FR3021218A1 - USE OF CINNAMALDEHYDE IN COMBINATION WITH EUGENOL AND / OR CARVACROL TO COMBAT ANTIBIOTIC RESISTANCE - Google Patents

Abstract

The present invention relates to transcinnamaldehyde in combination with eugenol and / or carvacrol for use in preventing the emergence of a resistance phenotype in a pathogen, it also relates to transcinnamaldehyde in combination with eugenol and / or carvacrol for use in the prevention and / or treatment of infection by a pathogen, wherein the occurrence of a resistance phenotype in a pathogen is prevented.

Description

USE OF CINNAMALDEHYDE IN COMBINATION WITH EUGENOL AND / OR CARVACROL TO COMBAT ANTIBIOTIC RESISTANCE.

The present invention relates to a novel use of cinnamaldehyde in combination with eugenol and / or carvacrol for the control of resistance to antiobiotics. WHO recently published (April 30, 2014) a report on antibiotic resistance (WHO Global Report on Antimicrobial Resistance, website: www.who.int/mediacentre/news/release/2014/amrreport) "Antimicrobial Resistance: Global Report on Surveillance"), which gathers data from 114 countries. This report indicates a serious threat to human health due to the proven presence of antibiotic resistance in all regions of the world. According to Dr. Keiji Fukuda, WHO Assistant Director-General for Health Security, "the world is moving towards a post-antibiotic era, where common infections and minor injuries that have been treated for decades, could again, unless the many actors involved act urgently and in a coordinated manner. "

Overconsumption of antibiotics, especially antibacterials, is the main cause of nosocomial infections; the generalization of the use of antibiotics, especially in hospitals, creates a selection pressure that favors the selection of strains of bacteria resistant to certain antibiotics, and contributes to the emergence of multiresistant hospital strains, which can be transmitted from one patient to another. This phenomenon has reached such a magnitude that some diseases, which were initially treated successfully with antibiotics, become incurable. WHO is calling attention to the need to develop new diagnostics, antibiotics and other tools to enable health professionals to stay ahead of the progression of resistance. Among these tools, the avoidance of antibiotic resistance is a path explored by many researchers and industrialists. Co-therapies may also be an effective way of counteracting certain resistance to a given antibiotic. As part of the work on the search for therapeutic alternatives to the treatment of antibiotic-resistant tuberculosis (www.inserm.fr, press release of March 23, 2010, citing the work of Jean-Emmanuel Hugonnet), we will mention for example the therapeutic efficacy of the combination of a carbapenem, namely meropenem (family of 8-lactam inhibitors of L, D-transpeptidases), with an inhibitor of S-lactamase, namely clavulanic acid, in order to treat tuberculosis resistant to antibiotics.

Other authors have tried to use essential oils or mixtures of essential oils known for their anti-bacterial activities, but they have encountered two major difficulties. On the one hand, because of their lipophilic nature, the difficulty is to find a formulation allowing optimal bioavailability. On the other hand, because of their natural origin, they may have quantitative or qualitative composition differences from one batch to another, and always present a risk of uncontrolled toxicity. Indeed, essential oils contain in their totum, molecules in very small quantities that can express a high toxicity. This is the case of clove oil eugenia caryophyllata (clove) which contains mainly eugenol (75%) and methyl eugenol in small quantities which is muta-carcinogenic. Essential oils are therefore difficult to implement in pharmaceutical compositions due to regulatory requirements. Some teams (Benoit JP et al: WO 201211420) have implemented a specific encapsulation formulation in lipids of essential oils associated with antibacterials with a description of a broad spectrum of antibacterial, antifungal and in vitro antiparasitic activities. . This solution has shown its efficacy in vitro but in vivo tests on a murine model have revealed its limits, the reproducibility of the results having proved random, in particular because of the random stability of the nanocapsules. Other authors have tried to prepare nanoemulsions without the addition of an emulsifier by imposing a vibratory regime using a transducer operating at a frequency of more than 900 kHz, but this technique has not proved to be reproducible industrially. formation and stability of the emulsions not being reproducibly ensured (WO2010 / 149668). In order to overcome random and non-transposable results in vivo, the authors of the present invention decided to work on molecules of high chemical purity to eliminate the presence of known or suspected toxic. They also decided to explore original dosage forms in order to obtain reliable and reproducible results both in vitro and in vivo. The authors have discovered that by making a selection among the pure molecules of natural origin present in the most common essential oils, it was possible to obtain active compositions on a large number of pathogens. The present inventors have concentrated their research on molecules of high chemical purity derived from plant essential oils, namely trans-cinnamaldehyde, eugenol and carvacrol, and have discovered quite surprisingly that trans-cinnamaldehyde, possibly associated with eugenol and / or carvacrol, prevented the development of a resistance phenotype in different pathogens. They have also discovered that in vivo a trans-cinnamaldehyde combination 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 first subject, the present invention relates to trans-cinnamaldehyde, optionally in combination with eugenol and / or carvacrol, intended for use in the prevention of the emergence of a resistance phenotype in a pathogen. According to another subject, the invention also relates to trans-cinnamaldehyde in combination with eugenol and / or carvacrol intended for use in the prevention and / or treatment of infection by a pathogen, wherein appearance of an antibiotic resistance phenotype in a pathogen is prevented. According to a third subject, the invention relates to trans-cinnamaldehyde in combination with eugenol and / or carvacrol for use as an antibiotic that does not develop a resistance phenotype. Finally, another subject of the invention is a method for preventing the appearance of an antibiotic resistance phenotype in a pathogenic agent characterized in that it comprises the steps of: providing trans-cinnamaldehyde, optionally in combination with eugenol and / or carvacrol; contact trans-cinnamaldehyde, optionally in combination with eugenol and / or carvacrol, with said pathogen. Finally, the invention also relates to a particular dosage form of trans-cinnamaldehyde, optionally in combination with eugenol and / or carvacrol, for use in preventing the occurrence of resistance phenotype in pathogens.

DETAILED DESCRIPTION OF THE INVENTION According to a first aspect, the present invention relates to trans-cinnamaldehyde for use in preventing the emergence of a resistance phenotype in a pathogen.

It also relates to trans-cinnamaldehyde in combination with eugenol and / or carvacrol for use in preventing the emergence of a resistance phenotype in a pathogen.

By "trans-cinnamaldehyde in combination with eugenol and / or carvacrol" is meant the following different combinations: trans-cinnamaldehyde and eugenol; trans-cinnamaldehyde and carvacrol; trans-cinnamaldehyde and eugenol and carvacrol. In the various combinations above, the following volume ratios are used: trans-cinnamaldehyde / eugenol: 20/80 to 80/20, preferably 30/70 to 70/30 and more preferably 50/50; trans-cinnamaldehyde / carvacrol: 20/80 to 80/20, preferably 30/70 to 70/30 and more preferably 50/50; trans-cinnamaldehyde / eugenol / carvacrol: 10/10/80 to 80/10/10, preferably 15/15/70 to 70/15/15 and more preferably 33/33/33. The constituents of these various combinations may be in one or the same galenic form, that is to say they may be simultaneously present in the same composition or formulation. Said composition or formulation may comprise excipients. However, advantageously, the amount of excipients and their number will be as small as possible.

They may also be in separate forms so that they can be administered separately or sequentially. In the present invention, trans-cinnamaldehyde is a molecule obtained by extraction of essential oil of Chinese cinnamon and purification to a purity of at least 98%, preferably at least 99% and more preferably more than 99.5%.

Trans-cinnamaldehyde may also advantageously be a synthetic molecule. In the present invention, the term cinnamaldehyde may also be used to designate the trans isomer of cinnamaldehyde. The cis-isomer and the racemic mixtures are not incorporated by the general term "cinnamaldehyde" in the present invention. In the same way, caravacrol is a molecule obtained by extraction of essential oil of oregano and purification to a purity of at least 98%, preferably at least 99%, and more preferably more than 99.5%. Carvacrol may also advantageously be a synthetic molecule.

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

Eugenol may also advantageously be a synthetic molecule. For the purposes of the present invention, there is "emergence of an antibiotic resistance phenotype in a pathogen" if an increase in the Minimal Inhibition Concentration (MIC) of the antibiotic by at least 2 times the MIC starting point appears before 50 passages in the following test: The pathogen is cultured 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 of a resistance corresponds to a stable increase of the MIC equal to at least 2 times the initial MIC.

The MIC characterizes the bacteriostatic effect of a product, especially an antibiotic. The MIC of a product for a given strain is the lowest concentration that completely inhibits the growth of said strain under standardized conditions in vitro. By "pathogen" in the present invention is meant bacteria, fungi or parasites. In particular, the present invention makes it possible to combat the development of resistance phenotype in bacteria. The term "bacterium", "bacterial strain" or "strain of bacteria" as used herein means any strain of bacteria capable of causing infection and, in particular, any strain of bacteria potentially pathogenic to a mammal and especially for a human being. Also included are mycobacteria. By way of example of a bacterium, mention may be made of a gram-positive bacterial strain chosen from the following families: the family of Staphylococcaceae, in particular the genus Staphylococcus, represented by the species Staphylococcus aureus (S.aureus or Staphylococcus aureus) and Staphylococcus epidermidis; - the Enterococcaceae family, in particular the genus Enterococcus, represented by the species Enterococcus faecalis, Enterococcus faecium, Enterococcus avium and Enterococcus gallinarum; - the family Clostridiaceae, in particular the genus Clostridium, represented by the species Clostridium difficile and Clostridium sp; the family of Streptococcaceae, including hemolytic Streptococci A and B, in particular the genus Streptococcus, represented by the species Streptococcus pneumoniae, Streptococcus agalactiae, Streptococcus viridans, Streptococcus and Streptococcus pyogenes; the Aerococcaceae family, in particular the genus Aerococcus represented by the species Aerococcus viridans; the family Micrococcaceae, in particular the genus Micrococcus; - the family Lactobacillaceae, in particular the genus Lactobacillus; - the family Nocardiaceae, in particular the genus Nocardia, represented by the species Nocardia asteroids, Nocardia brasiliensis and Nocardia caviae - the family Listeriaceae, in particular the genus Listeria, represented by the species Listeria monocytogenes; - the family Corynebacteriaceae, especially the genus Corynebacterium; - the Bacillaceae family, in particular the genus Bacillus, represented by the species Bacillus anthracis and Bacillus cereus; the family Propionibacteriaceae, in particular the genus Propionibacterium, represented by the species Propionibacterium acnes; and - the family Peptococcaceae, in particular the genus Peptococcus, represented by the species Peptococcus magnus. By way of example, mention may also be made of a gram-negative bacterial strain chosen from the following families: - the family Moraxellaceae, in particular the genus Acinetobacter, represented by the species Acinetobacter baumannii and Acinetobacter calcoaceticus - the family Legionellaceae, in particular the genus Legionella, represented by the species Legionella Pneumophila, Legionella longbeachae, Legionella bozmanii, and Legionella micdader; - the Enterobacteriaceae family, in particular the genus Enterobacter, represented by the species Enterobacter cloacae, the genus Escherichia, represented by the species Escherichia neck and Escherichia hermannii, the genus Klebsiella, represented by the species Klebsiella pneumoniae and Klebsiella oxytoca, the genus Serratia, represented by the species Serratia marcescens, the genus Citrobacter, represented by the species Citrobacter freundii and Citrobacter koseri, and the genera Salmonella, Shigella and Proteus; the family Pseudomonadaceae, in particular the genus Pseudomonas, represented by the species Pseudomonas aeruginosa (P. aeruginosa), Pseudomonas fluorescens, Pseudomonas putida and Pseudomonas stutzeri; the Alcaligenaceae family, in particular the genus Achromobacter, represented by the species Achromobacter xylosoxidans and Achromobacter denitrificans; - the Sphingomonadaceae family, in particular the genus Sphingomonas, represented by the species Sphingomonas paucimobilis; - the family of Pasteurellaceae, in particular the genus Haemophilus, represented by the species Haemophilus influenzae and Haemophilus parainfluenzae; - the family Moraxellaceae, in particular the genus Moraxella, represented by the species Moraxella catarrhalls; - the Neisseriaceae family, in particular the genus Kingella, represented by the species Kingella kingae - the family of Pasteurellaceae, in particular the genus Pasteurella, represented by the species Pasteurella multicida; the family Flavobacteriaceae, in particular the genus Capnocytophaga, represented by the species Capnocytophaga canimorsus; the family Neisseriaceae, in particular the genus Neisseria, represented by the species Neisseria gonorrhoeae and Neisseria lactamica; - the genus Campylobacter, represented by C. jejuni species, and C. neck; the family Bacteroidaceae, in particular the genus Bacteroides, represented by the species Bacteroides fragilis; - the family Stenotrophomonas, in particular, the species S. nitritireducens and S. maltophilia; the family of meningococci, in particular the Neisseria meningitidis species; and - the family Fusobacteriaceae, in particular the genus Fusobacterium. Particular examples of strains of interest are selected from strains belonging to the following genera: - genus Acinetobacter, represented by the species Acinetobacter baumannii and Acinetobacter calcoaceticus; Staphylococcus genus, represented by the species Staphylococcus aureus (S.aureus or Staphylococcus aureus) and Staphylococcus epidermidis; - Escherichia genus, represented by the species Escherichia neck and Escherichia hermannii; - genus Klebsiella, represented by the species Klebsiella pneumoniae and Klebsiella oxytoca; Listeria genus, represented by the species Listeria monocytogenes; - genus Salmonella, represented by the species Salmonella enteritidis; and genus Pseudomonas, represented by the species Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas putida and Pseudomonas stutzeri. Particularly advantageously, transcinnamaldehyde alone and in combination with eugenol and / or carvacrol has been shown to be effective in preventing the development of a resistance phenotype in bacteria of the following families - the family Pseudomonadaceae, in particular the genus Pseudomonas, represented by the species Pseudomonas aeruginosa (P. aeruginosa), Pseudomonas fluorescens, Pseudomonas putida and Pseudomonas stutzeri; - the family Moraxellaceae, in particular the genus Acinetobacter, represented by the species Acinetobacter baumannii and Acinetobacter calcoaceticus and - the family Staphylococcaceae, in particular the genus Staphylococcus, represented by the species Staphylococcus aureus (S. aureus or Staphylococcus aureus) and Staphylococcus epidermidis. As specific examples of strains, there may be mentioned Acinetobacter baumannii RCH Acinetobacter baumannii SAN008 Acinetobacter baumannii strain 12 Acinetobacter baumannii AYE Acinetobacter baumannii CIP7034 Acinetobacter baumannii CIP107292 Acinetobacter baumannii CIP5377 Staphylococcus aureus ATCC25923 Methicillin-resistant Staphylococcus aureus (MRSA) 070600025 SARM 0702E0196 SASM 0703H0036 SASM 070170095 Escherichia neck ATCC25922 Escherichia neck 0705A0434 Enterobacter cloacae 0705A1743 Enterobacter aerogenes 0705A0867 Klebsiella oxytoca 0705C0187 Salmonella enteritidis 4 Pseudomonas aeruginosa ATCC27853 Pseudomonas aeruginosa 0704C0134 Pseudomonas aeruginosa 0703CO259 Listeria monocytogenes No. 58 Internal strain. In particular, trans-cinnamaldehyde in combination with eugenol and / or carvacrol makes it possible to prevent the appearance of the resistance phenotype in Pseudomonas aeruginosa, Acinetobacter baumannii and Staphylococcus aureus. Surprisingly, transcinnamaldehyde alone prevents the emergence of a resistance phenotype in all bacterial strains, but it also allows, when combined with eugenol and / or carvacrol to prevent the emergence of a resistance which develops when carvacrol on the one hand or eugenol on the other hand are used alone. Thus, it has been demonstrated firstly that carvacrol does not prevent the emergence of carvacrol resistance in P. aeruginosa and secondly that eugenol does not prevent the emergence resistance to eugenol in P. aeruginosa, but that the combination trans-cinnamaldehyde with eugenol and / or carvacrol can prevent the emergence of resistance in P. aeruginosa. In another aspect, the invention relates to trans-cinnamaldehyde in combination with eugenol and / or carvacrol for use in the prevention and / or treatment of infection by a pathogen, including a bacterial infection. wherein the occurrence of an antibiotic resistance phenotype in a pathogen is prevented.

It also relates to a method for preventing and / or treating an infection by a pathogen, in particular a bacterial infection, in which the appearance of a resistance phenotype in a pathogen is prevented by the administration of a therapeutically effective amount. of transcinnamaldehyde in combination with eugenol and / or carvacrol to a mammal, particularly to humans, having an infection. A "bacterial infection" in the sense of the present invention means an infection caused by a bacterial strain as defined above and includes both the earliest phases of bacterial contamination, in particular the colonization of the host (for example a mammal , especially a human) by said bacterial strain, as the later phases, including the various pathologies that are the consequence of colonization by said bacterial strain; once a bacterial strain has colonized a host or host tissue, its proliferation usually results in cellular, tissue or general reactions, which usually result in an inflammatory syndrome. The expression "bacterial infection" therefore encompasses any adverse effect, clinical sign, symptom or any disease occurring in a mammal and in particular in a human following colonization of said mammal or said patient by a bacterial strain.

The term "treatment" includes the destruction of the microorganism, as well as the improvement of the clinical signs or symptoms observed in a mammal, in particular in a human, as well as the improvement of the condition of said mammal. It also covers slowing down, stopping, and stopping the progression of infection as well as inhibiting, mitigating, or preventing the adverse consequences of infection such as cellular or physiological damage caused by the infection. toxins produced by certain bacterial strains in infected or surrounding tissues. Thus, the term "treatment" applies to the primary point of infection and / or the point (s) of secondary infection (s), as well as to the symptoms arising from the infection. The present invention finds application in the treatment of so-called "sensitive", "intermediate" or "resistant" strains to a given antibiotic, as well as "multidrug-resistant" or "pan-resistant" strains. The susceptibility of a given bacterial strain to one or more antibiotics suspected or known may be determined in vitro in particular by performing an antibiogram. "Sensitive" means that a bacterium or a strain of bacteria can be killed or that its growth can be inhibited by the antibiotic tested. "Intermediate" means that the antibiotic is effective only under certain conditions, in high doses, against said bacterial strain. "Resistant" means that the antibiotic is ineffective against said bacterial strain. The term "multidrug-resistant" (or MDR) refers to a bacterium or strain of bacteria resistant to all antibiotics tested in at least two classes of antibiotics; following the accumulation of natural resistances (mutations) and / or acquired resistances (in particular by the acquisition of plasmids), a bacterium or a strain of bacteria is no longer sensitive to a small number of usually active antibiotics in therapy. More specific, "pan resistant" (or "toto resistant") means that the bacterium or bacterial strain is resistant to all conventional antibiotics tested.

Surprisingly, transcinnamaldehyde in combination with eugenol and / or carvacrol makes it possible to treat an infection due to a multidrug-resistant strain and even toto-resistance without developing new resistance.

A "therapeutically effective amount" of active ingredient is an amount sufficient to achieve a significant effect and in particular to provide a significant benefit to a mammal and, in particular, to a human as part of an application for prevention or treatment as defined in this application. Bacterial infection may be, for example, selected from respiratory tract infection (such as pneumonitis or pneumonia), urinary tract infection, skin or soft tissue infection, nosocomial legionellosis, system infection. central nervous system, invasive aspergillosis, meningoencephalitis, empyema, gastrointestinal infection, cardiopulmonary complication (such as endocarditis), bacteremia, surgical site infection, or systemic infection (such as sepsis) ).

Thus, according to the invention, trans-cinnamaldehyde in combination with eugenol and / or carvacrol is intended to be used as an antibiotic that does not develop a resistance phenotype. The trans-cinnamaldehyde combination with eugenol and / or carvacrol acts as an antibiotic, ie it is active on pathogenic bacterial strains, but it has the considerable advantage over antibiotics currently known, to develop no phenotype of resistance in the pathogenic bacterial strain. This effect is obtained quite advantageously on bacterial strains multi- or toto-resistant. In all aspects of the invention described above, trans-cinnamaldehyde in combination with eugenol and / or carvacrol may be formulated to facilitate its administration and in particular may be formulated with one or more carriers ( s) or pharmaceutically acceptable diluent (s). In the case of an injectable administration, it is possible in particular to choose a formulation in an aqueous, non-aqueous or isotonic solution. The vehicles used may be, for example, water, saline, serum albumin, Ringer's solution, polyethylene glycol, water-miscible solvents, sugars, binders, excipients, vegetable or mineral oils, water-soluble polymers, surfactants, thickeners or gelling agents, solubilizers, stabilizers, preservatives, or a combination thereof. According to a particular embodiment, a minimum of excipients or vehicles are used. Advantageously, the only excipients are a pH regulating agent, optionally with a nonionic emulsion stabilizer with surfactant properties, such as Poloxamer. Most advantageously, no excipient is added.

Particularly advantageously, transcinnamaldehyde in combination with eugenol and / or carvacrol is provided as an emulsion, and preferably as a nanoemulsion. The emulsion is an oil-in-water emulsion which has hydrophobic droplets of average size less than 10 to 500 nm, preferably 20 to 350 nm and more preferably still 50 to 250 nm and having a zeta potential ranging from 2 mV at -80 mV, preferably from -5 mV to -60 mV and more preferably from -10 mV to -40 mV. The Zeta potential is measured by electrophoresis using a Malvern device of the zeta-sizer type. In particular, the emulsion is devoid of any stabilizing agent.

The average particle size is measured by dynamic or viscoelastic scattering of light or photon correlation spectrometry using a Malvern-type apparatus. According to another aspect, the present invention relates to a nanoemulsion which is obtained by: contacting water with cinnamaldehyde, optionally in combination with eugenol and / or carvacrol, in order to obtain immiscible phases optionally, adjusting the pH to 7.0 +/- 0.3 by means of a pH regulator, preferably a mineral base; optionally adding an emulsion stabilizer with surface-active properties, such as poloxamer; homogenization of the immiscible phases thus obtained in a high-pressure homogenizer, said pressure ranging from 300 to 5000 bar, preferably from 500 to 3000 bar and even more preferably from 800 to 2000 bar. The mineral base allows pH adjustment at physiological pH of 7.0 +/- 0.3. It is selected from sodium hydroxide, lithium hydroxide, potassium hydroxide, magnesium hydroxide or calcium hydroxide.

Without wishing to be bound by any theory, the inventors are of the opinion that the presence of the inorganic cation introduced by the pH regulator also contributes to the stabilization of the nanoemulsion finally obtained.

In order to increase the effects of treatment, successive administrations may be repeated on one or more occasions, after a particular time interval. For example, it is possible to carry out several administrations a day or a week.

The routes of administration and dosages vary depending on a variety of parameters, for example depending on the condition of the patient, the type of infection and the severity of the infection to be treated. Trans-cinnamaldehyde in combination with eugenol and / or carvacrol is administered enterally, parenterally (intravenously, intramuscularly or subcutaneously), transcutaneously (or transdermally or percutaneously), cutaneous, oral, mucosal, especially transmucosal- oral, nasal, ophthalmic, otological (in the ear), vaginal, rectal, or intragastric, intracardiac, intraperitoneal, intrapulmonary or intratracheal.

Preferably, the administration is made parenterally with the nanoemulsion described above. Doses ranging from 20 to 100 mg / kg body weight, preferably from 40 to 80 mg / kg per day divided into one or more doses can be used. In another aspect, the invention relates to a method for preventing occurrence of a resistance phenotype in a pathogen characterized in that it comprises: - providing trans-cinnamaldehyde, optionally in combination with eugenol and / or carvacrol; contact trans-cinnamaldehyde, optionally in combination with eugenol and / or carvacrol, with said pathogen. All the features described above in connection with the various aspects of the invention are also applicable to this latter aspect of the invention. The invention will be described in detail below with the aid of examples which are given by way of illustration only and can not limit the scope of the invention. EXAMPLES In the examples the following products are used: EP1010: Eugenol marketed by Merck under the name "eugenol for synthesis", lot 56393655 301 - Density: 1.0670 g / ml - purity greater than or equal to 99%. EP1011: Carvacrol sold by Sigma-Aldrich under the designation Carvacrol-Kosher, lot STBD2891V - density: 0.9901 g / ml - purity greater than or equal to 98%. EP1012: trans-Cinnamaldehyde sold by Merck under the designation "trans-cinnamaldehyde for synthesis", lot 56154305 121 - density: 1.0723 g / ml - purity greater than or equal to 98%. EP1020: Cinnamaldehyde: Eugenol 50:50 v / v, Lot E1775 prepared by simple mixing of the above products - density: 1.076 g / ml. EP1021: Carvacrol: Cinnamaldehyde 50:50 v / v, Lot E1776 - prepared by simple mixing of the above products - density 1.087 g / ml. EP1030: Carvacrol: Cinnamaldehyde: Eugenol 33:33:33 v / v. Lot E1777 - prepared by simple mixing of the above products - density: 1,035 g / ml.

POLOXAMER: poloxamer marketed by BASF under the name Kolliphor P 407; Example 1: eugenol / carvacrol / trans-cinnamaldehyde emulsion 70/15/15 (by weight): A eugenol / carvacrol / transcinnamaldehyde 70/15/15 (by weight) mixture is prepared. 12 g of this mixture are added to 200 g of water (6% by weight). The two-phase medium is homogenized at 25,000 rpm on IKA ultraturrax stirrer. The pH, initially 4.6 is brought between 7.0 and 7.2 with 0.1M potassium hydroxide. The pH is adjusted over a period of 2 hours. The temperature of the medium is maintained between 25 and 30 ° C. After stabilizing the pH, the milk obtained is homogenized at 1000 bar with a GEA niro Soasi Panda plus 1000 apparatus, for 30 minutes, while maintaining the temperature inside the homogenizer at 25-30 ° C. A nanoemulsion having the following characteristics is obtained: Average particle size: 250 nm measured by a Malvern device of the zeta-sizer type.

Zeta potential: -47 mV measured by Malvern apparatus at 25 ° C. The medium is then packaged and then used as is for testing in the mammal.

Example 2 Eugenol / carvacrol / cinnamaldehyde emulsion 70/15/15 with 6% POLOXAMER A eugenol / carvacrol / transcinnamaldehyde mixture 70/15/15 (by weight) is prepared. 12 g of this mixture are added to 200 g of water (6% by weight). The two-phase medium is homogenized at 25,000 rpm on IKA ultraturrax stirrer. The pH, initially 4.6 is brought between 7.0 and 7.2 with 0.1M potassium hydroxide. The pH is adjusted over a period of 2 hours. The temperature of the medium is maintained between 25 and 30 ° C. After pH stabilization, 12 g POLOXAMER Kolliphor P 407 from BASF (6% by weight) are added. The medium is stirred until a homogeneous milk (about 30 min). Then this milk is homogenized for 30 minutes at 1000 bar with a GEA Niro Soasi panda plus 1000. A nanoemulsion is obtained having the following characteristics: Average particle size: 44 nm measured in volume using a Malvern apparatus zeta size type at 25 ° C. Zeta potential: -64mV measured by a Malvern device of zeta size at 25 ° C. The medium obtained is translucent and has a temperature of 72 ° C. The medium is cooled to ambient temperature, packaged and then used as for testing in the mammal. EXAMPLE 3 Emulsion of Trans-Cinnamaldehyde 100% with 6% Poloxamer To 200 g of water are added 12 g (6% by weight) of trans-cinnamaldehyde. The two-phase medium is homogenized at 25,000 rpm on IKA ultraturrax stirrer. The pH, initially 3.8 is brought between 7.0 and 7.2 with 0.1M potassium hydroxide. The pH is adjusted over a period of 2 hours. The temperature of the medium is maintained between 25 and 30 ° C. After stabilizing the pH, 12 g of POLOXAMER (6% by weight) are added to the milk obtained. The medium is stirred until a homogeneous milk is obtained (approximately 30 minutes), then is homogenized for 30 minutes at 1000 bar with a GEA niro Soasi Panda plus 1000. The medium obtained is translucent and present. a temperature of 72 ° C. A nanoemulsion having the following characteristics is obtained: Average particle size: 44 nm measured by volume using a Malvern zeta typeizer at 25.degree. Potential Zeta: -5mV measured by a Malvern device type zeta sizer at 25 ° C. The medium is cooled to room temperature, packaged and then used as for testing in the mammal.

Example 4: Emergence of resistance in vitro in Acinetobacter baumannii, Pseudomonas aeruginosa and Staphylococcus aureus.

The objective of this example is to evaluate the emergence of in vitro resistance in the 3 bacterial strains Acinetobacter baumannii, Pseudomonas aeruginosa and Staphylococcus aureus in the presence of Carvacrol, eugenol, trans-cinnamaldehyde alone and in the mixtures EP1020, EP1021 and EP1030 . As a positive control, a different antibiotic for each strain was chosen: imipenem for Pseudomonas aeruginosa, tigecycline for Acinetobacter baumannii and rifampicin for Staphylococcus aureus.

The emergence of resistance will be induced by culturing the bacteria in the presence of the test product at sub-inhibitory concentrations (1/4 of the MIC) (Luz et al., 2012). The IJC will then be determined regularly every 4 to 6 passages. The emergence of resistance over 50 passes will correspond to a stable increase in the MIC corresponding to at least 2 times the starting MIC. Materials and Methods culture medium - 0.85% NaCl, 2 ml, ref. 20070, BioMerieux - BHI medium (BHI-T), 9 ml, ref. 42081, BioMerieux - Mueller-Hinton Medium (MH), 200 ml, ref. 64884, BioRad Blood agar, ref. PB5039A, Oxoid Mueller-Hinton agar, ref. 63824, BioRad - Type 1 water in 10 ml glass tubes, ELGA PURELAB ref. 526000, BioMerieux - E-test rifampicin, - E-test imipenem, ref. MA0115F, Oxoid - Tigecycline E-test, ref. 412475, BioMerieux - NaCl 0.9% Versol, ref. 600020, Laboratoire Aguettant Test products For each product tested, proceed as described below for trans-cinnamaldehyde: extemporaneous reconstitution of the solution in 40% DMSO (400 mg / ml solution) add 400 μl of cinnamaldehyde to 600 μl of DMSO. Add 1 ml of the 400 mg / ml solution to 9 ml of BHI, so as to obtain a concentration of 40 mg / ml.

For antibiotics, they are diluted in NaCl. Bacteria Acinetobacter baumannii: reference of the strain CIP 7034, susceptible to tigecycline. Pseudomonas aeruginosa: Clinical strain 0703CO259, susceptible to imipenem. Staphylococcus aureus: reference of the ATCC 25923 strain, susceptible 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 MICs in carvacrol, cinnamaldehyde and Eugenol alone and in the EP1020, EP1021 and EP1030 mixtures of the different bacterial strains were measured by Mueller-Hinton culture. The initial MICs of the 3 bacterial strains for antibiotics were measured using standard E-test. For each product to be tested, the procedure was as follows: Exposure of the strains to the product at subinhibitory doses (1/4 MIC) in a liquid medium (BHI medium) per passage every 24 hours for 50 passages. Exposure of the strains to the product at 2 times its MIC every 4 to 6 passages in agar medium (medium MH) to determine the appearance of a resistant strain.

Determination of the new MIC in agar medium if appearance of a resistant strain. If the MIC is increased, the selected strain will be exposed to a sub-inhibitory dose corresponding to 1/4 of the new MIC. The cultures are made at 37 ° C. All manipulations are done under Microbiological Safety Station. At the end of the tests, the bacterial strains were inoculated on Cryo-Billes (AES Laboratoires) and stored at -80 ° C. The Cryo-Billes system consists of a small tube containing beads in which the microorganisms adhere, surrounded by a cryoconservative hypertonic solution. Results The results for each bacterial strain are shown separately below Acinetobacter baumannii The results are shown in Tables 1 and 2 below.

After 50 passages, there was no increase in MIC for carvacrol, eugenol or cinnamaldehyde for A. baumannii. After 50 passages, there was no increase in MIC in the combinations of cinnamaldehyde / Eugenol, carvacrol / cinnamaldehyde, or carvacrol / cinnamaldehyde / eugenol for A. baumannii. For tigecycline, the MIC for A. baumannii increased from 1.5 mg / l to 8 mg / l (passage 8), to 16 mg / l (passage 19), to 48 mg / l (passage 24).

Table 1. MIC in carvacrol, eugenol, cinnamaldehyde, alone and in admixture, and tigecycline for A. baumannii. A. baumannii CMI No. CMI Passage Methodology Initial Final Carvacrol 50 0.25 mg / ml 0.25 mg / ml 1/4 Initial MIC Eugenol 50 0.75 mg / ml 0.75 mg / ml 1/4 Initial MIC Cinnamaldehyde EP1020 50 0.5 mg / ml 0.5 mg / ml 1/4 Initial MIC 50 0.5 mg / ml 0.5 mg / ml 1/4 Initial MIC 50 0.5 mg / ml 0.5 mg / ml 50 0.5 mg / ml 0.5 mg / ml 8 1.5 mg / 1 8 mg / 1 24 8 mg / 1 48 mg / 1 TIGECycline 1/4 Initial MIC Initial 1/4 Initial MIC 1/4 Initial MIC 1/4 Increased MIC Table 2. Evolution of MICs to Tigecycline for Acinetobacter baumannii (MIC in mg / I) MIC Initial Number of Passages CMI 1.5 (P) P3 1.5 P10 8 P21 16 P8 P13 P18 P22 P27 48 P36 P41 P46 P50 Increased CMI 8 P25 8 8 8 8 8 P31 8 8 8 8 (P8) 1 48 P15 P19 P24 P28 P33 8 P43 P47 Increased CMI 16: P32 8 16 48 48 48 P38 48 48 (P19) 2 48 P25 P30 P35 P40 P44 48 Increased CMI MIC '16 48 48 48 48 48 (P24) 3 P30 P35 P40 P44 48 48 48 P37 P41 48 48 Increased MIC 48 (P30) 4 1 new sub-series with initial MIC of 8 mg / 1 Each box in the Table below Us shows 2 new sub-series with initial MIC of 16 mg / 1 The number of the passages and the MIC 3 new subseries with initial MIC of 48 mg / 1 4 new subseries with initial MIC of 48 mg / 1 shaded area shows where the MIC has increased Number of passes 1.5 <----_______ CMI Pseudbmonas aeruginosa The results are summarized in Tables 3 and 4 below. Table 3. MIC of carvacrol, eugenol, cinnamaldehyde, alone and in admixture, and imipenem for P. aeruginosa. P. aeruginosa Initial MIC No CMI Final Passage Methodology Carvacrol 18 1 mg / ml 10 mg / ml 1/4 Initial MIC 22 mg / ml 10 mg / ml 1/4 CMO 18 3 mg / ml 10 mg / ml increased 1/4 Initial MIC Eugenol 50 0.75 mg / ml 0.75 mg / ml 1/4 Initial MIC Cinnam aldehyde EP1020 50 1 mg / ml 1 mg / ml 1/4 Initial MIC (cin / 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 / 1 16 mg / 1 1/4 increased MIC Table 4. Evolution of MIC for imipenem and Pseudomonas aeruginosa (MIC in mg / I) Initial MIC Number of Passages MIC 0.25 (P0) Increased MIC 2 (P3) 1 Increased MIC 4 (P5) 2 Increased CMI 8 (P8) 3 Increased CMI 8 (P10) 4 Increased CMI 8 (P7) 5 P3 P8 P13 P18 P22 2 8 8 8 P5 P10 P15 P19 4 8 8 16 P7 P12 8 16 P10 16 P11 P15 8 16 P8 16 16 1 new subset of passages with initial MIC of 2 mg / 1 2 new subset of passages with initial MIC of f 4 mg / 1 3 new sub-series series of passages with initial CMI of 8 mg / 1 4 new subset of passages with initial CMI of 8 mg / 1 The shaded area shows where the MIC has increased Each box in the Table above shows the number of passages and MIC MIC p3 <Number of Passages 2 <--- _ MIC The carvacrol MIC for P. aeruginosa increased from the initial value of 1 mg / ml to 2 mg / ml at pass 18 and 10 mg / ml at pass 22 .

MIC for Eugenol for P. aeruginosa increased from an initial value of 3 mg / ml to 10 mg / ml at pass 18. There was no change in the MIC of Cinnamaldehyde for P. aeruginosa in 50 passages.

There was no increase in the MIC of the E1020, E1021 and E 1030 mixtures for P. aeruginosa in the 50 passages. For imipenem, the MIC for P. aeruginosa increased from 0.25 mg / l to 2 mg / l (pass 3), 8 mg / l (pass 8), and 16 mg / l (pass 22). Staphylococcus aureus The results are shown in Table 5 below. Table 5. MIC of carvacrol, eugenol, cinnamaldehyde, alone and in mixture and rifampicin against S. aureus. S. aureus Initial MIC Number Final MIC Methodology passages 50 0.5 mg / m1 0.5 mg / m1 1/4 Initial MIC Carvacrol 50 0.5 mg / m1 0.5 mg / m1 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 (cin / 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 Rifampicin There was no increase in the MIC of carvacrol, cinnamaldehyde or Eugenol for S. aureus on the 50 passages. There was no increase in the MIC of the EP1020, EP1021 and EP1030 mixtures for P. aeruginosa over the 50 passages. For rifampicin, the MIC for S. aureus increased from 0.016 mg / L to 32 mg / L after 8 passages, an increase of a factor of 2000.

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 was found in any of 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 any of A. baumannii, P. aeruginosa and S. aureus after 50 passages. EP1021 carvacrol / Cinnamaldehyde 50/50: No resistance developed in any of A. baumannii, P. aeruginosa and S. aureus after 50 passages. EP1030 carvacrol / Cinnamaldehyde / Eugenol 33/33/33: No resistance developed in any of 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 / l after 24 passages. Imipenem: P. aeruginosa showed initial resistance to imipenem after 3 passages. P. aeruginosa was resistant to imipenem after 8 passages and the MIC increased to 16 mg / l after 22 passages. Rifampicin: S. aureus became resistant to rifampicin after 8 passages and the MIC increased to 32 mg / l. Example 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) vis-à-vis cinnamaldehyde following example 4 , on 50 additional passages, ie 100 passages in total (from the 50th passage to the 100th passage).

The emergence of resistance will be induced by culturing the bacteria in the presence of the product at sub-inhibitory concentrations (1/4 of the MIC) (Luz et al., 2012). The IJC will then be determined regularly every 4 to 6 passages. The emergence of resistance during an additional 50 passes will correspond to a stable increase in the MIC corresponding to at least 2 times the initial MIC.

Materials and Methods Bacterial strains - Acinetobacter baumannii: reference strain CIP 7034, stored on cryobille at the end of Example 4 - Pseudomonas aeruginosa: clinical strain 0703CO259, stored on cryobille at the end of Example 4 - Staphylococcus aureus strain ATCC reference 25923, stored on cryobile at the end of Example 4.

Reagents and culture media - Ampoules Api NaC1 0.85% Medium, 2 ml, ref.20070, BioMérieux - Medium BHI-T, 9 ml, ref.42081, BioMérieux - Mueller-Hinton Medium (MH), 200 ml, ref .64884, BioRad - Blood agar plates, ref.PB5039A, Oxoid - Quality water 1 in autoclaved 10 ml glass tubes from the ELGA tank coupled to a PURELAB - NaCl NaCl system 0.9% for irrigation, ref.600020, Laboratory Aguettant Products to be tested Extemporaneous reconstitution of transcinnamaldehyde solution in 40% DMSO (400 mg / ml solution): Add 400 μl of cinnamaldehyde to 600 μl of DMSO. Add 1 ml of the 400 mg / ml solution to 9 ml of BHI, so as to obtain a concentration of 40 mg / ml. Methods In order to study the emergence of resistance, the 3 bacterial strains are cultured in the presence of cinnamaldehyde from passage 50 to passage 100 in the same manner as in example 4.

All the manipulations are done under PSM. Results No emergence of cinnamaldehyde resistance was observed after 100 passages, for the 3 bacterial strains. Example 6 Effectiveness of the EP1030 Mixture in Mice Having Acinetobacter Baumannil-Induced Septicemia In this example, the product used is the emulsion of Example 1 which is used without delay after its preparation. The strain used Acinetobacter baumannii is a multidrug-resistant clinical reference strain, ref. SAN 005, CHU Angers, France. This strain causes 90-100% mortality in a mouse model of sepsis. The mice used are 12 female C3H / HeN SPF mice from January LABS, aged 6 weeks and weighing 20g (+/- 4g). They were acclimated for 7 days before the start of the study. The mice were randomly divided into 2 groups of 6, each group of 6 being in separate cages under conditions of controlled temperature, humidity and brightness.

The emulsion prepared in Example 1 was immediately diluted in a physiological solution for injection at 8 mg / ml, hereinafter referred to as EP1031-P. A. baumannii is stored frozen at -80 ° C. on CryoBeads.

On day D1, a cryobead was placed on blood agar (Blood agar, PB5039A ref, Oxoid) and incubated for 24 h at 37 ° C under aerobic conditions. A suspension of the bacteria in physiological saline was then prepared to obtain a concentration of 5.6 CFU of A.baumannii in 50 μl. Infection: Group 1: A TO, in 6 mice, in the right part of the abdomen was injected intraperitoneally 5.6 CFU A.baumannii in 50p1. The same procedure was followed for Group 2.

Treatment: Group 1: At TO, that is to say immediately after infection with A.baumannii, the 6 mice were injected intraperitoneally with 100 μl of EP1031-P, ie 40 mg / kg, in the left side of the 'abdomen. At T-3 hours, the 6 mice were injected intraperitoneally with 100 μl EP1031-P, ie 40 mg / kg, into the right side of the abdomen. The total dose was therefore 80 mg / kg.

Group 2: At TO, i.e. immediately after infection with A.baumannii, the 6 mice were intraperitoneally injected with 100 μL of sterile saline solution in the left side of the abdomen. At 3:00 am, the 6 mice were injected intraperitoneally with 100 μl of sterile saline solution into the right side of the abdomen. The total dose was therefore 80 mg / kg.

Mortality was observed at 24 and 48 hours. The results are presented in the table below: Group Number of Mortality of deaths to death at 48 hours total survivors at 48h 24h EP1031-P 0 0 0/6 100% (group 1) Control (group 2) 1 3 3/6 50% This test was duplicated and the same results were obtained.

Intraperitoneal injection of a nanoemulsion containing trans-cinnamaldehyde, eugenol and carvacrol at 80mg / kg protected the mice against A.baumannii-induced acute septicemia resistant to conventional antibiotics.

EXAMPLE 7 Evaluation of Nanoemulsions in an Acinetobacter Baumannii Sepsis Model The objective is to evaluate the efficacy of several products derived from pharmaceutical research as an antibiotic therapeutic, administered intraperitoneally, in a model of Acinetobacter sepsis. baumannii. Sepsis is defined as a systemic reaction in response to the aggression of a microorganism whose symptoms reflect endothelial and tissue damage, resulting in organ failure. Severe sepsis is a leading cause of morbidity and mortality worldwide, with an estimated incidence of 0.3% in the United States, France, and Germany (Angus et al, 2001, Schuerholz et al, 2008).

The first experimental animal model of sespsis was validated and published in 1989 (Obana et al). The choice of C3H / HeN mice explained in the 1997 publication (Jolly-Guillou et al) is based on a particular sensitivity of these mice to Acinetobacter baumannii MPS, with the expectation of a good reproducibility of the results and therefore of a statistical analysis. reliable. Acinetobacter baumannii is an opportunistic germ responsible for various and sometimes severe nosocomial infections: pneumonia, urinary tract infection and soft tissue infection. It is inoculated intraperitoneally. Persistent sepsis is observed. The infection progresses rapidly, causing death of animals between 24 and 48 hours. 1. Materials and Methods Animals 36 female C3H / HeN SPF mice, from the January LABS breeding and 6 weeks old, weigh on average 20 grams +/- 4 grams. They are chosen because of their particular sensitivity to Acinetobacter baumannii, and because of the reproducibility of the results due to the consanguinity of the breed. The acclimation time of the animals is 7 days before any experimentation. The mice are randomized (randomized in the different therapeutic groups) in groups of 6 mice per cage. The cages are placed in ventilated enclosures and controlled in atmosphere (temperature, hygrometry), in a room specifically dedicated to the housing of animals reserved for animal experimentation.

Bacterial strain Acinetobacter baumannii: reference clinical strain of the microbiology laboratory of the University Hospital of Angers: SAN 005, 5 generating a mortality between 80% and 100% and multiresistant to antibiotics. Acinetobacter baumannii SAN is resistant to: -beta-lactams: amoxicillin and augmentin 10-third-generation cephalosporins: cefixime and ceftriaxone-first-generation cephalosporin: cefalotin -monobactams: aztreonam -quinolones: nalidixic acid, ofloxacin, ciprofloxacin, norfloxacin -aminosides : tobramycin, gentamycin, netilmicin, kanamycin-carbapenems: ertapenem-cyclins: doxycycline 20 -furans -carboxypenicillin: temocillin-cephamycin: cefotixin -fosfomycin -cotrimoxazole 25 Reagents and culture media - Ampoules Api NaC1 0.85% Medium, 2 ml, ref.20070, BioMérieux 30 - Blood agar plates, ref.PB5039A, Oxoid - Quality water 1 in autoclaved 10 ml glass tubes from the ELGA tank coupled to a PURELAB - Versol NaC1 0.9% system for irrigation, ref.600020 , Aguettant Laboratory Products to be tested EP 1031 is a combination of active ingredients: eugenol (70%), carvacrol (15%), cinnamaldehyde (15%). A2: EP1031 at 30 mg / ml of active ingredients + 10 mg / ml of stabilizer (poloxamer). White formulation having an oily dephasing start to the bottom at reception A3: EP1031 at 30 mg / m 2 of active agents + 30 mg / m 2 of stabilizer (poloxamer). Stable white formulation upon reception. A4: EP1031 at 60 mg / ml of active ingredients + 60 mg / ml of stabilizer (poloxamer). Translucent formulation stable on reception. Formulations A1 to A4 were prepared using the same method as in Example 2 above. The amounts of active ingredients and stabilizer (Poloxamer) are adapted. EP1032 is a combination of active ingredients: carvacrol (70%), eugenol (15%), cinnamaldehyde (15%). B1: EP1032 at 60 mg / ml + 60 mg / ml of stabilizer (Poloxamer). Stable white formulation upon reception. EP1012 contains Cinnamaldehyde (100%) Cl: EP1012 at 60 mg / ml of active ingredient + 60 mg / ml of stabilizer (Poloxamer). Formulation white out of phase at reception: presence of a large quantity of white powder at the bottom of the bottle.

Methods The aim of the Acinetobacter baumannii sepsis model is to work on bacterial strains responsible for nosocomial infections that are possibly multi-resistant or even toto-resistant. This model makes it possible to screen anti-infectious molecules in 48 hours and thus to quickly evaluate the therapeutic efficacy of new anti-microbial agents. Preparation of the bacterial strains The strains are stored in the form of cryobeads at -80 ° C. On D-1, each strain was taken out of the freezer and seeded on a still frozen bead on a blood agar using a needle. The agar is incubated at 37 ° C and aerobically grown for 24 hours.

Procedure of the study 36 female CH3 / HeN mice are divided into 6 groups of 6 animals: Group 1: 6 mice receive two injections per day for 1 day per IP at 3h intervals of 100 μl of physiological saline 3h before inoculation and just after inoculation. Group 2: 6 mice receive two injections per day for 1 day per PI at 3h intervals of A2 (40mg / kg in 100 μl, ie 80 mg / kg per day), 3h before inoculation and just after inoculation. Group 3: 6 mice receive two injections per day for 1 day by IP at 3h intervals of A3 (40 mg / kg in 100 μl, ie 80 mg / kg per day), 3h before inoculation and just after inoculation. Group 4: 6 mice receive two injections per day for 1 day per IP at a 3 hour interval of A4 (40 mg / kg in 100 μl, ie 80 mg / kg per day), 3 hours before inoculation and just after inoculation. Group 5: 6 mice receive two injections per day for 1 day per IP at 3h intervals of B1 (40mg / kg in 100 μl, ie 80 mg / kg per day), 3h before inoculation and just after inoculation. Group 6: 6 mice receive two injections per day for 1 day per IP at 3h intervals of Cl (40 mg / kg in 100 μl, ie 80 mg / kg per day), 3h before inoculation and just after inoculation. 15 a. TO Administration of the products: Group 1: 100 μl of physiological saline are injected by IP into each of the 6 mice of group 1 in the right flank. Group 2: 266 μl of A2 at 30 mg / ml are added at 734 μl. Sterile saline (8 mg / ml solution) and 100 μl were IP injected into each of the 6 group 2 mice (40 mg / kg) in the right flank. Group 3: 266 μl of A3 at 30 mg / ml are added to 734 μl of sterile saline (8 mg / ml solution) and 100 μl are injected ip to each of the 6 mice in group 3 (40 mg / ml). mg / kg) in the right flank. Group 4: 133 μl of A4 at 60 mg / ml are added to 867 μl of sterile saline solution (8 mg / ml solution) and 100 μl are injected ip to each of the 6 mice in group 4 (40 mg / ml). mg / kg) in the right flank. Group 5: 133 μl of B1 at 60 mg / ml are added to 867 μl of sterile saline (8 mg / ml solution) and 100 μl are injected ip to each of the 6 mice in group 5 (ie 40 mg). / kg), in the right side. Group 6: 133 μl of Cl at 60 mg / ml are added to 867 μl of sterile saline (8 mg / ml solution) and 100 μl are injected ip to each of the 6 mice in group 6 (ie 40 mg / kg), in the right side. b. T3: 3 hours after TO - bacterial inoculation: the bacterial inoculum is a saline suspension of 5 × 10 6 CFU in 50 μl, administered intraperitoneally in the right flank, to each of the 12 mice. Administration of the products: group 1: 100 μl of physiological saline are injected IP into each of the 6 group 1 mice in the left flank; group 2: 266 μl of A2 to 30 mg / ml are added at 734 μl; 30 μl of sterile saline (8 mg / ml solution) and 100 μl are injected ip to each of the 6 group 2 mice (40 mg / kg) in the left flank. Group 3: 266 μl of A3 at 30 mg / ml are added to 734 μl of sterile saline (8 mg / ml solution) and 100 μl are injected ip to each of the 6 mice in group 3 ( 40 mg / kg) in the left flank. Group 4: 133 μl of A4 at 60 mg / ml are added to 867 μl of sterile saline (8 mg / ml solution) and 100 μl are injected ip to each of the 6 mice in group 4 (40 mg). / kg), in the left flank. Group 5: 133 μl of B1 at 60 mg / ml are added to 867 μl of sterile saline solution (8 mg / ml solution) and 100 μl are injected ip to each of the 6 mice in group 5 (ie 40 mg). / kg), in the left flank. Group 6: 133 μl of Cl at 60 mg / ml are added to 867 μl of sterile saline solution (8 mg / ml solution) and 100 μl are injected ip to each of the 6 mice in group 6 (ie 40 mg). / kg), in the left flank. Mortality is recorded at 24 hours and 48 hours. 2. Deaths in deaths in 24 hours 48 hours Witnesses (Group 1) 3 3 6 of 6 A2 (Group 2) 0 1 1 of 6 A3 (Group 3) 0 1 1 of 6 A4 (Group 4 ) 0 0 0 out of 6 B1 (Group 5) 0 2 2 out of 6 Cl (Group 6) 1 3 4 out of 6 -In the control group, 3 out of 6 mice die within 24 hours, and 3 out of 6 mice die in 48 hours. In the batch of mice treated with A2 at the dosage of 40 mg / kg, 3 hours before the bacterial inoculation and just after inoculation, 1 mouse out of 6 dies within 48 hours. In the batch of mice treated with A3 at the dosage of 40 mg / kg, 3 hours before the bacterial inoculation and just after inoculation, 1 mouse out of 6 dies within 48 hours. In the batch of mice treated with A4 at the dosage of 40 mg / kg, 3 hours before the bacterial inoculation and just after inoculation, no mouse dies. In the batch of mice treated with B1 at a dosage of 40 mg / kg, 3 hours before bacterial inoculation and just after inoculation, 2 out of 6 mice die within 48 hours. In the batch of mice treated with Cl at a dosage of 40 mg / kg, 3 hours before bacterial inoculation and just after inoculation, 1 in 6 mice die within 24 hours, and 3 out of 6 mice die in 48 hours. All the mice in the control group died after 48 hours. The treatment with A2 at the dosage of 40 mg / kg, 3 hours before the bacterial inoculation and just after inoculation, saves 83% of the mice. Treatment with A3 at the dosage of 40 mg / kg, 3 hours before bacterial inoculation and just after inoculation, saves 83% of the mice. Treatment with A4 at a dosage of 40 mg / kg, 3 hours before bacterial inoculation and just after inoculation, makes it possible to save 100% of the mice. The treatment with B1 at a dosage of 40 mg / kg, 3 hours before bacterial inoculation and just after inoculation, makes it possible to save 66% of the mice. The treatment with A4 at the dosage of 40 mg / kg, 3 hours before the bacterial inoculation and just after inoculation, saves 33% of the mice. Conclusion Administration of A4 saved 100% of the mice, and no mice showed any sign of disease or failure throughout these 48 hours.

Claims (13)

REVENDICATIONS1. Trans-cinnamaldehyde, optionally in combination with eugenol and / or carvacrol for use in preventing the emergence of a resistance phenotype in a pathogen. 2. Trans-cinnamaldehyde in combination with eugenol and / or carvacrol for use in the prevention and / or treatment of a pathogen infection, wherein the occurrence of a resistance phenotype in said pathogen is prevented. 3. Trans-cinnamaldehyde in combination with eugenol and / or carvacrol for use as an antibiotic that does not develop a resistance phenotype. 15 4. In vitro method for preventing the appearance of a resistance phenotype in a pathogen characterized in that it comprises the steps of: providing trans-cinnamaldehyde, optionally in combination with eugenol and / or carvacrol; Contacting trans-cinnamaldehyde, optionally in combination with eugenol and / or carvacrol, with said pathogen. 5. A process according to claim 4, trans-cinnamaldehyde for use according to claim 1, or trans-cinnamaldehyde in combination with eugenol and / or carvacrol for use according to any one of claims 1 to 3, characterized in that transcinnamaldehyde, optionally in combination with eugenol and / or carvacrol, is provided in the form of an emulsion, and preferably in the form of a nanoemulsion. 6. A process according to claim 5, trans-cinnamaldehyde for use according to claim 5, or transcinnamaldehyde in combination with eugenol and / or non-carvacrol nitrite for use according to claim 5, characterized in that nanoemulsion is obtained by: - contacting water with cinnamaldehyde, optionally in combination with eugenol and / or carvacrol to obtain immiscible phases; optionally, the pH is adjusted to 7.0 ± 0.3 with the aid of a mineral base; optionally adding a stabilizing agent, preferably a nonionic stabilizing agent with surfactant properties; Homogenizing the immiscible phases thus obtained in a high pressure homogenizer, said pressure ranging from 300 to 5000 bar, preferably from 500 to 3000 bar and more preferably still from 800 to 2000 bar. 7. Process according to any one of claims 4 to 6, or trans-cinnamaldehyde in combination with eugenol or carvacrol for use according to any one of claims 1 to 6, characterized in that transcinnamaldehyde in combination with eugenol or carvacrol is provided in a weight ratio of 20/80 to 80/20, preferably 30/70 to 70/30 and more preferably 50/50. 8. Process according to any one of claims 4 to 6 or trans-cinnamaldehyde in combination with eugenol and carvacrol for use according to any one of claims 1 to 6, characterized in that transcinnamaldehyde in combination with eugenol and carvacrol is provided in a mass ratio of 10/10/80 to 80/10/10, preferably of 15/15/70 to 70/15/15 and more preferably of 33/33 / 33. 30 9. A process according to any one of claims 4 to 8, trans-cinnamaldehyde for use according to claim 1, or trans-cinnamaldehyde in combination with eugenol and / or carvacrol for use according to one of the preceding claims. any of claims 1 to 8, characterizedNotif irr_Sept14 48 that the pathogen is a bacterium selected from the following families: - the family Staphylococcaceae, in particular the genus Staphylococcus, represented by the species Staphylococcus aureus (S.aureus or staphylococcus walleye) and Staphylococcus epidermidis; the family Enterococcaceae, in particular the genus Enterococcus, represented by the species Enterococcus faecalis, Enterococcus faecium, Enterococcus avium and Enterococcus gallinarum; - the family Clostridiaceae, in particular the genus Clostridium, represented by the species Clostridium difficile and Clostridium sp; the family of Streptococcaceae, including hemolytic Streptococci A and B, in particular the genus Streptococcus, represented by the species Streptococcus pneumoniae, Streptococcus agalactiae, Streptococcus viridans, Streptococcus and Streptococcus pyogenes; the Aerococcaceae family, in particular the genus Aerococcus represented by the species Aerococcus viridans; the family Micrococcaceae, in particular the genus Micrococcus; - the family Lactobacillaceae, in particular the genus Lactobacillus; The family Nocardiaceae, in particular the genus Nocardia, represented by the species Nocardia asteroids, Nocardia brasiliensis and Nocardia caviae; - the family Listeriaceae, in particular the genus Listeria, represented by the species Listeria monocytogenes; The family Corynebacteriaceae, in particular the genus Corynebacterium; - the Bacillaceae family, in particular the genus Bacillus, represented by the species Bacillus anthracis and Bacillus cereus; Notif irr_Sept14 49 the Propionibacteriaceae family, in particular the genus Propionibacterium, represented by the species Propionibacterium acnes; the family Peptococcaceae, in particular the genus Peptococcus, represented by the species Peptococcus magnus; - the family Mbraxellaceae, in particular the genus Acinetobacter, represented by the species Acinetobacter baumannii and Acinetobacter calcoaceticus; the family Legionellaceae, in particular the genus Legionella, represented by the species Legionella Pneumophila, Legionella longbeachae, Legionella bozmanii, and Legionella micdader; the Enterobacteriaceae family, in particular the genus Enterobacter, represented by the species Enterobacter cloacae, the genus Escherichia, represented by the species Escherichia coli and Escherichia hermannii, the genus Klebsiella, represented by the species Klebsiella pneumoniae and Klebsiella oxytoca, the Serratia, represented by Serratia marcescens, Citrobacter, represented by Citrobacter freundii and Citrobacter koseri, and Salmonella, Shigella and Proteus; the family Pseudomonadaceae, in particular the genus Pseudomonas, represented by the species Pseudomonas aeruginosa (P. aeruginosa), Pseudomonas fluorescens, Pseudomonas putida and Pseudomonas stutzeri; the Alcaligenaceae family, in particular the genus Achromobacter, represented by the species Achromobacter xylosoxidans and Achromobacter denitrificans; the Sphingomonadaceae family, in particular the genus Sphingomonas, represented by the species Sphingomonas paucimobilis; - the family of Pasteurellaceae, in particular the genus Haemophilus, represented by the species Haemophilus influenzae and Haemophilus parainfluenzae; Notif irr_Sept14 50 - the family Moraxellaceae, in particular the genus Moraxella, represented by the species Moraxella catarrhalis; - the Neisseriaceae family, in particular the genus Kingella, represented by the species Kingella kingae; the family of Pasteurellaceae, in particular the genus Pasteurella, represented by the species Pasteurella multicida; the family Flavobacteriaceae, in particular the genus Capnocytophaga, represented by the species Capnocytophaga canimorsus; - the family Neisseriaceae, in particular the genus Neisseria, represented by the species Neisseria gonorrhoeae and Neisseria lactamica; - the genus Campylobacter, represented by C. jejuni species, and C. neck; the family Bacteroidaceae, in particular the genus Bacteroides, represented by the species Bacteroides fragilis; - the family Stenotrophomonas, in particular, the species S. nitritireducens and S. maltophilia; the family of meningococci, in particular the Neisseria meningitidis species; and - the family Fusobacteriaceae, in particular the genus Fusobacterium. 10. A process according to any one of claims 4 to 9, trans-cinnamaldehyde for use according to claim 1, or trans-cinnamaldehyde in combination with eugenol and / or carvacrol for use according to any one of claims 1 to 9, characterized in that the pathogen is a bacterium selected from strains belonging to the following genera: 30 - genus Acinetobacter, represented by the species Acinetobacter baumannii and Acinetobacter calcoaceticus; - genus Staphylococcus, represented by the species Staphylococcus aureus (S.aureus or Staphylococcus aureus) and Staphylococcus epidermidis; Notif irr_S ept14 51 - genus Escherichia, represented by the species Escherichia coli and Escherichia hermannii; - genus Klebsiella, represented by the species Klebsiella pneumoniae and Klebsiella oxytoca; - genus Listeria, represented by the species Listeria monocytogenes; - genus Salmonella, represented by the species Salmonella enteritidis; and - genus Pseudomonas, represented by the species Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas putida and Pseudomonas stutzeri. Trans-cinnamaldehyde for use according to claim 5, or trans-cinnamaldehyde in combination with eugenol and / or carvacrol for use according to claim 5, characterized in that the emulsion is administered by the parenteral, oral, transmucosal, percutaneous, pulmonary, and more particularly by the intravenous route. A process according to claim 5, trans-cinnamaldehyde for use according to claim 5 or 11, or trans-cinnamaldehyde in combination with eugenol and / or carvacrol for use according to claim 5 or 11, characterized in that the emulsion is an oil-in-water nanoemulsion which has hydrophobic droplets of average size less than 10 to 500 nm, preferably 20 to 350 nm and more preferably 50 to 250 nm and having a zeta potential ranging from -2 mV to -80 mV, preferably from -5 mV to -60 mV and still more preferably from -10 mV to -40 mV. 30 13. The method of claim 11 or 12, Transcinnamaldehyde for use according to any one of claims 5, 11 to 12, or transcinnamaldehyde in combination with eugenol and / or carvacrol for use according to one of Any of the above claims are subject to claim 5, 11 to 12, characterized in that the emulsion is free from any stabilizing agent.