WO2020174340A1 - Eutectic menthol-fatty acid combinations for wound healing - Google Patents

Eutectic menthol-fatty acid combinations for wound healing Download PDF

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Publication number
WO2020174340A1
WO2020174340A1 PCT/IB2020/051486 IB2020051486W WO2020174340A1 WO 2020174340 A1 WO2020174340 A1 WO 2020174340A1 IB 2020051486 W IB2020051486 W IB 2020051486W WO 2020174340 A1 WO2020174340 A1 WO 2020174340A1
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Prior art keywords
menthol
previous
eutectic composition
fatty acid
use according
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PCT/IB2020/051486
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French (fr)
Inventor
Joana Maria MARQUES DA SILVA
Carolina VELHINHO PEREIRA
Maria Francisca MANO
Rui Luis GONÇALVES REIS
Alexandre Babo DE ALMEIDA PAIVA
Ana Alexandra Figueiredo Matias
Ana Rita Cruz Duarte
Original Assignee
Universidade Nova De Lisboa
Association For The Advancement Of Tissue Engineering And Cell Based Technologies & Therapies (A4Tec) Associação
Nova ID FCT - Associação para a Inovação e Desenvolvimento da FCT
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Publication of WO2020174340A1 publication Critical patent/WO2020174340A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0014—Skin, i.e. galenical aspects of topical compositions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/14—Esters of carboxylic acids, e.g. fatty acid monoglycerides, medium-chain triglycerides, parabens or PEG fatty acid esters
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present disclosure relates to a eutectic composition of menthol-fatty acid as a THEDES (therapeutic deep eutectic system).
  • the present disclosure further relates to menthol-fatty acids for use in therapeutic purposes such as for wound treatment, and/or for the treatment or prevention of microbial infection.
  • DES can be easily prepared by mixing two or more compounds at specific molar ratios. At certain molar ratio, the compounds self-associate and form a eutectic mixture that has a melting point that is lower than the melting point of each individual compound. This phenomenon, depression of melting point, can be attributed to the strong hydrogen bond interaction between a hydrogen bond donor (HBD) and a hydrogen bond acceptor (HBA).
  • HBD hydrogen bond donor
  • HBA hydrogen bond acceptor
  • DES non-flammable, has a broad range or polarity, and low volatility. It is dipolar in nature, exhibits chemical and thermal stability. Further, it has water-compatibility, is biodegradable and has negligible toxicity profile.
  • the core characteristics of DES are similar to their analogues - ionic liquids (ILs). However, the green chemistry metrics of DES is better than ILs thus making DES a highly desirable and promising alternative solvent.
  • DES has attracted interests in several fields as solvents for organic synthesis, biocatalysis, electrochemistry, nanomaterials, polymer production, separation processes, CO 2 capture, foods, cosmetics, pharmaceutics and biomedical applications.
  • DES can be used to improve solubility, permeation and absorption of active pharmaceutical ingredients (API's).
  • API can be paired with a DES; this subclass of bioactive DES is termed therapeutic deep eutectic systems (THEDES).
  • THEDES therapeutic deep eutectic systems
  • Menthol is a terpene that can been extracted from Mentha species and it has already been used for THEDES preparation in combination with a wide range of compounds including ibuprofen, lidocaine, fluconazole, and among others, captopril.
  • DES based on menthol and fatty acid combination have been disclosed in prior art.
  • the therapeutic effect of menthol-fatty acid based THEDES has not been disclosed or evaluated by prior art.
  • the potential of menthol-fatty acid based THEDES for use in the biomedical field was not previously explored.
  • Document US20180093011 discloses ionic liquids that sterilize and prevent biofilm formation on skin wound healing devices.
  • the disclosure claims an ionic liquid with anti microbial potential.
  • the disclosure further claims that the ionic liquid comprises deep eutectic solvent and choline geranate. No reference or claim was made to the composition of the deep eutectic solvent or to compounds that potentially have deep eutectic solvent characteristics. No claims or references were made to the combination of menthol and fatty acids as a DES.
  • Document US8496950 B2 discloses a mixture containing menthol. However, no disclosure was made with regards to a mixture based on the combination of menthol and fatty acids. Furthermore, no disclosure was made regarding the anti-bacterial effect of menthol or any wound healing effect.
  • Document CN 101157612 B discloses 17 ester derivatives of menthol and their preparation method. Although, the derivatives are obtained after a chemical reaction which involves the presence of an organic solvent. The patent further discloses the 17 derivatives as suitable agents which enhances transdermal absorption of a range of pharmacological compounds. Specifically, the patent claims the use of heptanoate menthol as an agent which enhances the transdermal absorption of diclofenac potassium diclofenac indole or indole. However, the patent does not disclose and evaluate the optimal menthol:fatty acid ratio for optimal wound healing therapeutic effect.
  • thermophysical properties of menthol:fatty acid THEDES as well as its potential use for therapeutic purposes such as for wound treatment.
  • menthol Fatty acids, commonly extracted from vegetal and animal fats, are secreted from human sebaceous glands and allows the human skin to naturally disinfect itself.
  • the advantage of the menthol:fatty acid THEDES is that in addition to being an effective transdermal permeation enhancer, menthol and the fatty acid are also anti-inflammatory and antimicrobial. Additionally, the current disclosure discloses a menthol:fatty acid THEDES composition which is able to prevent skin flora, specifically Staphylococcus aureus, from developing resistance against the composition.
  • the present disclosure relates to a eutectic composition for use in medicine comprising menthol and a saturated fatty acid.
  • the eutectic composition can be used in the therapy or treatment of wounds and/or microbial infections.
  • the eutectic composition can be used in wound healing approaches, wherein the menthol and fatty acids promote the wound healing and the antibacterial effect.
  • the eutectic composition can prevent bacterial infection in wounds.
  • the eutectic composition of the present disclosure can be used for prevention, the treatment or therapy of infections caused by Candida albicans, Escherichia coli, Staphylococcus aureus, or combinations thereof.
  • An aspect of the present disclosure relates to a eutectic composition for use in medicine comprising menthol and a saturated fatty acid, preferably a saturated fatty acid comprising at least 10 carbons; more preferably wherein the saturated fatty acid comprises between 12 to 20 carbons.
  • Another aspect of the present disclosure relates to eutectic composition
  • eutectic composition comprising menthol and a saturated fatty acid, wherein the saturated fatty acid comprises at least 10 carbons, preferably wherein the saturated fatty acid comprises between 12 to 20 carbon.
  • the saturated fatty acid is selected from the following list: lauric acid (LA), stearic acid (SA), myristic acid (MA), or mixtures thereof.
  • the menthol and the saturated fatty acid have a molar ratio of 2:1 - 10:1, preferably a molar ratio of 4:1 - 9:1, more preferably 6:1 - 8:1.
  • the concentration of menthol may vary from 0.45 - 1.5 mM, preferably 0.5 to 1 mM.
  • the concentration of saturated fatty acid may be from 0.04 to 0.75 mM, preferably 0.06 to 0.5 mM.
  • the composition is a topical composition preferably a cream, a lotion or a gel.
  • An aspect of the present disclosure relates to a patch and/or kit comprising the eutectic composition from the previous embodiments.
  • the present disclosure relates to the use of the eutectic composition as an enhancer of wound healing and as a drug resistance preventer/prophylactic agent.
  • Figure 1 DSC thermograms obtained for powders (A) and THEDES, including: Menthol:LA (B) Menthol:MA (C) and Menthol:SA (D). Peaks arising above the baseline represent endothermic peaks.
  • FIG. 6 Figure 6 - Wound healing assay.
  • A Migration assessment of HaCaT cells after the treatment with menthol:SA (8:1), menthol and SA at 0 and 24 hours post-scratch. The lines indicated the boundary lines of the scratch at 0 hours.
  • B Results were expressed in terms of percentage of wound closure relatively to the control using the mean ⁇ SD of six independent experiments performed in duplicate. Statistically significant differences comparing the samples were calculated according to one-way ANOVA for multiple comparisons by Tukey's method.
  • FIG. 7 Wound healing assay.
  • A Migration assessment of HaCaT cells after the treatment with menthol:LA (2:1), menthol and LA at 0 and 19 hours post-scratch.
  • Figure 8 Representative images of disk diffusion assay plates obtained for individual counterparts, THEDES and controls. Plates are presented by bacterial strains tested. MIC/MBC values of individual counterparts and THEDES. Results are presented by bacterial strains tested. ND- Not dissolved.
  • Figure 9 A - Schematic representation of an embodiment comprising the antibiofilm properties of menthol:LA 4:1.
  • B Percentage of biofilm removed upon exposure to different concentrations of menthol:LA (625, 1250 and 2500 mg/mL) for a total period of 30 minutes.
  • FIG. 10 Figure 10 - Scanning electron microscope (SEM) images of surfaces after exposure with menthol:LA 4:1 from three distinct microorganisms seeded onto cover slips with different exposure times to menthol:LA eutectic blend. Results are presented by microorganism for the various exposure times applied. Scale bar is 10 mm.
  • Figure 11 Embodiment of the different molar ratios considered to prepare the THEDES compositions, with their respective visual aspect and POM micrographs. The scale bar is 200 mm.
  • the eutectic mixture was prepared.
  • the eutectic mixture was prepared using menthol (ref. M2772, Sigma Aldrich), lauric acid (LA; Ref.
  • the optical characterization of different formulations of THEDES was carried out at room temperature (RT - 22°C) using polarizing optical microscopy (POM).
  • the micrographs of the small droplets deposited on the cover glass were obtained using an Olympus BH2 transmission microscope (Olympus, UK) coupled with a Leica digital camera DFC 280 (Leica, UK).
  • the viscosity of THEDES was measured using a Kinexus Prot Rheometer (Kinuxus Prot, MaL 1097376, Malvern) fitted with parallel plate geometry (PU20 SR1740 SS). The viscosity of the different formulations was assessed under controlled stress conditions and at a shear rate of 10 s -1 . After equilibrating the sample temperature at 15°C, a temperature scan was performed from 50°C to 15°C at 2°C/min.
  • the Differential Scanning Calorimetry (DSC) experiments of the powders and THEDES were performed in a TA instrument DSC Q100 model (Thermal analysis & analysers, USA). The formulations were equilibrated at 40°C for 5 min followed by cooling to -40°C, an isothermal period for 5 min and a heating to 100°C at a 5°C /min.
  • NMR nuclear magnetic resonance
  • the HaCaT cell line (DFKZ, Germany) was cultured in DMEM medium supplemented with 10% (v/v) of heat-inactivated fetal bovine serum (FBS) and 1% (v/v) penicillin-streptomycin (PS).
  • FBS heat-inactivated fetal bovine serum
  • PS penicillin-streptomycin
  • Cells were maintained at 37°C with 5% CO 2 in a humidified incubator and routinely grown as a monolayer in 75 cm 2 culture flasks.
  • the cell culture medium and supplements were purchased from Invitrogen (Gibco, Invitrogen Corporation, UK).
  • cytotoxicity assay was performed using confluent and differentiated HaCaT cells which represent 80% of epidermal cells.
  • HaCaT cells were seeded into 96-well plates at a density of 4.5x10 4 cells/well and allowed to grow for 3 days.
  • cells were incubated with the different menthol:fatty acid formulations diluted in culture medium. Cells incubated with only culture medium were used as control.
  • cell migration was evaluated using HaCaT cells.
  • Cells were seeded at a density of 1x10 5 cell/cm 2 in a 12-well plate and allowed to grow until 100% confluence (48 hours).
  • a wound was created with a standard 200 mL pipette tip and each well washed twice with warm PBS to remove the non-adherent cells.
  • Menthol:SA mixture in 8:1 molar ratio was prepared by incubating 1.50mM of menthol with 0.19mM of SA or 0.75mM of menthol with 0.09mM of SA for 24 hours.
  • Menthol:LA mixture in 2:1 molar ratio was prepared by incubating 0.75mM of menthol with 0.375mM of LA for 18 hours.
  • THEDES antimicrobial activity was analysed using agar diffusion assay.
  • the antibacterial activity of eutectic blends was determined using Staphylococcus aureus ATCC 25923 and ATCC 700698 (Methicil lin-resista nt strain), Staphylococcus epidermis ATCC 35984 (Methicillin-resistant strain); Pseudomonas aeruginosa ATCC 27853 and Escherichia coli ATCC 25922, according to the guidelines of Clinical and Laboratory Standards Institute (CLSI).
  • Gentamicin sulphate (ref. G1914, Sigma Aldrich) at 50 mg/mL and sterile water were used as positive and negative control, respectively.
  • the discs (CT0998B, Oxford) were prepared by incubating them in THEDES and controls. Prior to the disk diffusion assay, suspension cultures were prepared by the inoculation of single colonies in 5 mL of tryptic soy broth (TSB, ref CM0337B, Frilabo). The cultures were grown for 24 h at 37°C in an orbital shaker at 180 rpm. Afterwards, bacteria cells were harvested and re-suspended in TSB and the absorbance at 640 nm was adjusted to a turbidity of 0.5 McFarland scale, which provide an equivalent concentration 1-2x10 8 colony forming units (CFUs)/mL. The inoculum was then distributed in Muller-Hinton agar (MHA, ref. CM0337B, Fisher Scientific) plates and allow to dry for 5 minutes. Then, the disks impregnated with the different formulations were placed on the inoculated agar.
  • MHA Muller-Hinton agar
  • the minimal inhibitory concentrations (MICs) and minimal bactericidal concentration (MBCs) were determined by microbroth dilution technique. Briefly, standard solutions of menthol and fatty acids in powder and THEDES form were prepared under sterile conditions by first dissolving the weighing compounds in DMSO (ref. 276855, Sigma), followed by serial dilution in Muller-Hinton Broth medium (MHB, ref 70192-500g, Sigma Aldrich) maintaining a 10%(V/V) percentage of DMSO. During the dilution process, the microtubes were warmed up to 50°C and homogenized using an ultrasonic bath until no precipitation was observed.
  • the microtubes containing the different formulations were inoculated with 500 mL of bacterial suspension at 1-2x10 6 CFUs/mL in MHB.
  • MHB i.e., bacteria-free
  • MHB with no added compounds
  • DMSO DMSO
  • the tubes were incubated at 37°C under shaking for 24 hours and the bacterial growth was monitored. The MICs of each compound were considered to be the concentrations at which the tested formulations prevent the turbidity in the well.
  • S. aureus ATCC 700698, C. albicans ATCC 90029 and E. coli ATCC 25922 were spread into TSA plates and grown overnight at optimal temperature for each organism, 37°C and 30°C for bacteria and yeast, respectively.
  • suspension cultures were prepared by inoculating single colonies into TSB and posterior incubation for 24 hours at optimal temperatures. Cultures were then harvested, resuspended in TSB and adjusted to approximately 1-2x10 4 CFUs/mL. Posteriorly, 200 mL of the previously prepared bacterial suspension was transferred into wells, in a 96-well polystyrene flat-bottom plate and incubated for an additional 20 hours, at optimal temperature for biofilm formation.
  • Biofilms were established in cover slips (ref. 83.1840.002, Sarstedt) for 20 hours and treated with the sample solutions for the prementioned time points. Posteriorly, sample solutions were gently removed, and microorganisms fixed using a 10% (v/v) formalin in PBS solution for 1 hour. Fixed biofilm was then dehydrated by immersion in solutions with increasing concentrations of ethanol (50%, 70%, 90% and 100%). Finally, samples were left air-drying to remove residual ethanol, followed by gold-sputtering for SEM image acquisition.
  • Figure 11 shows a summary of the different THEDES composition with their respective visual aspect and POM micrographs.
  • the scale bar is 200 mm.
  • DSC analysis was also performed to assess thermal events, namely the variations on the melting point of the eutectic mixture when compared with the parent species ( Figure 1).
  • a depression on the melting point represents a successful formation of THEDES.
  • the thermogram of racemic menthol presents two melting points at »28°C and »33°C, which have been ascribed to alpha and beta polymorphs, and is in good agreement with previous reported data.
  • a well-defined and sharp endothermic peak was obtained at » 46.6°C, »58.6°C, »73°C, for LA, MA, SA, respectively.
  • the thermograms of the individual starting materials corroborated previous data in the literature.
  • the peaks obtained in THEDES are different from the ones of the parent species, which further suggests the supramolecular rearrangement while the compounds are in THEDES form. Additionally, a clear depression on the melting point of the parent species can be observed.
  • the thermograms of THEDES indicate that, depending on the molar ratio, the intensity and shift of the peaks is different, which was also expected and previously reported for other THEDES.
  • menthol was combined with LA, MA, SA at 4:1, 8:1 and 8:1, respectively. In each case, a full black image was obtained with POM and a DSC thermogram with an endothermic peak was also achieved, showing a depression on the melting point of the individual compounds, proving the adequate molar ratio for the preparation of a eutectic mixture (THEDES).
  • the viscosity of the different THEDES was evaluated at constant shear rate and as a function of temperature.
  • the viscosity is an important feature in THEDES, as it dictates the mass transport phenomena and affecting their suitability for therapeutic applications.
  • the viscosity of the systems decreases as the temperature increase, which is in accordance with the Arrhenius equation ( Figure 4).
  • the menthol:fatty acid formulation with lowest viscosity was menthol:LA, followed by menthol:MA and menthol:SA.
  • menthol:SA it was only possible to evaluate the viscosity up to 30°C, at lower temperatures the system is in solid phase, as shown in the DSC thermogram.
  • the bioactivity of THEDES was assessed. Menthol-based THEDES were tested aiming at evaluating the cytotoxicity effect in order to select the concentrations tested in further assays. Comparing the effect of isolated compounds none of the fatty acids showed cytotoxicity, as Figure 5 (A, B, C) shows. Assessing THEDES cytotoxicity, menthol:LA at molar ratio of 4:1 showed higher cytotoxicity with EC 50 value of 5.569 ⁇ 0.326 mM of equivalent menthol ( Figure 5A and Table 1). Moreover, this system showed similar cytotoxicity with pure menthol.
  • Table 1 shows the EC 50 values for different THEDES systems using different molar ratios.
  • menthol:SA at molar ratio of 8:1 was selected to evaluate menthol:fatty acid THEDES wound healing properties.
  • Three non-cytotoxic concentrations of menthol:SA at molar ratio of 8:1 were selected and the ability of HaCaT cells to migrate was assessed using the wound healing assay.
  • the two highest menthol:SA (8:1) concentrations significantly induce cell migration leading to higher wound enclosure (areas of 66.00 ⁇ 5.92% and 70.50 ⁇ 4.28%) as compared to the control (53.23 ⁇ 5.35%), representing an increase of nearly 40%.
  • menthol:LA at molar ratio 2:1 was selected to evaluate the wound healing properties.
  • Non-cytotoxic concentrations were chosen and as depicted in figure 7 it is possible to observe that the systems lead to higher wound enclosure as compared to the control. Again, the system menthol:LA has shown a similar effect to menthol alone, but LA did not demonstrate any significant effect.
  • compositions demonstrate antimicrobial activity.
  • Menthol shows antimicrobial activity in all cases, being more efficient at higher concentrations, as expected.
  • the developed THEDES were also tested considering their antibiofilm properties (Figure 9).
  • the addition of THEDES formulation prepared from menthol:LA with a molar ratio of 4:1 caused a biofilm removal, whose extent was dependent on the bacterial strain tested.
  • Higher concentrations of THEDES resulted in a more effective removal of the biofilm.
  • coli being a gram-negative bacterium, requires a higher concentration (2500 mg/ml) and greater actuation time (10 minutes) for any relevant effect.
  • MRSA show itself as a mid-point in effectiveness when compared with E. coli and C. albicans. This is probably due to the organism's natural classification as a strong biofilm producer, when compared with the two other strains, which may in part explain the difference in results when compared with C. albicans.
  • menthol:LA showed efficacy against the used E. coli strain in the biofilm removal assay despite the fact that no antibacterial activity was verified in the preliminary assay. SEM images allowed a better visualization of the effect of THEDES on a formed biofilm.

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Abstract

The present disclosure relates to a eutectic composition comprising menthol and a saturated fatty acid as a THEDES (therapeutic deep eutectic system), for use in medicine. The present disclosure further relates to the eutectic composition of menthol-fatty acids for use in therapeutic purposes such as for wound treatment, and/or for the treatment or prevention of microbial infection. The use of the eutectic composition as an enhancer of wound healing and as a drug resistance preventer/prophylactic agent is also encompassed.

Description

D E S C R I P T I O N
EUTECTIC MENTHOL- FATTY ACID COMBINATIONS FOR WOUND HEALING
Technical field
[0001] The present disclosure relates to a eutectic composition of menthol-fatty acid as a THEDES (therapeutic deep eutectic system).
[0002] The present disclosure further relates to menthol-fatty acids for use in therapeutic purposes such as for wound treatment, and/or for the treatment or prevention of microbial infection.
Background
[0003] Interest in Deep Eutectic Solvents (DES) has grown exponentially in the last decade due to their unique and attractive properties.
[0004] DES can be easily prepared by mixing two or more compounds at specific molar ratios. At certain molar ratio, the compounds self-associate and form a eutectic mixture that has a melting point that is lower than the melting point of each individual compound. This phenomenon, depression of melting point, can be attributed to the strong hydrogen bond interaction between a hydrogen bond donor (HBD) and a hydrogen bond acceptor (HBA).
[0005] The cost of preparing DES is low. The preparation process is straightforward and there is no need for post-synthesis purification. DES is non-flammable, has a broad range or polarity, and low volatility. It is dipolar in nature, exhibits chemical and thermal stability. Further, it has water-compatibility, is biodegradable and has negligible toxicity profile. The core characteristics of DES are similar to their analogues - ionic liquids (ILs). However, the green chemistry metrics of DES is better than ILs thus making DES a highly desirable and promising alternative solvent.
[0006] One of the most attractive features of DES is the possibility of creating up to 106 number of different combinations. This makes them an environmentally friendly designer solvent. The physicochemical properties of DES can be tailored by modifying parameters such as the hydrogen bond donating/accepting component, molecular structure, chemical nature, ratio polarizability, temperature, and/or water content.
[0007] Since their emergence, DES has attracted interests in several fields as solvents for organic synthesis, biocatalysis, electrochemistry, nanomaterials, polymer production, separation processes, CO2 capture, foods, cosmetics, pharmaceutics and biomedical applications. In biomedical applications, DES can be used to improve solubility, permeation and absorption of active pharmaceutical ingredients (API's). The API can be paired with a DES; this subclass of bioactive DES is termed therapeutic deep eutectic systems (THEDES). The present disclosure discloses the therapeutic effects of THEDES based on menthol and fatty acids.
[0008] Menthol is a terpene that can been extracted from Mentha species and it has already been used for THEDES preparation in combination with a wide range of compounds including ibuprofen, lidocaine, fluconazole, and among others, captopril. DES based on menthol and fatty acid combination have been disclosed in prior art. However, the therapeutic effect of menthol-fatty acid based THEDES has not been disclosed or evaluated by prior art. The potential of menthol-fatty acid based THEDES for use in the biomedical field was not previously explored.
[0009] Document US20180093011 discloses ionic liquids that sterilize and prevent biofilm formation on skin wound healing devices. The disclosure claims an ionic liquid with anti microbial potential. The disclosure further claims that the ionic liquid comprises deep eutectic solvent and choline geranate. No reference or claim was made to the composition of the deep eutectic solvent or to compounds that potentially have deep eutectic solvent characteristics. No claims or references were made to the combination of menthol and fatty acids as a DES.
[0010] Document US8496950 B2 discloses a mixture containing menthol. However, no disclosure was made with regards to a mixture based on the combination of menthol and fatty acids. Furthermore, no disclosure was made regarding the anti-bacterial effect of menthol or any wound healing effect.
[0011] Document CN 101157612 B discloses 17 ester derivatives of menthol and their preparation method. Although, the derivatives are obtained after a chemical reaction which involves the presence of an organic solvent. The patent further discloses the 17 derivatives as suitable agents which enhances transdermal absorption of a range of pharmacological compounds. Specifically, the patent claims the use of heptanoate menthol as an agent which enhances the transdermal absorption of diclofenac potassium diclofenac indole or indole. However, the patent does not disclose and evaluate the optimal menthol:fatty acid ratio for optimal wound healing therapeutic effect.
[0012] These facts are disclosed in order to illustrate the technical problem addressed by the present disclosure.
General Description
[0013] This disclosure discloses the thermophysical properties of menthol:fatty acid THEDES as well as its potential use for therapeutic purposes such as for wound treatment.
[0014] Fatty acids, commonly extracted from vegetal and animal fats, are secreted from human sebaceous glands and allows the human skin to naturally disinfect itself. The advantage of the menthol:fatty acid THEDES is that in addition to being an effective transdermal permeation enhancer, menthol and the fatty acid are also anti-inflammatory and antimicrobial. Additionally, the current disclosure discloses a menthol:fatty acid THEDES composition which is able to prevent skin flora, specifically Staphylococcus aureus, from developing resistance against the composition.
[0015] In an embodiment, the present disclosure relates to a eutectic composition for use in medicine comprising menthol and a saturated fatty acid.
[0016] In an embodiment, the eutectic composition can be used in the therapy or treatment of wounds and/or microbial infections.
[0017] In a further embodiment, the eutectic composition can be used in wound healing approaches, wherein the menthol and fatty acids promote the wound healing and the antibacterial effect.
[0018] In an embodiment, the eutectic composition can prevent bacterial infection in wounds.
[0019] In a further embodiment, the eutectic composition of the present disclosure can be used for prevention, the treatment or therapy of infections caused by Candida albicans, Escherichia coli, Staphylococcus aureus, or combinations thereof. [0020] An aspect of the present disclosure relates to a eutectic composition for use in medicine comprising menthol and a saturated fatty acid, preferably a saturated fatty acid comprising at least 10 carbons; more preferably wherein the saturated fatty acid comprises between 12 to 20 carbons.
[0021] Another aspect of the present disclosure relates to eutectic composition comprising menthol and a saturated fatty acid, wherein the saturated fatty acid comprises at least 10 carbons, preferably wherein the saturated fatty acid comprises between 12 to 20 carbon.
[0022] In an embodiment, the saturated fatty acid is selected from the following list: lauric acid (LA), stearic acid (SA), myristic acid (MA), or mixtures thereof.
[0023] In an embodiment, the menthol and the saturated fatty acid have a molar ratio of 2:1 - 10:1, preferably a molar ratio of 4:1 - 9:1, more preferably 6:1 - 8:1.
[0024] In an embodiment, the concentration of menthol may vary from 0.45 - 1.5 mM, preferably 0.5 to 1 mM.
[0025] In an embodiment, the concentration of saturated fatty acid may be from 0.04 to 0.75 mM, preferably 0.06 to 0.5 mM.
[0026] In an embodiment, the composition is a topical composition preferably a cream, a lotion or a gel.
[0027] An aspect of the present disclosure relates to a patch and/or kit comprising the eutectic composition from the previous embodiments.
[0028] In an embodiment, the present disclosure relates to the use of the eutectic composition as an enhancer of wound healing and as a drug resistance preventer/prophylactic agent.
Brief Description of the Drawings
[0029] The following figures provide preferred embodiments for illustrating the description and should not be seen as limiting the scope of invention.
[0030] Figure 1 - DSC thermograms obtained for powders (A) and THEDES, including: Menthol:LA (B) Menthol:MA (C) and Menthol:SA (D). Peaks arising above the baseline represent endothermic peaks. [0031] Figure 2 - 1H NMR spectra of the powders; (A) menthol; (B) LA; (C) MA and (D) SA. All the resonances are attributed.
[0032] Figure 3 - 1H NMR spectra of the THEDES; (A) menthol:LA; (B) menthol:MA and (C) menthol:SA. All the resonances are attributed.
[0033] Figure 4 - Variation of the viscosity of the different formulations of THEDES as a function of the temperature.
[0034] Figure 5 - Cytotoxic effect of menthol:LA (4:1) (A); menthol:MA (8:1) (B); menthol:SA (8:1) (C) and using HaCaT cell model treated for 24 hours. Results were expressed relatively to the control as mean ± SD of at least three independent experiments performed in triplicate.
[0035] Figure 6 - Wound healing assay. (A) Migration assessment of HaCaT cells after the treatment with menthol:SA (8:1), menthol and SA at 0 and 24 hours post-scratch. The lines indicated the boundary lines of the scratch at 0 hours. (B) Results were expressed in terms of percentage of wound closure relatively to the control using the mean ± SD of six independent experiments performed in duplicate. Statistically significant differences comparing the samples were calculated according to one-way ANOVA for multiple comparisons by Tukey's method.
[0036] Figure 7 - Wound healing assay. (A) Migration assessment of HaCaT cells after the treatment with menthol:LA (2:1), menthol and LA at 0 and 19 hours post-scratch.
[0037] Figure 8 - Representative images of disk diffusion assay plates obtained for individual counterparts, THEDES and controls. Plates are presented by bacterial strains tested. MIC/MBC values of individual counterparts and THEDES. Results are presented by bacterial strains tested. ND- Not dissolved.
[0038] Figure 9 - A - Schematic representation of an embodiment comprising the antibiofilm properties of menthol:LA 4:1. B - Percentage of biofilm removed upon exposure to different concentrations of menthol:LA (625, 1250 and 2500 mg/mL) for a total period of 30 minutes.
[0039] Figure 10 - Scanning electron microscope (SEM) images of surfaces after exposure with menthol:LA 4:1 from three distinct microorganisms seeded onto cover slips with different exposure times to menthol:LA eutectic blend. Results are presented by microorganism for the various exposure times applied. Scale bar is 10 mm. [0040] Figure 11 - Embodiment of the different molar ratios considered to prepare the THEDES compositions, with their respective visual aspect and POM micrographs. The scale bar is 200 mm.
Detailed Description
[0041] The present disclosure is further described, in particular, using embodiments of the disclosure. Therefore, the disclosure is not limited to the descriptions and illustrations provided. These are used so that the disclosure is sufficiently detailed and comprehensive. Moreover, the intention of the drawings is for illustrative purposes and not for the purpose of limitation.
[0042] The breakthroughs achieved in recent years by deep eutectic solvents boost the appearance of different subclasses of these green solvents, among which are therapeutic deep eutectic solvents. These open intriguing possibilities on their use in biomedical field.
[0043] In the present disclosure is described the biomedical potential of hydrophobic THEDES based in menthol and fatty acids, preferably lauric acid, myristic acid and stearic acid. The present disclosure also discloses different formulations, with an optimal molar ratio, as well as the intermolecular interactions behind the successful formation of THEDES. The evaluation of their biological performance was also performed towards bacteria and HaCaT cells. Among the different formulations of THEDES, the one based on menthol and SA, establishes stronger hydrogen bonding interactions, being also the most promising formulation as did not elicit any relevant cytotoxicity, and potentiate wound healing while presenting antibacterial properties against Staphylococcus aureus. This work provides clues on the future use of THEDES based on menthol:SA as potential candidates for the preparation of wound dressings.
[0044] In the present disclosure, a mixture based on menthol:fatty acids was used due to their effectiveness as permeation enhancer together with their well-known anti-inflammatory and antimicrobial properties. Despite the biological activity of the isolated compounds both compounds have never been used in THEDES form. This disclosure provides relevant information on the thermophysical properties of eutectic composition of the present disclosure as well as on their potential use for therapeutic purposes, namely for wound treatment. [0045] In an embodiment, the eutectic mixture was prepared. In one embodiment, the eutectic mixture was prepared using menthol (ref. M2772, Sigma Aldrich), lauric acid (LA; Ref. W261408-SAMPLE-K, Sigma Aldrich), myristic acid (MA; ref.70082, Sigma Aldrich) and stearic acid (SA; ref. 175366, Sigma Aldrich). The eutectic mixtures were prepared by gently mixing the two components (menthol and fatty acid) at different molar ratios. The mixture was heated to 70°C, with constant stirring, until a clear liquid solution was formed. The liquid solution is obtained after 30 min.
[0046] In one embodiment, the optical characterization of different formulations of THEDES was carried out at room temperature (RT - 22°C) using polarizing optical microscopy (POM). The micrographs of the small droplets deposited on the cover glass were obtained using an Olympus BH2 transmission microscope (Olympus, UK) coupled with a Leica digital camera DFC 280 (Leica, UK).
[0047] In one embodiment, the viscosity of THEDES was measured using a Kinexus Prot Rheometer (Kinuxus Prot, MaL 1097376, Malvern) fitted with parallel plate geometry (PU20 SR1740 SS). The viscosity of the different formulations was assessed under controlled stress conditions and at a shear rate of 10 s-1. After equilibrating the sample temperature at 15°C, a temperature scan was performed from 50°C to 15°C at 2°C/min.
[0048] In one embodiment, the Differential Scanning Calorimetry (DSC) experiments of the powders and THEDES were performed in a TA instrument DSC Q100 model (Thermal analysis & analysers, USA). The formulations were equilibrated at 40°C for 5 min followed by cooling to -40°C, an isothermal period for 5 min and a heating to 100°C at a 5°C /min.
[0049] In an embodiment, nuclear magnetic resonance (NMR) experiments were recorded using 400 MHz Bruker Advance II. 1H chemical shifts are expressed in ppm with the residual HOD solvent signal as reference. Mestrenova 9.0 software (Mestrelab Research) was used for spectral processing. THEDES and raw materials were dissolved in dimethyl sulfoxide-d6 (DMSO-d6, 99.9 atom %, LOT. STBH4385, Sigma Aldrich) to identify the OH vibrational state. All the experiments were performed when the systems were in equilibrium and no further change in their properties were observed.
[0050] In an embodiment, the HaCaT cell line (DFKZ, Germany) was cultured in DMEM medium supplemented with 10% (v/v) of heat-inactivated fetal bovine serum (FBS) and 1% (v/v) penicillin-streptomycin (PS). Cells were maintained at 37°C with 5% CO2 in a humidified incubator and routinely grown as a monolayer in 75 cm2 culture flasks. The cell culture medium and supplements were purchased from Invitrogen (Gibco, Invitrogen Corporation, UK).
[0051] In an embodiment, cytotoxicity assay was performed using confluent and differentiated HaCaT cells which represent 80% of epidermal cells. HaCaT cells were seeded into 96-well plates at a density of 4.5x104 cells/well and allowed to grow for 3 days. On the day of the assay, cells were incubated with the different menthol:fatty acid formulations diluted in culture medium. Cells incubated with only culture medium were used as control. After 24 hours of incubation, the cells were washed once with PBS (Sigma-Aldrich, USA) and cell viability was assessed, according to manufacturer's instructions, using CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega, USA) containing MTS reagent. Absorbance was measured at 490 nm using a Spark® 10M Multimode Microplate Reader (Tecan Trading AG, Switzerland). Cell viability was expressed in terms of percentage of living cells relative to the control. At least three independent experiments were performed in triplicates.
[0052] In one embodiment, cell migration was evaluated using HaCaT cells. Cells were seeded at a density of 1x105 cell/cm2 in a 12-well plate and allowed to grow until 100% confluence (48 hours). A wound was created with a standard 200 mL pipette tip and each well washed twice with warm PBS to remove the non-adherent cells. Menthol:SA mixture in 8:1 molar ratio was prepared by incubating 1.50mM of menthol with 0.19mM of SA or 0.75mM of menthol with 0.09mM of SA for 24 hours. Menthol:LA mixture in 2:1 molar ratio was prepared by incubating 0.75mM of menthol with 0.375mM of LA for 18 hours. Photos were taken by inverted phase contrast microscope (Leica DM6000, Germany) at two different time points: 0 and 24 hours (menthol:SA) or 18 hours (menthol:LA). Image analysis was performed using ImageJ software and wound area was measured between the borderlines. Wound area recovered was expressed in terms of percentage using the following equation:
Figure imgf000009_0001
[0053] The results were expressed in terms of mean ± Standard Deviation of six independent experiments performed in duplicates.
[0054] In an embodiment, THEDES antimicrobial activity was analysed using agar diffusion assay. The antibacterial activity of eutectic blends was determined using Staphylococcus aureus ATCC 25923 and ATCC 700698 (Methicil lin-resista nt strain), Staphylococcus epidermis ATCC 35984 (Methicillin-resistant strain); Pseudomonas aeruginosa ATCC 27853 and Escherichia coli ATCC 25922, according to the guidelines of Clinical and Laboratory Standards Institute (CLSI). Gentamicin sulphate (ref. G1914, Sigma Aldrich) at 50 mg/mL and sterile water were used as positive and negative control, respectively. The discs (CT0998B, Oxford) were prepared by incubating them in THEDES and controls. Prior to the disk diffusion assay, suspension cultures were prepared by the inoculation of single colonies in 5 mL of tryptic soy broth (TSB, ref CM0337B, Frilabo). The cultures were grown for 24 h at 37°C in an orbital shaker at 180 rpm. Afterwards, bacteria cells were harvested and re-suspended in TSB and the absorbance at 640 nm was adjusted to a turbidity of 0.5 McFarland scale, which provide an equivalent concentration 1-2x108 colony forming units (CFUs)/mL. The inoculum was then distributed in Muller-Hinton agar (MHA, ref. CM0337B, Fisher Scientific) plates and allow to dry for 5 minutes. Then, the disks impregnated with the different formulations were placed on the inoculated agar.
[0055] The minimal inhibitory concentrations (MICs) and minimal bactericidal concentration (MBCs) were determined by microbroth dilution technique. Briefly, standard solutions of menthol and fatty acids in powder and THEDES form were prepared under sterile conditions by first dissolving the weighing compounds in DMSO (ref. 276855, Sigma), followed by serial dilution in Muller-Hinton Broth medium (MHB, ref 70192-500g, Sigma Aldrich) maintaining a 10%(V/V) percentage of DMSO. During the dilution process, the microtubes were warmed up to 50°C and homogenized using an ultrasonic bath until no precipitation was observed. Afterwards, the microtubes containing the different formulations (500 mL /tube) were inoculated with 500 mL of bacterial suspension at 1-2x106 CFUs/mL in MHB. As control, MHB (i.e., bacteria-free), MHB (with no added compounds), a solution of 10% DMSO in MHB and the pure components were used. The need for the usage of microtubes instead of traditional 96-well plates arose from menthol's volatility which could possibly lead to cross-well contamination. The tubes were incubated at 37°C under shaking for 24 hours and the bacterial growth was monitored. The MICs of each compound were considered to be the concentrations at which the tested formulations prevent the turbidity in the well. However, to confirm such results and to avoid underestimated values (i.e., the compounds itself may cause turbidity), subcultures of each concentration were performed onto trypticase soy agar (TSA, Ref. 610052, Frilabo) during 24 hours. This assay allows to confirm the MICs values and also the determination of the MBCs of each compound. The MBCs were considered as the minimal concentration of each formulation required to kill S. aureus CFUs. Experiments were carried out in triplicate and repeated in three independent occasions.
[0056] The statistical analysis was performed with all data expressed as mean ± standard deviation (SD). GraphPad Prism 6 software was used to calculate EC50 values (the concentration of sample necessary to decrease 50% of cell population) and to analyse significant differences between data set through One-Way Analysis of Variance (ANOVA) following Tukey post hoc test. A p-value < 0.05 was considered significant.
[0057] The capability to induce biofilm degradation and removal was studied using the menthol:LA formulation. In an embodiment, S. aureus ATCC 700698, C. albicans ATCC 90029 and E. coli ATCC 25922 were spread into TSA plates and grown overnight at optimal temperature for each organism, 37°C and 30°C for bacteria and yeast, respectively. Posteriorly, suspension cultures were prepared by inoculating single colonies into TSB and posterior incubation for 24 hours at optimal temperatures. Cultures were then harvested, resuspended in TSB and adjusted to approximately 1-2x104CFUs/mL. Posteriorly, 200 mL of the previously prepared bacterial suspension was transferred into wells, in a 96-well polystyrene flat-bottom plate and incubated for an additional 20 hours, at optimal temperature for biofilm formation.
[0058] Following biofilm formation, microbial suspensions were carefully removed, and sample solutions added. After different exposure times (5, 10, 20 and 30 minutes) sample solutions were removed and 200 mL of MHB media containing 10%(v/v) alamar blue dye (ref. BUF012B, Bio-Rad) was added into the wells, followed by plate incubation for 1 hour, at either 37°C or 30°C, protected from light. Posteriorly, the absorbance of each sample was acquired at 570 and 600 nm using a microplate reader (SYNERGY HT™), and used to calculate removal efficiency based on values obtained for biofilm treated with deionized water. SEM images were also obtained using a JEOL JSM-6010 LV (JEOL, Japan). Biofilms were established in cover slips (ref. 83.1840.002, Sarstedt) for 20 hours and treated with the sample solutions for the prementioned time points. Posteriorly, sample solutions were gently removed, and microorganisms fixed using a 10% (v/v) formalin in PBS solution for 1 hour. Fixed biofilm was then dehydrated by immersion in solutions with increasing concentrations of ethanol (50%, 70%, 90% and 100%). Finally, samples were left air-drying to remove residual ethanol, followed by gold-sputtering for SEM image acquisition.
[0059] In an embodiment, different molar ratios of the compounds in their solid state have been prepared by simple mixing of menthol and fatty acid either at equimolar or imbalanced ratios (Figure 11).
[0060] In an embodiment, Figure 11 shows a summary of the different THEDES composition with their respective visual aspect and POM micrographs. The scale bar is 200 mm.
[0061] In an embodiment, DSC analysis was also performed to assess thermal events, namely the variations on the melting point of the eutectic mixture when compared with the parent species (Figure 1). A depression on the melting point represents a successful formation of THEDES. The thermogram of racemic menthol presents two melting points at »28°C and »33°C, which have been ascribed to alpha and beta polymorphs, and is in good agreement with previous reported data. In the thermogram of each fatty acid, a well-defined and sharp endothermic peak was obtained at » 46.6°C, »58.6°C, »73°C, for LA, MA, SA, respectively. The thermograms of the individual starting materials corroborated previous data in the literature. The peaks obtained in THEDES are different from the ones of the parent species, which further suggests the supramolecular rearrangement while the compounds are in THEDES form. Additionally, a clear depression on the melting point of the parent species can be observed. The thermograms of THEDES indicate that, depending on the molar ratio, the intensity and shift of the peaks is different, which was also expected and previously reported for other THEDES. Thus, menthol was combined with LA, MA, SA at 4:1, 8:1 and 8:1, respectively. In each case, a full black image was obtained with POM and a DSC thermogram with an endothermic peak was also achieved, showing a depression on the melting point of the individual compounds, proving the adequate molar ratio for the preparation of a eutectic mixture (THEDES).
[0062] The establishment of hydrogen bonding between each parent species was then assessed using NMR spectroscopy. NMR is commonly used to elucidate the types of interactions, as well as the atoms of each counterpart involved, allowing insights on hydrogen bonding network. Figures 2 and 3 show the 1H NMR spectra of powders and THEDES. One of the differences between the spectrum of powders and the one of THEDES is the chemical signals of the hydroxyl groups of menthol. The powder spectrum (Figure 2A) of menthol presents a well-defined doublet (d= 3.9 ppm), whereas in the THEDES spectrum (Figure 3) a larger singlet was obtained without any further upward or downward chemical displacement. Additionally, the other evidence of the establishment of hydrogen bonding is the signal from proton (H9) bonded to the same carbon (C9) of the hydroxyl group from menthol. The establishment of hydrogen bonding is further proven by the disappearance of the hydroxyl group of fatty acids in THEDES spectrum compared, while in the powder's spectrum of fatty acids the sharp and defined signals were obtained in the expected chemical shift (d=11.5-11.8 ppm). (Figure 2). The overall data indicates that the hydrogen bonds are established between the hydroxyl groups from menthol (hydrogen bond donor) and the carboxyl group from fatty acids (hydrogen bond acceptor). The NMR data supports the POM and DSC, as an extensive hydrogen bonding network was observed for the evaluated molar ratios.
[0063] After this initial screening, the viscosity of the different THEDES was evaluated at constant shear rate and as a function of temperature. The viscosity is an important feature in THEDES, as it dictates the mass transport phenomena and affecting their suitability for therapeutic applications. As expected, the viscosity of the systems decreases as the temperature increase, which is in accordance with the Arrhenius equation (Figure 4). The menthol:fatty acid formulation with lowest viscosity was menthol:LA, followed by menthol:MA and menthol:SA. In menthol:SA, it was only possible to evaluate the viscosity up to 30°C, at lower temperatures the system is in solid phase, as shown in the DSC thermogram. The higher viscosity of menthol:SA can be attributed to their extensive hydrogen bonding interactions, which corroborated with the 1H NMR data. The data obtained also corroborated with literature, where the ability to adjust the viscosity of THEDES by the nature of starting compounds and temperature has been described. Additionally, the viscosity of these THEDES are relatively low, which is a valuable feature as it allows their manipulation and facilitate their potential applications without the need to, for example, add water in the formulations. The tailoring of viscosity of THEDES is usually performed by the addition of water, which gradually weakens the hydrogen bonding interactions and may eventually lead to their complete disappearance.
[0064] In an embodiment, the bioactivity of THEDES was assessed. Menthol-based THEDES were tested aiming at evaluating the cytotoxicity effect in order to select the concentrations tested in further assays. Comparing the effect of isolated compounds none of the fatty acids showed cytotoxicity, as Figure 5 (A, B, C) shows. Assessing THEDES cytotoxicity, menthol:LA at molar ratio of 4:1 showed higher cytotoxicity with EC50 value of 5.569 ± 0.326 mM of equivalent menthol (Figure 5A and Table 1). Moreover, this system showed similar cytotoxicity with pure menthol. Comparing menthol with other formulation of THEDES (menthol:MA (8:1) and menthol:SA (8:1)), the isolated compounds had higher cytotoxic activity (Figures 3B and 3C). The presence of MA and SA compounds in the systems decrease the cytotoxicity, which further suggest the establishment of hydrogen bonding between menthol and fatty acid and the successful formation of a eutectic mixture.
[0065] Table 1 shows the EC50 values for different THEDES systems using different molar ratios.
Figure imgf000014_0001
[0066] In an embodiment, menthol:SA at molar ratio of 8:1 was selected to evaluate menthol:fatty acid THEDES wound healing properties. Three non-cytotoxic concentrations of menthol:SA at molar ratio of 8:1 were selected and the ability of HaCaT cells to migrate was assessed using the wound healing assay. As Figures 6A and 6B show, the two highest menthol:SA (8:1) concentrations significantly induce cell migration leading to higher wound enclosure (areas of 66.00 ± 5.92% and 70.50 ± 4.28%) as compared to the control (53.23 ± 5.35%), representing an increase of nearly 40%. Moreover, isolated menthol and SA did not show strong effect on cell migration, with the exception of menthol at a concentration of 0.75 mM. The THEDES (0.75 mM menthol + 0.09 mM SA) is shown to be more effective than isolated menthol. This suggests that the hydrogen bonding interaction between SA and menthol potentiates cell migration activity. Menthol is a terpene highly explored as anti- inflammatory, antiseptic and antipruritic, therefore has a strong application in skin disorders. These results may reflect the potential bioactive properties of menthol:SA (8:1) over menthol for topical applications due to its high capacity of inducing cell migration.
[0067] In an embodiment, menthol:LA at molar ratio 2:1 was selected to evaluate the wound healing properties. Non-cytotoxic concentrations were chosen and as depicted in figure 7 it is possible to observe that the systems lead to higher wound enclosure as compared to the control. Again, the system menthol:LA has shown a similar effect to menthol alone, but LA did not demonstrate any significant effect.
[0068] In an embodiment, after confirming the biological activity of menthol:SA in wound healing by analysing migration of HaCaT cells, the antibacterial properties were analysed using the most promising concentrations of these THEDES, for carrying out the agar diffusion method. The results are depicted in Figure 8. According to the performed experiments menthol has a MIC value for S. aureus of 625 ug/ml. Stearic acid does not have any values reported in literature, which might be due to its low solubility. Literature only validates that for concentrations lower than 1 mg/mL, stearic acid does not have antimicrobial effect.
[0069] In general, all compositions demonstrate antimicrobial activity. Menthol shows antimicrobial activity in all cases, being more efficient at higher concentrations, as expected.
[0070] In embodiment, according to the molar ratio, 1 mg of Menthol:SA (8:1) composed of about 0.82 mg of menthol and 0.18 mg of stearic acid. Hence the amount of menthol required for the same effect is lower than that the pure component, suggesting a synergistic effect of menthol and the fatty acid. [0071] In an embodiment, when the THEDES is screened for antibacterial activity, it is possible to observe two different effects. For the bacteria tested, THEDES has shown promising antimicrobial activity against the gram-positive strains employed while against the gram- negative strains, at tested concentrations, no significant antibacterial effect was found. However, such results are not surprising as several instances in literature report greater difficulty in dealing with gram-negative bacteria due to their more complex membrane structure. As the systems comprising stearic acid tend to precipitate, no diffusion of the system is observed to the media, hence limiting the conclusions that can be taken from the test. The determination of the MIC and MIB can evidence the differences in the systems (Figure 8, table). Despite the fact that the MIC and MBC concentrations are similar when we compare menthol with the THEDES form, the concentration of menthol present is lower in the THEDES form, proving that the presence of stearic acid in the system potentiates the antimicrobial activity and leads to a decrease in menthol concentration for the same observed effect.
[0072] In an embodiment, the developed THEDES were also tested considering their antibiofilm properties (Figure 9). The addition of THEDES formulation prepared from menthol:LA with a molar ratio of 4:1 caused a biofilm removal, whose extent was dependent on the bacterial strain tested. Overall, higher concentrations of THEDES resulted in a more effective removal of the biofilm. Regardless, it was noticed a temporal dependency of the removal capacity, particularly when the smallest concentration was used. Analysing the obtained results, it becomes clear that menthol:LA is able to actuate overtime upon the tested organism's biofilm leading to its death/dispersion. This effect is more pronounced on C. albicans which is gram-positive like. On the other hand, E. coli being a gram-negative bacterium, requires a higher concentration (2500 mg/ml) and greater actuation time (10 minutes) for any relevant effect. Furthermore, MRSA show itself as a mid-point in effectiveness when compared with E. coli and C. albicans. This is probably due to the organism's natural classification as a strong biofilm producer, when compared with the two other strains, which may in part explain the difference in results when compared with C. albicans. Additionally, it should be highlighted that menthol:LA showed efficacy against the used E. coli strain in the biofilm removal assay despite the fact that no antibacterial activity was verified in the preliminary assay. SEM images allowed a better visualization of the effect of THEDES on a formed biofilm. As depicted in Figure 10, the addition of the DES reduced the surface area covered by bacteria, as compared to non-treated surfaces. The importance of the temporal factor on DES antibiofilm properties is also clearly showed. A progressive dispersion of the biofilm, over time, in all microorganisms subjected to DES exposure can be observed. This is possibly the result of a perturbation of the biofilm's polymer matrix, as highlighted before, leading both to detachment and enhanced permeation of menthol:LA.
[0073] Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and/or the understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. It is also to be understood that unless otherwise indicated or otherwise evident from the context and/or the understanding of one of ordinary skill in the art, values expressed as ranges can assume any subrange within the given range, wherein the endpoints of the subrange are expressed to the same degree of accuracy as the tenth of the unit of the lower limit of the range.
[0074] The term "comprising" whenever used in this document is intended to indicate the presence of stated features, integers, steps, components, but not to preclude the presence or addition of one or more other features, integers, steps, components or groups thereof.
[0075] The above described embodiments are combinable. The following claims further set out particular embodiments of the disclosure.
[0076] References
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[0078] M.A.R. Martins, E.A. Crespo, P.V. Pontes, L.P. Silva, M. Büllow, G.J. Maximo, E.A.C. Batista, C. Held, S.P. Pinho, J.A. Coutinho, Tunable hydrophobic eutectic solvents based on terpenes and monocarboxylic acids, ACS Sustainable Chemistry & Engineering (2018). [0079] B.D. Ribeiro, C. Florindo, L.C. Iff, M.A. Coelho, I.M. Marrucho, Menthol-based eutectic mixtures: hydrophobic low viscosity solvents, ACS Sustainable Chemistry & Engineering 3(10) (2015) 2469-2477.
[0080] U. Juergens, M. Stöber, H. Vetter, The anti-inflammatory activity of L-menthol compared to mint oil in human monocytes in vitro: a novel perspective for its therapeutic use in inflammatory diseases, European journal of medical research 3(12) (1998) 539-545.
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1642.

Claims

C L A I M S
1. A eutectic composition for use in medicine comprising menthol and a saturated fatty acid.
2. The eutectic composition according to the previous claim for use in the therapy or treatment of wounds and/or microbial infection.
3. The eutectic composition for use according to any of the previous claims for use in wound healing.
4. The eutectic composition for use according to any of the previous claims wherein the menthol and fatty acids promote the wound healing and the antibacterial effect.
5. The eutectic composition for use according to any of the previous claims for the prevention of bacterial infection in wounds.
6. The eutectic composition for use according to any of the previous claims for the prevention, the treatment or therapy of infections caused by Candida albicans, Escherichia coli, Staphylococcus aureus, or combinations thereof.
7. The eutectic composition for use according to any of the previous claims consisting of menthol and a saturated fatty acid.
8. The eutectic composition for use according to any of the previous claims, wherein the saturated fatty acid comprises at least 10 carbons.
9. The eutectic composition for use according to any of the previous claims wherein the saturated fatty acid comprises between 12 and 20 carbons.
10. The eutectic composition for use according to any of the previous claims wherein the saturated fatty acid is selected from the following list: lauric acid, myristic acid, stearic acid, or combinations thereof.
11. The eutectic composition for use according to any of the previous claims wherein the menthol and the saturated fatty acid have a molar ratio of 2:1 - 9:1.
12. The eutectic composition for use according to any of the previous claims wherein the menthol and the saturated fatty acid has a molar ratio of 4:1- 8:1.
13. The eutectic composition for use according to any of the previous claims wherein the concentration of menthol is 0.45 to 1.5 mM, preferably 0.5 to 1 mM.
14. The eutectic composition for use according to any of the previous claims wherein the concentration of saturated fatty acid is 0.04 to 0.75 mM, preferably 0.06 to 0.5 mM.
15. The eutectic composition for use according to any of the previous claims wherein said composition is a topical composition.
16. The eutectic composition for use according to any of the previous claims wherein said composition is a cream, a lotion or a gel.
17. A patch comprising the eutectic composition described in any of the previous claims.
18. A kit comprising the eutectic composition described in any of the previous claims.
19. Use of the composition according to any of the previous claims as an enhancer of wound healing and as a drug resistance preventer/prophylactic agent.
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CN115449229A (en) * 2022-10-17 2022-12-09 广东省农业科学院蚕业与农产品加工研究所 Modified chitosan/gelatin composite film and its preparation method and application

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