Larch turpentine emulsion

Abstract

The present invention relates to an emulsion comprising an aqueous phase A and a nonaqueous phase B emulsified with phase A, wherein phase B comprises larch turpentine as component B1 and at least one emulsifier as component B2. The present invention further relates to a topical spray comprising said emulsion, the use of said emulsion in a method of treatment of wounds of a subject, and a process for producing said emulsion.

Description

[0001] Larch turpentine emulsion

[0002] The field of the invention

[0003] The present invention relates to an emulsion comprising an aqueous phase A and a nonaqueous phase B emulsified with phase A, wherein phase B comprises larch turpentine as component Bl and at least one emulsifier as component B2. The present invention further relates to a topical spray comprising said emulsion, use of said emulsion in a method of treatment of wounds of a subject, and a process for producing said emulsion.

[0004] Background of the invention

[0005] Chronic wounds pose a significant economic and healthcare challenge globally (Mitura 2021). The global advancement in the wound care market has been estimated to reach US$16.2 Billion by 2020, growing at a compound annual growth rate of 5.5% over the analysis period 2022-2030 (ReportLinker, 2024). The lifetime prevalence of chronic wounds is described to be approximately 2.2-10 per 1000 population in developed countries (Martinengo et al., 2019). The wound healing process involves a series of overlapping and well-defined events, such as haemostasis, inflammation, proliferation and remodelling (Rodrigues et al., 2019; Pils et al., 2021). When the process is altered in one or more phases, an impaired healing process is observed, and chronic wounds arise, which can lead to severe complications (Enoch and Leaper, 2008). Most chronic wounds are derived from chronic leg ulcers but also from other conditions such as diabetes, vascular disorders, or prolonged inflammation, mainly affecting the adult and elderly populations (Falanga et al., 2022). Finding effective treatments for chronic wounds is crucial to alleviate patient suffering and reduce the economic burden on healthcare systems.

[0006] Conventional approaches to treating chronic wounds cover four crucial topics: tissue (T), infection (I), moisture imbalance (M), and epithelial edge advancement (E), receiving the acronym TIME (Powers et al., 2016; Ward et al., 2019; Tottoli et al., 2020). TIME is recommended based on its simplicity and can be used as a practical guide for chronic wound management. Tissue debridement types are autolytic, surgical, mechanical, biological, or enzymatic, where the last can be papain or collagenase. Infection is prevented using antiseptic drugs that are topically applied, for example, chlorhexidine or honey. Moisture balance and exudate management are handled with absorbent medications, such as films and hydrogels, and negative pressure wound therapy (NPWT). Lastly, epithelial edge advancement indicates whether the epithelization is taking place and is supported by laser therapy and NPWT (Powers et al., 2016; Tottoli et al., 2020). However, these methods may not yield satisfactory results, prompting researchers to explore alternative therapeutic options. Thus, there is a need for the development of improved medications that are easy for application and offer an effective management of chronic wounds e.g. by accelerating healing processes and improving the overall quality of life for individuals suffering from chronic wounds.

[0007] Summary of the invention

[0008] The present invention relates to an emulsion comprising an aqueous phase A and a nonaqueous phase B emulsified with phase A, wherein phase B comprises larch turpentine as component Bl and at least one emulsifier as component B2. The present invention further relates to a topical spray comprising said emulsion, use of said emulsion or said spray in a method of treatment of wounds of a subject, and a process for producing said emulsion.

[0009] The inventors of the present invention have found that an emulsion comprising larch turpentine successfully encapsulates the larch turpentine and holds promise for treating chronic wounds. Surprisingly for topical administration of liquid dosage forms, the emulsion provided by the present inventions enables the formulation to adhere to the skin, ensuring sustained surface contact for an extended period. Furthermore, applying spray forms a pleasant film on the skin is built that ceases to be sticky after a brief period. Thus, the present emulsion offers a convenient means of application that adheres effectively to the site of action, delivering pharmacological benefits in a comfortable manner.

[0010] The inventors herewith provide the present invention in its following aspects.

[0011] In one aspect, the present invention provides an emulsion comprising an aqueous phase A and a nonaqueous phase B emulsified with phase A, wherein phase B comprises larch turpentine as component Bl and at least one emulsifier as component B2.

[0012] In a further aspect, the present invention provides a topical spray comprising the emulsion as described herein. In a further aspect, the present invention provides the emulsion as described herein for use in a method of treatment of wounds of a subject.

[0013] In a further aspect, the present invention provides a process for producing the emulsion as described herein, comprising the steps of a) separately producing phases A and B as described herein, b) mixing phases A and B from step a) for production of an emulsion of phases A and B. In a further aspect, the present invention provides an emulsion obtainable by said process.

[0014] Brief description of the figures

[0015] Figure 1 shows time-dependent analysis of pH changes. Emulsions were stored at room temperature (25±2 °C). Four different formulations were evaluated: filtered and not-filtered with and without X-ray exposure. pH values were measured right after production (time 0), and after 1 week, 9 weeks and 14 weeks of X-ray exposure. Values are expressed as mean ± SD (n=3). Two-way ANOVA with Tukey multiple comparison test (* p<0.0001) between the evaluated times; n.s. - not statistically different.

[0016] Figure 2 shows droplet size (A) and poly dispersity index (PDI) (B) measurements. Four different diluted formulations were evaluated: filtered (F) and not-filtered (NF), with and without X-ray exposure. Droplet size and PDI values were measured after 1 week, 9 weeks and 14 weeks of X-ray exposure procedure. Dash line: threshold value of a monodisperse particle distribution. Values are expressed as mean ± SD (n=3). Two-way ANOVA with Tukey multiple comparison test (** p<0.01; *** p<0.001; ****p<0.0001) between the evaluated samples.

[0017] Figure 3 shows representative cryo-TEM micrographs of Larix decidua oleoresin emulsions. Samples were analyzed before and after sterilization processes at the indicated points in time. The employed sterilization processes were 1) X-ray exposure, and 2) membrane filtration (0.2 pm). Structures observed are comparable to the size and PDI measured by DLS (Figure 3). F - filtered; NF - not-filtered. Scale bar - 200 nm.

[0018] Figure 4 shows Chromatogram of Larix decidua oleoresin volatile compounds after GC-MS. Compounds are enumerated according to elution time and are described in Table 1.

[0019] Figure 5 shows in vivo assessment of wound healing. Quantitative analysis of wound contraction index (%) after seven days, expressed as mean ± standard deviation. Statistical significance compared to the vehicle emulsion group (CTR) is indicated by ** (p < 0.01) and *** (p < 0.001), determined by one-way ANOVA followed by Tukey’s post hoc test. Gl- CTR: Negative control treated with vehicle emulsion. G2-FIB: Positive control treated with Fibrinase. G3-ELD: Emulsion containing Larix decidua 10%.

[0020] Figure 6 shows D: Quantification of inflammatory cells per field, expressed as mean ± standard deviation. H: Quantification of fibroblast numbers per field, expressed as mean ± standard deviation. L: Quantification of new blood vessels per field, expressed as mean ± standard deviation. Statistical significance is indicated by * (p < 0.05) versus control group, and # (p < 0.05) versus fibrinase group, with *** and ### denoting p < 0.001, respectively, based on one-way ANOVA followed by Tukey's post hoc test.

[0021] Figure 6a shows photomicrographs of dermal wound sections after seven days of treatment, demonstrating the effects of different therapies on key parameters of wound healing. A-C: Representative images showing inflammatory cells (indicated by black arrows). E-G: Representative images illustrating fibroblast proliferation (black arrows). I-K: Representative images demonstrating angiogenesis (black arrows). A-C, E-G, I-K: 40* magnification (H&E staining). CTR: Negative control (vehicle emulsion); FIB: Positive control (Fibrinase); ELD: Emulsion containing Larix decidua 10%.

[0022] Figure 7 shows wound healing process of a patient from the first day of treatment with Larix decidua oleoresin emulsion (day 1, 09 / 08 / 2024) to the day the patient was discharged (day 28, 06 / 09 / 2024). Final check up of the wound on day 77 (26 / 10 / 2024) after beginning of treatment revealed that the wound was fully closed.

[0023] Detailed description of the invention

[0024] As outlined above, the present invention relates to an emulsion comprising an aqueous phase A and a nonaqueous phase B emulsified with phase A, wherein phase B comprises larch turpentine as component Bl and at least one emulsifier as component B2.

[0025] For the purposes of interpreting this specification, the following definitions will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. Features, integers, characteristics, compounds described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and / or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive. The invention is not restricted to the details of any foregoing embodiments.

[0026] The term “comprise” and variations thereof, such as, “comprises” and “comprising” as used herein is generally used in the sense of include, that is, as “including, but not limited to”, that is to say permitting the presence of one or more features or components.

[0027] The term “contain” and variations thereof, such as, “contains” and “containing” as used herein is generally used in the sense of "composed of substantially only," or "composed of only", that is to say not permitting the presence of other features or components than explicitly mentioned.

[0028] The singular forms “a,” “an,” and “the” as used herein include plural referents unless the content clearly dictates otherwise.

[0029] The term "about" as used herein refers to a range of values ± 10% of a specified value. For example, the phrase "about 200" includes ± 10% of 200, or from 180 to 220.

[0030] The term “emulsifier” as used herein refers to an additive that encourage the suspension of one liquid in another, as in the mixture of oil and water. A number of emulsifiers are derived from algae, among them algin, carrageenan, and agar. Lecithins, such as those found in egg yolk, can also be used as emulsifying agents. The basic structure of an emulsifying agent includes a hydrophobic portion, usually a long-chain fatty acid, and a hydrophilic portion that may be either charged or uncharged. The hydrophobic portion of the emulsifier dissolves in the oil phase, and the hydrophilic portion dissolves in the aqueous phase, forming a dispersion of small oil droplets. Emulsifiers thus form and stabilize oil-in-water emulsions. Emulsifiers comprise nonionic emulsifiers and ionic emulsifiers. Nonionic emulsifiers are e.g. polysorbate, sorbitan, glycerol laurate, pol oxamer 188, cetyl alcohol, cetostearyl alcohol, lauryl glucoside. Ionic emulsifiers are e.g., sodium stearate, sodium cholate, sodium lauryl sulphate, cetylpyridinium chloride, octenidine dihydrochloride. Emulsifiers of the present invention preferably do not comprise ethoxy diglycol.

[0031] The term “larch turpentine” is synonymously used herein with the terms “venice turpentine”, “larch oleoresin” and “Larix decidua oleoresin” and refers to turpentine obtained from Larix decidua, Miller 1768. Larc turpentine from Larix decidua, Miller 1768 is commercially available and can be obtained according to the method as described by Tschirch (Tschirch, 1900)(Tschirch, A.W., G., Untersuchungen Uber die Sekrete. Ueber den Harzbalsam von Larix decidua. (Ldrchenterpentin). Archiv der Pharmazie, 1900. 238(5): p. 387-400).

[0032] The term “aqueous phase” as used herein refers to a phase comprising water, preferably deionized water, as sole component or comprising water, preferably deionized water, and a water-soluble component selected from the group consisting of ethanol, glycerol, sorbitol, and propylene glycol, wherein ethanol is preferred as water soluble component.

[0033] The term “nonaqueous phase” as used herein refers to a phase which does not contain water or an aqueous fluid, and comprises larch turpentine as component Bl and at least one emulsifier as component B2. The nonaqueous phase of the present invention preferably contains larch turpentine as component Bl and at least one emulsifier as component B2.

[0034] Thus, in a first aspect the present invention provides an emulsion comprising an aqueous phase A and a nonaqueous phase B emulsified with phase A, wherein phase B comprises larch turpentine as component Bl and at least one emulsifier as component B2.

[0035] In one embodiment the larch turpentine as component Bl comprises at least 50 percent alphapinene, preferably at least 60 percent alpha-pinene, more preferably at least 70 percent alphapinene based on total relative abundance across the elution profile of the larch turpentine volatile compounds, preferably measured by HS-CG-MS, more preferably measured by HS- GC-MS analysis performed in a Shimadzu GCMS-QP2010 SE equipped with a Shimadzu Headspace auto-sampler.

[0036] In a further embodiment the larch turpentine as component Bl comprises at least 50 percent alpha-pinene, preferably at least 60 percent alpha-pinene, more preferably at least 70 percent alpha-pinene and at least 5 percent beta-pinene, preferably at least 10 percent alpha-pinene, more preferably at least 15 percent alpha-pinene, based on total relative abundance across the elution profile of the larch turpentine volatile compounds, preferably measured by HS-CGMS, more preferably measured by HS-GC-MS analysis performed in a Shimadzu GCMS-QP2010 SE equipped with a Shimadzu Headspace auto-sampler.

[0037] In a further embodiment the larch turpentine as component Bl comprises between 50 percent and 75 percent alpha-pinene and between 5 percent and 15 percent of beta-pinene, preferably between 54 percent and 72 percent alpha-pinene and between 6 percent and 15.5 percent of beta-pinene, based on total relative abundance across the elution profile of the larch turpentine volatile compounds, preferably measured by HS-GC-MS, more preferably measured by HS- GC-MS analysis performed in a Shimadzu GCMS-QP2010 SE equipped with a Shimadzu Headspace auto-sampler.

[0038] In a further embodiment the larch turpentine as component B 1 has an alpha-pinene to betapinene ratio molar ratio ranging from about 7: 1 to about 10: 1, preferably ranging from about 7.5: 1 to about 9.5: 1, more preferably ranging from about 8: 1 to about 9.5: 1, most preferably has an alpha-pinene to beta-pinene ratio molar ratio of about 9: 1.

[0039] In a further embodiment the larch turpentine as component Bl comprises alpha-pinene, betapinene, D-limonene, 3 -carene, P-myrcene, and camphene.

[0040] In a further embodiment the larch turpentine as component Bl comprises between 50 percent and 75 percent alpha-pinene and between 5 percent and 15 percent of beta-pinene, preferably between 54 percent and 72 percent alpha-pinene and between 6 percent and 15.5 percent of beta-pinene based on total relative abundance across the elution profile of the larch turpentine volatile compounds, and further comprises D-limonene, 3-carene, P-myrcene, and / or camphene, preferably between 4 percent and 6 percent of D-limonene, between 3 percent and 4 percent of 3-carene, between 3 percent and 4 percent of P-myrcene, and / or between 1 percent and 2 percent of camphene based on total relative abundance across the elution profile of the larch turpentine volatile compounds, preferably measured by HS-GC-MS, more preferably measured by HS-GC-MS analysis performed in a Shimadzu GCMS-QP2010 SE equipped with a Shimadzu Headspace auto-sampler.

[0041] In a peferred embodiment the larch turpentine as component Bl comprises between 50 percent and 75 percent alpha-pinene and between 5 percent and 15 percent of beta-pinene, preferably between 54 percent and 72 percent alpha-pinene and between 6 percent and 15.5 percent of beta-pinene based on total relative abundance across the elution profile of the larch turpentine volatile compounds, and further comprises D-limonene, 3-carene , P-myrcene, camphene , preferably between 4 percent and 6 percent of D-limonene, between 3 percent and 4 percent of 3-carene, between 3 percent and 4 percent of P-myrcene, between 1 percent and 2 percent of camphene based on total relative abundance across the elution profile of the larch turpentine volatile compounds, preferably measured by HS-GC-MS, more preferably measured by HS- GC-MS analysis performed in a Shimadzu GCMS-QP2010 SE equipped with a Shimadzu Headspace auto-sampler.

[0042] In a further preferred embodiment the larch turpentine as component Bl comprises alphapinene, beta-pinene, D-limonene, 3-carene, P-myrcene, and camphene and further comprises, larixyl acetate, isopimaric acid, abietic acid isomer (n.i. 3), abietic acid, and / or epimanool. In a more preferred embodiment the larch turpentine as component Bl comprises alpha-pinene, beta-pinene, D-limonene, 3-carene, P-myrcene, camphene, larixyl acetate , isopimaric acid, abietic acid isomer (n.i. 3), abietic acid, and epimanool.

[0043] The non-volatile compounds in larch turpentine as component Bl comprises larixyl acetate, isopimaric acid, abietic acid isomer (n.i. 3), abietic acid, and / or epimanool, preferably between 20 percent and 30 percent of larixyl acetate , between 15 percent and 25 percent of isopimaric acid, abietic acid isomer (n.i. 3), between 10 percent and 15 percent of abietic acid, between 8 percent and 12 percent of abietic acid, and / or between 7 percent and 11 percent of epimanool by weight based on total relativeabundance across the elution profile of the larch turpentine, preferably measured by GC-MS, more preferably measured by GC-MS analysis performed in a Shimadzu GCMS-QP2010 SE on a non-polar HB-5 column after derivatization.

[0044] In one embodiment phase B comprises larch turpentine as component Bl and two emulsifiers as component B2, preferably phase B comprises larch turpentine as component B 1 and two emulsifiers as component B2, wherein the two emulsifiers have a Hydrophilic-lipophilic balance (HLB) value of between 12 and 18, preferably a HLB value of 15.75. Hydrophilic- lipophilic balance (HLB) is the balance of the size and strength of the hydrophilic and lipophilic moieties of an emulsifier molecule. The HLB scale ranges from 0 to 20. According to this invention, the HLB value is determined according to the method described in Griffin, William C., "Classification of Surface-Active Agents by 'HLB'", Journal of the Society of Cosmetic Chemists. (1949), 1(5), 311-326 and Griffin, William C., "Calculation of HLB Values of NonIonic Surfactants", Journal of the Society of Cosmetic Chemists (1954), 5(4), 249-256.

[0045] In a further embodiment phase B comprises larch turpentine as component Bl and two nonionic emulsifiers as component B2, wherein the two nonionic emulsifiers are preferably selected from the group consisting of polysorbate, sorbitan, glycerol laurate, poloxamer 188, cetyl alcohol, cetostearyl alcohol, and lauryl glucoside.

[0046] In a preferred embodiment phase B comprises larch turpentine as component B 1 and a polysorbate and a sorbitan as component B2. In a more preferred embodiment phase B comprises larch turpentine as component Bl and polysorbate 20 and sorbitan trioleate as component B2.

[0047] In one embodiment phase A comprises water as sole component or comprises water and a water-soluble component selected from the group consisting of ethanol, glycerol, sorbitol, and propylene glycol, wherein ethanol is preferred as water soluble component. In a preferred embodiment phase A comprises or consists of water as sole component, preferably phase A comprises or consists of deionized water as sole component.

[0048] In one embodiment the aqueous phase A amounts from 20 percent to 90 percent, usually from 35 percent to 90 percent, preferably from 40 percent to 80 percent, more preferably from 50 percent to 80 percent, by weight based on total weight of the emulsion, while phase B provides the remainder of the emulsion. In a further embodiment the aqueous phase A amounts to 80 percent by weight based on total weight of the emulsion while phase B amounts to 20 percent by weight based on total weight of the emulsion. In one embodiment the larch turpentine as component Bl amounts from 2 percent to 35 percent based on total weight of the emulsion.

[0049] In one embodiment the at least one emulsifier, preferably two emulsifiers, more preferably two nonionic emulsifiers, in particular two nonionic emulsifiers selected from the group consisting of polysorbate, sorbitan, glycerol laurate, pol oxamer 188, cetyl alcohol, cetostearyl alcohol, and lauryl glucoside, more particular a polysorbate and a sorbitan, most particular polysorbate 20 and sorbitan trioleate amounts from 3 percent to 45 percent based on total weight of the emulsion.

[0050] In one embodiment the larch turpentine as component Bl amounts from 2 percent to 60 percent by weight of phase B, while the at least one emulsifier, preferably two emulsifiers, more preferably two nonionic emulsifiers, in particular two nonionic emulsifiers selected from the group consisting of polysorbate, sorbitan, glycerol laurate, poloxamer 188, cetyl alcohol, cetostearyl alcohol, and lauryl glucoside, more particular a polysorbate and a sorbitan, most particular polysorbate 20 and sorbitan trioleate, provides the remainder of phase B. In one embodiment the larch turpentine as component Bl amounts from 2 percent to 35 percent by weight of phase B, while the at least one emulsifier, preferably two emulsifiers, more preferably two nonionic emulsifiers, in particular two nonionic emulsifiers selected from the group consisting of polysorbate , sorbitan, glycerol laurate, poloxamer 188, cetyl alcohol, cetostearyl alcohol, and lauryl glucoside, more particular a polysorbate and a sorbitan, most particular polysorbate 20 and sorbitan trioleate, provides the remainder of phase B. In a preferred embodiment the larch turpentine as component Bl amounts from 10 percent to 60 percent by weight of phase B, while the at least one emulsifier, preferably two emulsifiers, more preferably two nonionic emulsifiers, in particular two nonionic emulsifiers selected from the group consisting of polysorbate , sorbitan, glycerol laurate, poloxamer 188, cetyl alcohol, cetostearyl alcohol, and lauryl glucoside, more particular a polysorbate and a sorbitan, most particular polysorbate 20 and sorbitan trioleate, provides the remainder of phase B. In a more preferred embodiment the larch turpentine as component Bl amounts from 30 percent to 60 percent by weight of phase B, while the at least one emulsifier, preferably two emulsifiers, more preferably two nonionic emulsifiers, in particular two nonionic emulsifiers selected from the group consisting of polysorbate, sorbitan, glycerol laurate, poloxamer 188, cetyl alcohol, cetostearyl alcohol, and lauryl glucoside, more particular a polysorbate and a sorbitan, most particular polysorbate 20 and sorbitan trioleate, provides the remainder of phase B. In the most preferred embodiment the larch turpentine as component Bl amounts to 50 percent by weight of phase B, while the at least one emulsifier, preferably two emulsifiers, more preferably two nonionic emulsifiers, in particular two nonionic emulsifiers selected from the group consisting of polysorbate , sorbitan, glycerol laurate, pol oxamer 188, cetyl alcohol, cetostearyl alcohol, and lauryl glucoside, more particular a polysorbate and a sorbitan most particular polysorbate 20 and sorbitan trioleate, amount to 50 percent by weight of phase B.

[0051] In one embodiment the at least one emulsifier as component B2 are polysorbate and sorbitan, preferably polysorbate 20 and sorbitan trioleate, which are present in a weight ratio in the range from 10:1 to 20: 1, preferably 15: 1 to 16:1 in component B2.

[0052] In a preferred embodiment the emulsion comprises or contains an aqueous phase A and a nonaqueous phase B emulsified with phase A, wherein phase B comprises larch turpentine as component Bl and at least one nonionic emulsifier as component B2, wherein the aqueous phase A amounts from 35 percent to 90 percent by weight based on total weight of the emulsion, the larch turpentine as component Bl amounts from 2 percent to 35 percent by weight based on total weight of the emulsion and the at least one emulsifier as component B2 amounts from 3 percent to 45 percent by weight based on total weight of the emulsion, provided that the combined weight percentage of all components of the emulsion does not exceed 100 weight percentage of the emulsion.

[0053] In a preferred embodiment the emulsion comprises or contains an aqueous phase A and a nonaqueous phase B emulsified with phase A, wherein phase B comprises larch turpentine as component Bl and at least one nonionic emulsifier as component B2, wherein the aqueous phase A amounts from 35 percent to 90 percent by weight based on total weight of the emulsion, the larch turpentine as component Bl amounts from 2 percent to 35 percent by weight based on total weight of the emulsion and the at least one emulsifier as component B2 amounts from 3 percent to 45 percent by weight based on total weight of the emulsion, provided that the combined weight percentage of the aqueous phase and the nonaqueous phase of the emulsion does not exceed 100 weight percentage of the emulsion. In a preferred embodiment the emulsion comprises or contains an aqueous phase A and a nonaqueous phase B emulsified with phase A, wherein phase B comprises larch turpentine as component Bl and at least one nonionic emulsifier as component B2, wherein the aqueous phase A amounts from 35 percent to 90 percent by weight based on total weight of the emulsion, the larch turpentine as component Bl amounts from 2 percent to 35 percent by weight based on total weight of the emulsion and the at least one emulsifier as component B2 amounts from 3 percent to 45 percent by weight based on total weight of the emulsion, provided that the combined weight percentage of the aqueous phase and component Bl and component B2 of the nonaqueous phase B of the emulsion does not exceed 100 weight percentage of the emulsion.

[0054] In a more preferred embodiment the emulsion comprises or contains an aqueous phase A and a nonaqueous phase B emulsified with phase A, wherein phase B comprises larch turpentine as component Bl and two nonionic emulsifiers as component B2, wherein the two nonionic emulsifiers are preferably selected from the group consisting of polysorbate ,sorbitan, glycerol laurate, poloxamer 188, cetyl alcohol, cetostearyl alcohol, and lauryl glucosideand are more preferably a polysorbate and a sorbitan , most preferably polysorbate 20 and sorbitan trioleate, wherein the aqueous phase A amounts from 40 percent to 80 percent by weight based on total weight of the emulsion, while phase B provides the remainder of the emulsion, wherein the larch turpentine as component Bl amounts from 30 percent to 60 percent by weight of phase B, while the two nonionic emulsifiers provides the remainder of phase B.

[0055] In a particular preferred embodiment the emulsion comprises or contains an aqueous phase A and a nonaqueous phase B emulsified with phase A, wherein phase B comprises larch turpentine as component Bl and two nonionic emulsifiers as component B2, wherein the two nonionic emulsifiers are preferably selected from the group consisting of polysorbate , sorbitan, glycerol laurate, poloxamer 188, cetyl alcohol, cetostearyl alcohol, and lauryl glucoside and are more preferably a polysorbate and a sorbitan , most preferably polysorbate 20 and sorbitan trioleate, wherein the aqueous phase A amounts to 80 percent by weight based on total weight of the emulsion, while phase B provides the remainder of the emulsion, wherein the larch turpentine as component Bl amounts to 50 percent by weight of phase B, while the two nonionic emulsifiers-amount to 50 percent by weight of phase B. In the most preferred embodiment the emulsion comprises or contains an aqueous phase A and a nonaqueous phase B emulsified with phase A, wherein phase B comprises larch turpentine as component Bl and polysorbate 20 and sorbitan trioleate as component B2, wherein the aqueous phase A amounts to 80 percent by weight based on total weight of the emulsion, while phase B provides the remainder of the emulsion, wherein the larch turpentine as component Bl amounts to 50 percent by weight of phase B, while polysorbate 20 and sorbitan trioleate amount to 50 percent by weight of phase B, wherein polysorbate 20 and sorbitan trioleate are present in a weight ratio in the range from 15: 1 to 16: 1 in component B2.

[0056] In one embodiment the emulsion of the present invention does not comprise ethoxy diglycol.

[0057] In one embodiment phase A has a pH in the range from 3 to 7, preferably from 4 to 5.

[0058] In one embodiment the emulsion is a sterile emulsion. A sterile emulsion of the present invention can be prepared by exposing the emulsion of the present invention to X-ray radiation, preferably exposing the emulsion to a total dose of between 10 and 50 kGy or by filtration of the emulsion of the present invention, preferably by membrane filtration of the emulsion of the present invention, more preferably by membrane filtration of the emulsion of the present invention through a 0.2 pm filter.

[0059] In one embodiment the emulsion is a nanoemulsion comprising droplets which have a size of between 20 and 300 nanometers, preferably between 100 and 300 nanometers. Preferably, the emulsion is a nanoemulsion comprising droplets which have a size of between 20 and 300 nanometers, preferably between 100 and 300 nanometers, wherein the size of the droplets is measured as mean droplet size using a dynamic light scattering (DLS) instrument, preferably a Zetasizer Nano-ZS Zen3600 DLS instrument (Malvern Panalytical, Worcestershire, UK). Measurements are usually conducted at room temperature (25 °C) using a quartz cuvette.

[0060] In a further aspect, the present invention provides a topical spray comprising the emulsion as described herein. The term “topical” or “topical application” refers to the spray being applied to a surface such as skin. “Topically active” refers to the emulsion or spray for topical application which treats predominately the surface on which it is applied.

[0061] The topical spray of the present invention comprises the emulsion as described herein above in a vehicle optionally containing a polymer or combination of polymers which, when sprayed on the surface of the skin, forms a film on the skin. The composition may further contain one or more film former e.g poloxamer, CMC, PVP, chitosan; solubilizer e.g caprylyl glucoside, PEG, polysorbate; permeation enhancer e.g diethylene glycol monoethyl ether; and / or plasticizer e.glanolin wax, PEG-12, glyceryl triacetate. The composition may contain one or more of these additives in amounts of up to about 10% film-former (e.g., 0.0001% to about 10%), up to about 10% solubilizer (e.g., 0.0001% to about 10%), up to about 8% permeation enhancer (e.g., 0.0001% to about 8%), and up to about 10% plasticizer (e.g., 0.0001% to about 10%). The inventive emulsion may be sprayed on a topical site to form a stable, breathable film on the site, from which film the larch turpentine acts locally on the surface or is transdermally available. The emulsion can be dispensed from any dispenser, preferably a dispenser which provides the composition as a spray, and may be used for topical action. Preferably, the emulsion is dispensed from a pump dispenser or from an aerosol dispenser. In the latter case, the composition additionally comprises from about 10% to 90% of propellant in order to provide a suitable pressure within the aerosol dispenser. Generally, propellant is not required for compositions dispensed from a pump dispenser. However, if desired, such compositions may also comprise from about 10% to 90% of a propellant which is liquid at room temperature, for example, tri chloromonofluoromethane (PH).

[0062] The emulsion or the spray as described herein is preferably a pharmaceutical emulsion or a spray comprising a pharmaceutical emulsion, preferably a pharmaceutical emulsion or a spray comprising a pharmaceutical emulsion for the treatment of wounds of a subject.

[0063] In a further aspect, the present invention provides the emulsion or the spray as described herein for use in a method of treatment of wounds of a subject.

[0064] Also provided is the use of the emulsion or the spray as described herein for the manufacture of a medicament for the treatment of wounds of a subject.

[0065] Also provided is the use of the emulsion or the spray as described herein for the treatment of wounds of a subject. Also provided is a method for the treatment of wounds of a subject, comprising administering to said subject a therapeutically effective amount of the emulsion or the spray as described herein.

[0066] The terms “treatment” / ” treating” as used herein includes: (1) delaying the appearance of clinical symptoms of the state, disorder or condition developing in an animal, particularly a mammal and especially a human, that may be afflicted with or predisposed to the state, disorder or condition but does not yet experience or display clinical or subclinical symptoms of the state, disorder or condition; (2) inhibiting the state, disorder or condition (e.g. arresting, reducing or delaying the development of the disease, or a relapse thereof in case of maintenance treatment, of at least one clinical or subclinical symptom thereof); and / or (3) relieving the condition (i.e. causing regression of the state, disorder or condition or at least one of its clinical or subclinical symptoms). The benefit to a patient to be treated is either statistically significant or at least perceptible to the patient or to the physician. However, it will be appreciated that when a medicament is administered to a patient to treat a disease, the outcome may not always be effective treatment.

[0067] The expression “effective amount” or “therapeutically effective amount” as used herein refers to an amount capable of invoking one or more of the following effects in a subject receiving the emulsion of the present invention: (i) better healing, (ii) faster healing compared to wounds which are not treated by the emulsion of the present invention, (iii) avoid bacterial growth, (iv) improve quality of life, and / or (v) close the wound injury. Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.

[0068] The emulsion of the present invention may be administered directly topically or assisted by a gel matrix.

[0069] An exemplary treatment regime entails administration of the emulsion once daily, twice daily, three times daily, every second day, every third day, twice per week, once per week, once every other week, once every three weeks, once per month, or once every 6 weeks. The emulsion of the invention is usually administered on multiple occasions. The emulsion of the invention may be given as a continous uninterrupted treatment. The emulsion of the invention may also be given in a regime in which the subject receives cycles of treatment interrupted by a drug holiday or period of non-treatment. Administration of the emulsion of the present invention can be combined with conventional treatment such as topical administration of alginate containing dressing e.g. Biatain®, carboxymethylcellulose containing dressing e.g. Comfeel®, Aquacel®, polyurethan containing dressing e.g. Suprasorb®, Opsite flexifix®, which can also contain silver in their composition.

[0070] In one embodiment, the wounds to be treated are preferably open wounds, chronic wounds or surgical wounds, more preferably chronic wounds, even more preferably chronic wounds derived from chronic leg ulcers, diabetes, vascular disorders, amputation, and / or prolonged inflammation.

[0071] In a further aspect, the present invention provides a process for producing the emulsion as described herein, comprising the steps of a) separately producing phases A and B as described herein, b) mixing phases A and B from step a) for production of an emulsion of phases A and B.

[0072] In one embodiment step a) comprises putting larch turpentine as component Bl and at least one emulsifier as component B2 of phase B together, heating the mixture to between 30° C and 50°C, and agitating the mixture for 2 to 10 min; step b) comprising mixing phases A and B from step a) by heating the aqueous phase to between 30° C and 50°C and dropping the aqueous phase to the phase B from step a), wherein the mixture of phases A and B are agitated for 10 to 60 min; optionally as step c) filtering the obtained emulsion.

[0073] In a preferred embodiment step a) comprises putting larch turpentine as component Bl and at least one emulsifier as component B2 of phase B together, heating the mixture to 37° C, and agitating the mixture for 5 min at 500 rpm; step b) comprising mixing phases A and B from step a) by heating the aqueous phase to 37° C and dropping the aqueous phase to the phase B from step a), wherein the mixture of phases A and B are agitated for 30 min at 1000 rpm; optionally as step c) filtering the obtained emulsion, preferably filtering the obtained emulsion through a membrane filter, more preferably filtering the obtained emulsion through a 0.2 pm PES filter. In a further aspect, the present invention provides an emulsion obtainable by the process described above.

[0074] Examples

[0075] 1. Material and Methods

[0076] 1.1. Materials and reagents

[0077] Oleoresins of Larix decidua Mill, were obtained from Schusser OG (S, Austria). It was collected in Carinthia, Austria, in August 2020 (Batch 204601). Polysorbate 20 (Tween® 20, Sigma) and sorbitan trioleate (Span® 85, Fluka) were of pharmaceutical grade.

[0078] 1.2. HS-GC-MS

[0079] 1.2.1. Sample preparation

[0080] About 40 mg of each resin was accurately weighed, added to a 20 mL headspace (HS) vial, and sealed for HS-GC-MS analysis.

[0081] A 500 pL of saturated / / -alkanes mixture C7-C40 (Sigma-Aldrich, Germany) were placed into a 10 mL volumetric flask and diluted to the volume with / / -hexane to make a 50 pg / mL concentration. Then, 3 mL of the alkane solution was transferred to a 20 mL headspace vial for the HS-GC-MS analysis (Zhang and Liang, 2019).

[0082] 1.2.2. Analysis conditions

[0083] The HS-GC-MS analysis was performed in a Shimadzu GCMS-QP2010 SE equipped with a Shimadzu Headspace auto-sampler (Shimadzu, Country). A capillary column ZB-5Plus of 30 m X 0.25 mm X 0.25 pm was used for the separation. The oleoresin in a 20 mL headspace vial was heated at an equilibrium temperature of 60 °C for 10 min, and the gas phase was injected into the GC-MS for analysis. The injection time was 1 minute. A low shaker mode of the headspace vial was applied during sample heating.

[0084] GC parameters were as follows: the carrier gas was high pure helium. The carrier gas (helium) was set at a 1 mL / min flow rate. The split ratio was 20: 1, and the pressure was 49.7 kPa. The column oven temperature was initially set at 40 °C for 1 min, and then ramped to 250 °C for 50 min, and kept at 250 °C for 5 min.

[0085] MS parameters were as follows: data were acquired in the electron impact (El) mode at 70 eV, using the full scan mode from m / z 40 to 750. The ion source and quadrupole temperatures were 200 °C and 300 °C, respectively. The identification of the volatile compounds was based on a comparison of their GC retention time and mass spectra with the retention index of n- alkane-saturated alkanes and the reference spectra from the US National Institute of Standards and Technology (NIST23). Data was analysed using Shimadzu LabSolution Postrun software.

[0086] 1.3. GC-MS

[0087] 1.3.1. Sample preparation

[0088] Samples were derivatised prior to GC-MS analysis (Isca et al., 2014). Ten milligrams of each resin were mixed with 125 pL of pyridine, 125 pL of N,O-bis(trimethylsilil) trifluoroacetamide (BSTFA) and 25 pL of trimethylchlorsilane (TMCS). The tubes were heated at 70 °C for 30 min and the derivatised samples injected into the GC-MS system.

[0089] 1.3.2. Analysis conditions

[0090] Samples were analysed by gas chromatography (ThermoFisher Scientific, Trace 1300 Series GC) coupled to a mass spectrometer (ThermoFisher Scientific, TSQ8000). One pL of each sample was injected (ThermoFisher Scientific, Triplus RSH), with a split ratio of 1 :50, with an injector at 250 °C. The initial oven temperature was at 80 °C, increasing 15 °C / min up to 180 °C, which was kept for 5 min, followed by an increase of 10 °C / min up to 285 °C, maintained for 12 min, totalising 34 min of chromatographic analysis. A non-polar HP-5 column (Agilent Technologies; 50m x 0.200 mm x 0.33pm) was used to separate compounds. Helium was used as carrier gas at 1.2 mL / min, and the transfer line was kept at 290 °C. Electronic ionization was carried out at 70 eV with the ion source at 250 °C. Detection was performed over the m / z range of 33 to 750.

[0091] 1.4. Formulation development

[0092] 1.4.1. Construction of pseudo-ternary phase diagram (PTD)

[0093] PTD was constructed according to the methodology described by Barradas et al. (2015). The emulsifier mixture was composed of polysorbate 20 and sorbitan trioleate 85 in a constant proportion of 94:6 (HLB 15.75). Larch oleoresin was the oily phase constituent, and the aqueous phase was composed of fresh deionized water. Firstly, emulsifiers were mixed with the oily phase and subsequently titrated with aqueous phase. The aqueous phase concentration ranged, yielding 99 formulations in which oil / emulsifier ratio (OSR) ranged from 1 :9 to 9: 1. After each titration, samples were homogenized with magnetic stirrer for 1 h at 37 °C and then left in rest for 24 h. After the diagram was built, some formulations of the best areas (without phase separation, with opaque or translucent appearance) were further tested and the most promising was selected.

[0094] 1.4.2. Macroscopic analysis

[0095] Visual inspection evaluated the macroscopic aspects of formulations and their stability variation (presence of creaming, coalescence, or phase separation). According to their macroscopic aspects, the formulations were classified as transparent, translucent, opaque, and phase separation.

[0096] 1.4.3. Emulsion preparation

[0097] The emulsion of the present study was prepared using a low-energy method described by Borges et al. (2018). The composition was as follows: 80% of water, 10% of oleoresin, 9.4% of polysorbate 20 (16.7), and 0.6% of sorbitan trioleate (1.8). Initially, the oily phase was mixed with emulsifiers for 5 min at 500 rpm in a glass beaker in a water bath at 37 °C using a pharmaceutical mixer (Witeg, Germany). Next, water at 37 °C was added dropwise at 1000 rpm, and when completed, the mixture was agitated for 30 min at 1000 rpm. In addition, a mixture of emulsifiers and water was prepared as a blank formulation. The final formulations were filtered (F) or not (NF) with a 0.2 pm PES filter (Sartolab P20 Plus, GF Prefilter, Sartoris AG, Germany). The emulsions were kept at room temperature (25±2 °C) in a spray bottle (30 mL, Semadeni AG, Switzerland).

[0098] 1.4.4. Dynamic light scattering (DLS)

[0099] The samples’ droplet size distribution, mean droplet size and poly dispersity index (PDI) were measured using a dynamic light scattering (DLS) instrument (Zetasizer Nano-ZS Zen3600, Malvern Panalytical, Worcestershire, UK). Measurements were conducted at room temperature (25 °C) using a quartz cuvette. All values reported correspond to the mean ± standard deviation of three measurements of each formulation diluted ten times in distilled water.

[0100] 1.4.5. pH determination

[0101] The formulations' pH values were determined in triplicate directly in the samples (Mettler Toledo FiveGo™, electrode LE422).

[0102] 1.4.6. X-Ray sterilisation

[0103] Emulsions with or without X-ray radiation were characterised by particle size, pH, and drug content. Sterilization of the bottled emulsion was done by X-ray irradiation using a total dose of 28.5 kGy (Synergy Health, Daniken, Switzerland). 1.4.7. Cryogenic Transmission Electron Microscopy (Cryo-TEM)

[0104] A 4 |iL aliquot of sample was adsorbed onto a holey carbon-coated grid (Lacey, Ted Pella, USA), blotted with Whatman 1 filter paper and vitrified into liquid ethany at -178 °C using a Leica GP plunger (Leica, Austria). Frozen grids were transferred onto a Talos electron microscope (FEI, USA) using a Gatan 626 cryo-holder. Electron micrographs were recorded at an accelerating voltage of 200 kV and a nominal magnification of 73000x, using a low-dose system (20 e- / A2) and keeping the sample at a low temperature. Micrographs were recorded on a CETA camera (Thermo Fisher Scientific, Waltham, MA).

[0105] 1.5. Wistar Rat Wound Healing Experiment

[0106] 1.5.1 Animals and Treatments

[0107] The study involving Rattus norvegicus Wistar was submitted to the Animal Use Ethics Committee of the Federal University of Amapa (CEUA / UNIFAP) and approved under protocol No. 008 / 2017. Wistar rats, obtained from the Multidisciplinary Centre for Biological Research at UNICAMP, aged 21 days, were used. They were divided into groups of five animals per cage and maintained under controlled temperature conditions (25° ± 2 °C). Fifteen adult rats were randomly divided into three groups, with treatments applied as follows: Group 1 : Negative control - Vehicle of the nanoemulsion, 80 mg / kg (CTR) Group 2: Positive control - Fibrinase® 150 mg / kg (FIB)

[0108] Group 3: Larix decidua emulsion 10% - 80 mg / kg (ELD)

[0109] Treatments were applied immediately after the surgical procedure to the wounds, daily for 7 days, according to the groups and doses established.

[0110] 1.5.2. Wound Induction and Evaluation Procedure

[0111] Animals were anesthetized with sodium thiopental (45 mg / kg) via intraperitoneal injection. After shaving and asepsis, an 8 mm2circular lesion was induced using a skin biopsy punch, removing tissue until exposing the dorsal muscle fascia.

[0112] The lesion areas were recorded with an iPhone 12 digital camera. Digital images were taken immediately after the surgical procedure and on the 7thday of treatment and stored for wound area analysis using the Imaged program.

[0113] Macroscopic evaluation was performed by calculating the wound contraction percentage, comparing the initial and final wound areas using the following formula: (Ao - Af) x 100

[0114] Wound contraction (%) — -

[0115] Ao

[0116] Where:

[0117] Ao = Initial Area

[0118] Af = Final Area

[0119] At the end of treatment, animals underwent biopsy for histological analysis and were euthanized following the procedure.

[0120] 1.5.3. Histopathological Analysis

[0121] For histopathological analysis, tissues were fixed in 10% buffered formalin for 24 hours. The samples were dehydrated through a series of alcohols (70%, 80%, 90%, and 100%). They were then cleared with xylene and embedded in paraffin. Sections of 5 pm thickness were cut using a microtome (Brand Rotary Microtome, Slee Medical, Germany). Histopathological analysis was conducted after the tissue sections were stained with haematoxylin and eosin, as described by Souza et al. (2016). Images were examined using an Olympus BX41 Microscopy and photographed with an iPhone 12 camera. The histological criteria for wound healing areas included: inflammatory cells, fibroblastic proliferation, and angiogenesis, which were quantified based on focus / cut.

[0122] 1.6. Statistical analyses

[0123] Statistical analyses were performed using GraphPad Prism 10.3.1. Data are presented as mean ± standard deviation. Differences between groups were assessed using t-tests (for two groups) or ANOVA with Tukey's post-hoc test (for multiple groups). Statistical significance was defined as p < 0.05.

[0124] 2. Results

[0125] 2.1. GC-MS

[0126] In this study, we first investigated the volatile and non-volatile compounds from an oleoresin from Larix decidua Mill., as a quality parameter for the developed pharmaceutical formulation. The results of the HS-GC-MS and GC-MS revealed the presence of volatile and non-volatile compounds, respectively. Monoterpenes hydrocarbons, monoterpenes oxygenated, and sesquiterpenes hydrocarbons were identified by HS-GC-MS using a ZB-5Plus column (Figure 4). The HS-GC-MS provided the basis for a rapid analytical approach to the resulting pattern of the volatile components. A total of 25 compounds were identified. Most of the identified compounds were monoterpenes hydrocarbons (99.45%), whilst oxygenated monoterpenes (0.31%) and sesquiterpenes hydrocarbons (0.09%) were found only as traces (Table 1).

[0127] Table 1. Retention time, molecular formula, retention indices and concentration in percentage of volatile compounds in Larix decidua oleoresin via HS-GC-MS. Compounds are shown in order of elution on a ZB-5Plus column.

[0128] RI Oleoresin

[0129] Number tR Compound Formula Lit.aExp.b% area ID**

[0130] 1 7.52 cyclene CioHie 931 931 0.45 RI, MS

[0131] 2 7.69 a-thujene CioHie 932 933 0.17 RI, MS

[0132] 3 7.95 a-pinene CioHie 940 942 74.07 RI, MS

[0133] 4 8.27 camphene CioHie 956 957 1.75 RI, MS

[0134] 5 9.00 sabinene CioHie 978 981 0.70 RI, MS

[0135] 6 9.08 P-pinene CioHie 983 987 8.14 RI, MS

[0136] 7 9.53 -myrcene CioHie 992 996 3.41 RI, MS

[0137] 8 9.91 a-phellandrene CioHie 1013 1013 0.23 RI, MS

[0138] 9 10.07 3-carene CioHie 1013 1018 3.45 RI, MS

[0139] 10 10.28 a-terpinene CioHie 1019 1024 0.22 RI, MS

[0140] 11 10.50 o-cymene CI0HI41029 1031 0.39 RI, MS

[0141] 12 10.62 D-limonene CioHie 1034 1035 5.42 RI, MS

[0142] 13 11.54 y-terpinene CioHie 1066 1065 0.27 RI, MS

[0143] 14 12.41 a-terpinolen CioHie 1089 1097 0.78 RI, MS

[0144] 15 13.17 fenchol Ci0Hi8O 1119 1122 tr RI, MS

[0145] 16 14.09 camphor CioHieO 1153 1152 tr RI, MS

[0146] 17 14.71 a-phellandren-8-ol CioHieO 1170 1174 tr RI, MS

[0147] 18 15.07 terpinen-4-ol Ci0Hi8O 1184 1185 0.12 RI, MS

[0148] 19 15.45 a-terpineol CI0HI8O 1197 1198 0.12 RI, MS

[0149] 20 15.68 myrtenal C10H14O 1196 1205 tr RI, MS

[0150] 21 16.71 thymol methyl ether CnHieO 1235 1240 0.07 RI, MS

[0151] 22 18.16 bornyl acetate C12H20O 1289 1293 tr RI, MS

[0152] 23 19.88 a-terpinyl acetate C12H20O2 1351 1356 tr RI, MS

[0153] 24 23.31 germacrene D CI5H241485 1492 0.09 RI, MS

[0154] 25 25.16 germacrene B CieH241569 1569 tr RI, MS

[0155] All 99.85

[0156] Monoterpenes hydrocarbons 99.45 oxygenated 0.31 Sesquiterpenes hydrocarbons 0.09aCompounds identification: retention index (RI) data compared against commercially available MS library NIST 23.bRI: retention index experimentally determined on a ZB-5Plus column relative to the tR of n- alkanes (C7-C40); compounds are listed in order of elution.

[0157] ID: identification of compounds - RI and mass spectrometry (MS) a-pinene (74.07%), P-pinene (8.14%), D-limonene (5.42%), 3-carene (3.45%), P-myrcene (3.41%), and camphene (1.75%) were the major identified compounds and accounted for 96.24% of the total content (Table 2).

[0158] Table 2. The 6 main metabolites of Larix decidua oleoresin obtained by HS-GC-MS. Compounds are shown in order of elution on a ZB-5Plus column.

[0159] Retention Index Oleoresin

[0160] Number tR(min) Compound Formula Lit.aExp.b% area ID

[0161] 3 7.95 a-pinene CioHie 940 942 74.07 RI, MS

[0162] 4 8.27 camphene CioHie 956 957 1.75 RI, MS

[0163] 6 9.08 P-pinene CioHie 983 987 8.14 RI, MS

[0164] 7 9.53 p-myrcene CioHie 992 996 3.41 RI, MS

[0165] 9 10.07 3-carene CioHie 1013 1018 3.45 RI, MS

[0166] 12 10.62 D-limonene CioHie 1034 1035 5.42 RI, MS

[0167] Total 96.24 a Compounds identification: retention index (RI) data compared against commercially available MS library NIST 23. b RI: retention index experimentally determined on a ZB-5Plus column relative to the tR of n- alkanes (C7-C40); compounds are listed in order of elution.

[0168] ID: identification of compounds - RI and mass spectrometry (MS)

[0169] In addition to the volatiles’ identification and semi-quantitative estimations from the obtained peak sizes, the non-volatiles were investigated after the oleoresin’ s derivatization. Diterpenes were identified using a non-polar HP-5 column and expressed in elution order. From a total of 15 compounds, 11 were identified according to their retention indices or with a reference standard, while four compounds were described as isomers of abietic acid (Table 3).

[0170] Table 3. Non-volatile compounds (diterpenes) of Larix decidua oleoresin identified by GC-MS after derivatization. Compounds are shown in oder of elution on a non-polar HB-5 column.

[0171] Retention

[0172] For-Ex bOleo-

[0173] Number tR(min) Compound mula Litaresin ID

[0174] 1 11.79 Epimanool C20H34O 2316 2218 10.06 RI, MS

[0175] 2 14.46 Neoabietyl acetate C22H34O2 2512 2372 1.64 RI, MS

[0176] C20H34O2 SD, RI,

[0177] 3 14.61 Larixol - 2384 5.46 MS 4 15.01 n.i. 1 - abietic acid isomer C20H30O2 - 2413 2.02 RI, MS

[0178] 5 15.78 n.i. 2 - abietic acid isomer C20H30O2 - 2480 2.58 RI, MS

[0179] 6 16.08 Sandaracopimaral C20H30O 2114 2507 1.44 RI, MS

[0180] C20H30O2 SD, RI,

[0181] 7 16.21 Isopimaric acid 2315 2524 17.58 MS

[0182] 8 16.35 Palustral C20H30O 2245 2535 2.85 RI, MS

[0183] 9 16.61 n.i. 3 - abietic acid isomer C20H30O2 - 2562 12.58 RI, MS

[0184] C24H38O SD, RI,

[0185] 10 16.85 Larixyl acetate - 2587 23.49 MS

[0186] 11 17.03 Neoabietyl alcohol, acetate C22H34O2 2578 2604 0.81 RI, MS

[0187] C20H30O2 SD, RI,

[0188] 12 17.37 Abietic acid 2410 2641 10.09 MS

[0189] C2oH2802SD, RI,

[0190] 13 17.4 Dehydroabietic acid 2371 2643 0.38 MS

[0191] 14 18.18 Neoabietinal C20H30O 2320 2732 0.79 RI, MS

[0192] 15 18.27 n.i. 4 - abietic acid isomer C2QH3O02- 2744 8.23 RI, MS

[0193] Total 100.00aCompounds identification: retention index (RI) data compared against commercially available MS library NIST 23.bRI: retention index experimentally determined on a nonpolar HP-5 column relative to the tR of n- alkanes (C7-C40); compounds are listed in order of elution.

[0194] ID: identification of compounds - chemical standard (SD), RI, mass spectrometry (MS) n.i. not identified compound

[0195] Larixyl acetate (23.49%), isopimaric acid (17.58%), abietic acid isomer (n.i. 3; 12.58%), abietic acid (10.09%), and epimanool (10.06%) are the major compounds and account for 73.80% of the total content. The sum of abietic acid and its isomers accounts for 35.5%.

[0196] Larixol, an exclusive compound for this genus, was identified at a percentage of 5.46%. Compounds with concentrations lower than 1% were neoabietyl alcohol acetate (0.81%), neoabietinal (0.79%) and dehydroabietic acid (0.38%).

[0197] 2.2. Pseudo-ternary phase diagram (PTD)

[0198] In formulating the pharmaceutical product, we opted for non-ionic emulsifiers to create European Larch emulsions, aligning with their HLB values and phase behaviour. The HLB value, determined to be 15.75 using the method described by Griffin (see Griffin, William C., "Classification of Surface-Active Agents by 'HLB'", Journal of the Society of Cosmetic Chemists. (1949), 1(5), 311-326 and Griffin, William C., "Calculation of HLB Values of NonIonic Surfactants", Journal of the Society of Cosmetic Chemists (1954), 5(4), 249-256), guided the proportion of emulsifiers to maintain this balance during the formulation process. To delve deeper into the complexity of emulsioned systems, particularly the equilibrium among the oily phase, water phase, and emulsifier mixture, we strategically employed the construction of a Pseudo-Ternary Phase Diagram (PTD). This approach was necessary to comprehend and fine-tune the interplay of these mixtures. The aqueous titration method, conducted at 37 °C, played a pivotal role in this comprehensive approach, allowing the observation of 99 formulations (Barradas et al., 2015).

[0199] The used mixtures produced a variety of dispersions. PTD was built with Larix decidua oleoresin as the oily phase, polysorbate 20 and sorbitan trioleate as emulsifiers (ratio 15.7: 1), and water as the aqueous phase to determine the ideal concentration range of the components to form the optimal emulsion. While using lower amounts of water, turbid or translucid formulations were obtained. However, the low water content and high emulsifier content translate into an increased chance of skin irritation, especially by anionic and cationic emulsifiers (Seweryn, 2018; Leanpolchareanchai and Teeranachaideekul, 2023). Therefore, instead of focusing on the turbid or translucid regions, we focused on the low-viscosity region with opaque emulsion. Furthermore, these obtained emulsions required less emulsifier and higher water contents.

[0200] An emulsion-containing region was successfully achieved ranging from 35-90% of water, 2- 35% of turpentine, and 3-45% of a mixture of emulsifiers. Notably, this successfully achieved emulsion-containing region exhibited a predominant water content higher than 50%, with emulsifier levels below 40% and oil content below 25%. An accomplishment was the attainment of an emulsion with an oil-to-emulsifier ratio of 1 : 1. This emulsion comprised 10% oleoresin, 10% emulsifiers (including 9.4% polysorbate 20 and 0.6% sorbitan trioleate), and 80% water.

[0201] 2.3. Formulation development, sterility processes and stability evaluation The selected emulsion was prepared using a low-energy phase-inversion composition (PIC) method, employing a pharmaceutical mixer with a three-bladed propeller. The resulting emulsions were subjected to two distinct sterilization methods: membrane filtration and X-ray exposure. Given the intended use of this formulation for treating open wounds, sterility is imperative (Ph.Eur., 2024). The primary objective was to discern the impact of these sterilization techniques on emulsion stability. The assessments were conducted at three specific intervals — after one week, nine weeks, and fourteen weeks of X-ray sterilization. To comprehensively evaluate stability, we focused on key parameters, including pH, droplet size, and morphological aspects (cryo-TEM). These chosen criteria were deemed crucial for understanding the enduring effects of sterilization methods on the emulsion's overall stability over an extended period.

[0202] Significant variations in pH (p<0.05) were notably observed after X-ray exposure for filtered and unfiltered emulsions, as shown in Figure 1. Interestingly, the pH remained stable over time for emulsions that did not undergo X-ray sterilization, whether filtered (4.31±0.11 [time 1], 4.28±0.09 [time 9], 4.24±0.06 [time 14]) or unfiltered (4.37±0.04 [time 1], 4.21±0.07 [time 9], 4.21±0.06 [time 14]). However, a distinct trend emerged for emulsions subjected to X-ray sterilization, where a noticeable decrease in pH was evident. In both filtered (3.75±0.01 [time 1], 3.14±0.02 [time 9], 3.25±0.04 [time 14]) and unfiltered (3.76±0.00 [time 1], 3.19±0.03 [time 9], 3.29±0.05 [time 14]) emulsions, the pH exhibited a significant reduction poststerilization. This shift in pH values underscores the influence of X-ray sterilization on the chemical properties of the emulsions, prompting further investigation into the potential implications for their stability and overall performance, which was evaluated by their droplet size and PDI. Given the observed pH instability following X-ray sterilization, subsequent stability evaluations focused on the filtered emulsion without X-ray treatment. Cryo-TEM was used to visualize the internal structure of emulsion droplets. The observed droplets exhibited a spherical form without any signs of aggregation. Additionally, at 16 months, slightly larger droplets were observed, aligning with the findings from size and PDI measurements.

[0203] Furthermore, this emulsion exhibited no alterations in pH.

[0204] The assessment of particle size and droplet stability emerges as a crucial parameter in the development of emulsions. The results elucidated a pattern wherein the formulations exhibited greater homogeneity initially, but over time, a gradual increase in droplet size occurred (Figure 2A), whilst there was no statistical difference for the PDI values, and they were below 0.2 (Figure 2B). The size increased over time for emulsions that did not undergo X-ray sterilization, whether filtered (163.01±2.21 nm [time 1], 187.53±9.59 nm [time 9], 212.23±33.15 nm [time 14]) or unfiltered (164.30±3.06 nm [time 1], 209.01±7.03 nm [time 9], 256.75±29.75 nm [time 14]). The same behaviour was observed for emulsions subjected to X- ray sterilisation, where an increase in the droplet size was found in both filtered (173.47±1.70 nm [time 1], 214.40±5.27 nm [time 9], 231.73±7.46 nm [time 14]) and unfiltered (173.73±2.46 nm [time 1], 209.90±4.01 nm [time 9], 224.53±1.62 [time 14]). There was an absence of phase separation, aggregation, precipitation, or creaming. Beyond measuring droplet size, we conducted a thorough analysis of the emulsions’ morphological aspects. This approach is essential, as it provides insights into droplet characteristics and serves as a stability indicator, capturing nuances undetectable by DLS. Cryo-TEM was employed, emerging as one of the most effective techniques for investigating emulsions, particularly in liquid specimens. This method offers detailed information about the internal structure in its natural state, enabling precise visualization of droplet shapes (Klang et al., 2012).

[0205] The observed droplets exhibited a spherical form without any signs of aggregation, as illustrated in Figure 3. Notably, a discernible difference in sizes was evident between the filtered and unfiltered samples, irrespective of X-ray exposure. Additionally, at 14 weeks, larger droplets were observed, aligning with the findings from size and PDI measurements. This observation raises the possibility of droplet aggregation and the formation of larger droplets.

[0206] Chromatogram of the analysed turpentine by HS-GC-MS (Figure 4). Compounds described in Tables 1 and 2 are shown according to their retention time based on their chromatogram peak. Compound 3 (alpha-pinene) appears as the major compound.

[0207] In summary, the formulation that underwent filtration and avoided X-ray sterilisation displayed the smallest instability over 14 weeks. Furthermore, this emulsion exhibited no alterations in pH, which was not the case for the ones that underwent X-ray exposure. The morphological analysis further complemented the overall understanding of the emulsion’s structural dynamics and stability characteristics. Consequently, the filtered emulsion without X-ray exposure was selected for further ongoing investigations.

[0208] 3. Assessment of antimicrobial effect of the emulsion

[0209] 3.1 Determination of minimum inhibitory concentration (MIC)

[0210] The MIC of the compounds was determined using the microdilution method in wells. The bacterial strains S. aureus (ATCC-6538P), S. epidermidis (ATCC-12228), E. coli (ATCC- 25922), P. aeruginosa (ATCC-9022) and C. albicans (ATCC-10231) were inoculated into wells with Muller Hinton (Kavski) broth medium for bacteria and Sabouraud Dextrose 2% (Kavski) for yeast. The colonies from these cultures were suspended in sterile Muller Hinton or Sabouraud media accordingly and the inoculum was standardised using the McFarland scale (1-5 x 106CFU / mL). Subsequently, 150 pL of the sterile vehicle control (formulation without the oleoresin) and 150 pL of the sterile Larix decidua nanoemulsion that underwent filtration as described in section 2.3 above were added to a 96-well plate, with an initial concentration of 50 mg / mL after which 150 pL were taken from the first line to the second line (1 :2) with successive serial dilutions, up to a final concentration of 0.39 mg / mL (1:64). Then 20 pL of the inoculum was added, at a concentration of approximately 1.5 x 108CFU / mL for bacteria and 1.5 x 107CFU / mL for yeasts). The wells were incubated at 37 °C for 24 hours. The MIC was defined as the lowest concentration capable of inhibiting the growth of the inoculum. A 0.15% resarzurin solution was used as a growth indicator.

[0211] 3.2 Determining the Minimum Bactericidal Concentration (MBC)

[0212] The MBC, defined as the minimum concentration that eliminates the majority (99.9%) of viable microorganisms, was obtained by sowing 10 pL aliquots obtained from the wells in which there was no microbial growth onto MH or SB agar plates and incubating for 24 h at 37°C. This was identified by the absence of colonies on the agar surface. All tests were carried out in triplicate under strictly aseptic conditions.

[0213] 3.3 Results

[0214] 3.3.1 Antimicrobial activity of nanoemulsion

[0215] The Larix decidua nanoemulsion demonstrated antimicrobial activity against all tested bacteria (Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Pseudomonas aeruginosa) with MICs of 0.39 mg / mL across the board (Table 1). The MBC varied: 0.39 mg / mL for S. epidermidis and E. coli, 25 mg / mL for S. aureus, and 12.5 mg / mL for P. aeruginosa. No activity was observed against Candida albicans. The vehicle control showed no antimicrobial activity against any of the tested microorganisms. This indicates that the observed antimicrobial effects are attributable to the nanoemulsion's active component Larix decidua) and not to any inherent properties of the vehicle control. The Larix decidua nanoemulsion exhibited notable antimicrobial activity against the tested Gram-positive and Gram-negative bacteria, with varying degrees of bactericidal activity.

[0216] Table 4 Antimicrobial Activity of 10% Larix decidua Nanoemulsion Against Selected Microorganisms

[0217] Larch turpentine Nanoemulsion Vehicle Control 10% Strain MIC (mg / mL) MBC (mg / mL) MIC MBC

[0218] Staphylococcus aureus 0.39 25.00 N N

[0219] Staphylococcus 0.39 0.39 N N epidermidis Escherichia coli 0.39 0.39 N N

[0220] Pseudomonas aeruginosa 0.39 12.50 N N

[0221] Candida albicans N N N N

[0222] 3.3.2. Macroscopic and Histopathological Analysis of Wounds, Inflammatory Cells, Fibroblasts, and Angiogenesis

[0223] The macroscopic evaluation of wound healing revealed that Wistar rat groups treated with formulations containing Larix decidua (ELD) and Fibrinase® (FIB) exhibited marked retraction of wound edges, evident re-epithelialization, and greater wound contraction compared to the control group (CTR) (Figure 5). The control group displayed irregular wound edges, erythema, and exudate, indicative of ongoing inflammation and delayed healing. Specifically, the ELD group showed well-defined, retracted edges and a dry wound bed, with a contraction index of 77.19 ± 1.53%, significantly higher than the control group (65.49 ± 1.06%, p < 0.05). The FIB group demonstrated an even greater contraction index of 82.44 ± 7.29%, accompanied by mild erythema at the wound edges and visible granulation tissue. Histopathological analysis performed after seven days of treatment confirmed these macroscopic findings, revealing notable differences in tissue repair among the groups. The control group presented disorganized tissue architecture with incomplete re-epithelialization, abundant exudate, a dense inflammatory infiltrate, and poorly structured granulation tissue with scant neovascularization. Conversely, the treated groups (FIB and ELD) exhibited complete re-epithelialization, well-organized epidermal layers, abundant fibroblasts, and extensive neovascularization, with a reduced inflammatory response compared to the control.

[0224] Further results are presented in Figure 6 and Figure 6a.

[0225] Quantitatively, the control group showed significantly higher inflammatory cell infiltration (148.5 ± 53.21 cells / field; p < 0.01 vs. FIB and ELD; Figure 6, D), indicating ongoing inflammation. Fibrinase® and Larix decidua emulsion groups demonstrated a substantial decrease in inflammatory cells (16.5 ± 5.95 and 44.0 ± 13.31 cells / field, respectively), reflecting effective modulation of the inflammatory process.

[0226] Immunohistochemical evaluation (Figure 6a, A-C, E-G, LK) showed that black arrows identified inflammatory cells, fibroblasts, and new blood vessels. The number of inflammatory cells was significantly lower in the Fibrinase® and Larix decidua emulsion groups, confirming the anti-inflammatory effects of the treatments. Additionally, fibroblast proliferation was markedly increased in the Larix decidua emulsion group (ELD), with an average of 30.5 ± 7.15 fibroblasts per field, compared to 20 ± 7.45 in the Fibrinase® group (Figure 6, H). This suggests enhanced cellular proliferation conducive to tissue regeneration.

[0227] Angiogenesis analysis revealed a significant increase in new vessel formation, particularly in the ELD group (35 ± 8.5 vessels / field), surpassing the control group (9.5 ± 3.65; p < 0.001). The Fibrinase® group also showed increased vascularization (20.5 ± 5.07 vessels / field), but significantly less than the ELD group, indicating a pro-angiogenic effect of Larix decidua even at lower concentrations (Figure 6, L).

[0228] Overall, these findings demonstrate that the Larix decidua emulsion not only accelerates wound contraction and re-epithelialization but also effectively reduces inflammation, promotes fibroblast proliferation, and enhances neovascularization, contributing to improved tissue regeneration during the healing process.

[0229] 4. Case report

[0230] Person (47 years), female patient started treatment at the clinic on June 28, 2024 after presenting with a chronic wound that developed on May 25, 2024. She was diagnosed with hypertension after her second pregnancy (2002), with fibromyalgia in 2019, and with rheumatoid arthritis since 2024, in addition to ongoing chronic migraine. As a result of her chronic illnesses, she developed depression. As treatment, she takes daily pregabalin 150 mg, cyclobenzaprine 10 mg, metoprolol 100 mg, and hydroxychloroquine 400 mg. For her pain, Paco® (paracetamol+ codeine phosphate; twice / day), tramadol (twice / day), and Dorflex® (orphenadrine; once / day). A comprehensive wound care protocol was implemented, including thorough cleansing and debridement. Several treatments were applied throughout her visits, such as Kerlix™, Papain 10% Gel, Copaiba oil 1%, calcium alginate, and silver alginate. No evident amelioration was observed under this treatment. On August 09, 2024, the novel Larch Turpentine nanoemulsion was introduced into her treatment regimen by administering the nanoemulsion topically every three days to the wound area. The initial assessment revealed inflammation and pain associated with the wound before starting with the Larch turpentine nanoemulsion. However, by August 22, 2024 (approx. 14 days), the patient showed marked epithelialization and granulation tissue formation. Continued care with the nanoemulsion resulted in excellent healing progress, with notable improvements observed. By September 06, 2024 (28 days), the wound had fully closed, leading to the patient’s discharge and she presented a fully healing by October 26, 2024 (77 days) (Figure 7). The patient's polypharmacy, including pregabalin, cyclob enzaprine, tramadol, paracetamol with codeine phosphate, and orphenadrine, warrants consideration. While crucial for pain management, these medications may have an indirect impact on wound healing. For example, pregabalin, while effective for acute pain, has been associated with decreased wound healing in an in vivo study, and opioid exposure has been linked to impaired healing in chronic wounds in a longitudinal observational study. Furthermore, in a retrospective observational study, hypertension has been linked to an increased risk of prolonged wound healing and drainage in some surgical populations. The results confirm that the novel nanoemulsion not only improved the healing process of the chronic wound without apparent adverse effects and despite the patient’s complex medication regimen, but also demonstrated good tolerability in the patient.

[0231] 5. Conclusion

[0232] The current study demonstrated the effectiveness of analysing essential oils using solid-phase microextraction headspace coupled with gas chromatography. The study also revealed the presence of the main diterpenes in the oleoresin, with larixyl acetate, larixol, and epimanool being abundant compounds. Additionally, isopimaric acid was identified as the predominant resin acid, distinguishing the European larch oleoresin from other pine oleoresins. From a pharmaceutical point of view, our findings suggest that the emulsion prepared with Larch oleoresin possesses thermodynamic stability, making it suitable for short-term applications, such as magistral preparations for patient use within three months. A successful sterile procedure was achieved through membrane filtration. The emulsion's composition suggests non-allergenic properties due to using non-ionic emulsifiers, antimicrobial properties, antiinflammatory and wound healing activities which makes it beneficial for the wound healing process. References

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Claims

35Claims1. Emulsion comprising an aqueous phase A and a nonaqueous phase B emulsified with phase A, wherein phase B comprises larch turpentine as component Bl and at least one emulsifier as component B2.

2. Emulsion according to claim 1, wherein phase B comprises larch turpentine as component Bl and two emulsifiers, preferably two nonionic emulsifiers, as component B2.

3. Emulsion according to claim 1, wherein phase B comprises larch turpentine as component Bl and a polysorbate and a sorbitan, preferably polysorbate 20 and sorbitan trioleate as component B2.

4. Emulsion according to any of claims 1 to 3, wherein the aqueous phase A amounts from 20 percent to 90 percent, preferably from 40 percent to 80 percent, by weight based on total weight of the emulsion, while phase B provides the remainder of the emulsion, preferably wherein the aqueous phase A amounts to 80 percent by weight based on total weight of the emulsion while phase B amounts to 20 percent by weight based on total weight of the emulsion.

5. Emulsion according to any of claims 1 to 4, wherein the larch turpentine as component Bl amounts from 2 percent to 60 percent by weight of phase B, while the polysorbate and the sorbitane provides the remainder of phase B, preferably wherein the larch turpentine as component Bl amounts to 50 percent by weight of phase B, while the polysorbate and the sorbitan-amount to 50 percent by weight of phase B.

6. Emulsion according to any of claims 1 to 3, wherein the aqueous phase A amounts from 35 percent to 90 percent by weight based on total weight of the emulsion, the larch turpentine as component Bl amounts from 2 percent to 35 percent by weight based on total weight of the emulsion and the at least one emulsifier as component B2 amounts from 3 percent to 45 percent by weight based on total weight of the emulsion, provided that the combined weight36 percentage of all components of the emulsion does not exceed 100 weight percentage of the emulsion.

7. Emulsion according to any of claims 3 to 6, wherein the polysorbate and the sorbitan are present in a weight ratio in the range from 10: 1 to 20: 1, preferably 15: 1 to 16: 1 in component B2.

8. Emulsion according to any of claims 1 to 7, wherein phase A has a pH in the range from 3 to 7, preferably from 4 to 5.

9. Emulsion according to any of claims 1 to 8, wherein the emulsion is a sterile emulsion.

10. Emulsion according to any of claims 1 to 9, wherein the emulsion is a nanoemulsion comprising droplets which have a size of between 20 and 300 nanometers, preferably between 100 and 300 nanometers,.

11. A topical spray comprising the emulsion according to any of claims 1 to 10.

12. The emulsion according to any of claims 1-10 or the spray of claim 11 for use in a method of treatment of wounds of a subject.

13. Process for producing the emulsion according to any of claims 1 to 10, comprising the steps of a) separately producing phases A and B as defined in any of claims 1 to 10, b) mixing phases A and B from step a) for production of an emulsion of phases A and B.

14. The process according claim 13, wherein step a) comprises putting larch turpentine as component Bl and at least one emulsifier as component B2 of phase B together, heating the mixture to 37° C, and agitating the mixture for 5 min at 500 rpm; step b) comprising mixing phases A and B from step a) by heating the aqueous phase to 37° C and dropping the aqueous phase to the phase B from step a), wherein the mixture of phases Aand B are agitated for 30 min at 1000 rpm; optionally as step c) filtering the obtained emulsion.

15. Emulsion obtainable by the process according to claim 13 or 14.

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