps ( about 5% to 30% by weight in total, e.g. about 5 to 15% by weight in total), methylparaben and propylparaben (about 0.005% to 0.2% by weight in total, e.g. 0.005 to 0.2% by weight in total), emulsifiers such as Span® 60 and Tween® 60 (about 0.5 to 10% by weight in total, e.g. about 0.5 to 5% by weight in total ) and one or more solvents, such as propylene glycol (about 2 to 20% by weight, for example, about 4 to 10% by weight). The extracts were formulated into a cream-based topical formulation that additionally contains liquid petrolatum, propylene glycol, cetyl stearyl alcohol, white petrolatum, Span® 60, Tween® 60, propylparaben, methylparaben and water.

[00101] A total of 12 patients completed the study. Each subject received one dose of the cream formulation on Day 1 followed by blood sample collection, then twice daily from Day 2 to Day 13 and an additional blood sample collection was scheduled on Day 14 after the final dose. Blood samples were analyzed to measure plasma concentrations of salvigenin and asiaticoside, two active ingredients of the cream, by a validated LC/MS/MS methodology. The results obtained on day 1 showed that in the 12 patients, 2 patients had detectable plasma salvigenin concentrations < 12,403 pg/mL; and 5 patients had detectable asiaticoside plasma concentrations < 9,276 ng/mL.

[00102] The results obtained on day 14 showed that 4 of the 12 patients had detectable plasma concentrations of salvigenin < 16,972 pg/mL; and 5 of 12 patients had detectable asiaticoside plasma concentrations <6,154 ng/mL.

[00103] Overall, the results obtained in this study showed that topical administrations of the topical formulation used in this clinical study resulted in limited systemic distribution of both salvigenin and asiaticoside.

[00104] A total of 6 adverse events (AEs) were reported by 3 patients. All AEs were considered mild in intensity and AEs were considered “unrelated” or “hardly related” to study treatment by the Investigator. In the end, 4 AEs were improved and 2 AEs were unchanged. There were no reports of AEs, deaths or serious AEs.

[00105] Conclusion: This 14-day study revealed that local application of the topical formulation used in this study resulted in limited systemic distribution of at least the active ingredients salvigenin and asiaticoside and is safe in patients with DFU.

EXAMPLE 6: Phase III clinical trial using a topical cream comprising an extract of Plectranassim Amboinicus and an extract of Centella asiatica

[00106] In a preliminary analysis, a total of 124 patients with DFU were randomized into a multicenter trial to evaluate the wound healing effect of the topical cream formulation described in Example 5 above. The inclusion or exclusion criteria for participation in the trial are listed below. Aquacel® Hidrofiber® dressing (ConvaTec, Princeton, NJ, USA) was used as a control treatment.

Selection criteria Inclusion criteria for patients

[00107] Patients were eligible for participation if all of the following criteria were met: 1. signed a written informed consent prior to the first study assessment; 2. at least 20 and less than 80 years of age; 3. diabetes mellitus (type 1 or 2) with an HbA1c < 12.0% measured during selection or within three months before randomization; 4. an ankle brachial index in the target limb of at least 0.8 measured during selection or in the three months before randomization; 5. a target ulcer with the following characteristics: a. grade 1 or 2 for the Wagner Ulcer Classification System; B. none higher than the ankle; w. no active infection; d. cross-sectional area between 1 and 25 cm2 post-debridement; and is. present for at least 4 weeks prior to randomization; 6. people of pregnancy potential had a negative pregnancy test and were not breastfeeding at the screening visit; and 7. able and willing to receive scheduled visits and consent to study procedures.

Exclusion criteria

[00108] Patients were excluded from participation if any of the following were applicable: 1. presence of necrosis, purulence or sinus tracts not removable by debridement; 2. acute Charcot neuroarthropathy as determined by clinical and/or radiographic examination; 3. underwent revascularization procedure designed to increase blood flow in the target limb of treatment < 4 weeks prior to randomization; 4. poor nutritional status defined as albumin < 2.5 g/dL; 5. AST and/or ALT >3 times the upper limit of normal; 6. serum creatinine >2 times the upper limit of normal; 7. treatment with immunosuppressive or chemotherapeutic agents, radiotherapy or systemic corticosteroids within 4 weeks before randomization; 8. use of any investigational drug or therapy in the 4 weeks before randomization; 9. a psychiatric condition (e.g., suicidal ideation), recent or chronic alcoholism or drug abuse problem or considered a threat to treatment suitability; or 10. considered by the investigator to be unsuitable for the study for any other reason.

Discontinuation Criteria for Individual Patients

[00109] Patients were free to withdraw from the study for any reason and at any time without giving an explanation when doing so and without penalty or prejudice.

[00110] Additionally, patients are discontinued from the study if any of the following apply: 1. violation and/or significant deviation from the study protocol; 2. lack of satisfactory efficacy (defined as a worsening of the Wagner grade to level 3); 3. security concerns; 4. loss of follow-up; 5. canceled consent; 6. if the patient's best interests are considered; or 7. termination of the study by the sponsor.

Objectives and results of the study

[00111] The main objective was to evaluate the use of the cream-based topical formulation in wound healing in human patients suffering from DFU.

[00112] The primary variable was the number of target ulcers healed in each group in 16 weeks. The primary efficacy outcome was the comparison of the incidence of complete healing of the target ulcer between the two treatment groups at the end of treatment.

[00113] For the purpose of this study, complete healing was defined as complete epithelialization that was maintained without any drain for at least 2 weeks and confirmed by a blinded evaluator.

[00114] The secondary efficacy outcomes were: time to complete ulcer healing (the original healing time was taken as the time to heal); percentage of change in ulcer surface area from baseline; percentage of individuals with a 50% reduction in ulcer surface area; and incidence of target ulcer infection.

[00115] Safety outcomes included assessment of the incidence of treatment-emergent adverse effects, clinical laboratory values, and vital signs.

[00116] The exploratory parameter was the recurrence of the target ulcer in the follow-up period evaluated in individuals who demonstrated complete wound healing at the end of the comparison period.

Results

[00117] The wound healing efficacy of diabetic patients treated with the topical cream formulation described in Example 5 or Aquacel® was analyzed in the full analysis set (FAS) and the modified intention-to-treat population (mITT). In the FAS and general mITT population, the results shown in Table 3 indicate that a greater percentage of patients treated with the topical formulation achieve complete ulcer healing compared to patients treated with Aquacel®. This result showed that the topical formulation used in this study was superior to Aquacel®, an existing product to treat wound healing, in at least complete healing of the ulcer, which is the primary endpoint of the study.

[00118] Additionally, subgroup analysis was performed on a patient population with baseline ulcer areas > 5cm2 and plantar ulcers. This type of ulcer has been known in the art to be difficult to recover. The results in Table 3 indicate that the complete healing rate of patients treated with the topical formulation was greater than the complete healing rate of patients treated with Aquacel®. Table 3: Main parameter: Complete healing rates *: Chi-square test

[00119] Thus, the interim results of this phase III clinical trial indicated that the topical cream formulation tested therein, comprising both PA and CA extracts, showed superior therapeutic effects in promoting wound healing in DFU patients with respect to to Aquacel® Hidrofiber® control.

OTHER MODALITIES

[00120] All features described in this patent application can be combined in any way. Each feature described in this patent application may be replaced by an alternative feature that serves the same or equivalent or similar purposes. Therefore, unless expressly stated otherwise, each characteristic described is only one example of a generic series of equivalent or similar characteristics.

[00121] In the foregoing description, one skilled in the art can easily determine the fundamental characteristics of the present invention and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various uses and conditions. Thus, other embodiments are also in the claims.

EQUIVALENTS AND SCOPE

[00122] In the claims, articles, such as “a”, “an”, “the” and “a”, may mean one or more than one, unless otherwise indicated or otherwise evident in the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the members of the group are present in, employed in, or otherwise relevant to a given product or process, unless otherwise indicated or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one or all members of the group are present in, employed in, or otherwise relevant to a given product or process.

[00123] Furthermore, the invention encompasses all variations, combinations and permutations in which one or more limitations, elements, clauses and descriptive terms of one or more of the listed claims is introduced into another claim. For example, any claim that depends on another claim may be modified to include one or more limitations found in any other claim that depends on the same underlying claim. Where elements are presented as lists, for example in Markush group format, each subgroup of the elements is also described and any element(s) may be removed from the group. It should be understood that, in general, where the invention or aspects of the invention are referred to as comprising particular elements and/or features, certain embodiments of the invention or aspects of the invention consist or consist essentially of such elements and/or features. For the sake of simplicity, the modalities have not been specifically presented in haec verba here. It should also be noted that the terms “comprising” and “containing” must be open-ended and allow for the inclusion of additional elements or steps. Where ranges are given, parameters are included. Furthermore, unless otherwise indicated or otherwise evident in the context and understanding of one skilled in the art, the values that are expressed as ranges may assume any specific value or subrange within the ranges established in the different embodiments of the invention, while tenth of a unit of the lower limit of the range, unless the context clearly indicates otherwise.

[00124] This patent application refers to the various published patents and patent applications filed, all of which are incorporated herein by reference. If there is a conflict between any of the incorporated references and this patent application, the patent application shall govern. Furthermore, any particular embodiment of the present invention that falls within the prior art may be explicitly excluded from any one or more of the claims. Because such embodiments are considered known by one skilled in the art, they may be excluded even if the exclusion is not explicitly stated here. Any particular embodiment of the invention may be excluded from any claim for any reason, whether or not related to the existence of the prior art.

[00125] Those skilled in the art will recognize or be able to determine, using no more than routine experiment, many equivalents for the specific modalities described herein. The scope of the present embodiments described herein is not to be limited to the foregoing description, but rather is as set forth in the appended claims. Those skilled in the art will appreciate that various changes and modifications to this description can be made without departing from the spirit and scope of the present invention, as defined in the following claims.

Claims (21)

1. Topical formulation, characterized by the fact that it comprises: (i) an active agent to promote wound healing in an amount of 0.1 to 30% (w/w), wherein the active agent comprises salvigenin in an amount of 0.0001% to 0.5% (w/w) and asiaticoside in an amount of 0.05% to 5% (w/w); (ii) a viscosity increasing agent in an amount of 1.0 to 10% (w/w); (iii) an ointment base in an amount of 5 to 30% (w/w); (iv) an antimicrobial preservative in an amount of 0.005 to 0.2% (w/w); and (v) an emulsifying agent in an amount of 0.5 to 10% (w/w). 2. Topical formulation according to claim 1, characterized by the fact that the active agent is a combination of an extract of PLECTRANTHUS AMBOINICUS and an extract of CENTELLA ASIATICA. 3. Topical formulation according to claim 1 or 2, characterized by the fact that the topical formulation comprises salvigenin ranging from 0.0001% to 0.1% (w/w) and asiaticoside ranging from 0.05% to 1% (w/w). 4. Topical formulation according to any one of claims 1 to 3, characterized in that the active agent additionally comprises cirsimaritin, madecassoside or a combination thereof. 5. Topical formulation according to any one of claims 1 to 4, characterized by the fact that in the topical formulation, the ointment base is 10 to 30% (w/w), the antimicrobial preservative is 0.01 to 0 .2% (w/w) and the emulsifying agent is 0.5 to 6% (w/w). 6. Topical formulation according to any one of claims 1 to 5, characterized by the fact that the Plectranthus AMBOINICUS extract is prepared by placing a total PLECTRANTHUS AMBOINICUS or a part thereof in contact with a solvent with a polarity index less than 7 to produce a solution and dry the solution to produce PLECTRANTHUS AMBOINICUS extract. 7. Topical formulation according to any one of claims 1 to 6, characterized by the fact that the topical formulation comprises both PLECTRANTHUS AMBOINICUS extract and CENTELLA ASIATICA extract in a weight ratio of 1:10 to 10:1, preferably from 1:5 to 5:1. 8. Topical formulation according to any one of claims 1 to 7, characterized in that 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. 9. Topical formulation according to any one of claims 1 to 8, characterized in that the emulsifying agent comprises natural emulsifying agents, colloidal clays, long-chain amino acid derivatives, high molecular weight alcohols, carbomers, carrageenan, cellulosic derivatives , sorbitan fatty acid esters, polyoxyethylene esters, sucrose fatty acid esters, polyethylene glycol fatty acid esters, polyoxyethylene ethers, and poly(vinyl pyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, Pluronic F 68, Poloxamer 188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride and sodium docusate and/or combinations thereof . 10. Topical formulation according to any one of claims 1 to 9, characterized in that the ointment base is a cream base. 11. Topical formulation according to any one of claims 1 to 10, characterized in that the formulation additionally comprises one or more solvents. 12. Topical formulation according to claim 11, characterized in that the one or more solvents comprise propylene glycol. 13. Topical formulation according to claim 1, characterized by the fact that the topical formulation comprises a combination of PLECTRANTHUS AMBOINICUS extract and CENTELLA ASIATICA extract in an amount of 0.1 to 30% (w/w), alcohol cetostearyl in an amount of 1.0 to 10% (w/w), a combination of white petrolatum and liquid petrolatum in a total amount of 5 to 30% (w/w), a combination of methyl paraben and propyl paraben in a total amount of 0.005 to 0.2% (w/w) and a combination of sorbitan monostearate and polysorbate 60 in a total amount of 0.5 to 10% (w/w). 14. Topical formulation according to claim 13, characterized in that it additionally comprises propylene glycol in an amount of 2 to 20% (w/w). 15. Topical formulation according to any one of claims 1 to 14, characterized in that the topical formulation comprises a combination of an extract of PLECTRANTHUS AMBOINICUS and an extract of CENTELLA ASIATICA in an amount of 0.1 to 30% ( w/w), cetostearyl alcohol in an amount of 3.0 to 7% (w/w), a combination of white petrolatum and liquid petrolatum in a total amount of 5 to 25% (w/w), a combination of methyl paraben and propyl paraben in a total amount of 0.005 to 0.2% (w/w) and a combination of sorbitan monostearate and polysorbate 60 in a total amount of 0.5 to 6% (w/w). 16. Topical formulation according to any one of claims 1 to 15, characterized by the fact that it is for use in promoting wound healing in an individual. 17. Topical formulation according to claim 16, characterized in that the formulation is for topical administration at least once a day, preferably twice a day. 18. Topical formulation according to claim 16 or claim 17, characterized by the fact that the subject is a human diabetic patient suffering from diabetic foot ulcers. 19. Topical formulation according to any one of claims 16 to 18, characterized by the fact that the subject is a human patient with an open wound, which optionally is an abrasion, incision, laceration, puncture or avulsion. 20. Topical formulation according to claim 16 or 17, characterized by the fact that the subject is a human patient with a chronic wound, which optionally is a surgical wound, a traumatic wound, a pressure ulcer, a venous ulcer, a diabetic ulcer or a wound caused by a carcinoma, burn, bedsore or atopic dermatitis. 21. Topical formulation according to claim 16 or 17, characterized by the fact that the subject is a human patient with acne.