Patent Description:
The use of molecules having antibacterial and/or antifungal activity for the treatment of infections in the urological-gynecological field is commonly associated with side and secondary effects that, very often, derive from a nontargeted action of the active molecules or from a systemic absorption thereof, even when administered with formulations for topical or external use, i.e. applied directly on the site of interest. Micro- and/or nanomedicine can provide the solution to this problem, due to the fact that human skin and mucosae commonly resist small-particle permeation, eliminating the concern regarding systemic absorption of the active molecules, if those are bound to nanoparticle carriers. It is therefore possible to bind an active molecule to a nanoparticle carrier modifying the molecule directing or absorption. Another strategy is that of using active agents that be themselves particles which, being unable to permeate skin barriers and mucosae, will carry out their pharmacological action directly and exclusively on the administration site, if suitably formulated, eliminating the problem of systemic side effects.

The use of nano- and microcapsules (NPs) of different nature, metallic, polymeric, lipidic, carbon-based, ceramic ones, etc., finds wide use in a biological and pharmaceutical field, such molecules can, e.g., be used as carriers to which one or more active molecules may be bound, or also for their inherent activities at a pharmaceutical level.

<NPL>, discloses gold nanoparticles coated with pegylated graphene oxide for the selective killing of uropathogenic E. coli that be used in the treatment of patients with urinary tract infections.

<NPL>, discloses various gold-graphene nanocomposites and their use for the photothermal ablation of bacteria. More particularly, gold nanoparticles coated with pegylated graphene oxide nanoparticles have been used tor the selective killing of uropathogenic E. coli and described as potential treatment of patients with urinary infections.

In the gynecological and urological field, the treatment of infections is, as mentioned above, carried out by the use of active molecules that are normally absorbed by the organism and have undesired side effects.

An example of drugs commonly used for the treatment of said infections is represented by antibiotics or by formulations containing them, or even by mixtures of plant essential oils such as, e.g., mint oil and others, which have a powerful antibacterial effect.

Such active principles, however, have undesired secondary effects like many drugs, as they also penetrate at a systemic level notwithstanding the topical application thereof. To date, no formulations for the topical treatment of urogenital infections exist which do not exhibit the above-reported technical problem.

The Authors of the present invention solve the technical problem linked to current formulations for topical use for the treatment of urogenital infections, using, as active principles, gold and graphene micro- or nanoparticles or derivatives thereof or mixtures thereof in suitable mixtures or compositions, using a particular mixture of components having an antimicrobial effect markedly improved compared to that observed with the individual components. Moreover, the components of the mixture are not known to have secondary effects following topical administration, not even when administered systemically. Surprisingly, the Authors of the present invention have found that the combination of gold particles with graphene particles or derivatives thereof or mixtures thereof carries out a greater bactericidal and antifungal activity compared to that carried out by the individual components, with a relevant synergistic effect (see Examples <NUM> and <NUM>). Moreover, as mentioned above, the use of particles of gold and of graphene (or a derivative thereof) of micro/nano sizes determines the absence/reduction of systemic absorption of the particles themselves, resulting in a localized therapeutic action limited to the site that is to be treated, i.e. the urogenital district.

Therefore, objects of the present invention are:.

In the present description, the terms "synergistic effect" or "synergistic activity" can be used indistinctly, as interchangeable; in both cases, in fact, an effect provided by the combination of two components that is greater than the sum of the individual effects of the individual components is denoted.

In the present description the term "dispersion", "suspension" and "solution" can be used indistinctly, as interchangeable at least in the micro- and nanometric sizes.

In the present description, "about" refers to the experimental error that may occur during conventional measurements. More particularly, when referring to a value it indicates ± <NUM>% of the indicated value, and, when referred to a range, ± <NUM>% of the ends thereof.

In the present description the term "topical use" denotes an external or an internal non-systemic use. Such use, therefore, also comprises an internal use by means of suppositories or creams with applicator or ovules or other medical devices, in which the active principles are intended for internal mucosae.

As mentioned above, one object of the present invention is represented by a mixture comprising:.

In one preferred embodiment of the invention, the mixture as described above and as claimed will further comprise an aqueous phase with an acid pH, said phase comprising at least one organic acid.

In particular, the pH of the mixture, in any one of the embodiments indicated above and below, could be from <NUM> to <NUM> or from <NUM> to <NUM> or, in a preferred embodiment, said pH could be of about <NUM>,<NUM>.

The pH as indicated above and claimed can be obtained by addition to the mixture of the at least one organic acid indicated above, or of an inorganic acid such as hydrochloric acid. Such organic acid could be any one organic acid with from one to eight carbon atoms. Such acid could be one or more among methanoic, acetic, ethanoic, ethanedioic, oxoethanoic, <NUM>-hydroxyethanoic, propanoic, prop-<NUM>-enoic, <NUM>-propionic, propanedioic, <NUM>-hydroxypropanedioic, oxopropanedioic, <NUM>,<NUM>-dihydroxypropanedioic, <NUM>-oxopropanoic, <NUM>-hydroxypropanoic (lactic acid), <NUM>-hydroxypropanoic, <NUM>,<NUM>-dihydroxypropanoic, butanoic, <NUM>-methylpropanoic, <NUM>-oxobutanoic, <NUM>-oxobutanoic, <NUM>-oxobutanoic, (E)-butenedioic, (Z)-butenedioic, but-<NUM>-enedioic, oxobutanedioic, hydroxybutanedioic, <NUM>,<NUM>-dihydroxybutanedioic, (E)-but-<NUM>-enoic, pentanoic, <NUM>-methylbutanoic, pentanedioic, <NUM>-oxopentanedioic, hexanoic, hexanedioic, <NUM>-hydroxypropane-<NUM>,<NUM>,<NUM>-tricarboxylic, prop-<NUM>-en-<NUM>,<NUM>,<NUM> tricarboxylic, <NUM>-hydroxypropane-<NUM>,<NUM>,<NUM> tricarboxylic, (2E,4E)-hexa-<NUM>,<NUM>-dienoic, heptanoic, cyclohexanecarboxylic, benzenecarboxylic, <NUM>-hydroxybenzoic, octanoic, benzene-<NUM>,<NUM>-dicarboxylic acid.

In one preferred embodiment, said acid will be <NUM>-hydroxypropanoic acid, better known as lactic acid.

According to the present invention, said lactic acid could be in any enantiomeric form or a racemate thereof, therefore the mixture could comprise S-(+)-lactic acid, L-(+)-lactic acid S-(-)-lactic acid, L-(-)-lactic acid, or mixtures thereof. In one embodiment the mixture comprises L-(+)-lactic acid and/or L-(-)-lactic acid.

According to the present invention, the mixture for the use described above and claimed, said gold particles can have sizes from <NUM> to <NUM>, or from <NUM> to <NUM> and said graphene particles or derivative thereof can have sizes from <NUM> to <NUM>, or from <NUM> to <NUM>. According to the invention, any combination of the particles as defined above is suitable.

Therefore, the mixture could contain gold particles of sizes from <NUM> to <NUM> in combination with graphene particles or derivative thereof of sizes from <NUM> to <NUM> or mixtures thereof; gold particles of sizes from <NUM> to <NUM> in combination with graphene particles or derivative thereof of sizes from <NUM> to <NUM> or mixtures thereof, graphene particles or derivative thereof of sizes from <NUM> to <NUM> in combination with gold particles of sizes from <NUM> to <NUM> or mixtures thereof.

In one embodiment of the invention mixtures are preferred in which at least one type of particles has colloidal sizes (from <NUM> to <NUM>) or in which both types of particles have sizes from <NUM> to <NUM>.

The mixture comprises a linker as defined below and the particles of gold and graphene or derivative thereof form a complex, in the examples defined as "gold/graphene complex". The mixture of the invention, therefore, comprises the gold-linker-graphene particle complex or derivative thereof. Said complex will preferably have sizes in the above-indicated ranges for the individual particles.

According to the present invention, the mixture could comprise gold particles and graphene particles (or derivative thereof or mixtures thereof) in different weight ratios. By way of a non-limiting example, the weight ratio between said gold particles and said graphene particles or derivative thereof or mixtures thereof could be from <NUM>:<NUM> a <NUM>:<NUM>, preferably from <NUM>:<NUM> to <NUM>:<NUM> or more preferably of <NUM>:<NUM>; or, the weight ratio between said graphene particles or derivative thereof or mixtures thereof and said gold particles could be from <NUM>:<NUM> to <NUM>:<NUM> or, preferably, from <NUM>:<NUM> to <NUM>:<NUM>. In one preferred embodiment, the weight ratio between the aforesaid particles is of about <NUM>:<NUM>.

In particular, in any embodiment disclosed in the present description, the concentration of said gold particles could be, e.g., from <NUM>% to <NUM>% w/w, preferably from <NUM> to <NUM>% w/w and more preferably from <NUM> to <NUM>% w/w and the concentration of said graphene particles or derivative thereof or mixtures thereof could be from <NUM>% to <NUM>% w/w, preferably from <NUM> to <NUM>% w/w or, more preferably, from <NUM> to <NUM>% w/w.

In one embodiment the concentration of said gold particles can be from <NUM> to <NUM>% w/w, preferably from <NUM> to <NUM>% w/w, and the concentration of said graphene oxide particles can be from <NUM> to <NUM>% w/w or from <NUM> to <NUM>% w/w.

According to the present invention, the mixture, in any one of the embodiments provided in the present description or claimed, comprises a linker binding said gold particles to said graphene particles or derivative thereof or mixtures thereof, wherein said linker is alpha lipoic acid. Examples of suitable linkers that are not part of the invention is represented by a natural or synthetic molecule comprising one or more portions capable of interacting with at least one gold particle, and one or more portions capable of interacting with at least one graphene particle or derivative thereof e.g. comprising, in said portion capable of interacting with at least one gold particle, one or more sulfur and/or nitrogen atoms and, in said portion capable of interacting with at least one graphene particle or derivative thereof, one or more chemical groups capable of forming hydrophobic interactions and/or hydrogen bonds.

Chemical groups capable of forming hydrogen bonds comprises chemical groups selected from carboxylic acids, esters thereof, amino groups and/or derivatives thereof (such as alkyl amines) and alcohol groups.

According to the invention, said linker is alpha lipoic acid.

According to the present invention, in one embodiment applicable to any of the above-described embodiments, said alpha lipoic acid could have a concentration ranging from <NUM> to <NUM>% w/w, preferably from <NUM> to <NUM>% w/w.

A further object of the invention is represented by a mixture, as such and not limited to the use, comprising:.

and a linker binding said gold particles to said graphene oxide particles wherein said linker is alpha lipoic acid
wherein said mixture has a pH of between <NUM> and <NUM>, or between <NUM> and <NUM>, or wherein said mixture has a pH of about <NUM>.

In the present description, the mixture as a product not limited to therapeutic use for the treatment and/or the prevention of urogenital tract infections is also simply referred to as "mixture as such" in order to distinguish it from the mixture for therapeutic use described above and object of the claims.

In one embodiment of the above-described mixture as such, said gold particles can have sizes from <NUM> to <NUM>, preferably from <NUM> to <NUM>, and said graphene oxide particles can have sizes from <NUM> to <NUM>, preferably from <NUM> to <NUM>.

In the mixture as such object of the invention, the weight ratio between said gold particles and said graphene oxide particles is from <NUM>:<NUM> to <NUM>:<NUM>, preferably of <NUM>:<NUM>; or the weight ratio between said graphene oxide particles and said gold particles is from <NUM>:<NUM> to <NUM>:<NUM>.

In the mixture as such object of the invention, in any embodiment provided in the present description, wherein the concentration of said gold particles is from <NUM>% to <NUM>% w/w, or from <NUM> to <NUM>% w/w, or from <NUM> to <NUM>% w/w, or from <NUM> to <NUM>% w/w, or from <NUM> to <NUM>% w/w and the concentration of said graphene oxide particles, is from <NUM>% to <NUM>% w/w, or from <NUM> to <NUM>% w/w and/or from <NUM> to <NUM>% w/w, or from <NUM> to <NUM>% w/w or from <NUM> to <NUM>% w/w.

In one embodiment the concentration of said gold particles can be from <NUM> to <NUM>% w/w, preferably from <NUM> to <NUM>% w/w and the concentration of said graphene oxide particles can be from <NUM> to <NUM>% w/w or from <NUM> to <NUM>% w/w.

Also the mixture as such as defined herein, in any one of the embodiments provided, comprises a linker binding said gold particles to said graphene oxide particles, wherein said linker is alpha lipoic acid.

Preferably, said alpha lipoic acid will have a concentration ranging from <NUM> to <NUM>, or also a concentration from <NUM> to <NUM>% w/w.

Non-limiting examples of said mixture are provided hereinafter.

mixture comprising, per <NUM> grams of final formulation:.

Object of the invention is also a pharmaceutical composition comprising the mixture (for the use described in the treatment and/or the prevention of urogenital tract infections, or as such), and at least one pharmaceutically acceptable excipient.

The technician in the field will know how to select said excipient according to the type of formulation to be made, and other further additives, on the basis of his/her general knowledge of the pharmacopoeia.

According to some embodiments, said composition could be made in liquid, solid or semi-solid form, in the form of cream, gel, cream-gel, emulgel, softgel, foam, emulsion, sprayable emulsion, suppository.

One example of pharmaceutical cream composition is provided hereinafter.

Another formulation example also object of the invention, is provided in the.

Object of the invention is also the mixture as such according to any one of the embodiments provided, for use as a medicament, e.g. for use in the treatment and/or prevention of urogenital tract infections.

Object of the invention is also the composition of the invention, e.g. for use in the treatment and/or prevention of urogenital tract infections.

According to the present invention, such infections may be bacterial and/or fungal and/or protozoan and/or mycoplasma infections.

In one embodiment of the invention said bacterial infections may be infections caused by gram-negative, gram-positive and/or gram-variable bacteria.

According to a further embodiment, said fungal infections may be infections caused by a fungus of the genus Candida.

The mixture or the composition of the invention are preferably meant for topical use, i.e. intended in particular for skin or mucosae (external or internal; both male and female ones).

In particular, said topical use can be carried out by applying said mixture or composition externally, internally, on the skin, on the mucosa, in the vagina, on the vulva or on the penis.

Object of the invention is the mixture or the composition according to any one of the embodiments of the invention for use for the prevention and/or the treatment of urogenital tract infections as defined above, by topical application (internal or external, as explained in the present description).

A further object of the invention is a mixture according to any of the embodiments described, for preparing a pharmaceutical composition or a medical device for use in the treatment and/or prevention of urogenital tract infections, as well as a process for preparing a pharmaceutical composition as described or claimed, wherein a mixture according to any of the embodiments described is additioned with at least one pharmaceutically acceptable excipient.

A further object of the invention are medical devices comprising the mixture or the composition according to the present description in any one of the embodiments indicated, or a kit for applying the aforesaid mixture or composition, said kit comprising aliquots of said mixture or composition introduced or to be introduced into suitable applicators, such as, e.g. vaginal applicators, single-dose sachets.

Finally, object of the invention are a vaginal capsule, vaginal tablet, vaginal softgel, ovum, vaginal films, bandage, plaster, wadding, spray, probe comprising the mixture or the composition according to the present description in any one of the embodiments indicated.

Anywhere in the present description the term comprising may be replaced by the term "consisting of" or "being consisting of".

The examples provided hereinafter illustrate in a non-limiting manner the invention and some embodiments thereof.

In the following examples the following active principles, based on identified colloidal gold and graphene, were used:.

The Inventors declare that all cell lines used are commercial cell lines, and that the provisions of the decrees cited in Art. <NUM>, specifically related to the confined use of genetically modified microorganisms, were complied with.

Stabilization test of the graphene- and gold-based colloidal mixture and characterization thereof by dynamic light scattering (DLS) characterization subdivided in the following sections:.

A preliminary analysis was carried out on the colloidal gold sample provided by Sigma Aldrich Company (<NUM> Gold nanoparticles, Code <NUM>) in order to determine the amount of colloidal gold per volume unit of colloidal dispersion. For that purpose, the sample was subjected to UV-Vis analysis at a <NUM>-nm wavelength, and absorbance was compared to that of a standard of known concentration. The analysis revealed that the sample of commercial colloidal gold has a composition of <NUM>/ml of colloidal gold. For the sample of commercial colloidal graphene (Graphene oxide nanocolloids <NUM>/mL, cod. <NUM>) the weight/volume composition is provided by the manufacturer. The colloidal dispersions, both of gold and of graphene, were analyzed by DLS in order to confirm mean sizes, dispersity, and surface potential. The DLS graphs, reported hereinafter, confirmed the colloidal sizes and a good polidispersity index (see Table <NUM> and Graph <NUM> reported hereinafter). The mean hydrodynamic radius detected for the gold sample proved consistent with that declared by the manufacturer in the technical specifications (Mean Hydrodynamic Diameter (Z) <NUM>-<NUM>). At DLS, the mean sizes of the graphene particles proved, as expected, greater than those detected by the manufacturing company (nanocolloid= <<NUM>) owing to the high scattering due to the morphology of graphene itself (<NPL>)*. However, this feature proved to be advantageous for this study, as it allowed to effectively discriminate the two colloid populations. The graphene polydispersity index proved to be very good:.

Exactly measured volumes of the colloidal dispersions of gold and graphene, respectively, were suitably mixed so as to obtain a gold/graphene weight ratio of <NUM>/<NUM>. Specifically, <NUM> of gold colloidal dispersion (containing <NUM> of gold) was mixed with <NUM> of graphene colloidal dispersion (containing <NUM> of graphene). The obtained dispersion was acidified to a pH value of <NUM> using a <NUM>% lactic acid aqueous solution. The final dispersion proved to be homogeneous and of a dark pink color (<FIG>).

DLS analysis of the colloidal dispersion showed the presence of two distinct populations having sizes consistent with those of the gold particles and the graphene particles, respectively (<FIG>). Gold and graphene particle aggregation was induced by adding to the acidified colloidal mixture increasing aliquots of a saturated solution of alpha lipoic acid at pH <NUM> in bidistilled water. The formation of a stable gold/graphene complex was obtained by using a volume ranging between <NUM>-<NUM> of alpha lipoic acid solution, at the concentration of <NUM>/mL, in <NUM> of the starting colloidal mixture. Alpha lipoic acid binds to the surface of gold, generating hydrophobic interactions and hydrogen with the graphene. DLS analysis highlighted the appearance of a new population of particles (gold/graphene complex) with mean sizes of <NUM> and higher intensity (<FIG>), in equilibrium with the original colloidal species. The forming of the complex does not alter the color of the colloidal dispersion. However, the further addition of alpha lipoic acid solution destabilizes the system, generating the appearance of a dark precipitate. In confirmation of the gold/graphene complex forming, also aliquots of sole gold or of sole graphene were analyzed in the presence of alpha lipoic acid, without the occurrence of interaction phenomena. Therefore, without being bound by theory, it may be assumed that the <NUM>-nm peak be actually comprised of a "near-stoichiometric" association of a gold particle with a graphene particle, as illustrated in <FIG>.

The colloidal gold- and graphene-based active principles used in the examples are the following:.

The exactly weighed amount of Ceteareth was solubilized in bidistilled water under stirring and in a water bath at <NUM>. Upon solubilization, the gold and graphene solutions, and subsequently the solid-state antimicrobials were added. The exactly weighed amount of liquid paraffin and caprylic/capric triglyceride were put in a water bath at the temperature of <NUM>. Subsequently, Cetearyl alcohol was added until complete melting. The mixing of the two phases occurred by using an Ultraturrax VDI <NUM> (VWR): the aqueous phase was slowly added to the oily phase under the action of the machinery at <NUM> rpm for <NUM>, and when the emulsion temperature had reached <NUM> said emulsion was left to cool down under constant stirring at <NUM> rpm using a blade stirrer. At room temperature the cream is stable, has a homogeneous appearance, fluid consistency and white color.

The exactly weighed amounts of Tween <NUM> and PEG were solubilized in bidistilled water under stirring and in a water bath at <NUM>. Upon solubilization, the gold and graphene solutions, and subsequently the solid-state antimicrobials were added. The exactly weighed amounts of liquid paraffin and cetearyl alcohol were put in a water bath at the temperature of <NUM> until complete melting. The mixing of the two phases occurred by using an Ultraturrax VDI <NUM> (VWR): the aqueous phase was slowly added to the oily phase under the action of the machinery at <NUM> rpm for <NUM>, and slight foaming was observed at this stage. When the emulsion temperature had reached <NUM> said emulsion was left to cool down under constant stirring at <NUM> rpm using a blade stirrer. At room temperature the cream is stable, has a homogeneous appearance, fluid and soft consistency, white color.

The exactly weighed amounts of Tween <NUM> and PEG were solubilized in bidistilled water under stirring. Upon solubilization, the solid-state antimicrobials and the gold and graphene solutions were added, and lactic acid was added until obtaining a final pH of the aqueous phase equal to <NUM>. Upon reaching a pH value equal to <NUM> the forming of a dark precipitate was observed, which increased with pH lowering.

In the subsequent tests the gold-graphene mixture was stabilized in an acid solution with alpha lipoic acid, and their concentrations were balanced in order to obtain a stable formulation, with required color and pH.

The exactly weighed amounts of Tween <NUM> and PEG were solubilized in bidistilled water under stirring. Upon solubilization, the gold and graphene solutions were added and lactic acid was added until obtaining a final pH of the aqueous phase equal to <NUM>. The exactly weighed amounts of liquid paraffin and cetearyl alcohol were put in a water bath at the temperature of <NUM> until complete melting. The mixing of the two phases occurred by using an Ultraturrax VDI <NUM> (VWR) and thermostating the process at <NUM>: the aqueous phase (previously heated at <NUM>) was slowly added to the oily phase under the action of the machinery at <NUM> rpm for <NUM>. When the emulsion temperature reached <NUM> said emulsion was let to cool down under constant stirring at <NUM> rpm using blade stirrer, thermostating the process at <NUM>. At room temperature the cream is stable, has a homogeneous appearance, fluid consistency, pink color.

The exactly weighed amount of Phospholipon <NUM> was solubilized in bidistilled water under stirring and in a water bath at <NUM>. Upon solubilization, the gold and graphene solutions, and subsequently the solid-state antimicrobials were added. The exactly weighed amount of white vaseline was put in a water bath at the temperature of <NUM>. Subsequently, liquid paraffin, caprylic/capric triglyceride and cetearyl alcohol were added. Upon reaching the total melting of the components a homogeneous liquid phase was obtained and soy lecithin (the oily phase turns deep yellow) and the emulgator (emulsifier) were added.

The mixing of the two phases occurred by using an Ultraturrax VDI <NUM> (VWR): the aqueous phase was slowly added to the oily phase under the action of the machinery at <NUM> rpm for <NUM>, and when the emulsion temperature reached <NUM> said emulsion was left to cool down under constant stirring at <NUM> rpm using a blade stirrer. At room temperature the cream is stable, has a homogeneous appearance, fluid consistency and yellow color.

Microbiological testing of the gold-graphene-lipoic acid mixture according to the invention, and of the superiority thereof compared to the single active agents (gold or graphene) individually, in an aqueous phase:.

The antibacterial and antifungal activity of the gold + graphene + lipoic acid (nanogold particles <NUM>/ml + graphene <NUM>/ml + lipoic acid <NUM>/ml) aqueous mixture, prepared by mixing <NUM> of aqueous solution of nanogold particles <NUM>/MI + <NUM> of graphene oxide nanocolloid in water (graphene <NUM>/mL) + <NUM> of lactic acid + <NUM> of a <NUM>/ml alpha lipoic acid aqueous solution was assessed in terms of MIC (Minimum Inhibitory Concentration) in % v/v or in weight concentration (m/ml), by using a <NUM>-well plate micromethod, said method briefly consists in a <NUM>:<NUM> serial dilution of the sample in the suitable culture medium (MH in case of bacteria or Sabouraud Broth in case of fungi), in the adding of the microbial inoculum, incubating at <NUM> for <NUM>, reading the optical density (OD) of the wells, comparing between the OD of the sample wells and of the positive control wells (untreated growth control) and the negative control wells (sole culture medium and sample at the different concentrations, but without microbial inoculum) and finally in the determining of the MIC.

Micromethod by dilution in Mueller Hinton liquid medium.

The gold+graphene+lipoic acid mixture (gold nanoparticles <NUM>/ml + graphene <NUM>/ml + lipoic acid <NUM>/ml), (Examples B4 and B5), exhibited an interesting antimicrobial activity against a free-living strain of S. Agalactiae ATCC <NUM>.

The mixture tested in the bacterial inhibition studies reported below constitutes a part of the components of the aqueous phase of Examples B4 and B5, i.e.: <NUM> of aqueous solution of nanogold particles <NUM>/ml + <NUM> of nanocolloid graphene oxide in water (graphene <NUM>/mL) + <NUM> of lactic acid + <NUM> of an aqueous solution of <NUM>/ml alpha lipoic acid.

Said mixture corresponds to (<NUM>/ml nanogold particles + graphene <NUM>/ml + <NUM>/ml lipoic acid). The lactic acid was used to bring the pH to <NUM>.

The tested mixture constitutes a part of the components of the aqueous phase of Examples B4 and B5, i.e.: <NUM> of aqueous solution of nanogold particles <NUM>/ml + <NUM> of nanocolloid graphene oxide in water (graphene <NUM>/mL) + <NUM> of lactic acid + <NUM> of a <NUM>/ml alpha lipoic acid aqueous solution.

The gold-graphene-lipoic acid mixture (gold nanoparticles <NUM>/ml + graphene <NUM>/ml + lipoic acid <NUM>/ml), (Examples B4 and B5), exhibited an interesting antimicrobial activity against a free-living strain of E. Coli ATCC <NUM>.

The gold-graphene-lipoic acid mixture (gold nanoparticles <NUM>/ml + graphene <NUM>/ml + lipoic acid <NUM>/ml), (Examples B4 and B5), exhibited an interesting antimicrobial activity against a free-living strain of E. Faecalis ATCC <NUM>.

Micromethod by dilution in Sabouraud liquid medium.

The gold-graphene-lipoic acid mixture (gold nanoparticles <NUM>/ml + graphene <NUM>/ml + lipoic acid <NUM>/ml), (Examples B4 and B5), exhibited antifungal activity against free-living strains of C. albicans ATCC <NUM> and C. krusei ATCC <NUM>.

The results of the microbiological tests obtained to date demonstrate the superiority of the gold-graphene-lipoic acid mixture (gold nanoparticles <NUM>/ml + graphene <NUM>/ml + lipoic acid <NUM>/ml), aqueous phase of the final formulation in cream (Examples B4 and B5), compared to the single active agents (gold or graphene) at the same (gold) or higher (graphene) concentration.

The gold-graphene-lipoic acid formulation (gold nanoparticles <NUM>/ml + graphene <NUM>/ml + lipoic acid <NUM>/ml (aqueous phase of the cream), in fact, has an interesting antimicrobial activity against reference E. coli ATCC <NUM> and E. faecalis ATCC <NUM> strains and has a MIC of <NUM>% v/v against both strains, said MIC in % v/v corresponds to a MIC in concentration by weight equal to <NUM>µg/ml+<NUM>µg/ml.

The MIC against a reference S. agalactiae ATCC <NUM> strain is equal to <NUM>% v/v, corresponding to a MIC by weight of <NUM>µg/ml+<NUM>µg/ml.

The gold-graphene-lipoic acid mixture (gold nanoparticles <NUM>/ml + graphene <NUM>/ml + lipoic acid <NUM>/ml, aqueous phase of the cream, has an interesting antifungal activity against C. albicans ATCC <NUM> and exhibits a MIC equal to <NUM>% v/v (<NUM>. 2µg/ml+<NUM>. 2µg/ml by weight). krusei ATCC <NUM> it exhibits a MIC of <NUM>% v/v (<NUM>µg/ml+<NUM>µg/ml by weight).

In view of the important role in chronic and persistent infections and in the resistance to antibiotics of the complex microbial communities referred to as biofilm, the Inventors proceeded to assess the antibiofilm activity of the gold+graphene complex or mixture (aqueous phase of the cream).

The testing performed comprises biofilm formation inhibition assessment, using concentrations lower than the MIC of the gold-graphene-lipoic acid mixture (gold nanoparticles <NUM>/ml + graphene <NUM>/ml + lipoic acid <NUM>/ml) and the ability to disrupt a <NUM>-preformed biofilm at concentrations equal to (in case of S. agalactiae) or higher than (in case of E. coli and E. faecalis) the MIC.

The method used is a Micromethod in a <NUM>-well plate with crystal violet staining of the adhered biomass and measurement of the optical density of sample wells, and comparison with growth control wells (untreated) and negative control wells (culture medium and sample at different concentrations only, but without microbial inoculum).

Obtained results are reported in Tables <NUM> and <NUM>.

Biofilm formation inhibition activity was performed at two different sub-MIC concentrations: <NUM>% and <NUM>% v/v (values lower than the MIC obtained toward planktonic strains). Biofilm formation inhibition is reported in terms of inhibition percent compared to untreated growth control.

Mean values ± standard deviation of two independent tests in triplicate.

The gold-graphene-lipoic acid mixture (gold nanoparticles <NUM>/ml + graphene <NUM>/ml + lipoic acid <NUM>/ml) interferes with the forming of the biofilm of E. coli ATCC <NUM>, E. faecalis ATCC <NUM> and S. agalactiae ATCC <NUM> strains, particularly at the concentration of <NUM>% v/v (lower than the MIC against free-living strains).

The activity disrupting a <NUM>-preformed biofilm was performed at a concentration of <NUM>% v/v, equal to the MIC in the case of S. agalactiae or higher than the MIC (<NUM>% and <NUM>% v/v) in the case of E. coli and E.

Mean values ± standard deviation of two independent experiments in triplicate; nt= not tested.

The gold-graphene-lipoic acid mixture (gold nanoparticles <NUM>/ml + graphene <NUM>/ml + lipoic acid <NUM>/ml) (aqueous phase) interferes with the formation of the biofilm of E. coli ATCC <NUM>, E. faecalis ATCC <NUM> and S. agalactiae ATCC <NUM> reference strains, particularly at a concentration of <NUM>% v/v (lower than the MIC against the free-living strains).

The gold-graphene-lipoic acid mixture(gold nanoparticles <NUM>/ml + graphene <NUM>/ml + lipoic acid <NUM>/ml) at concentrations higher than the MIC (<NUM>% and <NUM>% v/v) has an interesting activity disrupting a <NUM>-preformed biofilm, particularly toward E. coli ATCC <NUM>.

In order to determine the synergistic effect between the active agents gold and graphene, joined to form the gold-graphene complex, the FIC (Fractional Inhibitory Concentration) index was used, which measures the interaction between two or more active agents, joined to form a complex, starting from the MIC (Minimal Inhibitory Concentration) values of the same active agents considered individually or in combination, according to the following formula: <MAT> <MAT> <MAT>.

FIC index analysis reveals a synergism of the gold-graphene complex vs the single active agents gold and graphene considered individually; specifically, a Marked Synergism toward Escherichia Coli and Enterococcus Faecalis; a Weak Synergism toward Candida Albicans and a subadditive Synergism toward Streptococcus Agalactiae and Candida Krusei are highlighted. In all cases, markedly in additive synergism ones, but also in weak and subadditive synergism cases, by analyzing the MICs a superiority of the gold-graphene complex is detected compared to the individually considered active agents, that can be highlighted from the concentrations expressed in the Table. The calculation was performed according to what described in <NPL>.

An additional validation study of the effectiveness of the gold-graphene complex according to the present description, by means of in vivo tests on animal model for vaginal candidiasis, is under way.

Animals are treated in compliance with European directives and national regulations on the management of laboratory animals.

The selected animals are Wistar rats or adult female mice.

Candidiasis is induced in the animals, followed by validation thereof by means of analysis of the state of candidiasis (fungal count).

One or more applications of the gold-graphene complex as defined herein to the animal models at periodic time intervals,.

Assessment of infection reduction after a single application and after multiple applications over one week,.

The animals are split into four (<NUM>) groups, each comprised of six (<NUM>) animals, differentiated as follows:.

Claim 1:
A mixture comprising:
- gold particles, and
- graphene particles or a derivative thereof or mixtures thereof,
and a linker binding said gold particles to said graphene particles or derivative thereof or mixtures thereof, wherein said linker is alpha lipoic acid,
for use in the treatment and/or prevention of urogenital tract infections,
wherein said derivative is graphene oxide.