WO2015181084A1

WO2015181084A1 - Use of a nanoemulsion of cinnamaldehyde and/or a metabolite thereof, possibly in association with eugenol and/or carvacrol, to prevent resistance to antibiotics

Info

Publication number: WO2015181084A1
Application number: PCT/EP2015/061429
Authority: WO
Inventors: Raphaël Georges DUVAL; Elisabeth ROSSINES
Original assignee: Eydo Pharma
Current assignee: Eydo Pharma
Priority date: 2014-05-26
Filing date: 2015-05-22
Publication date: 2015-12-03
Anticipated expiration: 2016-11-26
Also published as: FR3021218A1

Abstract

The present invention relates to an oil-in-water nanoemulsion of trans-cinnamaldehyde and/or a metabolite thereof, optionally in association with eugenol and/or carvacrol, for a use in the prevention and/or treatment of an infection caused by a pathogen, wherein the emergence of a phenotype of resistance in said pathogen is prevented; it also relates to said nanoemulsion for a use as an antibiotic which does not develop phenotype of resistance.

Description

USE OF A NANOEMULSION OF CINNAMALDEHYDE AND/OR A METABOLITE THEREOF, POSSIBLY IN ASSOCIATION WITH EUGENOL AND/OR CARVACROL, TO PREVENT RESISTANCE TO ANTIBIOTICS.

The present invention concerns the use of a nanoemulsion of cinnamaldehyde and/or a metabolite thereof, possibly in association with eugenol and/or carvacrol, to prevent resistance to antibiotics.

On April 30 2014, WHO published a report on antimicrobial resistance ("Antimicrobial resistance: global report on surveillance" www. who . int/mediacentre/news/release/2014/amr-report) , encompassing data from 114 countries. This report mentions a serious threat for human health due to the confirmed presence of a antimicrobial resitance in every region of the world. According to Dr Keiji Fukuda, WHO'S Assistant Director-General for Health Security, «Without urgent, coordinated action by many stakeholders, the world is headed for a post-antibiotic era, in which common infections and minor injuries which have been treatable for decades can once again kill,"

The overconsumption of antibiotics, in particular of antimicrobials, is the main cause of the nosocomial infections; the generalization of the use of antibiotics in hospitals, creates a selection stress that promotes the selection of bacteria strains that are resistant to some antibiotics, and contributes to the emergence of hospital multiresistant strains, which can be transmitted from one patient to another one. This phenomenon is so developed that some illness which were initially successfully treated, become incurable.

WHO points on the fact that it is necessary to design new diagnostic products, new antibiotics and other tools to allow health professional to maintain their advance on the development of resistances.

Among these tools, many researchers and industrials explore the avoidance of antibioresistance . Co-therapies can also be an efficient way to fight some resistances to a given antibiotic.

The therapeutical efficiency of the association of a carbapenem, the meropenem (which pertains to the family of L, D-transpeptidase inhibitor lactamins) with a β- lactamase inhibitor, clavulanic acid for treating antibiotic resistant tuberculosis can be cited as an example of therapeutical alternatives to the treatment of resistant (www.inserm.fr, press release March 23, 2010, on researches made by Jean-Emmanuel Hugonnet)

Other authors tried to use essential oils or mixtures of essential oils known for their antibacterial activities, but they were faced to the two following major difficulties. From the one hand, because of the lipophilic nature of the essential oils, the difficulty is to find a formulation allowing an optimal bioavailability. On the other hand, due to their natural origin, they can differ on quantity or quality from one batch to another one, and they always present a risk of uncontrolled toxicity. Indeed, essential oils contain in their totum molecules in very low concentration which can present a high toxicity. It is the case of the essential oil of eugenia caryophyllata clove which mainly contains eugenol (75%) and low amounts of methyl eugenol which is mutacarcinogen . Thus, it is difficult to provide for pharmaceutical compositions containing essential oils because of the regulatory requirements. Some teams (Benoit J. P. et al : WO 201211420) have prepared a specific encapsulation formulation in the lipids of essential oils associated to antibacterials with a description of a broad spectrum of antibacterial, antifungal and antiparasitic in vitro activities. In vitro this solution was demonstrated as efficient, but in vivo trials on a murine model have demonstrated its limits, repeatability of the results was random, in particular because of the random stability of the nanocapsules .

Other authors, tried to prepare nanoemulsions with no addition of emulsifier by applying a vibratory regiment using a transducer at a frequency of more than 900 kHz, but this technique could not be used in industry since the formation and stability of the emulsions were not reproducible (WO2010/149668) .

In order to get rid of the random and not transposable in vivo results, the authors of the present invention decided to work on molecules of high chemical purity in order to eliminate the presence of known or suspected toxics. They also decided to explore original galenic forms in order to obtain reliable and repeatable results in vitro as well as in vivo. The present inventors found that by selecting pure molecules of natural origin present in the most common essential oils, it was possible to obtain compositions which are active on a great number of pathogens. The authors of the present invention concentrated their researches on molecules of high chemical purity from plant essential oils, i.e. trans-cinnamaldehyde, eugenol and carvacrol, and surprisingly found that the trans-cinnamaldehyde, optionally associated to eugenol and/or carvacrol, prevented the appearance of a phenotype of resistance in different pathogens. They also found that, in vivo, a nanoemulsion of trans-cinnamaldehyde, optionally with eugenol and/or carvacrol, allowed to avoid any appearance of a resistance phenotype in a pathogen while treating the infection caused by said pathogen. Summary of the invention

Thus, according to a fist object, the present invention relates to a nanoemulsion of trans- cinnamaldehyde and/or a metabolite thereof, optionally in association with eugenol and/or carvacrol, for a use in the prevention of the emergence of a phenotype of resistance in a pathogen.

According to another object, the invention also relates to a nanoemulsion of trans-cinnamaldehyde and/or a metabolite thereof, optionally in association with eugenol and/or carvacrol, for a use in the prevention and/or treatment of an infection by a pathogen, wherein the appearance of a resistance pathogen to an antibiotic in a pathogen is prevented.

According to a third object, the invention relates to a nanoemulsion of trans-cinnamaldehyde and/or a metabolite thereof in association with eugenol and/or carvacrol for a use as an antibiotic which does not develop phenotype of resistance.

Further, another object of the invention is an in vitro process for preventing the appearance of a phenotype of resistance to an antibiotic in a pathogen, characterized in that it comprises the steps consisting in :

providing trans-cinnamaldehyde and/or a metabolite thereof, optionally in association with eugenol and/or carvacrol; - putting into contact trans-cinnamaldehyde and or its metabolite, optionally in association with eugenol and/or carvacrol, with said pathogen.

Finally, the invention also relates to a nanoemulsion of trans-cinnamaldehyde and/or a metabolite thereof, optionally in association with eugenol and/or carvacrol, for use to prevent and/or treat an infection by a pathogen, even a multi-resistant pathogen, while preventing the appearance of a phenotype of resistance in said pathogen, by intraperitoneal administration.

Detailed description of the invention

According to a first aspect, the present invention relates to a nanoemulsion of trans-cinnamaldehyde and/or a metabolite thereof, for a use in the prevention of the emergence of a resistance phenotype in a pathogen.

It also relates on trans-cinnamaldehyde and/or a metabolite thereof, in association with eugenol and/or carvacrol, for a use in the prevention of the emergence of a phenotype of resistance in a pathogen.

In the present invention, the term cinnamaldehyde can be used also for the trans-isomer of cinnamaldehyde. Cis-isomer and racemic mixtures are not incorporated in the general term « cinnamaldehyde » in the present invention.

In the present invention, cinnamaldehyde can be used as such or in combination with a metabolite thereof, in particular cinnamic alcohol. Also, in the present invention, a metabolite of cinnamaldehyde can be used, in the absence of cinnamaldehyde. In particular, said metabolite is cinnamic alcohol. By « trans-cinnamaldehyde and/or a metabolite thereof in association with eugenol and/or carvacrol », the following different associations are encompassed:

trans-cinnamaldehyde and/or a metabolite thereof and eugenol;

trans-cinnamaldehyde and/or a metabolite thereof and carvacrol ;

trans-cinnamaldehyde and/or a metabolite thereof and eugenol and carvacrol.

In the different associations above, the following volume ratios are used:

trans-cinnamaldehyde and/or a metabolite thereof /eugenol : 20/80 to 80/20, preferably from 30/70 to 70/30 and more preferably of 50/50;

trans-cinnamaldehyde and/or a metabolite thereof

/carvacrol: 20/80 to 80/20, preferably from

30/70 to 70/30 and more preferably of 50/50 ;

trans-cinnamaldehyde and/or a metabolite thereof /eugenol/carvacrol : 10/10/80 to 80/10/10, preferably from 15/15/70 to 70/15/15 and more preferably of 33/33/33.

Any ratio between trans-cinnamaldehyde to a metabolite thereof can be used.

The constituents of these different associations can be under a same and alone galenic form, i.e. they are simultaneously present in the same composition or formulation. Said composition or formulation can comprise excipients. However, advantageously, the amount of excipients and their number will be as low as possible. They can also be under separated forms in order to be administrable separately or sequentially.

In the present invention, trans-cinnamaldehyde is a molecule obtained by extraction of the essential oil of China cinnamon and purification until a purity of at least 98%, preferably at least 99% and still more preferably more than 99.5%.

Advantageously, trans-cinnamaldehyde can also be a synthesis molecule. Metabolites of trans-cinnamaldehyde are preferably synthesis molecules. In particular, cinnamic alcohol is a synthetic product.

In the same way, carvacrol is a molecule obtained by extraction of essential oil of origan and purification until a purity of at least 98%, preferably at least 99% and still more preferably more than 99.5%.

Advantageously, carvacrol can also be a synthesis molecule .

Eugenol is a molecule obtained by extraction of essential oil of eugenia caryophyllata clove and purification until a purity of at least 98%, preferably at least 99% and still more preferably more than 99.5%.

Advantageously, eugenol can also be a synthesis molecule .

In a particularly advantageous embodiment, trans- cinnamaldehyde and/or a metabolite thereof, possibly in association with eugenol and/or carvacrol is provided under the form of an emulsion, and more specifically a nanoemulsion .

The emulsion is an oil-in-water emulsion which has hydrophobic droplets presenting a mean size ranging 10 to 500 nm, preferably 20 to 350 nm and still more preferably 30 to 250 nm. The mean size of the particles is measured by dynamic or viscoelastic diffusion of the light or by spectrometry of photon correlation with a Malvern apparatus .

It has a zeta potential ranging -2 mV to -80 mV, preferably -5 mV to -60 mv and still more preferably - lOmV a -40mV. The zeta potential is measured by electrophoresis with a Malvern zeta-sizer apparatus.

According to a specific embodiment, the emulsion is free of any stabilizer.

The nanoemulsion according to the present invention may be obtained by any process able to provide stable nanoemulsion. For example it can be prepared by sonication. It may also be obtained by:

contacting water with cinnamaldehyde and/or a metabolite thereof, optionally in association with eugenol and/or carvacrol, in order to obtain non-miscible phases ,

- optionally adjusting the pH to 7.0 +/- 0.3 with a pH regulator, preferably a mineral base ;

- optionally adding a stabilizer, preferably a non- ionic stabilizer presenting surfactant properties, such as poloxamer ;

homogenization of the non-miscible phases thus obtained in a high pressure homogenizer, said pressure ranging 300 to 5000 bars, preferably 500 to 3000 bars and still more preferably 800 a 2000 bars.

The mineral base allows the adjustment of the pH to the physiological pH of 7.0 +/-0.3. It is selected from sodium, lithium, potassium, magnesium or calcium hydroxide.

Without being linked by any theory, the inventors believe that the presence of the mineral cation introduced by the pH regulator, also participates in the stabilisation of the finally obtained nanoemulsion.

In the meaning of the present invention, there is

« emergence of a phenotype of resistance to antibiotics in a pathogen » if the increase of the Inhibitory Concentration (MIC) of the antibiotic of at least twice the initial MIC appears before 50 passages during the following test:

The pathogen is cultivated in the presence of sub^¬ inhibitory concentrations (1/4 of the MIC) of the product to be tested, the MIC being regularly determined every 4 to 6 passages. The emergence a resistance corresponds to a stable increase of the MIC equal to at least twice the initial MIC.

The MIC characterizes the bacteriostatic effect of a product, in particular an antibiotic. The MIC of a product for a given strain is the lowest concentration which totally inhibits the growth of said strain in standardized in vitro conditions.

By « pathogen » in the present invention it is meant bacteria, fungi or parasites. In particular, the present invention allows to fight against the development of phenotype of resistance in bacteria.

The term "bacteria", "bacterial strain" or "strain of bacteria" as used in the present invention designates any strain of bacteria able to cause an infection, in particular, any strain of bacteria possibly pathogen for a mammal and more particularly for a human being. The mycobacteria are also included.

As example of bacteria, a gram positive strain selected from the following families can be cited:

Staphylococcaceae, in particular genus

Staphylococcus, represented by the species Staphylococcus aureus (S. aureus ou golden staphylococcus) and

Staphylococcus epidermidis;

- Enterococcaceae, in particular genus Enterococcus, represented by the species Enterococcus faecalis,

Enterococcus faecium, Enterococcus avium and Enterococcus gallinarum; Clostridiaceae, in particular genus Clostridium, represented by the species Clostridium difficile and Clostridium sp;

Streptococcaceae, including A and B haemolytic streptococcus, in particular genus Streptococcus, represented by the species Streptococcus pneumoniae, Streptococcus agalactiae, Streptococcus viridans, Streptococcus and Streptococcus pyogenes;

Aerococcaceae, in particular genus Aerococcus represented by the specie Aerococcus viridans;

Micrococcaceae, in particular genus Micrococcus;

Lactobacillaceae, in particular genus Lactobacillus; Nocardiaceae, in particular genus Nocardia, represented by the species Nocardia asteroides, Nocardia brasiliensis and Nocardia caviae;

- Listeriaceae, in particular genue Listeria, represented by the specie Listeria monocytogenes;

Corynebacteriaceae, in particular genus

Cory/1ebacteri urn;

Bacillaceae, in particular genus Bacillus, represented by the species Bacillus anthracis and Bacillus cereus;

Propionibacteriaceae, in particular genus Propionibacterium, represented by the specie

Propionibacterium acnes; and

Peptococcaceae, in particular genus Peptococcus, represented by the specie Peptococcus magnus .

As example, a gram negative bacterial strain selected from the strains of the following families can also be cited :

Moraxellaceae, in particular genus Acinetobacter, represented by the species Acinetobacter baumannii and Acinetobacter calcoaceticus; Legionellaceae, in particular genus Legionella, represented by the species Legionella Pneumophila , Legionella longbeachae, Legionella bozmanii , and Legionella micdader;

- Enterobacteriaceae, in particular genus Enterobacter, represented by the specie Enterobacter cloacae, genus Escherichia, represented by the species Escherichia coli and Escherichia hermannii , genus Klebsiella, represented by the species Klebsiella pneumoniae and Klebsiella oxytoca, genus Serratia, represented by the specie Serratia marcescens, genus Citrobacter, represented by the species Citrobacter freundii and Citrobacter koseri, and genus Salmonella, Shigella and Proteus;

Pseudomonadaceae, in particular genus Pseudomonas, represented by the species Pseudomonas aeruginosa (P. aeruginosa) , Pseudomonas fluorescens, Pseudomonas putida and Pseudomonas stutzeri;

- Alcaligenaceae, in particular genus Achromobacter, represented by the species Achromobacter xylosoxidans and Achromobacter denitrifleans;

- Sphingomonadaceae, in particular genus Sphingomonas, represented by the specie Sphingomonas paucimobilis;

Pasteurellaceae, in particular genus Haemophilus, represented by the species Haemophilus influenzae and Haemophilus parainfluenzae;

Moraxellaceae, in particular genus Moraxella, represented by the specie Moraxella catarrhalis;

Neisseriaceae, in particular genus Kingella, reprssented by the specie Kingella kingae;

Pasteurellaceae, in particular genus Pasteurella, represented by the specie Pasteurella multicida; - Flavobacteriaceae, in particular genus Capnocytophaga, represented by the specie Capnocytophaga canimorsus; Neisseriaceae, in particular genus Neisseria, represented by the species Neisseria gonorrhoeae and Neisseria lactamica;

genus Campylobacter, represented by the species C. jejuni, et C. coli;

Bacteroidaceae, in particular genus Bacteroides, represented by the specie Bacteroides fragilis;

Stenotrophomonas, in particular, species S. nitritireducens and S. maltophilia ;

meningococcus, in particular the species Neisseria meningi tidis ;

- Fusobacteriaceae, in particular genus Fusobacterium.

Specific examples of strains of interest are selected among the strains pertaining to the following genus :

genus Acinetobacter, represented by the species Acinetobacter baumannii and Acinetobacter calcoaceticus ; genus Staphylococcus, represented by the species Staphylococcus aureus and Staphylococcus epidermidis;

genus Escherichia, represented by the species Escherichia coli , Escherichia coli BLSE and Escherichia hermannii;

- genus Klebsiella, represented by the species Klebsiella pneumoniae and Klebsiella oxytoca;

genus Listeria, represented by the specie Listeria monocytogenes;

- genus Salmonella, represented by the specie Salmonella enteritidis ;

genus Pseudomonas, represented by the species Pseudomonas aeruginosa , Pseudomonas fluorescens, Pseudomonas putida and Pseudomonas stutzeri.

Advantageously, trans-cinnamaldehyde alone as well as in association with eugenol and/or carvacrol was found efficient to prevent the development of a phenotype of resistance in bacteria of the following families:

Pseudomonadaceae, in particular genus Pseudomonas, represented by the species Pseudomonas aeruginosa (P. aeruginosa) , Pseudomonas fluorescens,

Pseudomonas putida and Pseudomonas stutzeri ;

Moraxellaceae, in particular genus Acinetobacter, represented by the species Acinetobacter baumannii and Acinetobacter calcoaceticus and

- Staphylococcaceae, in particular genus

Staphylococcus, represented by the species Staphylococcus aureus and Staphylococcus epidermidis .

As specific examples of strains, are cited:

Acinetobacter baumannii RCH

Acinetobacter baumannii SAN008

Acinetobacter baumannii souche 12

Acinetobacter baumannii AYE

Acinetobacter baumannii CIP7034

Acinetobacter baumannii CIP107292

Acinetobacter baumannii CIP5377

Staphylococcus aureus ATCC25923

Meticillin resistant Staphylococcus aureus (SARM)

0706C0025

SARM 0702E0196

SASM 0703H0036

SASM 070170095

Escherichia coli ATCC25922

Escherichia coli 0705A0434

Enterobacter cloacae 0705A1743

Enterobacter aerogenes 0705A0867

Klebsiella oxytoca 0705C0187

Salmonella enteritidis 4 Pseudomonas aeruginosa ATCC27853

Pseudomonas aeruginosa 0704C0134

Pseudomonas aeruginosa 0703C0259

Listeria monocytogenes N° 58 (internal strain) .

In particular, nanoemulsion of trans-cinnamaldehyde alone or in association with eugenol and/or carvacrol allows to prevent the appearance of the phenotype of resistance in Pseudomonas aeruginosa, Acinetobacter baumannii and Staphylococcus aureus.

Very surprisingly, trans-cinnamaldehyde and/or a metabolite thereof allows to prevent the emergence of a phenotype of resistance in any bacterial strain but it also allows, when associated with eugenol and/or caravacrol, to prevent the emergence of a resistance which is developed when carvacrol or eugenol is used alone .

It was thus demonstrated that, from the one hand, carvacrol does not allow to prevent the emergence of a resistance to carvacrol in P. aeruginosa and, in the other hand, that eugenol does not allow to prevent the emergence of a resistance to eugenol in P. aeruginosa, but that the association trans-cinnamaldehyde and/or a metabolite thereof with eugenol and/or carvacrol allows to prevent the emergence of a resistance in P. aeruginosa .

According to a second aspect, the invention relates on Trans-cinnamaldehyde and/or a metabolite thereof, possibly in association with eugenol and/or carvacrol for a use in the prevention and/or the treatment of an infection caused by a pathogen, in particular a bacterial infection, in which the appearance of a phenotype of resistance to an antibiotic in the pathogen is prevented. It also relates on a method for preventing and/or treating of an infection caused by a pathogen, in particular a bacterial infection, in which the appearance of a phenotype of resistance in the pathogen is prevented by the administration of a therapeutically efficient amount of trans-cinnamaldehyde and/or a metabolite thereof, possibly in association with eugenol and/or carvacrol to a mammal, in particular to a human being suffering from said infection.

In the meaning of the invention, a "bacterial infection" designates an infection caused by a bacterial strain as previously defined and in particular resistant bacterial strains. It includes the early phases of the bacterial contamination, in particular the colonization of the host (for example a mammal, in particular a human being) by said bacterial strain, as well as the latest phases, in particular the various pathologies which are the consequences of the colonization by said bacterial strain ; when a host or a host tissue has been colonized by a bacterial strain, its proliferation results in general cellular or tissular reactions which are manifested by inflammatory syndrome. The term "bacterial infection" thus encompasses any detrimental effect, clinical manifestation, symptoms or illness which appear in a mammal, in particular in a human being, further to the colonization of said mammal or said patient by a bacterial strain.

The term "treatment" includes the destruction of the microorganism, as well as the amelioration of the clinical manifestations or symptoms observed in a mammal, in particular in a human being, as well as the amelioration of the condition of said mammal. It also covers the slowdown, the interruption and the stop of the progression of the infection as well as the inhibition, the attenuation or the prevention of the detrimental consequences of the infection such as the cellular or physiological damages caused by the toxins produced by some bacterial strains on or around the infected tissues.

Thus, the term "treatment" is applied on the main point and/or on the secondary point (s) of infection, as well as to the symptoms resulting from the infection.

The present invention can be applied in the treatment of strains which are

"sensitive", "intermediate" or "resistant" to a given antibiotic, as well as "antibiotic multiresistant " or "pan resistant" strains.

The susceptibility of a given bacterial strain to one or several antibiotics may be determined in vitro by carrying out an antibiogram. "Sensitive" means that a bacteria or a bacterial strain can be killed or its growth can be inhibited by the tested antibiotic. "Intermediate" means that the antibiotic is efficient against said bacterial strain, only in certain conditions, at high doses. "Resistant" means that the antibiotic is not efficient against said bacterial. The term "multiresistant to antibiotics" (or MRS) applies to a bacteria or a bacterial strain resistant to all the tested antibiotics in at least two antibiotic classes; further to the accumulation of natural resistances (mutations) and/or adquired resistances (in particular by plasmide acquisition) , a bacteria or a bacterial strain is sensitive to only a small number of antibiotics which were conventionally therapeutically used. More specific, the term "pan resistant" (or "toto resistant") means that the bacteria or bacterial strain is resistant to all the conventional tested antibiotics. Very surprisingly, trans-cinnamaldehyde, possibly in association with eugenol and/or carvacrol allows the treatment of an infection caused by a multiresistant and even toto-resistant strain without developing new resistance.

A "therapeutically efficient amount" of active substance is an amount which is enough to obtain a significant effect and in particular to give a significant benefit to a mammal, in particular to a human being, in the context of the application for the prevention and/or the treatment as defined in the present invention .

The bacterial infection can be selected for example among an infection of the respiratory tractus (such as a pneumopathy or a pneumonia) , an urinary infection, an infection of the skin or of the soft tissues, a nocosomial legionellose, an infection of the central neural, an invasive aspergillosis, a meningo^¬ encephalitis, an empyema, a gastro-intestinal infection, a cardiopulmonary complication (such as an endocarditis) , a bacteremia, an infection of the operative site or a general infection (such as a septicemia) .

Thus, according to the invention, trans- cinnamaldehyde and/or a metabolite thereof, possibly in association with eugenol and/or carvacrol is for a use as an antibiotic which does not develop phenotype of resistance .

The trans-cinnamaldehyde and/or a metabolite thereof, possibly in association with eugenol and/or carvacrol, plays the role of an antibiotic, i.e. it is active on the pathogen bacterial strains, but it presents the considerable advantage with respect to the presently known antibiotics, of developing no phenotype of resistance in the pathogen bacterial strain. This effect is obtained very advantageously on multi- or toto- resistant bacterial strains. In other words, thanks to the nanoemulsion according to the invention, it is possible to prevent or treat patients suffering from infections caused by pathogens which are resistant, even multi- or tot-resistant to classical antibiotics while no developing new phenotype of resistance.

In all the aspects of the invention above-described, trans-cinnamaldehyde and/or a metabolite thereof, possibly in association with eugenol and/or carvacrol can be formulated in order to facilitate its administration and in particular, it can be formulated with one or more pharmaceutically acceptable carrier (s) or diluent (s). In the case of an injectable administration, a formulation in an aqueous or non-aqueous solution may be used.

The used carriers can be, for example, water, saline solution, serum albumin, Ringer' s solution, polyethylene glycol, water miscible solvents, sugars, binders, excipients, vegetal or mineral oils, water soluble polymers, surfactants, thickeners or gelling agent, solubilisers , stabilizers, preservatives, or a mixture thereof .

According to a specific embodiment, a minimum number and amount of excipients or carriers is used.

Advantageously, the only excipients are a pH regulator, optionally with a non ionic emulsion stabilizer with surfactant properties, such as Poloxamer. In an advantageous embodiment, no excipient is added.

In order to increase the effects of the treatment, successive administrations can be administered at on or several moments, after a specific lapse of time. There can be several administrations a day or a week. Administration routes and dosages will vary depending on various parameters, for example the patient's condition, the type of infection and the seriousness of the infection to be treated.

Trans-cinnamaldehyde and/or a metabolite thereof, possibly in association with eugenol and/or carvacrol is administrated by enteral, parenteral (intravenous, intramuscular or sub-cutaneous) , transcutaneous (or transdermal or percutaneous, cutaneous, mucosal, in particular transmucoso-buccal, nasal, ophtalmic, otologic (in the ear), vaginal, rectal route, or still by intragastric, intracardiac, intraperitoneal, intrapulmonar (by inhalation) or intratracheal route.

Preferably, the administration is by parenteral route or by pulmonary route with the above-described nanoemulsion, in particular by intravenous route or by inhalation .

Doses from 1 to 100 mg/kg of body weight, preferably from 2 to 80 mg/kg per day in one or several administrations .

According to another aspect, the invention relates on an in vitro process for preventing the appearance of a phenotype of resistance in a pathogen characterized in that it comprises:

- providing trans-cinnamaldehyde and/or a metabolite thereof, optionally in association with eugenol and/or carvacrol;

- putting into contact trans-cinnamaldehyde and/or a metabolite thereof, optionally in association with eugenol and/or carvacrol, with said pathogen. All the features described before with respect to the different aspects of the invention are also applicable to this last aspect of the invention.

The invention will be described in more details thereafter in the examples which are given for illustrative purposes only and are not limitative.

EXAMPLES

In the examples, the following products are used:

EP1010: Eugenol marketed by Merck under the name « eugenol for synthesis », lot S6393655 301 - Density: 1.0670 g/ml - purity: equal to or more than 99%.

EP1011: Carvacrol marketed by Sigma-Aldrich under the name « Carvacrol-Kosher », lot STBD2891V - density: 0.9901 g/ml - purity equal to or above 98%.

EP1012: Trans-Cinnamaldehyde marketed by Merck under the name « Trans-cinnamaldehyde for synthesis », lot S6154305 121 - density: 1.0723 g/ml - purity equal to or above 98%.

EP1020: Cinnamaldehyde/Eugenol 50/50 v/v, Lot E1775 - prepared by mixing the above products - density: 1.076 g/ml .

EP1021: Carvacrol/Cinnamaldehyde 50/50 v/v, Lot E1776 - prepared by mixing the above products - density: 1.087 g/ml .

EP1030: Carvacrol /Cinnamaldehyde/Eugenol 33/33/33 v/v. Lot E1777 - prepared by mixing the above products density: 1.035 g/ml. POLOXAMER: Poloxamer marketed by BASF under the name «

Kolliphor P 188 »; Example 1 : emulsion eugenol/carvacrol/trans-cinna- maldehyde 70/15/15 (by weight) :

A mixture eugenol/carvacrol/trans-cinnamaldehyde 70/15/15 (by weight) is prepared. 12 g of said mixture is added on 200g of water (6% by weight) . The bi-phase medium is homogenized at 25000 t/mn on a IKA ultra- turrax® disperser. The pH, which initially was 4.6 is increased between 7.0 and 7.2 with potassium hydroxide 0.1M. The pH is adjusted during 2 hours. The temperature of the medium is maintained between 25 and 30°C. After stabilisation of the pH, the obtained milk is homogenised at 1000 bars with a GEA niro Soasi type Panda plus 1000 apparatus during 30 minutes, while maintaining the temperature inside the homogenizer at 25-30°C.

A nanoemulsion presenting the following features was obtained :

Mean size of the particles: 250 nm measured by a Malvern zeta-sizer apparatus.

Zeta potential: -47 mV measured by a Malvern zeta-sizer apparatus at 25°C.

The medium is then packaged and used as such in tests on mammals.

Example 2 : emulsion eugenol/carvacrol/cinnamaldehyde 70/15/15 with 6% of POLOXAMER

A mixture eugenol/carvacrol/trans-cinnamaldehyde 70/15/15 (by weight) is prepared. 12 g of this mixture is added 200g of water (6% by weight) . The bi-phasic medium is homogenized at 25000 t/mn on a IKA ultra-turrax® disperser. The pH, which initially was of 4.6 is increased between 7.0 and 7.2 with potasse 0.1M. The pH is adjusted during 2 hours. The temperature of the medium is maintained between 25 and 30°C. After stabilisation of the pH, 12g of POLOXAMER Kolliphor P 188 from BASF (6% by weight) is added. The medium is maintained under agitation until a homogeneous milk is obtained (about 30 min) . Then, this milk is homogenized during 30 minutes at 1000 bars with a GEA Niro Soasi type panda plus 1000 apparatus .

A nanoemulsion presenting the following features is obtained :

Mean size of the particles: 44 nm measured in volume with a Malvern zetasizer apparatus at 25°C.

Zeta potential: -64 mV measured by a Malvern zeta-sizer apparatus at 25°C.

The obtained medium is translucent and presents a temperature of 72 °C. The medium is cooled to the room temperature, packaged and used such as for tests on mammals.

Example 3 : emulsion of trans-cinnamaldehyde 100% with 6% Poloxamer On 200 g of water are added 12g (6% by weight) of trans-cinnamaldehyde. The bi-phasic medium is homogenized at 25000 t/mn on a IKA ultra-turrax® disperser. The pH, initially of 3.8 is increased between 7.0 and 7.2 with magnesium hydroxide 0.1M. The pH is adjusted on 2 hours. The temperature of the medium is maintained between 25 and 30°C. After stabilisation of the pH, on the obtained milk 12g of POLOXAMER (6% by weight) are added. The medium is maintained under agitation until a homogeneous milk is obtained (about 30 minutes) , then it is homogenized during 30 minutes, at 1000 bars with a GEA niro Soasi type Panda plus 1000 apparatus.

A nanoemulsion presenting the following features is obtained :

Mean size of the particles: 44 nm measured in volume with a Malvern zeta sizer apparatus at 25°C.

Zeta potential: -5mV measured by a Malvern zeta-sizer apparatus at 25°C.

The obtained medium is translucent and has a temperature of 72 °C.

The medium is cooled to the room temperature, packaged and used as such on mammals.

Example 4 : Emergence of in vitro resistance in

Acinetobacter baumannii , Pseudomonas aeruginosa and

Staphylococcus aureus. The objective of this example is to evaluate in vitro the emergence of resistance in the three bacterial strains Acinetobacter baumannii , Pseudomonas aeruginosa and Staphylococcus aureus in the presence of carvacrol, eugenol, trans-cinnammaldehyde alone and in the mixtures EP1020, EP1021 and EP1030. As positive control, a different antibiotic for each strain was selected:

imipenem for Pseudomonas aeruginosa,

tigecycline for Acinetobacter baumannii and

rifampicin for Staphylococcus aureus.

The emergence of the resistance will be induced by cultivating bacteria in the presence of the product to be tested at sub-inhibitory concentrations (1/4 of the MIC) (Luz et al. 2012). The MIC will then be regularly determined every 4 to 6 passages. The emergence of a resistance during 50 passages will correspond to a stable increase of the MIC corresponding to at least twice the initial MIC.

Materials and Methods

Culture medium

0.85% NaCl, 2 ml, ref. 20070, BioMerieux

Medium BHI (BHI-T) , 9 ml, ref. 42081, BioMerieux Medium Mueller-Hinton (MH) , 200 ml, ref. 64884, BioRad

Blood agar, ref. PB5039A, Oxoid

Mueller-Hinton agar, ref. 63824, BioRad

Water Type 1 in glass tubes of 10 ml, ELGA PURELAB

E-test rifampicin, ref. 526000, BioMerieux

E-test imipenem, ref. MA0115F, Oxoid

E-test tigecycline, ref. 412475, BioMerieux

NaCl 0,9% Versol, ref. 600020, Laboratoire Aguettant

Products to be tested

For each tested product, it is proceeded as described below for trans-cinnamaldehyde : extemporaneous reconstitution of the solution in DMSO at 40% (solution at 400 mg/ml) : add 400 μΐ of cinnamaldehyde to 600 μΐ of DMSO. Add 1 ml of the solution at 400mg/ml to 9 ml of BHI, in order to obtain a concentration of 40 mg/ml.

For the antibiotics, they are diluted in NaCl.

Bacteria

Acinetobacter baumannii: strain reference CIP 7034, sensitive to tigecycline. Pseudomonas aeruginosa: clinical strain 0703C0259, sensitive to imipenem.

Staphylococcus aureus: strain reference ATCC 25923, sensitive to rifampicin.

Antibiotics

Imipenem (Tienam®, MSD, 500 mg) , lot 2094910.

Tigecycline (Tygacil®, Wyeth, 50 mg) , lot F47009.

Rifampicin (Rifadine®, Sanofi Aventis, 600 mg) , lot A2451.

The MIC in carvacrol, cinnamaldehyde and eugenol alone and in the mixtures EP1020, EP1021 and EP1030 of the different bacterial strains were measured by culture on Mueller-Hinton .

Initial MIC of the three bacterial strains for the antibiotics were measured with a conventional E-test.

For each product to be tested, the procedure was as follows :

- Exposition of the strains to the product at sub^¬ inhibitory doses (1/4 MIC) in liquid medium (BHI medium) by passage every 24 hours, during 50 passages .

Exposition of the strains to the product at twice its MIC every 4 to 6 passages in agar plate (MH agar) in order to determine the appearance of a resistant strain.

Determination of the new MIC in agar plate in case of appearance of a resistant strain.

- In case of MIC increase, the selected strain is submitted to an exposition of a sub-inhibitory dose corresponding to 1/4 of the new MIC. The cultures are carried out at 37°C. All manipulations were carried out in a Biosafety cabinet.

At the end of the tests, the bacterial strains were inoculated on CryoBeads® (AES Laboratoires ) and stored at -80°C.

The CryoBeads® system consists in a small tube containing balls in which the strains are adhered, surrounded by a cryo-preservative hypertonic solution.

Results

The results for each bacterial strain are presented separately hereunder.

Acinetobacter baumannii

The results are summarized in tables 1 and 2 hereunder .

After 50 passages, there was no increase of carvacrol, eugenol or cinnamaldehyde MIC for A. baumannii .

After 50 passages, there was no increase of the cinnamaldehyde/eugenol , carvacrol/cinnamaldehyde, or carvacrol/cinnamaldehyde/eugenol combination MIC for A. baumannii .

For tigecycline, the MIC for A. baumannii increased from 1.5 mg/1 to 8 mg/1 (passage 8), to 16 mg/1 (passage 19), to 48 mg/1 (passage 24).

Table 1. MIC of carvacrol, eugenol, cinnamaldehyde, alone and in mixture, and of tigecycline for A baumannii.

A. baumannii

Number Initial

Final MIC Method of passages MIC

50 0.25 mg/ml 0.25 mg/ml ¼ initial MIC

Carvacrol

1/4 Initial

Eugenol 50 0.75 mg/ml 0.75 mg/ml

MIC 1/4 initial

50 0.5 mg/ml 0.5 mg/ml

Cinnamaldehyde MIC

1/4 initial

EP1020 50 0.5 mg/ml 0.5 mg/ml

MIC

1/4 initial

EP1021 50 0.5 mg/ml 0.5 mg/ml

MIC

1/4 initial

EP1030 50 0.5 mg/ml 0.5 mg/ml

MIC

1/4 initial

8 1.5mg/l 8mg/l

MIC

Tigecycline

1/4 increased

24 8mg/l 48 mg/1

MIC

Table 2. Evolution of Tigecycline MIC for Acinetobacter baumannii (MIC in mg/1)

/ new subserie with initial MIC of 8 mg/1

Each cell in the above Table shows the number of

2 new subserie with initial MIC of 16 mg/1

passages and the MIC

3 new subserie with initial MIC of 48 mg/1

4 new subserie with initial MICC of 48 mg/1 assagi

Pseudomonas aeruginosa

The results are summarized in Table 3 and 4 hereunder. Table 3 . MIC of carvacrol, eugenol, cinnamaldehyde, alone and in mixture and imipenem for P. aeruginosa.

P. aeruginosa

Number of

Initial MIC Final MIC Method passages

18 1 mg/ml 10 mg/ml 1/4 initial MIC

Carvacrol

22 2 mg/ml 10 mg/ml 1/4 increased MIC

18 3 mg/ml 10 mg/ml 1/4 initial MIC

Eugenol

50 0.75 mg/ml 0.75 mg/ml 1/4 initial MIC

Cinnamaldehyde

EP1020 50 1 mg/ml 1 mg/ml 1/4 initial MIC

(cinn/eug) - - - -

EP1021 50 1 mg/ml 1 mg/ml 1/4 initial MIC

(carv/cinn) - - - -

EP1030 50 1.5 mg/ml 1.5 mg/ml 1/4 initial MIC

(carv/cinn/eug) - - - -

22 0.25 mg/1 16 mg/1 1/4 initial MIC

Imipenem

8 8 mg/l 16 mg/1 1/4 increased MIC

Table 4. Evolution of MIC for imipenem and Pseudomonas aeruginosa (MIC in mg/1)

/ new subserie of passages with an initial MIC of 2 mg/1

2 new subserie of passages with an initial MIC of 4 mg/1

3 new subserie of passages with an initial MIC of 8 mg/1 Each cell in the above Table shows

4 new subserie of assages with an initial MIC of 8 mg/1 The number of assages and the MIC

Carvacrol MIC for pour P. aeruginosa increased from the initial value of 1 mg/ml to 2 mg/ml at passage 18 and to 10 mg/ml at passage 22.

Eugenol MIC for P. aeruginosa increased from an initial value of 3 mg/ml to 10 mg/ml at passage 18.

There was no change in cinnamaldehyde MIC for P. aeruginosa after 50 passages.

There was no increase of MIC of the mixtures E1020, E1021 and E 1030 for P. aeruginosa during the 50 passages.

For imipenem, the MIC for P. aeruginosa increased from

0.25 mg/1 to 2 mg/1 (passage 3), to 8 mg/1 (passage 8), and to 16 mg/1 (passage 22) .

Staphylococcus aureus

The results are given in Table 5 hereunder.

Table 5. MIC of carvacrol, eugenol, cinnamaldehyde, alone and in mixture and of rifampicine against S. aureus.

S. aureus

Number of

Initial MIC Final MIC Method passages

50 0.5 mg/ml 0.5 mg/ml 1/4 initial MIC

Carvacrol

50 0.5 mg/ml 0.5 mg/ml 1/4 initial MIC

Eugenol

50 1 mg/ml 1 mg/ml 1/4 initial MIC

Cinnamaldehyde

EP1020 50 1 mg/ml 1 mg/ml 1/4 initial MIC

(cinn/eug) - - - -

EP1021 50 0.75 mg/ml 0.75 mg/ml 1/4 initial MIC

(carv/cinn) - - - -

EP1030 50 0.75 mg/ml 0.75 mg/ml 1/4 initial MIC

(carv/cinn/eug) - - - -

8 0.016 mg/1 32 mg/1 1/4 initial MIC

Rifampicine There was no increase of the MIC of carvacrol, cinnamaldehyde or eugenol for S. aureus on the 50 passages .

There was no increase of the MIC of the mixtures EP1020, EP1021 and EP1030 for P. aeruginosa on the 50 passages. For rifampicin, the MIC for S. aureus increased from 0.016 mg/1 to 32 mg/1 after 8 passages, i.e. a 2000 fold increase .

Conclusions

Carvacrol: no resistance developed in A. baumannii, and S. aureus after 50 passages. Resistance developed in P. aeruginosa after 18 passages.

Cinnamaldehyde: no resistance developed in the strains A. baumannii, P. aeruginosa and S. aureus after 50 passages.

Eugenol: no resistance developed in A. baumannii, and S. aureus after 50 passages. Resistance developed in P. aeruginosa after 18 passages.

EP1020 cinnamaldehyde/eugenol 50/50: no resistance developed in A. baumannii , P. aeruginosa and S. aureus after 50 passages.

EP1021 carvacrol/cinnamaledhyde 50/50: no resistance developed in A. baumannii , P. aeruginosa and S. aureus after 50 passages.

EP1030 carvacrol/cinnamaldehyde/eugenol 33/33/33: no resistance developed in A. baumannii , P. aeruginosa and S. aureus after 50 passages. Tigecycline: A. baumannii became resistant to tigecycline after 8 passages and the MIC increased to 48 mg/1 after 24 passages.

Imipenem: P. aeruginosa show an initial resistance to imipenem after 3 passages. P. aeruginosa was resistant to imipenem after 8 passages and the MIC increased to 16 mg/1 after 22 passages.

Rifampicin: S. aureus became resistant to rifampicin after 8 passages and the MIC increased to 32 mg/1.

Exemple 5 :

The objective of this example is to evaluate in vitro the emergence of the resistance of 3 bacterial strains (Acinetobacter baumannii , Pseudomonas aeruginosa and Staphylococcus aureus) towards cinnamaldehyde further to example 4, on 50 additional passages, i.e. 100 passages (from the 50^th passage to the 100^th passage) .

The emergence of the resistance will be induced by cultivating the bacteria in the presence of the product at sub-inhibitory concentrations (1/4 of the MIC) (Luz et al . 2012) . MIC is then regularly measured every 4 to 6 passages. The emergence of a resistance during the additional 50 passages will correspond to a stable increase of the MIC corresponding to at least twice the initial MIC.

Materials and Methods Bacterial strains

- Acinetobacter baumannii: reference CIP 7034, stored on CryoBeads® at the end of example 4

- Pseudomonas aeruginosa: clinical strain 0703C0259, stored on CryoBeads® at the end of example 4

- Staphylococcus aureus: reference strain ATCC 25923, stored on CryoBeads® at the end example 4.

Reactant and culture media

- Vials Api NaCl 0,85% Medium, 2 ml, ref.20070,

BioMerieux

- Medium BHI-T, 9 ml, ref.42081, BioMerieux

- Medium Mueller-Hinton (MH) , 200 ml, ref.64884, BioRad

- Blood agar, ref.PB5039A, Oxoid

Water quality 1 in glass tubes 10 ml autoclaved from ELGA reservoir coupled to a PURELAB system

- Versol NaCl 0,9% for irrigation, ref.600020, Laboratoire Aguettant

Products to be tested

Extemporaneous reconstitution of the solution of trans-cinnamaldehyde in DMSO at 40% (solution at 400 mg/ml) : add 400 μΐ of cinnamaldehyde to 600 μΐ of DMSO. Add 1 ml of the solution of 400mg/ml to 9 ml of

BHI, in order to obtain a concentration of 40 mg/ml.

Methods

In order to study the emergence of resistance, the 3 bacterial strains are cultivated in the presence of the cinnamaldehyde from passage 50 to passage 100 in the same way as in example 4.

All the manipulations are made under PSM. Results

No emergence of resistance to cinnamaldehyde is observed after 100 passages, for the 3 bacterial strains.

Example 6 : Efficiency of the mixture EP1030 in mice having a septicemia caused by Acinetobacter baumannii In this example, the used product is the emulsion of example 1 which is used immediately after its preparation .

The used Acinetobacter baumannii strain is a clinical multiresistant reference strain, ref. SAN 005, CHU Angers, France. This strain causes 90-100% of mortality in a septicemia murine model.

The mice are 12 female mice C3H/HeN SPF from Janvier LABS, aged 6 weeks and weighing 20g ( +/-4g) . They were acclimated during 7 days before the beginning of the study. Mices were separated randomly into two groups of 6 mice each, each group being placed in different cages, under controlled temperature, humidity and light conditions .

The emulsion prepared in example 1 was immediately diluted in a physiologic solution for injection of

8mg/ml, named thereafter EP1031-P

A. baumannii is stored frozen at -80°C on CryoBeads®.

At day 1 Dl, a CryoBead® was placed on blood agar (Blood agar, ref PB5039A, Oxoid) and incubated during 24 h at 37°C under aerobic conditions. A suspension of bacteria in a saline physiologic solution was prepared to obtain a concentration of 5.6 CFU of A. baumannii in 50μ1. Infection :

Group 1: At TO, 6 mice were injected intraperitoneally in the right part of the abdomen, with 5.6 CFU de A. baumannii 50μ1.

The same was administered to Group 2.

Treatment :

Group 1 :

At TO, that is immediately after the infection by A. baumannii , the 6 mice were intraperitoneally injected with lOOyL of EP1031-P i.e. 40mg/kg, in the left side of the abdomen.

At T-3hours, 6 mice were intraperitoneally injected, with lOOyL de EP1031-P i.e. 40mg/kg, in the right side of the abdomen.

The total dose was thus of 80 mg/Kg.

Group 2 :

At TO, i.e. immediately after the infection by A. baumannii , 100yL of sterile physiological solution were intraperitoneally injected to 6 mice, in the left side of the abdomen.

At T-3hours, 6 mice were intraperitoneally injected with 100yL of sterile physiological solution, in the right side of the abdomen.

The total dose was thus of 80 mg/Kg.

The mortality was observed at 24 and 48 hours.

The results were given in the hereunder table: Group Number of Number of Total

deads at deads at mortality survival 24h 48 h at 48h

EP1031-P 0 0 0/6 100% (group 1)

Model 1 3 3/6 50% (group 2)

This test was repeated once and the same results were obtained .

The intraperitoneal injection of a nanoemulsion containing trans-cinnamaldehyde, eugenol and carvacrol at

80mg/kg protected the mice against the acute septicemia caused by A. baumannii resistant to conventional antibiotics . EXEMPLE 7 : Evaluation of nanoemulsions in a Acinetobacter baumannii sepsis model

The objective is to evaluate the efficiency of several products from the pharmaceutical research as antibiotic active ingredients, administered intraperitoneally in a

Acinetobacter baumannii sepsis model.

Sepsis is defined as a systemic reaction in response to the aggression of a micro-organism, the symptoms of which show the damages to endothelium and tissues, resulting in the organ failure. Severe sepsis is a main cause of morbidity and mortality throughout the world, with an incidence estimated to 0.3% in the United States, in France and in Germany (Angus et al, 2001, Schuerholz et al, 2008) .

The first experimental sepsis animal model was validated and published in 1989 (Obana et al) . The selection of the mice C3H/HeN explained in the publication of 1997 (Joly- Guillou et al) is based on a specific sensibility of these mice to Acinetobacter baumannii MPS auguring well for repeatability of the results and thus for a reliable statistic analysis.

Acinetobacter baumannii is an opportunistic germ, responsible of diverse and sometimes severe, nosocomial infections: pneumonia, urinary infection and infection of soft tissues. It is inoculated intraperitoneally . A persistant septic state is observed. The infection rapidly spreads, causing the death of the animals between 24 and 48 hours.

1. Materials and Methods Animals

36 female C3H/HeN SPF mice, from Janvier LABS, aged 6 weeks and weighing 20 grammes +/- 4 grammes. They were selected because of their particular sensitivity to Acinetobacter baumannii , and because of the repeatability of the results due to the consanguinity of the race. They were acclimated for 7 days before starting the study. The mice were allocated to 6 groups (random separation in different therapeutic groups) . They were housed under temperature/humidity/light controlled conditions.

Bacterial strains

Acinetobacter baumannii: reference clinical strain, ref SAN 005 from the Laboratoire de Microbiologie, CHU Angers, France. SAN 005, causes 60%-80% mortality and is multiresistant to antibiotics.

Acinetobacter baumannii SAN is resistant to: -beta-lactamines : amoxicillin and augmentin (amoxicillin + clavulanic acid)

-third generation cephalosporins : cefixime and ceftriaxone

-first generation cephalosporins: cefalotin

-monobactarns : aztreonam

-quinolones : nalidixic acid, ofloxacin, ciprofloxacin, norfloxacin

-aminosides : tobramycin, gentamycin, netilmicin, kanamycin

-carbapenems : ertapenem

-cyclines : doxycycline

-furanes

-carboxypenicillin : temocillin

-cephamycins : cefotixin

-fosfomycin

-cotrimoxazole

Reactants and culture media

- Vials Api NaCl 0,85% Medium, 2 ml, ref.20070, BioMerieux

- Blood agar, ref.PB5039A, Oxoid

Autoclaved purified water in 10 ml glass test tubes; sourced from ELGA + PURELAB system

- Versol 0.9% NaCl, ref.600020, Laboratoire Aguettant

Products to be tested

EP1031 is a combination of actives: eugenol (70%), carvacrol (15%), cinnamaldehyde (15%).

A2 : EP1031 at 30 mg/ml of actives + 10 mg/ml of stabilizer (poloxamer) . White formulation presenting a beginning of oily dephasing in the bottom at receipt. A3: EP1031 at 30 mg/ml of actives + 30 mg/ml of stabilizer (poloxamer) . White formulation stable at receipt .

A4 : EP1031 at 60 mg/ml of actives + 60 mg/ml of stabilizer (poloxamer) . Stable and translucent formulation at receipt.

Formulations Al to A4 were prepared using the same process as the one use in the above example 2. The amounts of actives and stabilizer (Poloxamer) are adapted.

EP1032 is a combination of actives: carvacrol (70%), eugenol (15%), cinnamaldehyde (15%).

Bl: EP1032 at 60 mg/ml + 60 mg/ml of stabilizer (Poloxamer) . White and stable formulation at receipt.

EP1012 contains cinnamaldehyde (100%)

CI: EP1012 at 60 mg/ml of active + 60 mg/ml of stabilizer (Poloxamer). De-phased and white formulation at receipt: presence of an important amount of white powder in the bottom of the vial.

Methods

The objective of Acinetobacter baumannii sepsis model is to allow working on bacterial strains responsible of nosocomial infections, which may be multi- or toto-resistant . This model allows screening anti- infectious molecules in 48 hours and thus to rapidly evaluate the therapeutic value of new antimicrobial agents.

Preparation of the bacterial strains

Strains are stored in CryoBeads® at -80°C. At D-l, each strain is seeded on the still frozen ball on a blood agar with a loop. The agar plate is placed in the oven at 37°C and cultivated aerobically during 24 hours. Study

36 mices CH3/HeN females are separated into 6 groups of 6 animals :

-Group 1 : The 6 mice were IP injected twice during 1 day with 100 μΐ physiological solution, 3h before inoculation and just after inoculation.

-Group 2 : The 6 mice were IP injected twice during 1 day with A2 (40mg/kg in 100 μΐ, i.e. 80 mg/kg per day), 3h before inoculation and just after inoculation.

-Group 3 : The 6 mice were IP injected twice during 1 day with A3 (40mg/kg in 100 μΐ, i.e. 80 mg/kg per day), 3h before inoculation and just after inoculation.

-Group 4 : The 6 mice were IP injected twice during 1 day with A4 (40mg/kg in 100 μΐ, i.e. 80 mg/kg per day), 3h before inoculation and just after inoculation.

-Group 5 : The 6 mice were IP injected twice during 1 day with Bl (40mg/kg in 100 μΐ, i.e. 80 mg/kg per day), 3h before inoculation and just after inoculation.

-Group 6 : The 6 mice were IP injected twice during 1 day with CI (40mg/kg in 100 μΐ, i.e. 80 mg/kg per day), 3h before inoculation and just after inoculation. a. TO

Administration of the products:

-Group 1 : The 6 mice of group 1 were IP injected in the right side of the abdomen with 100 μΐ of physiological solution .

-Group 2 : 266 μΐ of A2 at 30 mg/ml are added to 734 μΐ sterile physiological solution (solution at 8 mg/ml) and each of the 6 mice of group 2 are IP injected in the right side of the abdomen with 100 μΐ of said solution (i.e. 40 mg/kg) .

-Group 3 : 266 μΐ of A3 at 30 mg/ml are added to 734 μΐ sterile physiological solution (solution at 8 mg/ml) and each of the 6 mice of group 3 are IP injected in the right side of the abdomen with 100 μΐ of said solution (i.e. 40 mg/kg) .

-Group 4 : 133 μΐ of A4 at 60 mg/ml are added to 867 μΐ sterile physiological solution (solution at 8 mg/ml) and each of the 6 mice of group 4 are IP injected in the right side of the abdomen with 100 μΐ of said solution (i.e. 40 mg/kg)

-Group 5 : 133 μΐ of Bl at 60 mg/ml are added to 867 μΐ sterile physiological solution (solution at 8 mg/ml) and each of the 6 mice of group 5 are IP injected in the right side of the abdomen with 100 μΐ of said solution (i.e. 40 mg/kg) -Group 6 : 133 μΐ of CI at 60 mg/ml are added to 867 μΐ sterile physiological solution (solution at 8 mg/ml) and each of the 6 mice of group 6 are IP injected in the right side of the abdomen with 100 μΐ of said solution ( i . e . 40 mg/kg) . b. T3: 3 hours after TO

• Bacterial inoculation: the bacterial inoculum is a suspension in physiological solution of 5.10^s CFU in

50 μΐ, administered to each of the 12 mice intraperitoneally in the right part of the abdomen.

• Administration of the products:

-Group 1 : The 6 mice of group 1 were IP injected in the left side of the abdomen with 100 μΐ of physiological solution

-Group 2 : 266 μΐ of A2 at 30 mg/ml are added to 734 μΐ sterile physiological solution (solution at 8 mg/ml) and each of the 6 mice of group 2 are IP injected in the left side of the abdomen with 100 μΐ of said solution (i.e. 40 mg/kg) .

-Group 3 : 266 μΐ of A3 at 30 mg/ml are added to 734 μΐ sterile physiological solution (solution at 8 mg/ml) and each of the 6 mice of group 3 are IP injected in the left side of the abdomen with 100 μΐ of said solution (i.e. 40 mg/kg) . -Group 4 : 133 μΐ of A4 at 60 mg/ml are added to 867 μΐ sterile physiological solution (solution at 8 mg/ml) and each of the 6 mice of group 4 are IP injected in the left side of the abdomen with 100 μΐ of said solution (i.e. 40 mg/kg)

-Group 5 : 133 μΐ of Bl at 60 mg/ml are added to 867 μΐ sterile physiological solution (solution at 8 mg/ml) and each of the 6 mice of group 5 are IP injected in the left side of the abdomen with 100 μΐ of said solution (i.e. 40 mg/kg)

-Group 6 : 133 μΐ of CI at 60 mg/ml are added to 867 μΐ sterile physiological solution (solution at 8 mg/ml) and each of the 6 mice of group 6 are IP injected in the left side of the abdomen with 100 μΐ of said solution (i.e. 40 mg/kg) .

Mortality is measured at 24 hours and at 48 hours.

2. Results

· The treatment with A2 at a dose of 40 mg/kg, 3 hours before the bacterial inoculation and immediately after the inoculation, allows saving 83 % of the mice.

• The treatment with A3 at a dose of 40 mg/kg, 3 hours before the bacterial inoculation and immediately after the inoculation, allows saving 83 % of the mice.

•The treatment with A4 at a dose of 40 mg/kg, 3 hours before the bacterial inoculation and immediately after the inoculation, allows saving 100 % of the mice.

•The treatment with Bl at a dose of 40 mg/kg, 3 hours before the bacterial inoculation and immediately after the inoculation, allows saving 66 % of the mice.

•The treatment with A4 at a dose of 40 mg/kg, 3 hours before the bacterial inoculation and immediately after the inoculation, allows saving 33 % of the mice. Conclusion

The administration of allows saving 100% mice, and no mouse showed illness failure signals during these 48 hours.

Claims

REVEND I CAT I ONS

1. Oil-in-water nanoemulsion of trans-cinnamaldehyde and/or a metabolite thereof, optionally in association with eugenol and/or carvacrol for a use in the prevention and/or treatment of an infection caused by a pathogen, wherein the emergence of a phenotype of resistance in said pathogen is prevented.

2. Oil-in-water nanoemulsion of Trans-cinnamaldehyde and/or a metabolite thereof, optionally in association with eugenol and/or carvacrol, for a use as an antibiotic which does not develop phenotype of resistance.

3. Oil-in-water nanoemulsion for a use according to claim 1 or claim 2, wherein the mean size of the hydrophobic droplets ranges 10 to 500 nm, preferably 20 to 350 nm and still more preferably 30 to 250 nm.

4. Oil-in-water nanoemulsion for a use according to anyone of claims 1 to 3, wherein the zeta potential ranges -2mV to -80mV, preferably -5mV to -60mV and still more preferably -lOmv to -40mV.

5. Oil-in-water nanoemulsion for a use according to anyone of claims 1 to 4, wherein it is free of any stabilizer.

6. Oil-in-water nanoemulsion for a use according to anyone of claims 1 to 4, wherein the nanoemulsion is obtainable by:

- putting into contact water with cinnamaldehyde and/or a metabolite thereof, optionally in association with eugenol and/or carvacrol in order to obtain non-miscible phases ,

- optionally adjusting the pH to 7.0 +/- 0.3 with a mineral base;

optionally adding a stabilizer, preferably a non- ionic stabilizer presenting surfactant properties ; homogenization of the thus obtained non-miscible phases in a high pressure homogenizer, said pressure ranging 300 to 5000 bars, preferably 500 to 3000 bars and still more preferably 800 to 2000 bars.

7. Oil-in-water nanoemulsion for a use according to anyone of claims 1 to 6, wherein the pathogen is a bacteria selected from the following families:

Staphylococcaceae, in particular genus

Staphylococcus, represented by the species Staphylococcus aureus and Staphylococcus epidermidis;

- Enterococcaceae, in particular genus Enterococcus, represented by the species Enterococcus faecalis, Enterococcus faecium, Enterococcus avium and Enterococcus gallinarum;

Clostridiaceae, in particular genus Clostridium, represented by the species Clostridium difficile and Clostridium sp;

- Streptococcaceae, including haemolytic Streptococcus A and B, in particular genus Streptococcus, represented by the species Streptococcus pneumoniae, Streptococcus agalactiae, Streptococcus viridans, Streptococcus and Streptococcus pyogenes;

- Micrococcaceae, in particular genus Micrococcus;

- Lactobacillaceae, in particular genus Lactobacillus;

Nocardiaceae, in particular genus Nocardia, represented by the species Nocardia asteroides, Nocardia brasiliensis and Nocardia caviae;

- Listeriaceae, in particular genus Listeria, represented by the specie Listeria monocytogenes;

Corynebacteriaceae, in particular genus

Corynebacterium; Bacillaceae, in particular genus Bacillus, represented by the species Bacillus anthracis and Bacillus cereus;

Propionibacteriaceae, in particular genus Propionibacterium, represented by the specie Propionibacterium acnes;

Peptococcaceae, in particular genus Peptococcus, represented by the specie Peptococcus magnus;

Moraxellaceae, in particular genus Acinetobacter, represented by the species Acinetobacter baumannii and Acinetobacter calcoaceticus;

Legionellaceae, in particular genus Legionella, represented by the species Legionella Pneumophila , Legionella longbeachae, Legionella bozmanii , and Legionella micdader;

- Sphingomonadaceae, in particular genus Sphingomonas, represented by the species Sphingomonas paucimobilis; Pasteurellaceae, in particular genus Haemophilus, represented by the species Haemophilus influenzae and Haemophilus parainfluenzae;

Neisseriaceae, in particular genus Kingella, represented by the specie Kingella kingae;

Pasteurellaceae, in particular genus Pasteurella, represented by the specie Pasteurella multicida;

- Flavobacteriaceae, in particular genus Capnocytophaga, represented by the specie Capnocytophaga canimorsus;

Neisseriaceae, in particular genus Neisseria, represented by the species Neisseria gonorrhoeae and Neisseria lactamica;

- Campylobacter, represented by the species C. jejuni, and C. coli;

Stenotrophomonas, in particular, the species S. nitritireducens and S. maltophilia ;

meningoccocus , in particular the species Neisseria meningi tidis ;

- Fusobacteriaceae, in particular genus Fusohacterium.

8. Oil-in-water nanoemulsion for a use according to anyone of claims 1 to 7, wherein the pathogen is a bacteria selected among the strain pertaining to the following genus:

genus Acinetobacter, represented by the species Acinetobacter baumannii and Acinetobacter calcoaceticus; genus Staphylococcus, represented by the species Staphylococcus aureus and Staphylococcus epidermidis; genus Escherichia, represented by the species Escherichia coli , Escherichia coli BLSE and Escherichia hermannii;

genus Listeria, represented by the specie Listeria monocytogenes;

- genus Salmonella, represented by the specie Salmonella enteritidis ;

- genus Pseudomonas, represented by the species

Pseudomonas aeruginosa , Pseudomonas fluorescens, Pseudomonas putida and Pseudomonas stutzeri.

9. Oil-in-water nanoemulsion for a use according to anyone of claims 1 to 8, wherein it is administered by parenteral, oral, transmucosal , percutaneous, pulmonary, and more specifically by intravenous route or by inhalation .

10. Oil-in-water nanoemulsion for use according to anyone of claims 1 to 9, wherein the infection is caused by a multiresistant pathogen which does not develop any new phenotype of resistance.

11. Oil-in-water nanoemulsion for use according to anyone of claims 1 to 10, wherein the metabolite of trans-cinnamaldehyde is cinnamic alcohol.

12. In vitro method for preventing the appearance of a phenotype of resistance in a pathogen, characterized in that it comprises the steps of:

- putting into contact trans-cinnamaldehyde and/or a metabolite thereof, optionally in association with eugenol and/or carvacrol, with said pathogen.