EP3930772A1 - Medical aid for curing skin pathologies and corresponding production method - Google Patents

Medical aid for curing skin pathologies and corresponding production method

Info

Publication number
EP3930772A1
EP3930772A1 EP20714706.7A EP20714706A EP3930772A1 EP 3930772 A1 EP3930772 A1 EP 3930772A1 EP 20714706 A EP20714706 A EP 20714706A EP 3930772 A1 EP3930772 A1 EP 3930772A1
Authority
EP
European Patent Office
Prior art keywords
hydrogel
hydrolat
medical aid
volume
citrus aurantium
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP20714706.7A
Other languages
German (de)
French (fr)
Inventor
Maura DI VITO
Paola MATTARELLI
Monica MODESTO
Francesca BUGLI
Francesca Mondello
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Universita di Bologna
Universita Cattolica del Sacro Cuore
Original Assignee
Universita di Bologna
Universita Cattolica del Sacro Cuore
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Universita di Bologna, Universita Cattolica del Sacro Cuore filed Critical Universita di Bologna
Publication of EP3930772A1 publication Critical patent/EP3930772A1/en
Withdrawn legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
    • A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
    • A61L15/40—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons containing ingredients of undetermined constitution or reaction products thereof, e.g. plant or animal extracts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/75—Rutaceae (Rue family)
    • A61K36/752—Citrus, e.g. lime, orange or lemon
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
    • A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
    • A61L15/22—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons containing macromolecular materials
    • A61L15/34—Oils, fats, waxes or natural resins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
    • A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
    • A61L15/42—Use of materials characterised by their function or physical properties
    • A61L15/44—Medicaments
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
    • A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
    • A61L15/42—Use of materials characterised by their function or physical properties
    • A61L15/46—Deodorants or malodour counteractants, e.g. to inhibit the formation of ammonia or bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
    • A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
    • A61L15/42—Use of materials characterised by their function or physical properties
    • A61L15/60—Liquid-swellable gel-forming materials, e.g. super-absorbents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
    • A61L2300/30—Compounds of undetermined constitution extracted from natural sources, e.g. Aloe Vera
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
    • A61L2300/404—Biocides, antimicrobial agents, antiseptic agents

Definitions

  • the present invention concerns a medical aid for the treatment of skin pathologies and a method to produce the medical aid, generally able to be used to treat skin lacerations preventing the onset of microbial infections that are also resistant to conventional drugs.
  • Medical aids are known, typically in the form of gauzes or gauze compresses, used in the treatment of skin pathologies, such as, for example, lacerations, wounds, bums.
  • the gauzes used fall within the type of medical aid defined as pre-medicated, that is, they are prepared for use by soaking them with one or more drugs that have disinfectant, pain soothing and regenerative effects on the damaged skin tissues when applied in the area of the lesion, or in the so-called “bed” of the lesion.
  • the substances used to prepare these pre-medicated gauzes typically comprise small percentages of natural antimicrobials, such as, for example, essential or synthetic oils (for example, Bumshield or Bumcare gauzes) with which the gauzes are soaked before being used.
  • natural antimicrobials such as, for example, essential or synthetic oils (for example, Bumshield or Bumcare gauzes) with which the gauzes are soaked before being used.
  • pre-medicated gauzes use a hydrogel that allows to keep the lesion area humid and to promote debridement or autolytic debridement, granulation and epithelization.
  • a first disadvantage is that due to the use of a hydrogel to prepare the pre- medicated gauzes, the latter can become occlusive and, due to the saturation of water, the absorption of exudate is limited, favoring, as a consequence, microbial proliferation.
  • Another disadvantage is that the treatments to care for skin lesions performed using known pre-medicated gauzes produce skin maceration processes and the development of bad smells.
  • One purpose of the present invention is therefore to make available a medical aid for the treatment of skin pathologies that allows to medicate the damaged skin surface, to prevent the adhesion of the aid to wounds, to promote the immune response of patients, to prevent microbial superinfections also resistant to conventional drugs.
  • Another purpose of the present invention is to provide a medical aid for the treatment of skin pathologies which allows to use a medicament to replace essential or synthetic oils with another substance which is hydrophilic and economical, coming from waste from the production process of essential oils.
  • Another purpose of the present invention is to perfect a method to produce the medical aid which is simple and inexpensive.
  • the Applicant has devised, tested and embodied the present invention to overcome the shortcomings of the state of the art and to obtain these and other purposes and advantages.
  • a medical aid to treat skin pathologies, comprising a support for skin application and a medicament associated with the support and intended to be put in contact with an injured area of skin.
  • the medicament comprises a hydrogel which has the following composition: gellan gum gel, calcium acetate, hydrophilic hydrolat of Citrus aurantium flowers, preservatives for human topical use.
  • the gellan gum gel is comprised between 1% and 3% by volume with respect to a total volume of the hydrogel.
  • the calcium acetate is comprised between 0.03% and 0.05% by volume with respect to a total volume of the hydrogel.
  • the hydrophilic hydrolat of Citrus aurantium flowers is comprised between 45% and 55% by volume with respect to a total volume of the hydrogel.
  • the preservatives for human topical use are used in a mixture with a concentration comprised between 0.15% and 0.35% by volume with respect to a total volume of the hydrogel.
  • the preservatives are compounds known in the state of the art, for which the responsible authorities have authorized use for human topical use.
  • they can comprise phenoxyethanol 0.35%, benzoic acid 0.1%, dehydroacetic acid 0.05%.
  • the experimental tests conducted, described in detail below, have shown that the introduction of preservatives, which on their own at the final tested concentrations do not show any antibacterial or antimicrobial action, allows to enhance the antimicrobial action of the hydrolat, as well as guarantee the correct preservation thereof at least until the expected expiry date.
  • the hydrogel is in a solid state.
  • the support for skin application is a gauze made of fabric.
  • the support is configured as a suitable carrier intended to guarantee a cutaneous application of the medicament.
  • the carrier that is, the inactive basic substance which allows to administer and therefore use the medicament and, in some cases, also regulates its release on the site of action, can for example be a cream or an ointment.
  • the hydrophilic hydrolat of Citrus aurantium flowers comprises hydrophilic hydrolat of Citrus aurantium flowers in the subspecies amara.
  • a method is provided to produce a medicament associable with a support for skin application that provides to create a volume of hydrogel by dissolving a second quantity of gellan gum powder in a first quantity of water, by heating it to a temperature higher than 40°C.
  • the first and the second quantities form a solution which has a volume equal to 50% of the final volume of the hydrogel.
  • the third quantity has a volume equal to 50% of the final volume of the hydrogel.
  • the cooling takes place at a temperature lower than 40°C.
  • hydrophilic hydrolat of Citrus aurantium flowers in particular in its subspecies amara, to cure pathologies of the skin.
  • the medical aid according to the present invention prevents the proliferation of pathogenic microorganisms in skin lesions, and at the same time stimulates the local immune response of patients, thus accelerating their healing.
  • One advantage of the medical aid according to the present invention is that it counteracts the development of bad smells caused by the process of skin maceration that often occurs in the presence of lesions and/or bums, thanks to the presence of the hydrolat of Citrus aurantium flowers.
  • the medical aid according to the invention can be made in the form of a hydrogel with which to soak gauzes or compresses (or other similar supports).
  • a hydrogel with which to soak gauzes or compresses (or other similar supports).
  • it can easily be mixed with the hydrogel. The result is a method to produce the medicament that is economical and effective.
  • the packages that contain the pre-medicated gauzes have to be marketed in packages able to preserve their sterility and prevent their dehydration, able to also be stored at room temperature without there being a deterioration of the medical aid.
  • the medical aid according to the invention for example in the form of pre medicated gauze, is able to prevent the development of microbial infections by exerting a cytocidal action on a wide spectrum of microbial species generally involved in human skin infections.
  • hydrogels allow the gradual release over time of the active ingredients contained therein and therefore a continuous and gradual treatment of the damaged skin area.
  • the patient can heal quickly, reducing the convalescence period and avoiding healthcare costs correlated to the development of infected wounds.
  • Another advantage of the medical aid according to the present invention is that it is ecological, given the presence of hydrolat of Citrus aurantium flowers, which is actually a vegetable product obtained as a waste product during the processing of the corresponding essential oil of Citrus aurantium, in particular in its subspecies amara.
  • - fig. 1 is a histogram showing the results of the cytotoxicity tests conducted on gingival fibroblast cells treated with substances released over 24 hours by hydrogels formulated with 50% of hydrolat of Citrus aurantium flowers subspecies amara (a) or hydrolat obtained from flowering tips of Monarda fistulosa (b);
  • - fig. 2 a is a graph showing the overlapping of chromatograms for gellan with hydrolat of Citrus aurantium flowers subspecies amara at different times: lh, 3h (diluted 1 : 10), 6h, 24h, 30h (diluted 1 :5);
  • - fig. 2 b is a graph showing the overlapping of chromatograms for gellan with hydrolat of Monarda fistulosa MF at different times: lh, 3h, 6h (diluted 1 :3);
  • - fig. 3 shows a histogram showing the release of carvacrol from gellan made with hydrolat of Monarda fistulosa.
  • Embodiments described here concern a data processing method, of which particular forms of embodiment will be described below, as a non-limiting example.
  • the aim of this study was to identify the most effective antimicrobial hydrolates as cytocidal activity among some selected ones.
  • the activity of the hydrolates was tested against isolated clinical microbial strains and ATCC reference strains, sensitive and resistant to antibiotics.
  • Cytocidal capacity of the following hydrolates was tested by means of in vitro broth microdilution test, carried out in 96-well plates and according to EUCAST international guidelines: Monarda citriodora, Monarda fistulosa, Monarda didyma, Lavandula vera, Citrus aurantium subspecies amara from flowers, the latter marketed by Magentina srl.
  • the effectiveness values were expressed in terms of Minimum Inhibitory Concentration (MIC) and Minimum Fungicidal or Bactericidal Concentration (respectively, MFC or MBC), both identified respectively in the lower concentration of hydrolat present in the well able to inhibit microbial growth or have a cytocidal effect with a visible absence of turbidity (microbial growth index) of the medium.
  • MIC Minimum Inhibitory Concentration
  • MFC or MBC Minimum Fungicidal or Bactericidal Concentration
  • the MIC values were identified by means of visualization and confirmed with OD reading (450 nm) at the spectrophotometer, while the values of the cytocide concentrations were identified by seeding the contents of the wells on suitable growth medium (Sabouraud for fungi and Mueller Hinton for bacteria) and reading the result of seeding after 24h of incubation.
  • the aim of the study is to analyze the effective inhibiting and cytocidal effectiveness of the hydrolat of Citrus aurantium subspecies amara.
  • the broth microdilution analyses were repeated on the various microbial strains and according to the method indicated in Example 1, testing, in parallel with the hydrolates, the mixture of preservatives in the proportions used for the formulation.
  • the abbreviation CC indicates “with preservative”
  • the abbreviation SC indicates “without preservative”
  • the abbreviation C indicates the preservative on its own.
  • results obtained show that the preservative on its own, at the concentrations tested in the broth microdilution assay, is not able to inhibit microbial growth, nor to kill the tested strains.
  • the hydrolat of Citrus aurantium subspecies amara on its own is effective at higher concentrations than those identified in the association of hydrolates plus preservatives.
  • the action of the hydrolat is enhanced by the presence of preservatives; this indicates a synergy between the hydrolat and the preservatives when combined in the final formulation.
  • Example 3 STUDY OF THE TERPENE COMPONENTS OF THE HYDROLATES OF CITRUS AURANTIUM SUBSPECIES AMARA AND OF MONARDA FISTULOSA
  • the aim of the study was to identify the terpene components of the hydrolates of Citrus auratium subspecies amara and Monarda fistulosa.
  • a liquid-liquid extraction was carried out by mixing an amount of 0.5 ml of hydrolates with 0.5 ml of heptane with tridecane as internal standard.
  • a shaker (Retsch-MM300, Haan, Germany) was used for lh at 4 cycles per second.
  • the supernatants were used for the analysis after centrifugation at 4000 rpm for 10 minutes at 10°C in an Eppendorf mod. 5810R (Westbury, NY) centrifuge.
  • the heptane extracts were then filtered with 0.45 pm PTFE syringe filters and injected (3 pi) into the GC-MS system of Agilent Tech (Palo Alto, AC, USA).
  • the GC injection temperature was 250°C, splitless mode, and heating was programmed at 40°C for 1 min, followed by a ramp of 5°C / min at 200°C and of 10°C / min at 260°C.
  • VOC was made on the basis of the peak of correspondence with the spectral database of the library (NIST 08) and of the kovats indices.
  • the quantity of each monoterpene was expressed as a percentage of the total monoterpenes.
  • Table 3 is shown below, comprising the results of the GC analysis of the hydrolates of Citrus aurantium subspecies amara and of Monarda fistulosa, with the values expressed as a percentage.
  • the aim was to develop a pre-medicated gauze model to be used in in vitro studies aimed at evaluating antimicrobial effectiveness.
  • gauzes formulated with only 2% v/v gellan with gellan (2% v/v) + hydrolat at 50% v/v ⁇ Citrus aurantium subspecies amara or Monarda didyma or Monarda fistulosa ) were seeded with 10 microliters of a 0.5 McF suspension of each bacterium or fungus indicated in point 1.
  • Table 4 below indicates the bacterial and fungal growth detected on a culture medium after the application of gellan gels, modified or not, previously seeded with the strains (fungal or bacterial) under study.
  • CTR + Positive control; (-) no growth; (+) rare colonies; (++) moderate microbial growth; (+++) strong microbial growth; (++++) blanket growth.
  • the study aimed to evaluate the cytotoxic action of the gauzes formulated with gellan hydrogel + hydrolat of Citrus aurantium subspecies amara or hydrolat of Monarda fistulosa.
  • gingival fibroblast cells which were treated with culture medium previously incubated, for 24 hours, with gellan hydrogel at 2% v/v and hydrolat of Citrus aurantium subspecies amara or hydrolat of Monarda fistulosa.
  • the cytotoxicity of the hydrogels was assessed through the MST test (Promega, Madison, WI, USA test).
  • the treated culture medium was used unaltered (TQ) and diluted at 50% (1 :2), 25% (1 :4) and 12.5% (1 :8) in base medium.
  • the cells were resuspended in 200 m ⁇ of base medium and seeded in 96-well plates and incubated for 24h until a subconfluent monolayer was obtained.
  • the MTS test was performed in accordance with the operating protocol established by the manufacturer, and each experiment was done in triplicate. With reference to fig. 1, it can be observed that the substances released by the hydrogel into the culture medium after 24h incubation were toxic to the gingival fibroblasts treated with the medium conditioned by the hydrogel formulated with hydrolat of Monarda fistulosa, while they show no cytotoxicity for those treated with the hydrogel formulated with hydrolat of Citrus aurantium subspecies amara.
  • the stated goal was to evaluate the release kinetics of the active ingredients over 24 hours, the estimated time a medication remains in place.
  • hydrogel of gellan at 2% v/v as control and hydrogel of gellan 2% modified with hydrolat of Citrus aurantium subspecies amara or with hydrolat of Monarda fistulosa at 50% v/v were prepared in 6 cm diameter plates; 5 mL of physiological solution were deposited on the surface of the hydrogels and incubated for lh, 3h, 6h, 24h and 30h.
  • a THERMOQUEST HPLC system (produced by Shimadzu, Kyoto, Japan) was used for the analyses, equipped with two LCGA pumps and a UV/Vis SPD 10A detector (Shimadzu).
  • a UV-Vis lamp was connected at the end of the column, set to the absorbance wavelength of the analyzed compounds.
  • this shows the trend as a function of the time of release of the carvacrol (the two characteristic peaks at 5.7 minutes and 6.3 minutes were taken), which is the main component of the hydrolat of Monarda fistulosa identified in the sample obtained from the gellan hydrogel made with the same hydrolat.
  • the greatest release of the active ingredients occurs in the first 3 hours of contact, and then gradually decreases up to the 30 hours.
  • the figure shows how the intensity of the two peaks corresponding to the carvacrol is greatest after the first and third hour, while it tends to decrease in the following hours.
  • This datum confirms the controlled release over time of the active ingredient“carvacrol” when integrated, in the form of hydrolat, into the gellan hydrogel (Gelid made with hydrolat of M. fistulosa indicated with the acronym Gelid MF in fig. 3).

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Abstract

A medical aid is described comprising a support for skin application and a medicament associated with the support and intended to be put in contact with a damaged area of skin.

Description

“MEDICAL AID FOR CURING SKIN PATHOLOGIES AND CORRESPONDING PRODUCTION METHOD”
FIELD OF THE INVENTION
The present invention concerns a medical aid for the treatment of skin pathologies and a method to produce the medical aid, generally able to be used to treat skin lacerations preventing the onset of microbial infections that are also resistant to conventional drugs.
BACKGROUND OF THE INVENTION
Medical aids are known, typically in the form of gauzes or gauze compresses, used in the treatment of skin pathologies, such as, for example, lacerations, wounds, bums.
In particular, the gauzes used fall within the type of medical aid defined as pre-medicated, that is, they are prepared for use by soaking them with one or more drugs that have disinfectant, pain soothing and regenerative effects on the damaged skin tissues when applied in the area of the lesion, or in the so-called “bed” of the lesion.
The substances used to prepare these pre-medicated gauzes typically comprise small percentages of natural antimicrobials, such as, for example, essential or synthetic oils (for example, Bumshield or Bumcare gauzes) with which the gauzes are soaked before being used.
Other pre-medicated gauzes use a hydrogel that allows to keep the lesion area humid and to promote debridement or autolytic debridement, granulation and epithelization.
This type of gauze has some technical disadvantages.
A first disadvantage is that due to the use of a hydrogel to prepare the pre- medicated gauzes, the latter can become occlusive and, due to the saturation of water, the absorption of exudate is limited, favoring, as a consequence, microbial proliferation.
Another disadvantage is that, in general, since wounds are a fertile ground for the onset of infectious processes that can compromise wound healing and patient health, it is necessary to provide the application of secondary medications, meaning with this term a medication with the function of filling the injured cavity or securing the primary medication, aimed at preventing, or at least limiting, these risks.
Another disadvantage is that the treatments to care for skin lesions performed using known pre-medicated gauzes produce skin maceration processes and the development of bad smells.
Another disadvantage is that the pre-medicated gauzes that use small percentages of natural or synthetic antimicrobial agents do not always have specifically hydrophilic characteristics, which makes them have low compatibility with the hydrogel.
There is therefore the need to provide a medical aid that is able to overcome at least one of the disadvantages of the state of the art.
One purpose of the present invention is therefore to make available a medical aid for the treatment of skin pathologies that allows to medicate the damaged skin surface, to prevent the adhesion of the aid to wounds, to promote the immune response of patients, to prevent microbial superinfections also resistant to conventional drugs.
Another purpose of the present invention is to provide a medical aid for the treatment of skin pathologies which allows to use a medicament to replace essential or synthetic oils with another substance which is hydrophilic and economical, coming from waste from the production process of essential oils.
Another purpose of the present invention is to perfect a method to produce the medical aid which is simple and inexpensive.
The Applicant has devised, tested and embodied the present invention to overcome the shortcomings of the state of the art and to obtain these and other purposes and advantages.
SUMMARY OF THE INVENTION
The present invention is set forth and characterized in the independent claims. The dependent claims describe other characteristics of the invention or variants to the main inventive idea.
According to one aspect of the present invention, a medical aid is provided to treat skin pathologies, comprising a support for skin application and a medicament associated with the support and intended to be put in contact with an injured area of skin. According to a characteristic aspect of the present invention, the medicament comprises a hydrogel which has the following composition: gellan gum gel, calcium acetate, hydrophilic hydrolat of Citrus aurantium flowers, preservatives for human topical use.
According to one embodiment of the present invention, the gellan gum gel is comprised between 1% and 3% by volume with respect to a total volume of the hydrogel.
According to one embodiment of the present invention, the calcium acetate is comprised between 0.03% and 0.05% by volume with respect to a total volume of the hydrogel.
According to one embodiment of the present invention, the hydrophilic hydrolat of Citrus aurantium flowers is comprised between 45% and 55% by volume with respect to a total volume of the hydrogel.
According to one embodiment of the present invention, the preservatives for human topical use are used in a mixture with a concentration comprised between 0.15% and 0.35% by volume with respect to a total volume of the hydrogel.
In one embodiment, the preservatives are compounds known in the state of the art, for which the responsible authorities have authorized use for human topical use. By way of example, they can comprise phenoxyethanol 0.35%, benzoic acid 0.1%, dehydroacetic acid 0.05%. The experimental tests conducted, described in detail below, have shown that the introduction of preservatives, which on their own at the final tested concentrations do not show any antibacterial or antimicrobial action, allows to enhance the antimicrobial action of the hydrolat, as well as guarantee the correct preservation thereof at least until the expected expiry date.
According to one embodiment of the present invention, the hydrogel is in a solid state.
According to one embodiment of the present invention, the support for skin application is a gauze made of fabric.
According to alternative embodiments, the support is configured as a suitable carrier intended to guarantee a cutaneous application of the medicament. In these embodiments the carrier, that is, the inactive basic substance which allows to administer and therefore use the medicament and, in some cases, also regulates its release on the site of action, can for example be a cream or an ointment.
According to one embodiment of the present invention, the hydrophilic hydrolat of Citrus aurantium flowers comprises hydrophilic hydrolat of Citrus aurantium flowers in the subspecies amara.
According to another aspect of the invention, a method is provided to produce a medicament associable with a support for skin application that provides to create a volume of hydrogel by dissolving a second quantity of gellan gum powder in a first quantity of water, by heating it to a temperature higher than 40°C.
According to a characteristic aspect of the method according to the present invention, it is provided to add to the volume of hydrogel a third quantity of hydrophilic hydrolat of Citrus aurantium flowers, wherein the step of adding the hydrolat provides that the addition occurs by stirring it in the first step and during a cooling step.
According to one embodiment, the first and the second quantities form a solution which has a volume equal to 50% of the final volume of the hydrogel.
According to one embodiment, the third quantity has a volume equal to 50% of the final volume of the hydrogel.
According to one embodiment, the cooling takes place at a temperature lower than 40°C.
According to another aspect of the present invention, it is provided to use hydrophilic hydrolat of Citrus aurantium flowers, in particular in its subspecies amara, to cure pathologies of the skin.
Advantageously, the medical aid according to the present invention prevents the proliferation of pathogenic microorganisms in skin lesions, and at the same time stimulates the local immune response of patients, thus accelerating their healing.
One advantage of the medical aid according to the present invention is that it counteracts the development of bad smells caused by the process of skin maceration that often occurs in the presence of lesions and/or bums, thanks to the presence of the hydrolat of Citrus aurantium flowers.
The medical aid according to the invention can be made in the form of a hydrogel with which to soak gauzes or compresses (or other similar supports). Advantageously, thanks to the hydrophilic nature of the hydrolat of Citrus aurantium flowers, it can easily be mixed with the hydrogel. The result is a method to produce the medicament that is economical and effective.
In all cases, the packages that contain the pre-medicated gauzes have to be marketed in packages able to preserve their sterility and prevent their dehydration, able to also be stored at room temperature without there being a deterioration of the medical aid.
The medical aid according to the invention, for example in the form of pre medicated gauze, is able to prevent the development of microbial infections by exerting a cytocidal action on a wide spectrum of microbial species generally involved in human skin infections.
This allows to better manage the lesion.
The prevention of infections together with an attenuation of aggressive pro- inflammatory responses, allows to complete the healing process quickly without resorting to local or oral antimicrobial therapies that debilitate the patient and contribute to the development of antibiotic resistance.
Finally, the specific syneresis of the hydrogels allows the gradual release over time of the active ingredients contained therein and therefore a continuous and gradual treatment of the damaged skin area.
With a medication, the patient can heal quickly, reducing the convalescence period and avoiding healthcare costs correlated to the development of infected wounds.
Another advantage of the medical aid according to the present invention is that it is ecological, given the presence of hydrolat of Citrus aurantium flowers, which is actually a vegetable product obtained as a waste product during the processing of the corresponding essential oil of Citrus aurantium, in particular in its subspecies amara.
These and other aspects, characteristics and advantages of the present disclosure will be better understood with reference to the following description, drawings and attached claims. The drawings, which are integrated and form part of the present description, show some embodiments of the present invention, and together with the description, are intended to describe the principles of the disclosure. The various aspects and characteristics described in the present description can be applied individually where possible. These individual aspects, for example aspects and characteristics described in the description or in the attached dependent claims, can be the object of divisional applications.
It is understood that any aspect or characteristic that is discovered, during the patenting process, to be already known, shall not be claimed and shall be the object of a disclaimer.
BRIEF DESCRIPTION OF THE DRAWINGS
These and other characteristics of the present invention will become apparent from the following description of some embodiments, given as a non-restrictive example with reference to the attached drawings wherein:
- fig. 1 is a histogram showing the results of the cytotoxicity tests conducted on gingival fibroblast cells treated with substances released over 24 hours by hydrogels formulated with 50% of hydrolat of Citrus aurantium flowers subspecies amara (a) or hydrolat obtained from flowering tips of Monarda fistulosa (b);
- fig. 2 a) is a graph showing the overlapping of chromatograms for gellan with hydrolat of Citrus aurantium flowers subspecies amara at different times: lh, 3h (diluted 1 : 10), 6h, 24h, 30h (diluted 1 :5);
- fig. 2 b) is a graph showing the overlapping of chromatograms for gellan with hydrolat of Monarda fistulosa MF at different times: lh, 3h, 6h (diluted 1 :3);
- fig. 3 shows a histogram showing the release of carvacrol from gellan made with hydrolat of Monarda fistulosa.
To facilitate comprehension, the same reference numbers have been used, where possible, to identify identical common elements in the drawings. It is understood that elements and characteristics of one embodiment can conveniently be incorporated into other embodiments without further clarifications.
DETAILED DESCRIPTION OF SOME EMBODIMENTS
Embodiments described here concern a data processing method, of which particular forms of embodiment will be described below, as a non-limiting example.
Example 1 : STUDY OF THE INHIBITIVE AND CYTOCIDAL ACTION OF CERTAIN HYDROLATES ON CLINICAL MICROBIAL STRAINS AND ATCC (American Type Culture Collection) STRAINS
The aim of this study was to identify the most effective antimicrobial hydrolates as cytocidal activity among some selected ones.
The activity of the hydrolates was tested against isolated clinical microbial strains and ATCC reference strains, sensitive and resistant to antibiotics.
Specifically, both fungal strains and also GRAM+ and GRAM- bacterial strains most responsible for human skin infections were studied.
To achieve the goal, 11 clinical strains ( Candida albicans, C. parapsilosis, C. glabrata, Pichia sp, Staphyloccoccus aureus, Streptococcus pyogenes, Enterococcus faecalis, Enterococcus faecium, S. aureus MRSA, E. faecalis VRE) and 10 ATCC strains (C. albicans 24433, C. parapsilosis 22019, C. krusei 6258, S. aureus MSSA 29213, E. faecalis 29212, Pseudomonas aeruginosa 27853, S. aureus MRSA 43300, S. aureus 25923) were studied.
Cytocidal capacity of the following hydrolates was tested by means of in vitro broth microdilution test, carried out in 96-well plates and according to EUCAST international guidelines: Monarda citriodora, Monarda fistulosa, Monarda didyma, Lavandula vera, Citrus aurantium subspecies amara from flowers, the latter marketed by Magentina srl.
For all hydrolates, scalar dilutions comprised between 1/2 v/v and 1/32 v/v were tested.
The effectiveness values were expressed in terms of Minimum Inhibitory Concentration (MIC) and Minimum Fungicidal or Bactericidal Concentration (respectively, MFC or MBC), both identified respectively in the lower concentration of hydrolat present in the well able to inhibit microbial growth or have a cytocidal effect with a visible absence of turbidity (microbial growth index) of the medium.
The MIC values were identified by means of visualization and confirmed with OD reading (450 nm) at the spectrophotometer, while the values of the cytocide concentrations were identified by seeding the contents of the wells on suitable growth medium (Sabouraud for fungi and Mueller Hinton for bacteria) and reading the result of seeding after 24h of incubation.
The data obtained from this first analysis are summarized in Table 1.
Table 1
The most effective in vitro hydrolat proved to be that of Citrus aurantium subspecies amara. The others, although showing effectiveness, prove to be active at higher concentrations.
The hydrolat of Citrus aurantium subspecies amara was more active on fungal strains than on bacterial strains.
Among the latter, only E. faecalis - both the clinical strain and also the ATCC strain - showed greater resistance to cytocidal action while remaining strongly inhibited in growth at the lower concentrations tested (1/32).
In light of the results obtained, it is clear that the hydrolat of Citrus aurantium subspecies amara proved to be the most effective in terms of cytocidal action.
Example 2: IN-DEPTH STUDY OF THE EFFECTIVENESS OF THE HYDROLAT OF CITRUS A URANTJUM SUBSPECIES AMARA
The aim of the study is to analyze the effective inhibiting and cytocidal effectiveness of the hydrolat of Citrus aurantium subspecies amara.
This objective was outlined in consideration of the fact that the product showed, on the label, the addition of preservatives according to law inserted to increase the shelf-life of the product.
To perform the study, a pure sample of only hydrolat of Citrus aurantium subspecies amara was requested.
At the time of production, the industry supplied both the hydrolat with the addition of preservatives and also the unaltered hydrolat, both originating from the same harvest.
The broth microdilution analyses were repeated on the various microbial strains and according to the method indicated in Example 1, testing, in parallel with the hydrolates, the mixture of preservatives in the proportions used for the formulation.
In table 2 below, the results have been added of the study of the cytocidal action of only the hydrolat of Citrus aurantium subspecies amara, of only the mixture of preservatives and of the two combined together.
Table 2
In the table, the abbreviation CC indicates “with preservative”, the abbreviation SC indicates “without preservative”, and the abbreviation C indicates the preservative on its own.
The results obtained show that the preservative on its own, at the concentrations tested in the broth microdilution assay, is not able to inhibit microbial growth, nor to kill the tested strains.
The hydrolat of Citrus aurantium subspecies amara on its own is effective at higher concentrations than those identified in the association of hydrolates plus preservatives.
In the formulation, the action of the hydrolat is enhanced by the presence of preservatives; this indicates a synergy between the hydrolat and the preservatives when combined in the final formulation.
Example 3: STUDY OF THE TERPENE COMPONENTS OF THE HYDROLATES OF CITRUS AURANTIUM SUBSPECIES AMARA AND OF MONARDA FISTULOSA
The aim of the study was to identify the terpene components of the hydrolates of Citrus auratium subspecies amara and Monarda fistulosa.
A liquid-liquid extraction was carried out by mixing an amount of 0.5 ml of hydrolates with 0.5 ml of heptane with tridecane as internal standard.
A shaker (Retsch-MM300, Haan, Germany) was used for lh at 4 cycles per second.
The supernatants were used for the analysis after centrifugation at 4000 rpm for 10 minutes at 10°C in an Eppendorf mod. 5810R (Westbury, NY) centrifuge.
The heptane extracts were then filtered with 0.45 pm PTFE syringe filters and injected (3 pi) into the GC-MS system of Agilent Tech (Palo Alto, AC, USA).
An Agilent 7820 GC chromatograph equipped with an MSI 5977 A mass spectrometer with El ionization operating at 70 eV was used for the analysis.
A J&W Innovax 50 m chromatography column, 0.20 mm, ID 0.4 pm DF was used.
The GC injection temperature was 250°C, splitless mode, and heating was programmed at 40°C for 1 min, followed by a ramp of 5°C / min at 200°C and of 10°C / min at 260°C.
This high temperature was maintained for 5 minutes. Mass spectra were acquired in the 29-350 M/Z range with an Agilent 5977 MSD spectrometer with three s-1 scans.
The identification of VOC was made on the basis of the peak of correspondence with the spectral database of the library (NIST 08) and of the kovats indices.
The quantity of each monoterpene was expressed as a percentage of the total monoterpenes.
Table 3 is shown below, comprising the results of the GC analysis of the hydrolates of Citrus aurantium subspecies amara and of Monarda fistulosa, with the values expressed as a percentage.
Table 3
With reference to table 3, it can be noted that the hydrolat of Citrus aurantium subspecies amara has linalool (47.7%), a-terpineol (13.83%) and terpinolene (24.82%) as the largest components, while the hydrolat of Monarda fistulosa is essentially characterized by carvacrol (49.68%) and thymol (45.26%).
Example 4: DEVELOPMENT OF A PRE-MEDICATED GAUZE PROTOTYPE TO BE USED TO ASSESS ANTIMICROBIAL EFFECTIVENESS
The aim was to develop a pre-medicated gauze model to be used in in vitro studies aimed at evaluating antimicrobial effectiveness.
To achieve this purpose, some sterile gauzes were soaked in gellan gum hydrogel formulated at 50% with hydrolat of Citrus aurantium subspecies amara.
Specifically, an amount of powder equal to 2% of the established average final volume of the hydrogel as above was dissolved in heat in a quantity of water equal to 50% of the same final volume.
During the cooling step, a similar amount of hydrolat of Citrus aurantium subspecies amara was added to the solution of water and gellan powder.
Subsequently, the solution was poured onto sterile gauzes cut out and placed in a 6 cm diameter Petri dish, waiting for it to become solid.
After developing the prototype of pre-medicated gauze for the in vitro study of antimicrobial effectiveness, we proceeded with several experiments.
Specifically, gauzes formulated with only 2% v/v gellan with gellan (2% v/v) + hydrolat at 50% v/v {Citrus aurantium subspecies amara or Monarda didyma or Monarda fistulosa ) were seeded with 10 microliters of a 0.5 McF suspension of each bacterium or fungus indicated in point 1.
After incubating the gauze for 24 hours, we assessed the presence of microbial growth on each gauze and then we proceeded to make the gauze adhering on suitable culture media (Sabouraud for fungi and Mueller Hinton for bacteria).
After a further 24 hours of incubation, we assessed the growth of the strains seeded in order to identify their cytostatic or cytocidal activity.
At the end of the first incubation all the gauzes soaked in gellan only (control) showed a fungal growth, but not a bacterial one, while the gauzes soaked with gellan + hydrolat did not show any fungal or bacterial growth visible to the naked eye. Continuing with the experiment, and after another 24 hours of incubation, the plates to which gauzes soaked with gellan alone were adhered showed important fungal and bacterial growth.
This datum indicates that gellan alone has no cytocidal effect or inhibiting effect on the growth of the strains under study.
On the contrary, all the plates with hydrogel-soaked gauzes formulated with the hydrolates showed a strong inhibition or the total absence of microbial growth, as can be deduced from table 4.
Table 4 below indicates the bacterial and fungal growth detected on a culture medium after the application of gellan gels, modified or not, previously seeded with the strains (fungal or bacterial) under study.
The symbols shown in the table have the following meaning: CTR +: Positive control; (-) no growth; (+) rare colonies; (++) moderate microbial growth; (+++) strong microbial growth; (++++) blanket growth.
Table 4
Example 5: STUDY OF THE CYTOTOXIC ACTION OF THE GAUZES FORMULATED WITH HYDROGEL AND HYDROLAT
The study aimed to evaluate the cytotoxic action of the gauzes formulated with gellan hydrogel + hydrolat of Citrus aurantium subspecies amara or hydrolat of Monarda fistulosa.
The study was carried out on gingival fibroblast cells, which were treated with culture medium previously incubated, for 24 hours, with gellan hydrogel at 2% v/v and hydrolat of Citrus aurantium subspecies amara or hydrolat of Monarda fistulosa.
The cytotoxicity of the hydrogels was assessed through the MST test (Promega, Madison, WI, USA test).
The treated culture medium was used unaltered (TQ) and diluted at 50% (1 :2), 25% (1 :4) and 12.5% (1 :8) in base medium.
The cells were resuspended in 200 mΐ of base medium and seeded in 96-well plates and incubated for 24h until a subconfluent monolayer was obtained.
Cell viability was assessed at 24h and 48h after the addition of the previously conditioned medium.
The MTS test was performed in accordance with the operating protocol established by the manufacturer, and each experiment was done in triplicate. With reference to fig. 1, it can be observed that the substances released by the hydrogel into the culture medium after 24h incubation were toxic to the gingival fibroblasts treated with the medium conditioned by the hydrogel formulated with hydrolat of Monarda fistulosa, while they show no cytotoxicity for those treated with the hydrogel formulated with hydrolat of Citrus aurantium subspecies amara.
The data indicates a reasonable certainty in the use of the gauze formulated with hydrolat of Citrus aurantium subspecies amara.
Example 6: RELEASE KINETICS OF THE ACTIVE COMPONENTS PRESENT IN THE PRE-MEDICATED GAUZE
The stated goal was to evaluate the release kinetics of the active ingredients over 24 hours, the estimated time a medication remains in place.
For the study, hydrogel of gellan at 2% v/v as control and hydrogel of gellan 2% modified with hydrolat of Citrus aurantium subspecies amara or with hydrolat of Monarda fistulosa at 50% v/v were prepared in 6 cm diameter plates; 5 mL of physiological solution were deposited on the surface of the hydrogels and incubated for lh, 3h, 6h, 24h and 30h.
At the end of each incubation, the samples were taken and stored at +4°C until the qualitative-quantitative analysis performed by means of high performance liquid chromatography (HPLC), Infrared (IR).
A THERMOQUEST HPLC system (produced by Shimadzu, Kyoto, Japan) was used for the analyses, equipped with two LCGA pumps and a UV/Vis SPD 10A detector (Shimadzu).
A UV-Vis lamp was connected at the end of the column, set to the absorbance wavelength of the analyzed compounds.
The analyses were performed using the following isocratic conditions: (i) Solvent A: Acetonitrile/H20/Acetic Acid 95:34:1, (ii) Ambient T °, (iii) As =274 nm, (iv) flow rate = 1 ml/min.
The samples, if necessary, were diluted in acetonitrile at different V/V ratios according to their [C] and subsequently filtered on RephiQuik Phobic PTFE 0.45 pm filters.
With reference to figure 2, it can be observed that in the analyzed samples there is a growing trend of active ingredients of the hydrolat released in the physiological solution as a function of time. The maximum release peak is observed 3 hours after application, with a progressive decrease in active ingredients up to the 30 hours.
From the analysis, thanks to comparison with some standards, it was possible to identify the presence of carvacrol which is one of the main components of the hydrolat of Monarda fistulosa.
Therefore, in a time-dependent manner, the active ingredients of the hydrolates responsible for the described antimicrobial action are also released.
The data obtained from the analysis show that the“lh” sample has some quite intense peaks, an intensity that becomes even more relevant in the“3h” sample (for both types of sample tested), an indication of the increase in the concentration of this compound in the sample analyzed and therefore an indication of a greater release of the components of the hydrolat by the gellan hydrogel.
This increasing trend of the released material gradually begins to decrease, maintaining a trend that can be considered constant up to 30 hours. This datum confirms, also for the gellan hydrogel modified with hydrolates, what is already known in literature, that is, that the gellan hydrogel releases its aqueous component in a constant manner over time.
Therefore, in a time-dependent manner, the active ingredients of the hydrolates responsible for the described antimicrobial action are also released.
With reference to fig. 3, this shows the trend as a function of the time of release of the carvacrol (the two characteristic peaks at 5.7 minutes and 6.3 minutes were taken), which is the main component of the hydrolat of Monarda fistulosa identified in the sample obtained from the gellan hydrogel made with the same hydrolat.
As shown in fig. 3, the greatest release of the active ingredients occurs in the first 3 hours of contact, and then gradually decreases up to the 30 hours. The figure shows how the intensity of the two peaks corresponding to the carvacrol is greatest after the first and third hour, while it tends to decrease in the following hours. This datum confirms the controlled release over time of the active ingredient“carvacrol” when integrated, in the form of hydrolat, into the gellan hydrogel (Gelid made with hydrolat of M. fistulosa indicated with the acronym Gelid MF in fig. 3).

Claims

1. Medical aid comprising:
- a support for skin application;
- a medicament associated with said support and intended to be put in contact with an damaged area of skin;
characterized in that said medicament comprises a hydrogel which has the following composition:
- gellan gum gel;
- calcium acetate;
- hydrophilic hydrolat of Citrus aurantium flowers;
- preservatives for human topical use.
2. Medical aid as in claim 1, characterized in that said gellan gum gel is comprised between 1% and 3% by volume with respect to a total volume of the hydrogel.
3. Medical aid as in claim 1 or 2, characterized in that said calcium acetate is comprised between 0.03% and 0.05% by volume with respect to a total volume of the hydrogel.
4. Medical aid as in any claim from 1 to 3, characterized in that said hydrophilic hydrolat of Citrus aurantium flowers is comprised between 45% and 55% by volume with respect to a total volume of the hydrogel.
5. Medical aid as in any claim hereinbefore, characterized in that said preservatives for human topical use are comprised between 0.15% and 0.35% by volume with respect to a total volume of the hydrogel.
6. Medical aid as in any claim hereinbefore, characterized in that said hydrogel comprises a solid state hydrogel.
7. Medical aid as in any claim hereinbefore, characterized in that said support is a gauze made of fabric.
8. Medical aid as in any claim hereinbefore, characterized in that said hydrophilic hydrolat of Citrus aurantium flowers comprises hydrophilic hydrolat of Citrus aurantium flowers in the subspecies amara.
9. Method for the production of a medicament associable with a support for skin application, suitable to define a medical aid when reciprocally associated, where the method comprises the steps of: - creating a volume of hydrogel by dissolving a second quantity of gellan gum powder in a first quantity of water, by heating it to a temperature higher than 40°C;
the method being characterized in that it comprises adding to said volume of hydrogel a third quantity of hydrophilic hydrolat of Citrus aurantium flowers, and in that said addition comprises adding during a cooling step.
10. Method as in claim 9, characterized in that said first and second quantities form a solution which has a volume equal to 50% of the final volume of said hydrogel.
11. Method as in claim 9 or 10, characterized in that said third quantity is
50% of the final volume of said hydrogel.
12. Method as in any claim from 9 to 11, characterized in that said cooling takes place at a temperature lower than 40°C.
13. Use of hydrophilic hydrolat of Citrus aurantium in a medical aid for the treatment of skin pathologies in particular lacerations or wounds or burns.
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