Antimicrobial composition of polyunsaturated fatty acids

Abstract

The invention relates to a pharmaceutical composition comprising the combination of polyunsaturated fatty acids (AGPI). The invention also relates to the use of said composition as an antimicrobial agent for the treatment and prevention of an infection due to pathogenic agents involved in the infection of a surgical incision site (ISQ), and to a medical device comprising said composition.

Description

[0001] Antimicrobial composition of polyunsaturated fatty acids

[0002] The present invention relates to a pharmaceutical composition comprising a combination of polyunsaturated fatty acids (PUFAs). The present invention also relates to the use of said composition as an antimicrobial agent for the treatment and prevention of infection by pathogens involved in incisional surgical site infection (SSI), and to a medical device comprising said composition.

[0003] STATE OF THE ART

[0004] Currently, polyunsaturated fatty acids (PUFAs) are found in numerous dietary supplements and various medical devices such as sutures, catheters, prostheses, and ointments, playing a role in infection control, although their clinical use is not yet widespread (see Maicas VT, Rochina IJ. Linoleic acid emulsion on the peri-lesion skin of venal ulcers. Action and cicatrizant effect. Corpus study. Rev Enferm. 2008; 31 (4): 26-32; Bowler PG, Jones SA, Walker M, Parsons D. Microbicidal properties of a silver containing hydrofiber. Dressing against a variety of burn wound pathogens. J Burn Care Rehabil. 2004; 25:192-6; López A, Garcia F, Jareño P, García J, García N. Hyperoxygenated fatty acids effectiveness in the prevention of the pressure ulcers. Gerokomos 2007; 18 (4): 197-201; Elkhyat A, MacMary S, Degouy A. Évaluation biométrologique des effets de l'huile hyperoxygénée SANYRENE sur la peau.Journal des Paies et Cicatrisations 2003 ;7: 115-181 ; Chopra I. The increasing use of silver-based products as antimicrobial agents: a useful development or a cause for concern. J Antimicrob Chemoter. 2007; 59(4): 587-590; Sanders D, Lambie J, Bond P, Moate R, Steer JA. An in vitro study assessing the effect of mesh morphology and suture fixation on bacterial adherence. Hernia. 2013;17(6):779- 789; Lai NM, Chaiyakunapruk N, Lai NA, et al. Catheter impregnation, coating or bonding for reducing central venous catheter-related infection in adults. Cochrane Database Syst Rev. 2016; 3; y Fernández-Gutiérrez M, Olivares E, Pascual G, et al. Low-density polypropylene meshes coated with resorbable and biocompatible hydrophilic polymers as controlled release agents of antibiotics. Acta Biomater. 2013; 9(4):6006-6018).In recent years, PUFAs have demonstrated beneficial effects at the topical level through their application in patients with slow-healing and difficult-to-heal ulcers (Seth N, Chopra D, Lev-Tov H. Fish skin grafts with omega-3 for treatment of chronic wounds: Exploring the role of omega-3 fatty acids in wound healing and A review of clinical healing outcomes. Surg Technol Int. 2022; 40:38-46.), as well as for covering prostheses in direct contact with abdominal viscera (Armañanzas L, Ruiz-Tovar J, Arroyo A, et al. Prophylactic mesh vs suture in the closure of the umbilical trocar site after laparoscopic cholecystectomy in high-risk patients for incisional hernia. A randomized clinical trial. J Am Coll Surg.2014; 218(5): 960-968) among others, and beneficial systemic effects by reducing the inflammatory response in critically ill patients with cardiovascular disease, chronic kidney disease, sepsis, polytrauma, and acute pancreatitis (Tortosa-Caparrós E, Navas-Carrillo D, Marín F, Orenes-Piñero E. Anti-inflammatory effects of omega 3 and omega 6 polyunsaturated fatty acids in cardiovascular disease and metabolic syndrome. Grit Rev Food Sci Nutr. 2017; 57(16):3421-9; Minar E, Schillinger M. Innovative technologies for SFA occlusions: drug coated balloons in SFA lesions. J Cardiovasc Surg (Torino). 2012; 53(4): 481-486; Hung AM, Booker C, Ellis CD, Siew ED, Graves AJ, Shintani A, Abumrad NN, Himmelfarb J, Tkizler TA. Omega-3 fatty acids inhibit the up-regulation of endothelial chemokines in maintenance hemodialysis patients. 2014; 28; and Wang C, Han D, Feng X, Wu J.Omega- 3 fatty acid supplementation is associated with favorable outcomes in patients with sepsis: an updated meta-analysis. J Int Med Res. 2020;48(12)).

[0005] Entre la gran variedad de actividades biológicas de los AGPI se encuentra la capacidad bactericida o bacteriostática sobre el crecimiento de microorganismos (Desbois, A.P, Lawlor K.C. Antibacterial activity of long-chain polyunsaturated fatty acids against Propionibacterium acnés and Staphylococcus aureus. Mar Drugs. 2013; 11(11): 4544- 4557; Mil-Homens D, Bernardes N & Fialho A. The antibacterial properties of docosahexaenoic omega-3 fatty acid against the cystic fibrosis multiresistant pathogen Burkholderia cenocepacia. FEMS Microbiol Lett. 2011 ; 1-9; y Shin SY, Bajpai VK, Kim HR & Kang SC. Antibacterial activity of bioconverted eicosapentaenoic (EPA) and docosahexaenoic acid (DHA) against foodborne pathogenic bacteria. Int J Food Microbiol. 2007; 113: 233-236).Thus, their broad spectrum of activity, non-specific mode of action, and safety make them attractive as antimicrobial agents for various medical applications, particularly when the use of conventional antibiotics is undesirable or not tolerated. The search for new antimicrobial products has increased in recent years due to the rise in antibiotic resistance, and the use of natural products such as PUFAs deserves special consideration. On the other hand, it is known that PUFAs have a potential bactericidal or bacteriostatic function (Das, II. N. (2018). Arachidonic acid and other unsaturated fatty acids and some of their metabolites function as endogenous antimicrobial molecules: A review. J Adv Res 2018; 11: 57-66; and Chanda, W., Joseph, TP, Guo, XF, Wang, WD, Liu, M., Vuai, MS, Zhong, MT (2018). Effectiveness of omega-3 polyunsaturated fatty acids against microbial pathogens.2018; 79(4): 253-262).

[0006] Therefore, it would be desirable to have a polyunsaturated fatty acid composition capable of improving the properties of fatty acids against microorganisms that most frequently produce SSIs and that is also made up of natural elements, which lack described side effects, avoiding or minimizing the toxicity of the products currently used.

[0007] DESCRIPTION OF THE INVENTION

[0008] The present invention relates to a composition comprising two or more polyunsaturated fatty acids (PUFAs), particularly where the polyunsaturated fatty acids (PUFAs) are selected from docosahexaenoic acid (DHA), gamma-linolenic acid (GLA), and eicosapentaenoic acid (EPA).

[0009] Furthermore, the present invention relates to said composition for use in the treatment and prevention of an incisional surgical site infection (I SQI), particularly where the microbial infection is caused by Staphylococcus aureus, Staphylococcus epidermidis (Gram-positive germs) and Escherichia coli and Klebsiella pneumoniae (Gram-negative germs).

[0010] Finally, the invention relates to a medical device comprising said composition.

[0011] Therefore, a first aspect of the present invention relates to a composition comprising a mixture of two or more polyunsaturated fatty acids.

[0012] In another embodiment, the invention relates to the composition defined above, wherein the polyunsaturated fatty acids are selected from docosahexaenoic acid (DHA), gamma-linolenic acid (GLA), eicosapentaenoic acid (EPA) and mixtures thereof.

[0013] In another embodiment, the invention relates to the composition defined above, wherein the polyunsaturated fatty acids are selected from docosahexaenoic acid (DHA), gamma-linolenic acid (GLA), and eicosapentaenoic acid (EPA).

[0014] In another embodiment, the invention relates to the composition defined above, where the composition comprises: docosahexaenoic acid (DHA), gamma-linolenic acid (GLA); and eicosapentaenoic acid (EPA).

[0015] In another embodiment, the invention relates to the composition defined above, wherein the composition comprises: between 6.1% and 7.1% docosahexaenoic acid (DHA), between 10.1% and 11.0% gamma-linolenic acid (GLA); and between 81.8% and 83.8% eicosapentaenoic acid (EPA), wherein the percentages refer to the weight of the total polyunsaturated fatty acids.

[0016] In another embodiment, the invention relates to the composition defined above, where the composition comprises:

[0017] 6.5% by weight of docosahexaenoic acid (DHA)

[0018] 10.4% by weight of gamma-linolenic acid (GLA); and

[0019] 83.1% by weight of eicosapentaenoic acid (EPA), where the percentages refer to the weight of total polyunsaturated fatty acids.

[0020] In another embodiment, the invention relates to the composition defined above, which further comprises physiological saline solution.

[0021] In another embodiment, the invention relates to the composition defined above, which further comprises an organic solvent.

[0022] In another embodiment, the invention relates to the composition defined above, wherein the organic solvent is selected from ethanol, methanol, and / or mixtures thereof, and more preferably where the organic solvent is ethanol. Another aspect of the invention relates to a pharmaceutical composition comprising the composition defined above and one or more pharmaceutically acceptable excipients.

[0023] Another aspect of the invention relates to the composition defined above or to a pharmaceutical composition thereof, for use as a medicament.

[0024] Another aspect of the invention relates to the composition defined above or to a pharmaceutical composition thereof, for use in the treatment and / or prevention of a microbial infection, and preferably where the microbial infection occurs at the incisional surgical site.

[0025] In another embodiment, the invention relates to the composition for the use defined above, wherein the microbial infection is caused by one or more pathogens selected from Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, and Klebsiella pneumoniae.

[0026] Another aspect of the invention relates to a medical device comprising the composition defined above or a pharmaceutical composition thereof.

[0027] Another aspect of the invention relates to a device comprising:

[0028] - a vial prepared under sterile conditions containing the composition defined above or a pharmaceutical composition thereof; and

[0029] - a sterile applicator integrated into the vial for topical application of the composition.

[0030] In another embodiment, the invention relates to the device defined above, where the vial has a volume of between 8 and 12 ml.

[0031] In another embodiment, the invention relates to the device defined above, where the vial has a volume of 10 ml.

[0032] In another embodiment, the invention relates to the device defined above, wherein the composition of the invention is present at a concentration of between 10% and 30% v / v with respect to the total volume of the vial. In another embodiment, the invention relates to the device defined above, wherein the composition of the invention is present at a concentration of 20% v / v with respect to the total volume of the vial.

[0033] Another aspect of the invention relates to the use of the above-defined composition for the manufacture of a medicament for the treatment and / or prevention of a microbial infection, preferably where the microbial infection occurs at the incisional surgical site, and more preferably where the microbial infection is caused by one or more pathogens selected from Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, and Klebsiella pneumoniae.

[0034] Another aspect of the invention relates to a method of treating and / or preventing a microbial infection, preferably where the microbial infection occurs at the incisional surgical site, and more preferably where the microbial infection is caused by one or more pathogens selected from Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, and Klebsiella pneumoniae, in a subject in need, especially in humans, comprising administering to said subject an effective amount of the composition defined above.

[0035] Throughout this description, the term “treatment” refers to eliminating, reducing, or lessening the cause or effects of a disease. For the purposes of this invention, treatment includes, but is not limited to, relieving, lessening, or eliminating one or more symptoms of the disease; reducing the severity of the disease; stabilizing (i.e., not worsening) the disease state; delaying or slowing the progression of the disease; alleviating or improving the disease state; and achieving remission (either total or partial).

[0036] As used in the present invention, the term “prevention” refers to preventing the onset of a disease in a patient who is predisposed or has risk factors, but who does not yet exhibit symptoms of the disease. Prevention also includes preventing the recurrence of a disease in a person who has previously suffered from that disease.

[0037] Thus, the invention relates to the composition of the invention, which comprises a mixture of polyunsaturated fatty acids (PUFAs), as defined above, which, at the studied doses, exhibits a synergistic effect that is equal to or greater than that of the individual components of the composition. This synergistic effect has the advantage of allowing for reduced dosages and, consequently, results in a composition with lower toxicity and fewer side effects than those caused by the use of the individual components. This is particularly beneficial for patients undergoing multiple interventions and treatments, such as surgical patients, and also enhances the effect of the individual components.

[0038] The present invention also relates to a pharmaceutical composition comprising the compound of the invention and one or more pharmaceutically acceptable excipients. The excipients must be "acceptable" in the sense of being compatible with the other ingredients of the composition and not harmful to the person taking the composition.

[0039] The composition of the present invention is administered intraoperatively, after the completion of the surgical procedure, by applying the entire contents of the vial to the surgical wound. The route of administration of this composition is local and topical.

[0040] Throughout the description and claims, the word "comprises" and its variants are not intended to exclude other technical features, additives, components, or steps. For those skilled in the art, other objects, advantages, and features of the invention will become apparent partly from the description and partly from the practice of the invention. The following examples and figures are provided for illustrative purposes and are not intended to limit the scope of the present invention.

[0041] BRIEF DESCRIPTION OF THE FIGURES

[0042] Fig. 1. Shows the bactericidal / bacteriostatic activity of the synergy of the three fatty acids.

[0043] EXAMPLES

[0044] The invention will then be illustrated by means of tests carried out by the inventors, which demonstrate the effectiveness of the product of the invention.

[0045] Example 1: In vitro experimental study for the evaluation of the antimicrobial efficacy of three PUFAs against pathogens.

[0046] For this study, the minimum inhibitory concentration (MIC) of fatty acids (docosahexaenoic acid (DHA), linolenic acid (GLA) and eicosapentaenoic acid (EPA)) was determined against Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli and Klebsiella pneumoniae.

[0047] The results showed bactericidal activity (DHA .g / ml) against Gram-positive bacteria (Staphylococcus aureus, Staphylococcus epidermidis), while against Gram-negative bacteria (Escherichia coli and Klebsiella pneumoniae), the PUFA chains exerted a bacteriostatic effect. Additionally, synergistic interactions were detected among the three PUFAs, achieving an antimicrobial effect at lower doses against both Gram-positive and Gram-negative bacteria.

[0048] The fatty acids DHA, GLA, and EPA showed a bactericidal effect against Gram-positive microorganisms and a bacteriostatic effect against Gram-negative microorganisms. DHA was the most effective compound, exhibiting an antimicrobial effect at lower doses. Furthermore, the synergistic action of the fatty acid chains allowed for a reduction in dosage while achieving the same antimicrobial effect.

[0049] Example 2: In vitro experimental study for the evaluation of the bactericidal / bacteriostatic effect of PUFAs on the microorganisms that most frequently cause SSIs.

[0050] An in vitro experimental study was conducted to evaluate the bactericidal / bacteriostatic effect of the three PUFAs (DHA, GLA and EPA) on the microorganisms Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus and Staphylococcus epidermidis, which most frequently cause SSI.

[0051] The initial compound used in the first trial was obtained from the Microbiology Department of the Elche University General Hospital and consisted of a mixture of three different PUFAs, which had a concentration of 33% relative to the total product. Subsequently, in the remaining trials, the chains of the three selected PUFAs, purified and individually 100%, were used, and these were also obtained from the hospital's Microbiology Department.

[0052] 2.1) Preparation of microorganism colonies and antibiograms

[0053] Various culture media were prepared using Blood Agar in a laminar flow hood, and the microorganisms that most frequently cause SSI were seeded in the center of the Petri dishes separately, thus favoring their growth.

[0054] On the seventh day, 5 mm discs were cut from the edges of the colonies inoculated onto each Petri dish. A turbidity test was then performed by introducing the microorganism colonies into physiological saline solution and comparing them to another sterile tube of physiological saline solution alone.

[0055] The density of the first tube had to show turbidity values ​​between 0.5 McFarland, thus confirming the presence of an optimal concentration of microorganisms to perform the PUFA sensitivity tests in a standardized manner. Subsequently, the stock solution was obtained, composed of Mueller-Hinton medium (2 ml) and the microorganism colonies obtained after the turbidity test.

[0056] Antibiograms were performed where 2 pl of the stock solution was added only in well twelve of the antibiogram and 1 pl of Muellen Hinton Agar in the rest of the wells.

[0057] Serial dilutions of the stock solution were made, with a dilution factor of 1:2 in concentrations from 20% to 50%, from wells 2 to 12. A control (well 1) was also included where there was only culture medium without PUFAs, so that increasing doses of PUFAs were added after the control well or sample (without PUFAs) up to the last well (with the maximum concentration of PUFAs).

[0058] The initial volume was calculated using the formula [ ]¡ x Vi = []fx Vf, where Vi is the initial volume and Vf is the final volume, to determine and obtain the increasing concentrations of the PUFAs. The antibiogram was then covered, and the plates were incubated at 37°C for 18 hours. This procedure was performed before each of the study's assays, varying the doses and composition of the PUFAs according to the type of assay performed. 2.2) In vitro assays

[0059] Four consecutive in vitro assays were performed to study the antimicrobial properties of PUFAs and the required dosage. The initial dilution of the PUFAs was performed with ethanol. To eliminate potential confounding bias due to the inhibitory action of ethanol, a control test was conducted by culturing the studied microorganisms separately in ethanol and verifying their growth in the culture medium. The minimum inhibitory concentration (MIC) was defined as the lowest PUFA concentration that inhibits bacterial growth after incubation.

[0060] A bactericidal agent is defined as one that directly destroys bacteria. This bactericidal action was observed in the assay by the absence of bacterial growth in one of the wells of the antibiogram due to the action of the PUFAs. To confirm this finding, in Gram-positive bacteria, the corresponding well was cultured with a bactericidal MIC on a solid medium (cultured at 37°C for 24 hours), confirming the destruction of the microorganism. (Table 1).

[0061] Bacteriostatic agents are defined as those substances that inhibit bacterial reproduction, that is, they prevent bacterial proliferation by interrupting their growth. This bacteriostatic action was observed in the assay by noting the absence of bacterial proliferation in one of the wells of the antibiogram due to the action of the PUFAs. To confirm this finding, in Gram-negative bacteria, a culture was performed on the corresponding well with bacteriostatic MIC on a solid medium (culture at 37°C for 24 hours), confirming the inhibition of growth and thus its bacteriostatic effect. (Table 1).

[0062] Table 1: Antibiogram to check bacteriostatic and bactericidal effect.

[0063] The first trial was exploratory, designed to initially observe the biological behavior of PUFAs against the microorganisms that most frequently cause SSIs. Sequenced dilutions of the stock solution were prepared with a 1:2 dilution factor at concentrations ranging from 20% to 50%, from wells 2 to 12. A control (well 1) containing only culture medium without PUFAs was also included. Subsequently, increasing doses of PUFAs were added from the control well to the final well (well 12).

[0064] The second trial consisted of evaluating the antimicrobial behavior of individual PUFAs, once the antimicrobial potential of PUFAs had been confirmed. Serial dilutions of the stock solution were performed with a dilution factor of 1:2 at concentrations between 20% and 50%, from wells 2 to 12. Individual PUFAs were added at increasing concentrations after the control well (without PUFAs) up to the last well (number 12), similar to the previous study, with the distinction being the use of individual PUFA chains.

[0065] In the antibiograms performed in the second assay, the different PUFA chains were observed individually and organized in rows against Gram-positive microorganisms (Staphylococcus aureus and Staphylococcus epidermidis), noting the MICs of the PUFAs against the microorganism under study. The growth of the Gram-positive microorganism was observed up to a specific well where the PUFA had exerted its bactericidal effect (MIC) and where growth was no longer observed (disappearance of the central white spot).

[0066] The same procedure was performed with Gram-negative germs, observing an inhibition of the microorganism, so the central white spot did not disappear, but maintained its size.

[0067] Next, a third assay was performed based on the previously obtained results (bactericidal MIC for Gram-positive bacteria with each of the PUFAs). For this, the bactericidal dose of the most potent PUFA (DHA) was selected and fixed. Antibiograms were performed with the combined fatty acids (DHA+EPA and DHA+GLA) to obtain the MIC of the two combined fatty acids, thus assessing a possible synergistic and bactericidal / bactehostatic effect resulting from their combination.

[0068] In the antibiograms of the third assay, the different PUFA chains, combined in pairs and arranged in rows, were observed against Gram-positive (Staphylococcus aureus and Staphylococcus epidermidis) and Gram-negative (Escherichia coli and Klebsiella pneumoniae) microorganisms. The growth of the Gram-positive microorganism was observed up to a specific well where the PUFA combination had exerted its bactericidal effect (MIC) and where no further growth was observed.

[0069] The same procedure was performed with Gram-negative germs, observing an inhibition of the microorganism, so the central white spot did not disappear, but maintained its size.

[0070] Once the MICs of the PUFAs (combined in pairs) were obtained for each microorganism, a fourth test was performed, where a combination of the three PUFAs was made based on the results obtained previously (bactericidal / bacteriostatic MIC with the PUFAs combined in pairs).

[0071] To achieve this, the bactericidal dose of the most potent PUFA combination (DHA+EPA) was selected and fixed. Antibiograms were performed with the combined fatty acids (DHA+EPA+GLA) to obtain the MIC of the three fatty acids combined. This resulted in a final compound that allowed for a further reduction in the previous doses. This final product was approved as a medical device by the Spanish Agency for Medicines and Health Products (AEMPS) and named PrevOmega.

[0072] 2.3) Biological behavior of PUFAs against microorganisms causing SSI.

[0073] In the third trial, antibiograms were performed with PUFAs combined in pairs against the microorganisms that most frequently cause SSIs. The dose of DHA, which was the most potent PUFA in the previous trial, was fixed and combined with EPA and GLA at lower doses than in the previous trial. This yielded the MICs with bactericidal effect of PUFA 2 and PUFA 3 after their combination with DHA against Gram-positive microorganisms.

[0074] Against Gram-negative bacteria, the PUFAs tested showed no bactericidal effect, but doses with a bacteriostatic effect were obtained. The doses used in this paired assay were lower than those used in the previous assay with the PUFAs individually.

[0075] In the studies, against Gram-positive bacteria (Staphylococcus aureus, Staphylococcus epidermidis), a MIC with a bactericidal effect was obtained, while against Gram-negative bacteria (Escherichia coli and Klebsiella pneumoniae), the PUFAs had a bacteriostatic effect. Additionally, synergistic interactions were detected among the three PUFAs, resulting in both a bactericidal effect (against Gram-positive bacteria) and a bacteriostatic effect (against Gram-negative bacteria) with a composition using lower doses of PUFAs, thus defining the minimum dose necessary to achieve this effect.

[0076] The final product was approved as a medical device by the AEMPS and named PrevOmega.

[0077] Example 3: Clinical trial.

[0078] A prospective, randomized, double-blind study was conducted to evaluate the development of surgical site infections (SSIs) in high-risk patients undergoing laparoscopic cholecystectomy with umbilical stent placement and topical application of a polyunsaturated fatty acid composition to the surgical wound versus no application. The study protocol was approved by the hospital's Ethics Committee and is registered in the European Clinical Trials Database (EudraCT) under code 2018-002260-67. Authorization was obtained from the Spanish Agency for Medicines and Health Products (AEMPS) and granted as a medical device under file number 628 / 17 / EC, with the name PrevOmega.

[0079] The surgical procedure consisted of placing a 6.4 cm diameter circular prosthesis (BARD Hernia Patch®) intra-abdominally, following a laparoscopic cholecystectomy. The solution contained in the 10 ml opaque vial was then administered to the surgical wound according to a randomized schedule. The procedure was standardized for one minute before the excess solution was dried with a sterile compress. Finally, the skin was closed with staples.

[0080] The SSI rate was significantly lower in the PUFA group compared to the non-PUFA group. Multivariate analyses showed that only PUFA significantly reduced the SSI rate. Therefore, topical application of a PUFA solution at the umbilical trocar site after laparoscopic cholecystectomy reduced the SSI rate.

[0081] Example 4: Composition and characterization of the composition

[0082] Table 2. MIC with combination of the 3 polyunsaturated fatty acids.

[0083] The preparation and handling of the medical device was carried out in a cleanroom and under horizontal laminar flow hoods, under aseptic conditions. The preparation process was performed in collaboration with the Pharmacy Department of the Elche University General Hospital.

[0084] Under a laminar flow hood, the AGPI composition was prepared by performing the following steps:

[0085] - Starting with 25 mg of docosahexaenoic acid (DHA), 10 mg of eicosapentaenoic acid (EPA) and 500 mg of gamma-linolenic acid (GLA), where each of the PUFAs is contained in one ampoule.

[0086] - Open the three ampoules and add 1 ml of absolute ethanol to each one.

[0087] - Next, in a sterile 10 ml vial, add 1 ml of DHA and 9 ml of ethanol, obtaining a solution with a concentration of 2.5 mg of DHA.

[0088] - Repeat the previous step with a second sterile 10 ml vial in which 1 ml of EPA and 9 ml of ethanol are added, obtaining a solution with a concentration of 1.0 mg of EPA.

[0089] - Repeat the previous step with a third sterile 10 ml vial in which 1 ml of GLA and 9 ml of ethanol are added, obtaining a solution with a concentration of 50 mg of GLA.

[0090] Next, 0.32 ml of DHA, 1.28 ml of EPA, and 0.2 ml of GLA from the prepared solutions are loaded into a sterile 100 ml vial, and 98.2 ml of sterile physiological saline are added. The prepared PUFA composition contains 8 g / ml of DHA, 12.8 g / ml of EPA, and 102.4 pg / ml of GLA, with a total PUFA concentration of 123.2 g / ml.

[0091] The medical device is a sterile solution of the polyunsaturated fatty acid composition diluted in a sterile 10 ml vial. To prepare it, 2 ml of the PUFA composition are drawn into a sterile 10 ml vial and 8 ml of physiological saline solution are added. The composition of one 10 ml vial is: - 2 ml of the polyunsaturated fatty acid composition,

[0092] - 8 ml 0.9% sodium chloride (Physiological saline solution).

[0093] Example 5: Efficacy trials demonstrating the synergy of PUFAs.

[0094] Once the MICs of the PUFAs (combined in pairs) were obtained for each microorganism, a fourth test was performed, where a combination of the three PUFAs was made based on the results obtained previously (bactericidal / bacteriostatic MIC with the PUFAs combined in pairs).

[0095] For this purpose, the bactericidal dose of the most potent PUFA combination (DHA+EPA) was selected and fixed. Antibiograms were performed with the combined fatty acids (DHA+EPA+GLA) to obtain the MIC of the three combined fatty acids.

[0096] Thus, a bactericidal MIC was obtained for the three PUFAs against Staphylococcus aureus and Staphylococcus epidermidis. And, for Gram-negative microorganisms (Escherichia coli and Klebsiella pneumoniae), bacteriostatic concentrations were obtained (Table 2), resulting in a final compound that allowed for a further reduction in previous doses, confirming its synergistic effect.

[0097] Thus, the combination of the three PUFAs achieved the same bactericidal effect against Gram-positive bacteria and bacteriostatic effect against Gram-negative bacteria, using a much lower dose of each PUFA individually. This demonstrated the synergistic effect resulting from the combination of these PUFAs, allowing the determination of the lowest dose of each needed to achieve the desired effect.

[0098] In this way, the final composition of the product used in subsequent studies was established, which was approved by the AEMPS as a health product and named PrevOmega.

[0099] Materials and methods. Study design

[0100] This prospective study evaluated the in vitro bactericidal / bacteriostatic effect of three polyunsaturated fatty acids, EPA, GLA, and DHA, on the microorganisms Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, and Staphylococcus epidermidis, which are the most frequent causes of surgical site infections (SSIs). Various blood agar culture media were prepared in a laminar flow hood, and the microorganisms that most frequently cause SSIs were inoculated into the center of Petri dishes. On day seven, 5 mm discs were cut from the edges of the colonies to create different antibiograms, combining the stock solution composed of Mueller-Hinton medium (2 ml) with the colonies of the microorganisms.

[0101] A turbidity test was previously performed by introducing the microorganism colonies into physiological saline solution and comparing it to another sterile tube of physiological saline solution alone. The density of the first tube should show turbidity values ​​between 0.07 and 0.1.

[0102] Two units of the stock solution were added to well twelve of the antibiogram with the highest concentration of compound, and one unit of Mueller-Hinton agar was added to the remaining wells. Dilutions were made to one medium from the last well to the preceding wells until reaching the first well, where the control well (stock solution, but without the three hyperoxygenated fatty acids) was located.

[0103] Four in vitro assays were performed. The first was exploratory, initially observing the biological behavior of PUFAs (33% purity) against the microorganisms that most frequently cause surgical site infections (SSIs). The second assay consisted of observing this bactericidal / bacteriostatic behavior with the PUFAs individually, once the bactericidal potential of Omega-3 at 33% had been confirmed and the purified fatty acid chains had been obtained individually. In the third assay, a combination of the different PUFAs was performed in pairs to assess whether they exhibited a synergistic effect in inhibiting the growth of microorganisms. Finally, in the fourth assay, a combination of doses of the three fatty acids was carried out, achieving the synergistic antimicrobial effect at lower doses.

[0104] The initial volume was calculated using the formula [ ]¡ x Vi = [ ]fx Vf, where Vi is the initial volume and Vf is the final volume. 100 microliters of this solution were added to each well and the antibiogram was then covered. The plates were incubated at 37°C for 16–20 hours.

[0105] • EPA: [ ] f = 409.6 pg / ml ; V¡ 82 pl

[0106] GLA: [ ] f = 4096 pg / ml ; V¡ 16 pl • DHA: [ ] f= 128 pg / ml ; V¡ 10 l (Final concentration for Colli and Klebsiella)

[0107] The MIC was defined as the minimum antibacterial concentration that inhibits bacterial growth after incubation. The initial dilution of the fatty acids (DHA, EPA, and GLA) was performed with ethanol (control sample). A control was performed by culturing the microorganisms (Gram-positive and Gram-negative) in ethanol, verifying their growth in the culture medium, thus eliminating the potential confounding bias due to the inhibitory action of ethanol.

[0108] To verify the bactericidal and bacteriostatic effect, antibiograms were performed with increasing concentrations of PUFAs in the different wells, against the four microorganisms studied (Gram-positive and Gram-negative). In Gram-positive bacteria, when the well showed negative growth, a sample was taken from that well and cultured on a solid medium (at 37°C for 24 hours), confirming the bactericidal effect. In the case of Gram-negative bacteria, when neither negative growth nor growth was observed, the well was also cultured on a solid medium (at 37°C for 24 hours) to demonstrate the inhibition of growth and thus its bacteriostatic effect.

[0109] Biological behavior of PUFAs against microorganisms causing SSI

[0110] Against Gram-positive bacteria (Staphylococcus aureus, Staphylococcus epidermidis), a bactericidal MIC (DHA 8 pg / ml) was obtained, while against Gram-negative bacteria (Escherichia coli and Klebsiella pneumoniae), the PUFAs only had a bacteriostatic effect. Additionally, synergistic interactions were detected among the three PUFAs, resulting in a bactericidal effect at lower doses against Gram-positive bacteria. DHA was the most effective compound, exhibiting a bactericidal effect at lower doses.

[0111] The first antibiogram allowed the determination of the MIC using Omega 3 (fish extract with an Omega 3 mixture) at a purity of 33%, whose value was 256 pg / ml for Staphylococcus aureus, not inhibiting the rest of the microorganisms in the study. Table 3.

[0112] Table 3. CM I with Omega 3 at a purity of 33% and individualized PI AG.

[0113] Antibiograms were subsequently performed with increasing doses of the different purified and individualized PUFAs against the various microorganisms. In the case of Staphylococcus aureus and Staphylococcus epidermidis, a bactericidal MIC (fv of 8 pg / ml) was obtained for DHA. However, neither bactericidal nor bacteriostatic concentrations were achieved against the Gram-negative microorganisms (Escherichia coli and Klebsiella pneumoniae) with any of the PUFAs at these doses. Table 3.

[0114] Based on these results, where DHA was the most potent fatty acid (achieving a bactericidal MIC for Gram-positive bacteria at a lower concentration), its dose was set at 8 pg / ml. Therefore, antibiograms were performed with the combined fatty acids to obtain the MIC of the two remaining fatty acids (EPA and GLA). Table 4.

[0115] In this way, the doses of DHA are reduced sixteen times in relation to the results described by Desbois, (see Desbois, AP & Lawlor, KC “Antibacterial activity of long-chain polyunsaturated fatty acids against Propionibacterium acnes and Staphylococcus aureus”. Marine Drugs 2013, 11 (11), 4544-4557.

[0116] Table 4. MIC with combination of 2 fatty acids

[0117] From this second antibiogram, the MICs with bactericidal effect of EPA and GLA against Gram-positive microorganisms were obtained, while for Gram-negative microorganisms, no bactericidal doses were obtained, only inhibitory doses, thus increasing the required doses. This second antibiogram showed a reduction in the doses of both EPA and GLA compared to the aforementioned article by Desbois. 1

[0118] Finally, an antibiogram was performed using the bactericidal MICs of the three PUFAs, which had been obtained from the previous antibiograms. This resulted in a final compound consisting of the three PUFAs with bactericidal activity against Gram-positive bacteria and bacteriostatic activity against Gram-negative bacteria (Table 5 and Figure 1).

[0119] Table 5. MIC with combination of the 3 fatty acids.

[0120] The surprising effect and inventive nature of the product lies in the synergistic effect of the combination of the three PUFAs, which can reduce the dose of some of the component fatty acids by up to 20 times compared to studies described in the literature. In the study conducted by Desbois 1 The following bactericidal doses were found against Staphylococcus aureus (Table 6):

[0121] Table 6. MIC and MBC against Staphylococcus aureus.

[0122] The DHA MBC was reduced from 128 to 8 pg / ml in our study (Table 5), sixteen times lower than in the published study. The EPA dose was reduced from 256 to 12.8 pg / ml (Table 5), twenty times lower than in the previous study, and finally, GLA was reduced from 512 to 102.4 pg / ml (Table 5), five times lower against Staphylococcus aureus.

Claims

CLAIMS 1. Composition comprising a mixture of two or more polyunsaturated fatty acids selected from docosahexaenoic acid (DHA), gamma-linolenic acid (GLA), eicosapentaenoic acid (EPA) and mixtures thereof, wherein the composition comprises: between 6.1% and 7.1% docosahexaenoic acid (DHA), between 10.1% and 11.0% gamma-linolenic acid (GLA); and between 81.8% and 83.8% eicosapentaenoic acid (EPA), wherein the percentages refer to the weight of the total polyunsaturated fatty acids.

2. A pharmaceutical composition comprising the composition according to claim 1 and one or more pharmaceutically acceptable excipients.

3. The composition according to claim 1, or a pharmaceutical composition thereof, for use as a medicament.

4. The composition according to claim 1, or a pharmaceutical composition thereof, for use in the treatment and / or prevention of a microbial infection.

5. The composition for the use defined according to claim 4, wherein the microbial infection takes place at the incisional surgical site.

6. The composition for the use defined according to any of claims 4 or 5, wherein the microbial infection is caused by one or more pathogens selected from Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli and Klebsiella pneumoniae.

7. Medical device comprising the composition according to claim 1, or a pharmaceutical composition thereof.

8. Device comprising: - a vial prepared under sterile conditions containing the composition according to claim 1, or a pharmaceutical composition thereof; and - a sterile applicator integrated into the vial for topical application of the composition.

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