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    • 专利摘要:
    Endothelial microvesicles with microbicidal effect. The present invention relates to the use of microvesicles derived from endothelial cells (MVe) as a medicament, more specifically as an antibiotic. The MVe of the invention are useful against Gram + and Gram- bacteria, as well as against multiresistant bacteria to common antibiotics. Therefore, the present invention also relates to a pharmaceutical composition comprising the MVe of the invention for use in the treatment of bacterial infections that are preferably selected from any of the following sepsis, gangrene, salmonellosis, botulism, cholera, impetigo, meningitis, pneumonia, tetanus, tuberculosis, gonorrhea, syphilis, nephritis and cystitis. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2724215A1
    申请号:ES201800049
    申请日:2018-03-01
    公开日:2019-09-09
    发明作者:Magro Guillermo Bodega；Chamond Manuel Rafael Ramirez；Patino José Luis Copa；De Carranza Juan Soliveri；Aguilar Matilde Alique；Morales Miriam Moran；Magro M Lourdes Bohorquez
    申请人:Universidad de Alcala de Henares UAH；
    IPC主号:
    专利说明:

    [0001]
    [0002] Endothelial microvesicles with microbicidal effect.
    [0003]
    [0004] Technical sector
    [0005]
    [0006] The present invention relates to microvesicles derived from endothelial cells (MVe) with microbicidal effect and falls within the pharmaceutical sector, specifically in the design of new antibiotics.
    [0007]
    [0008] Background of the invention
    [0009]
    [0010] Sepsis is one of the leading causes of mortality worldwide [1]. The American College of Chest Physicians and The Society of Critica! Care Medicine defines it as a systemic inflammatory response syndrome (SIRS) in case of an infection [2]. This syndrome is characterized by a complex systemic response that leads to an excess of cellular activation and uncontrolled inflammation that injures the tissues and organs of their own in response to an infection mainly of bacterial type. Its most severe form can lead to septic shock and multiorgan dysfunction that causes the death of the patient [3].
    [0011]
    [0012] Worldwide, 20-30 million cases of sepsis are recorded annually, and it is the cause of the death of 50 people every hour. Sepsis affects about 50,000 people annually in Spain, of which 17,000 die. In the last ten years, cases of sepsis have doubled and studies consider that the trend will continue to rise [4].
    [0013]
    [0014] Some of the objective factors that explain this trend are the increasing use of catheters and other invasive equipment, chemotherapy, immunosuppression in patients with organ transplants or inflammatory diseases, and also the increase in the population over 65, many of the which suffer from chronic diseases, and, in general, are a group with a higher risk of infection [5].
    [0015]
    [0016] Despite several decades of study of this pathology, the standard treatment of bacterial sepsis remains the administration of fluids and vasopressors to restore blood pressure and blood flow of organs, oxygenation and administration of antibiotics. This has a great health cost, since the patient with sepsis remains hospitalized in the ICU. In Spain the approximate average economic cost of treatment and hospitalization in the ICU of a patient with sepsis is about € 17,000, although the figure is much lower than the cost of other nearby countries such as Germany where it amounts to € 25,000 - € 50,000 [4].
    [0017]
    [0018] The increasingly frequent use of broad-spectrum antibiotics has resulted in an increase in bacterial resistance to antimicrobials, which is a major problem worldwide [6] and especially for these patients, since it does not only prolong the duration of their stay in the ICU, but the effect on mortality is uncertain being the type of organism that causes sepsis determining the outcome.
    [0019]
    [0020] The increase in resistant bacteria has also contributed to the increase in the frequency of cases of sepsis due to Gram positive bacteria (Gram +), now almost as common as Gram negative infections (Gram-). The predominant organisms causing sepsis are Staphylococcus aureus (20.5%), Pseudomonas (19.9%), Enterobacteriaceae (mainly E. coli, 16.0%), and fungi (19%) [7]. In addition, these organisms are at the top of the WHO list of pathogens for which new antibiotics with high and critical priority are needed [8].
    [0021] Recent studies on sepsis and infection in vivo have observed a large increase in blood MV [9-12], Mostefai et al. (2008) [10] observed that the number of circulating MVs increased in septic patients compared to non-septic patients, and specifically observed a large increase in MVe in these patients. It has also been observed that patients with bacteraemia had higher levels of circulating MV than healthy subjects. Herrmann et al. (2015) [13] investigated the diagnostic potential of MVs derived from polymorphonuclear cells (PMN) to differentiate sepsis from systemic inflammatory response syndrome. The results obtained showed that MVs suffer phenotypic changes based on specific stimuli, such as infection, which leads to an increase in their bacterial aggregation capacity. So far, it is therefore known to increase blood MVs when there is a bacterial infection, as well as the usefulness of these MVs for differentiation between different pathologies of bacterial origin.
    [0022]
    [0023] Taking into account the above, there is a need in the state of the art to develop drugs, preferably antibiotics, alternative to those known in the state of the art, for the treatment of bacterial infections in general, where such drugs are broad spectrum, being effective also against multiresistant bacteria.
    [0024]
    [0025] Description of the invention
    [0026]
    [0027] The present invention relates to MVe that possess a potent antimicrobial capacity both on the main pathogens involved in sepsis and in multiresistant bacteria to conventional antibiotics. These MVs cause the destruction of bacteria but do not affect eukaryotic cells. Therefore, the invention provides an application of these microvesicles for obtaining a new medically, specifically an antibiotic.
    [0028] The MVe play an important role in the defense against pathogens being, therefore, beneficial as part of the natural response to the infection. This new function of the MVs is of great diagnostic utility and also offers a wide variety of therapeutic targets, as well as an alternative to antibiotics for the treatment of infectious diseases such as sepsis. Said bactericidal action of the MVe of the invention is carried out even at physiological concentrations thereof, and with a high concentration of microorganisms, specifically bacteria, this action being more apparent the higher the concentration of MVe is used.
    [0029]
    [0030] In addition, the antimicrobial activity associated with the MVe of the invention is not dependent on the complement pathway and is very stable, as it is resistant to freezing and heat treatments up to 85 ° C. Treatment with the MVe of the invention does not induce pathophysiological changes in eukaryotic cells.
    [0031]
    [0032] MVe have bactericidal activity against both Gram- and Gram + bacteria. These results suggest a relevant role of MVe in infectious pathologies such as sepsis, being promising therapeutic targets for their treatment.
    [0033]
    [0034] MVs are structures derived from the cell membrane, 0.2 to 1.5 pm in diameter. Plasma MVs are generated from a wide variety of cell types: endothelial cells, leukocytes and platelets. Due to their composition of proteins, lipids and other cytoplasmic components, MVs can transmit a large number of messages between cells and contribute to a wide variety of physiological and pathological processes [14], their function as reactive species eliminators has even been demonstrated of oxygen [15].
    [0035]
    [0036] This functional content varies according to the cell of origin of the MV and also depends on the physiological and pathological conditions existing at the time of its formation and secretion [16], which makes the definition of its function a challenge, since the different MV populations may have synergistic effects or even opposite effects on recipient cells.
    [0037]
    [0038] Thus, a first aspect of the present invention relates to the use of MV derived from endothelial cells for the preparation of a medicament. Alternatively, the present invention also relates to MV derived from endothelial cells for use as a medicament.
    [0039] The term medication, as used herein, refers to any substance used for prevention, diagnosis, relief, treatment or cure of diseases in man and animals. In the context of the present invention, the disease is a disease that occurs with bacterial infection.
    [0040]
    [0041] In a preferred embodiment of the use of the MVs of the present invention, these are characterized in that the medicament is preferably an antibiotic.
    [0042]
    [0043] In another preferred embodiment of the use of the MVs of the invention, said antibiotic is effective against Gram + and Gram- bacteria. In yet another more preferred embodiment, the Gram + bacteria are selected from the list consisting of: Staphilococcus aureus, Staphilococcus aureus resistant to methicillin, Streptococcus pneumoniae, Streptococcus pyogenes, Enterococcus faecalis, Enterococcus faecium, Listeria Closidtrium tertidium, Closettrium tertidum, Closettrium tertidum, Closettrium tertidum, Closettrium tertidum, Closettrium tertidum, Closettrium tertidum, Closettrium tertidum, Closettrium tertidum, Closettrium tertidum perfringens, Bacillus anthracis, Actinomyces israelii; or combinations thereof, and Gram- bacteria are selected from the list consisting of: Pseudomonas aeruginosa, Escherichia coli, Salmonella typhi, Haemophilus influenzae, Bordetella pertussia, Helicobacter pylori, Legionella pneumophila, Acinetobacter baumannii, Neisseria meningitidis or combinations of same.
    [0044]
    [0045] In another preferred embodiment of the use of the MVs of the invention, these are characterized in that the medicament, preferably the antibiotic, comprises the cytosolic fraction thereof.
    [0046] Another aspect of the present invention relates to the use of MVs derived from endothelial cells of the invention for the preparation of a medicament, preferably an antibiotic, for the treatment of pathologies that occur with bacterial infection. Alternatively, the present invention relates to MVs derived from endothelial cells of the invention for use in the treatment of pathologies that occur with bacterial infection.
    [0047]
    [0048] In a preferred embodiment, the use of the MVs of the invention for the treatment of pathologies that occur with bacterial infection is characterized in that said medicament, preferably an antibiotic, comprises the cytosolic fraction thereof.
    [0049]
    [0050] The term "treatment" as understood in the present invention refers to combating the effects caused as a result of a disease or pathological condition of interest in a subject (preferably mammal, and more preferably a human) that includes:
    [0051]
    [0052] (i) inhibit the disease or pathological condition, that is, stop its development;
    [0053]
    [0054] (ii) alleviate the disease or the pathological condition, that is, cause the regression of the disease or the pathological condition or its symptomatology;
    [0055]
    [0056] (iii) stabilize the disease or pathological condition.
    [0057]
    [0058] For the purposes of the present invention, the term "pathologies with bacterial infection" refers to those infectious diseases caused by bacteria. In another preferred embodiment, the pathologies that occur with bacterial infection are selected from the list consisting of: sepsis, gangrene, salmonellosis, botulism, cholera, impetigo, meningitis, pneumonia, tetanus, tuberculosis, gonorrhea, syphilis, nephritis and cystitis.
    [0059]
    [0060] In another preferred embodiment, the bacterial infection is caused by a bacterium that is selected from the list consisting of Gram + and Gram- bacteria. In other more preferred embodiments, the Gram + and Gram- bacteria are selected from the list consisting of any of those mentioned above throughout the present invention, or combinations thereof.
    [0061]
    [0062] Another aspect of the present invention relates to a composition, preferably a pharmaceutical composition comprising the MVs derived from endothelial cells of the invention, and at least one pharmaceutically acceptable carrier.
    [0063]
    [0064] The term "pharmaceutically acceptable vehicle" refers to a vehicle that must be approved by a federal government regulatory agency or a state government or listed in the United States Pharmacopoeia or the European Pharmacopoeia, or other pharmacopoeia generally recognized for use in animals, and more specifically in humans.
    [0065]
    [0066] The term "vehicle" refers to a diluent, adjuvant, excipient or carrier with which the MVe of the invention or the composition comprising them should be administered; obviously, said vehicle must be compatible with them.
    [0067]
    [0068] The pharmaceutical composition of the invention, if desired, may also contain, when necessary, additives to increase, control or otherwise direct the desired therapeutic effect of the MVe of the invention, which comprise said pharmaceutical composition, and / or auxiliary substances or pharmaceutically acceptable substances, such as buffering agents, surfactants, co-solvents, preservatives, etc. Such pharmaceutically acceptable substances that can be used in the pharmaceutical composition of the invention are generally known to those skilled in the art and are normally used in the preparation of pharmaceutical compositions. Examples of suitable pharmaceutical vehicles are described, for example, in "Remington's Pharmaceutical Sciences " by EW Martin. Additional information on these vehicles can be found in any pharmaceutical technology manual (Galenic Pharmacy).
    [0069]
    [0070] In another preferred embodiment, the composition of the invention may further comprise at least one active ingredient.
    [0071]
    [0072] As used herein, the term "active ingredient", "active substance", "pharmaceutically active substance", "active ingredient" or "pharmaceutically active ingredient" means any component that potentially provides a pharmacological activity or other different effect on the diagnosis, cure, mitigation, treatment, or prevention of a disease, or that affects the structure or function of the body of man or other animals. The term includes those components that promote a chemical change in the preparation of the drug and are present therein in a modified form intended to provide the specific activity or effect.
    [0073]
    [0074] For the purposes of the present invention, the active ingredient is selected from the list consisting of: antibiotics, anti-inflammatories, antipyretics and analgesics, more specifically the active ingredients are selected from the list consisting of acetylsalicylic acid, diclofenac, indomethacin, piroxicam, ibuprofen , naproxen, dihydrocodeine, dextropropoxyphene, fentanyl, tramadol, metamizole, paracetamol and / or any combination of the above.
    [0075]
    [0076] The pharmaceutical composition of the invention will contain a therapeutically effective or effective amount of the MVs of the invention, more preferably of the cytosolic fraction of said MVe, to provide the desired therapeutic effect. As used herein, the term "therapeutically effective amount" or "therapeutically effective amount" refers to the amount of MVe or the cytosolic fraction thereof contained in the pharmaceutical composition that is capable of producing the therapeutic effect. desired and, in general, will be determined, among other factors, by the desired therapeutic effect pursued. In general, the therapeutically effective amount of MVe or the cytosolic fraction thereof that must be administered will depend, among other factors, on the subject's own characteristics, the severity of the disease, the form of administration, etc. For this reason, the doses mentioned in this invention should be taken into account only as a guide for the person skilled in the art, who should adjust this dose depending on the factors described above. The pharmaceutical composition of the invention will be formulated according to the chosen form of administration. The pharmaceutical composition of the invention can be prepared in a liquid or gel dosage mode, for example, in the form of a suspension, to be injected or perfused to the individual.
    [0077]
    [0078] Administration of the pharmaceutical composition of the invention to the individual will be carried out by conventional means. For example, said pharmaceutical composition can be administered to said individual intravenously using suitable devices, such as syringes, catheters (a standard peripheral intravenous catheter, a central venous catheter or a pulmonary arterial catheter, etc.), trocars, cannulas, etc. In all cases, the pharmaceutical composition of the invention will be administered using the equipment, apparatus and devices suitable for the administration of such compositions and known to those skilled in the art.
    [0079]
    [0080] As the person skilled in the art understands, sometimes direct administration of the pharmaceutical composition of the invention to the site that is intended to benefit can be advantageous. Thus, the direct administration of the pharmaceutical composition of the invention to the desired organ or tissue can be achieved by direct administration (by injection, etc.) on the external surface of the affected organ or tissue by insertion of a suitable device, eg, an appropriate cannula, by arterial or venous perfusion (including retrograde flow mechanisms) or by other means mentioned in this description or known in the art.
    [0081]
    [0082] The pharmaceutical composition of the invention, if desired, can be stored until the moment of its application by conventional procedures known to those skilled in the art. This pharmaceutical composition can also be stored together with additional medications, useful in the treatment of diseases, in an active form comprising a combination therapy.
    [0083]
    [0084] Another aspect of the present invention relates to a method of treatment of pathologies that present with bacterial infection comprising the administration to a subject afflicted with said pathology, or in need of treatment, of a therapeutically effective amount of the MVe of the invention or of the composition of the invention.
    [0085]
    [0086] For the purposes of the present invention the term "subject in need of treatment" includes those cases that already have the disorder as well as those in which the disorder is to be prevented. Administration can be carried out "in combination with" one or more therapeutic agents including simultaneous (concurrent) and consecutive administration in any order.
    [0087]
    [0088] In a preferred embodiment of the method of treatment of the invention, this is characterized in that the pathologies that occur with bacterial infection are selected from the list consisting of: sepsis, gangrene, salmonellosis, botulism, cholera, impetigo, meningitis, pneumonia, tetanus , tuberculosis, gonorrhea, syphilis, nephritis and cystitis.
    [0089] Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be derived partly from the description and partly from the practice of the invention. The following examples and figures are provided by way of illustration, and are not intended to be limiting of the present invention.
    [0090]
    [0091] Brief description of the drawings
    [0092]
    [0093] Fig. 1. Antimicrobial assessment of MVe at a concentration of 100,000 MV / mL on S. aureus at inoculum concentrations of 2 x 104 CFU / mL (p <0.001) (A), and 2 x 107 CFU / mL (p = 0.008) (D). Mean ± SD, n = 3. ** p <0.01, *** p <0.001. Only the statistical significance data corresponding to the last hour of the trial has been included.
    [0094]
    [0095] Fig. 2. Antimicrobial assessment of MVe at a concentration of 100,000 MV / mL on P. aeruginosa at inoculum concentrations of 2 x 102 and 2 x 103 CFU / mL (p = 0.0075) (A) and 2 x 102 CFU / mL (p = 0.0064) (B). Mean ± SD, n = 3. ** p <0.01. Only the statistical significance data corresponding to the last hour of the trial has been included.
    [0096]
    [0097] Fig. 3. Antimicrobial assessment of MVe at a concentration of 500,000 MV / mL (p = 0.0048) (A) and 1,000,000 MV / mL (p <0.001) (B) on P. aeruginosa at inoculum concentration of 2 x 102 CFU / mL. Mean ± S d , n = 3. ** p <0.01, *** p <0.001. Only the statistical significance data corresponding to the last hour of the trial has been included.
    [0098]
    [0099] Fig. 4. Antimicrobial assessment of MVe at a concentration of 100,000 MV / mL on MRSA at inoculum concentrations of 2 x 105 CFU / mL (p <0.001) (A) and 2 x 106 CFU / MI (p = 0, 0018) (B). Mean ± SD, n = 3. * p <0.05; ** p <0.01, *** p <0.001. Only the statistical significance data corresponding to the last hour of the trial has been included.
    [0100]
    [0101] Fig. 5. Antimicrobial assessment of MVe at a concentration of 500,000 MV / mL on MRSA at inoculum concentrations of 2 x 106 CFU / mL (p <0.0001) (A) and 2 x 107 CFU / mL (p = 0 , 0001) (B). Mean ± SD, n = 3. **** p <0.0001. Only the statistical significance data corresponding to the last hour of the trial has been included.
    [0102]
    [0103] Fig. 6. Antimicrobial evaluation of the cytosolic and membrane fractions of the MVe obtained from 500,000 MV / mL on S. aureus (2 x 106 CFU / mL) (p <0.0001). Mean ± SD, n = 3. **** p <0.0001. Only the statistical significance data corresponding to the last hour of the trial has been included.
    [0104]
    [0105] Fig. 7. Assessment of the antimicrobial capacity of MVe (A) and its cytosolic fraction (B) after decomplementation (indicated as C) (p <0.0001). 500,000 MVe / mL. Mean ± SD, n = 3 . **** p <0.0001. Only the statistical significance data corresponding to the last hour of the trial has been included.
    [0106]
    [0107] Fig. 8. Interaction of MVe with P. aeruginosa at 30 min (A). Interaction of MVe with P. aeruginosa at 4 h (B). Interaction of MVe with S. aureus at 4 h (C). Interaction of MVe with S. aureus at 20 h (D). Images obtained with differential interference contrast (DIC) (1). Images obtained by overlapping the fluorescence images (light gray in the figure) and that obtained with DIC (2).
    [0108]
    [0109] Examples
    [0110]
    [0111] The following examples serve to illustrate the nature of the present invention. These examples are included for illustrative purposes only and should not be construed as limitations to the invention claimed herein. Therefore, the examples described below illustrate the invention without limiting its scope of application.
    [0112]
    [0113] Materials and methods
    [0114]
    [0115] Cultivation, isolation and quantification of MVe
    [0116]
    [0117] Umbilical cord endothelial cells (HUVEC; ATCC catalog number. PCS-100-010) were grown in endothelial growth medium (EGM) (Lonza, Basel, Switzerland) supplemented with 10% bovine fetal serum (FBS, Fetal Bovine Serum) (Sigma, St. Louis, USA) decomplemented. The cultures were maintained at 37 ° C in an atmosphere with 5% CO 2 and 95% humidity.
    [0118]
    [0119] MVe were obtained from the supernatants of said cell cultures. Supernatants from pass 4 to 7 were used to obtain a pool of young MVe. The isolation was performed by centrifuging the supernatants at 3100 rpm for 15 min at room temperature, followed by another centrifugation at 14000 rpm for 15 min at 4 ° C. Then the MVs were resuspended in PBS (Sigma, St. Louis, USA) and, after discarding the contamination of the different passes by conducting a contamination test, incubating a sample of each pass in Luria Bertani broth (LB) (Conda , Torrejón de Ardoz, Spain) at 37 ° C for 72 hours, the contamination-free passes were unified and the MVs were quantified.
    [0120]
    [0121] The MVe were quantified by flow cytometry with the BD FACSCalibur ™ cytometer. First, the population of MV was selected based on size (FSC / forward scatter) and complexity (SSC / side scatter) using a kit intended for this purpose (Flow Cytometry Sub-Micron Size Reference Kit, Green Fluorescent. Life Technologies, Paisley, RU) containing spheres of different sizes. Using the areas of 0.5 pm, 1 pm and 2 pm as reference, the area in which the MVs were located, which have a size of 0.2 to 2 pm in diameter, was determined in the dot-plot . Events less than 0.5 pm were excluded to correctly distinguish true events from cytometer noise, as well as events greater than 2 pm to avoid possible confusion with apoptotic bodies.
    [0122]
    [0123] Once located for their size, those positive events were selected for Annexin V (Alexa Fluor 647, Biolegend. San Diego, USA) and for CD31 (CD31 L133.1 PE, Becton Dickinson. Franklin Lakes, USA), since being MV of endothelial origin are positive for said marker.
    [0124]
    [0125] The absolute number of MV (events) / pL was determined using a known concentration of Flow Count Calibrator beads of 10 pm (Beckman Coulter. Brea, USA) and following the manufacturer's recommendations to use the CXP software (total events x standard spheres / L) / (counted standard spheres).
    [0126]
    [0127] MVe fractionation
    [0128]
    [0129] A sample of MVe was fractionated by sonication and then centrifuged at 50,000 rpm for 1 h and at 4 ° C. Thus, it was possible to separate the cytosolic content and the membrane of the MVe to assess, independently, its antimicrobial capacity in bioassays.
    [0130]
    [0131] Bacterial cultures and bioassays
    [0132]
    [0133] The choice of the appropriate culture medium for the bioassays was a complex process, since the bacteria needed an appropriate medium for optimal growth and the MVe also needed an appropriate medium given their origin of eukaryotic cells. Thus, the different combinations of the means were tested to establish the most suitable proportion of them to carry out the bioassays. In addition, the combination chosen was supplemented with fetal bovine serum (FBS, Fetal Bovine Serum) since it was observed that otherwise, the activity of the MVe was reduced.
    [0134]
    [0135] The culture media used for the bioassay were Mueller-Hinton Broth (MFI) 1X (Scharlau Microbiology, Barcelona, Spain) and Dulbecco's Modified Eagle Medium (DMEM) (Sigma, St. Louis, USA). They were used alone and also mixed in different proportions (50:50, 25:75 and 75:25 VA /). In addition, in all cases the culture medium was supplemented with 10% FBS. Finally, the medium of choice for bioassays consisted of 75% MFI broth and 25% DMEM, supplemented with 10% FBS. Once the medium was formulated, experiments were carried out to determine the minimum concentration of inoculum to be used for each microorganism (Table 1).
    [0136]
    [0137]
    [0138]
    [0139]
    [0140] Table 1.- Minimum concentration of inoculum for the optimal growth of the different microorganisms in the medium of choice for the bioassays (MH: DMEM 75:25 V / V, 10% FBS).
    [0141]
    [0142] Three different microorganisms have been used: Staphylococcus aureus (CECT 108, Gram positive), Pseudomonas aeruginosa (CECT 240, Gram negative), both from the Spanish Type Culture Collection (CECT), and methicillin- resistant Staphylococcus aureus and other antibiotics ( SARM 10225758) assigned by the Prince of Asturias Hospital (Alcalá de Henares, Spain). The generation used of the different microorganisms was always lower than the G-5. For the performance of the antimicrobial titration bioassays of the MVe, the culture and inoculum preparation of the microorganisms mentioned above was prepared according to the international standard methods ISO 20776-1 [18].
    [0143]
    [0144] The microorganisms were plated with a non-selective nutrient medium, plate count agar (PCA, Plate Count Agar) (Scharlau Microbiology. Barcelona, Spain), at 37 ° C for 24 h. Then several colonies were resuspended in 1X Mueller-Hinton broth (Scharlau Microbiology. Barcelona, Spain) and incubated at 37 ° C with shaking at 110 rpm for 24 h. Starting from the previous broth, a dilution was prepared and the absorbance was measured with the spectophotometer at 625 nm until a dilution in the range of 0.08-0.11 (0.05 McFarland) was achieved, corresponding to a concentration of 1 x 108 CFU / mL From this dilution, the concentration of microorganism required could be adjusted according to the type of microorganism and the method used for the administration of the inoculum. In this study, several dilutions were made to obtain the different inoculums with concentrations from 2 x 102 CFU / mL to 2 x 107 CFU / mL.
    [0145]
    [0146] The bioassays were performed in 96-well plates and the Ultra Microplate Reader ELx808 reader plate (BioTek. Winooski, USA) was used following an absorbance reading protocol at 630 nm every hour for a total of 20 h, incubation at 37 ° C and agitation.
    [0147]
    [0148] MVe: bactericidal or bacteriostatic effect
    [0149]
    [0150] After the bioassays, the effect that MVe had on bacteria was also analyzed to see if they had a bactericidal or bacteriostatic effect. To do this, samples were taken from some wells after the bioassays, placed on PCA plates and incubated for 48 h at 37 ° C. The results showed that the MVe, and more specifically the cytosolic fraction exert a bactericidal effect since there is no plaque growth.
    [0151]
    [0152] Confocal microscopy
    [0153]
    [0154] The MVe were stained with CelITracker CM-Dil (Life Technologies, Paisley, UK) to visualize their interaction with S. aureus and P. aeruginosa. Their interaction was observed after 30 min, at 4 h and at 20 h. Fluorescence was detected using a Leica TSC SP5 confocal microscope (Leica Microsystems. Wetzlar, Germany).
    [0155]
    [0156] Statistic analysis
    [0157]
    [0158] The MVe concentration was obtained from the analysis of the data of three independent measurements of the sample with the Cyflogic program (version 1.2.1) and the CXP software mentioned above. The bioassay data was analyzed with the Gen5 analysis software (version 2.00) and using the Microsoft Excel spreadsheet. Each condition was performed in triplicate (n = 3) and in the graphs each point is represented as mean ± SD. Statistical analysis was performed with the GraphPad Prism program (version 5.00); Differences between groups were analyzed using Student's t test and One-way ANOVA. A p <0.05 has been considered statistical significance.
    [0159]
    [0160] Example 1. Analysis of the bactericidal effect of the MVe of the invention on S. aureus.
    [0161]
    [0162] The study of the antimicrobial assessment of MVe was initially performed on S. aureus. The first bioassay was performed using a bacterial inoculum at a concentration of 2 x 104 CFU / mL and an MVe concentration of 10,000 MV / mL, which corresponds to a physiological concentration. The controls grew normally; however, there was no bacterial growth in any of the cases in which MVe were added. To corroborate these results, another bioassay was performed using the same concentration of MVe and bacterial inoculums of different concentrations, from 2 x 104 CFU / mL to 2 x 107 CFU / mL. As can be seen in Figure 1, there was no bacterial growth when MVe were added regardless of the inoculum concentration (p <0.001), except for those in which an inoculum of 2 x 107 CFU / mL was used. In this case, an increase in the number of microorganisms was detected, by multiplying them, 12 h after the entrance of the control in exponential phase of growth, and, at the end of the bioassay (t = 20 h) it was observed that the MVe inhibited 64% (p = 0.008) bacterial growth at said inoculum concentration.
    [0163]
    [0164] Example 2. Analysis of the bactericidal effect of the MVe of the invention on P. aeruginosa.
    [0165]
    [0166] Taking into account the results obtained on Gram + bacteria, it was studied whether MVe had the same effect on Gram- bacteria. To that end, P. aeruginosa inoculums were used at a concentration of 2 x 102 CFU / mL and 2 x 103 CFU / mL, and a physiological concentration of MVe (10,000 MV / mL). When an inoculum of 2 x 103 CFU / mL and MVe was used, the microorganism entered the exponential phase at 10 h, as well as the control, but at = 20 h the MVe inhibited bacterial growth by 14% (p = 0, 0075) (Figure 2A). When the inoculum of 1x102 CFU / mL and MVe was used, the microorganism entered an exponential phase of growth at 11 h, as well as the control, but at = 20 h the MVe inhibited bacterial growth by 13% (p = 0 , 0064) (Figure 2B). Another bioassay was performed using the lowest concentration of inoculum and increasing the concentration of MVe 5 (5X) and 10 (10X) times, to simulate the levels of MV found in pathological states such as sepsis. When the concentration of MVe was increased 5 times, an increase in the number of microorganisms was detected, by multiplying them 2 h after entry of the control in exponential phase of growth, and, at = 20 h, the MVe inhibited a 71 % (p = 0.0048) bacterial growth (Figure 3A). However, when the MVe concentration was increased 10 times, there was a total inhibition of bacterial growth (p <0.001) (Figure 3B).
    [0167]
    [0168] Example 3. Analysis of the bactericidal effect of the MVe of the invention on methicillin- resistant Staphylococcus aureus (MRSA).
    [0169]
    [0170] Subsequently, a bioassay was performed using MRSA, which is a microorganism of great importance in hospitals for its resistance to various antibiotics and against which new therapies are needed. MRSA inoculums of 2 x 105 CFU / mL and 2 x 106 CFU / mL and an MVe concentration of 10,000 MV / mL were used. Bacterial growth was totally inhibited (p <0.001) when the inoculum of 2 x 105 CFU / mL was used (Figure 4A). With the inoculum of 2 x 106 CFU / mL there was no total inhibition, but the microorganism entered the exponential phase of growth 1 h later than the control, and at = 20 h the MVe inhibited 13% (p = 0.018) bacterial growth (Figure 4B). Another bioassay was performed increasing the concentration of MVe 5 times and using MRSA inoculums of 2 x 106 CFU / mL and 2 x 107 CFU / mL. With this concentration of MVe there was a total inhibition of bacterial growth in both cases (p <0.001 and p = 0.0001) (Figure 5).
    [0171]
    [0172] Example 4. Analysis of the bactericidal effect of the cytosolic fraction and the membrane fraction isolated from the MVe of the invention on S. aureus.
    [0173]
    [0174] In order to elucidate in which fraction of the MVe the antimicrobial mechanism resides, a bioassay was performed by adding, independently, each of the fractions (cytosolic and membrane), previously separated by ultracentrifugation. An inoculum of 2 x 10 6 CFU / mL of S. aureus and a membrane and cytosol concentration equivalent to 50,000 MV / mL were used. A control with 50,000 MV / mL was also performed to see if the effect of the individual fractions was the same as that of the entire MVs. The cytosolic fraction produced a total inhibition of bacterial growth, as did the control with 50,000 MV / mL (p <0.0001), while the membrane fraction did not inhibit growth (Figure 6), although it delayed it.
    [0175]
    [0176] Example 5. Analysis of bactericidal activity of the MVe of the invention via complement inhibition.
    [0177]
    [0178] The MVe contain and / or carry associated complement system proteins (C1QBP, C3, C4-A, C5, C6 C9) defense mechanism whose main mission is to eliminate pathogens from the circulation. In fact, they present a large amount of C3 that is key in the activation of the different pathways of the complement system [17], which suggests the importance of these MVe in the defense against pathogens.
    [0179]
    [0180] In order to elucidate whether the microbicidal effects observed in the previous examples were due to the loading of complement system proteins containing these MVe, a bioassay was performed by decomplementing at 56 ° C for 30 min the FBS that was supplemented to the medium and the MVe themselves. A concentration of 10,000 MV / mL and an inoculum of 2 x 10® CFU / mL of S. aureus were used. The different possible combinations were tested: decomplemented MVe medium, MVe decomplemented FBS medium, and decomplemented MVe decomplemented FBS medium, with their respective controls. In all cases where there was MVe, both normal and decomplemented, there was a total inhibition of bacterial growth, while in controls without MVe there was adequate growth (p <0.0001) (Figure 7 A).
    [0181]
    [0182] Since the antimicrobial capacity of MVe resides in the cytosol, another bioassay was performed by decomplementing at 56 ° C for 30 min, the FBS and the cytosolic fraction. An inoculum of 2 x 10 6 CFU / mL of S. aureus and a cytosol concentration equivalent to 10,000 MV / mL were used, and the same combinations were tested as in the previous bioassay. In In all cases in which there was cytosol, there was a total inhibition of bacterial growth (p <0.0001), equal to that observed in bioassays with unfractionated MVe (Figure 7B).
    [0183] In addition to heating the MVe at 56 ° C to decomplement, a treatment at 85 ° C was performed, both at the MVe and the cytosolic fraction, in order to test its heat resistance. This treatment did not affect the antimicrobial activity of the components tested.
    [0184]
    [0185] Example 6. Analysis of the interaction of the MVe of the invention with P. aeruginosa and S. aureus.
    [0186] Finally, the interaction of MVe with P. aeruginosa and S. aureus at different times (30 min, 4 h and 20 h) was visualized by confocal microscopy. A high concentration of both bacteria and MV was used to easily visualize them. The MVe interact quickly with P. aeruginosa (Figure 8A), and at 4h large accumulations of MV and bacteria can be seen, the latter being dead since they have lost their characteristic shape and are broken (Figure 8B). At 8 pm P. aeruginosa had formed a biofilm that was completely filled with MVe. As for S. aureus, the MVe take a little longer to interact with the microorganism, but at 4 h accumulations of MV and S. aureus are already observed (Figure 8C). At 20 h it is observed that these accumulations are larger than those observed at 4h (Figure 8D). In each of the figures marked with 1 the image obtained with DIC is shown, in those marked with 2 the fluorescence image (CelITracker) is shown and the image obtained with overlapped DIC.
    [0187] REFERENCES
    [0188]
    [0189] 1. Ramachandran, G. 2014. Gram positive and gram negative bacterial toxins in sepsis.
    [0190] Virulence, 5 (1): 213-218.
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    [0192] 2. Hodgin, K.E. and M. Moss. 2008. The epidemiology of sepsis. Current Pharmaceutical Design, 14 (19): 1833-1839.
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    [0194] 3. Meziani, F., Delabranche, X., Asfar, P. and F. Toti. 2010. Bench to bedside review:
    [0195] Circulating microparticles a new player in sepsis Critical Care, 14 (5): 236-243.
    [0196]
    [0197] 4. Press Release Sepsis Day. 2016 (September 13). Spanish Society of Intensive, Critical Medicine and Coronary Units. http://www.semicyuc.org/temas/semicyuc/cornunicados-oficial/lasepsis-acaba-con-lavida-de-una-persona-cada-cuatro-segundos
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    [0199] 5. Van Amersfoort, E.S., Van Berkel, T. and J. Kuiper. 2003. Receptors, mediators, and mechanisms involved in bacterial sepsis and septic shock. Clinical Microbiology Reviews, 16 (3): 379-414.
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    [0206] 9. Zafrani, L., Gerotziafas, G., Bymes, C., Hu, X., Perez, J., Lévi, C., et al. 2012. Calpastatin Controls polymicrobial sepsis by limiting procoagulant microparticle release. American Journal of Respiratory and Critical Care Medicine, 185 (7): 744-755.
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    [0209] Circulating microparticles from patients with septic shock exert protective role in vascular function. American Journal of Respiratory and Critical Care Medicine, 178: 1148-1155.
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    [0211] 11. Schorey, J.S. and C.V. Harding. 2016. Extracellular vesicles and infectious diseases: New complexity to an oíd story. Journal of Clinical Investigated, 126 (4): 1181-1189.
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    [0213] 12. Souza, A.C.P., Yuen, P.S.T. and R.A. Star. 2015. Microparticles: Markers and mediators of sepsisinduced microvascular dysfunction, immunosuppression, and AKI. Kidney International, 87 (6): 1100-1108.
    [0214]
    [0215] 13. Hermann, I. K., Bertazzo, S., O'Callaghan, D.f Schlegal, A. A., et al. 2015. Differentiating sepsis from non-infectious systemic inflammation based on microvesicle bacteria aggregation. Nanoscale, 7: 13511-13520.
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    [0217] 14. Zafrani, L., Ince, C. and P. Yuen. 2013. Microparticles during sepsis: target, canary or cure Intensive Medicine Care, 39: 1854-1856.
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    [0219] 15. Bodega, G., Alique, M., Bohórquez, L., Ciordia, S., Mena, M.C. and M.R. Ramirez 2017
    [0220] The antioxidative role of young and senescent human umbilical vein endothelial cells and their microvesicles. Volume Article ID 7094781.12 pages. https://doi.Org/10.1155/2017/7094781.
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    [0222] Pitt, J.M., Kroemer, G. and L. Zitvogel. 2016. Extracellular vesicles: Masters of intercellular communication and potential clinical interventions. Journal of Clinical Investigation, 126 (4): 1139-1143.
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    权利要求:
    Claims (17)
    [1]
    1. Use of microvesicles derived from endothelial cells (MVe) for the preparation of an antibiotic.
    [2]
    2. Use according to claim 2 wherein the antibiotic is effective against Gram + and Gram- bacteria.
    [3]
    3. Use according to claim 2 wherein the Gram + bacteria are selected from the list consisting of: Staphilococcus aureus, Staphilococcus aureus resistant to methicillin, Streptococcus pneumoniae, Streptococcus pyogenes, Enterococcus faecalis, Enterococcus faecium, Listeria monoidtogenes, closidium, bottium tetanus Clostridium perfringens, Bacillus anthracis, Actinomyces israelii or combinations thereof;
    [4]
    4. Use according to claim 2 wherein the Gram-bacteria are selected from the list consisting of: Pseudomonas aeruginosa, Escherichia coli, Salmonella typhi, Haemophilus influenzae, Bordetella pertussia, Helicobacter pylori, Legionella pneumophila, Acinetobacter baumannii, Neisseria meningitidis or combination combinations the same.
    [5]
    5. Use according to any of claims 1 to 4 characterized in that the antibiotic comprises the cytosolic fraction of the MVe.
    [6]
    6. Use of MVe for the elaboration of a medicine for the treatment of pathologies that present with bacterial infection.
    [7]
    7. Use according to claim 6 characterized in that the medicament comprises the cytosolic fraction of the MVe.
    [8]
    8. Use according to any of claims 6 to 7 wherein the medicament is an antibiotic.
    [9]
    9. Use according to any of claims 6 to 8 wherein the bacterial infection is produced by a bacterium that is selected from the list consisting of Gram + and Gram- bacteria.
    [10]
    10. Use according to any of claims 6 to 9 wherein the Gram + bacteria are selected from the list consisting of: Staphylococcus aureus, Methicillin-resistant Staphilococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes, Enterococcus faecalis, Enterococcus faecium, Listeria monocyte, Listeria monocyte, Listeria monocyte, Listeria monocyte, Listeria monocyte, Listeria monocyte, Listeria monocyte, Listeria monocyte, Listeria monocyte, Listeria monocyte, Listeria monocyte, Listeria monocyte , Clostridium botulinum, Clostridium perfringens, Bacillus anthracis, Actinomyces israelii or combinations thereof.
    [11]
    11. Use according to any of claims 6 to 9 wherein the Gram bacteria are selected from the list consisting of: Pseudomonas aeruginosa, Escherichia coli, Salmonella typhi, Haemophilus influenzae, Bordetella pertussia, Helicobacter pylori, Legionella pneumophila, Acinetobacter baumannii, Neisseria meningitidis or combinations thereof.
    [12]
    12. Use according to any of claims 6 to 11, wherein the pathology with bacterial infection is selected from the list consisting of: sepsis, gangrene, salmonellosis, botulism, cholera, impetigo, meningitis, pneumonia, tetanus, tuberculosis, gonorrhea, syphilis, nephritis and cystitis.
    [13]
    13. Pharmaceutical composition comprising MVe and at least one pharmaceutically acceptable excipient and / or vehicle.
    [14]
    14. Pharmaceutical composition according to claim 13 characterized in that it comprises the cytosolic fraction of the MVe.
    [15]
    15. Pharmaceutical composition according to any of claims 13 to 14 further comprising at least one active ingredient.
    [16]
    16. Pharmaceutical composition according to claim 15 wherein the active ingredient is selected from the list consisting of: antibiotics, anti-inflammatories, antipyretics, analgesics and / or any combination thereof.
    [17]
    17. Pharmaceutical composition according to any of claims 15 to 16 wherein the active ingredient is selected from the list consisting of: acetylsalicylic acid, diclofenac, indomethacin, piroxicam, ibuprofen, naproxen, dihydrocodeine, dextropropoxyphene, fentanyl, tramadol, metamizol, paracetamol, and / or any combination of the above.
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    同族专利:
    公开号 | 公开日
    ES2724215B2|2020-04-14|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    ES2409180T3|2007-10-15|2013-06-25|Fresenius Medical Care Deutschland Gmbh|Use of microvesicles , to prepare a medicament that has adjuvant activity in endothelial cell transplantation, particularly in the treatment of diabetes through pancreatic islet transplantation, and related method|
    ES2423483T3|2007-10-29|2013-09-20|Fresenius Medical Care Deutschland Gmbh|Use of microvesicles derived from stem cells to prepare a drug for endothelial / epithelial regeneration of damaged or damaged tissues or organs, and related methods in vitro and in vivo|
    ES2446301T3|2008-02-01|2014-03-07|The General Hospital Corporation|Use of microvesicles in the diagnosis, prognosis and treatment of diseases and medical conditions|
    ES2604946T3|2011-07-28|2017-03-10|Cryo-Save Ag|Microvesicles isolated from mesenchymal stem cells for use as immunosuppressive agents|
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