WO2022162265A1 - Antimicrobials against pathogens of the genus streptococcus - Google Patents
Antimicrobials against pathogens of the genus streptococcus Download PDFInfo
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- WO2022162265A1 WO2022162265A1 PCT/ES2022/070042 ES2022070042W WO2022162265A1 WO 2022162265 A1 WO2022162265 A1 WO 2022162265A1 ES 2022070042 W ES2022070042 W ES 2022070042W WO 2022162265 A1 WO2022162265 A1 WO 2022162265A1
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- streptococcus
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Definitions
- the present invention relates to a series of compounds for antimicrobial use against pathogens of the Streptococcus genus.
- Streptococcus pneumoniae also known as pneumococcus, is a Gram-positive, facultatively anaerobic, non-sporulating, nonmotile coccoid bacterium whose main habitat is the upper respiratory tract of humans (Brooks L, Mias G. (2016) Streptococcus pneumoniae's virulence and host immunity: aging, diagnostics, and prevention. Front. Immunol. 9(1136): 1-29. https://doi.org/10.3389/fimmu.2018.01366).
- Pneumococcus is an important human pathogen responsible for both invasive and non-invasive pathologies, such as meningitis and otitis media, being the main causal agent of community-acquired pneumonia (Loughran AJ, Orihuela CJ, Tuomanen El. (2019). Streptococcus pneumoniae: Invasion and Inflammation. Microbiol Spectr. 7(2):GPP3-0004-2018. https://dx.doi.org/10.1128%2Fmicrobiolspec.GPP3-0004-2018).
- One of these strategies is the repositioning of drugs, based on using drugs already authorized by regulatory agencies in the treatment of new pathologies, or at least identifying bioactive molecules that allow their research and development. Being drugs already authorized by regulatory agencies, their pharmacokinetic and pharmacodynamic properties are known, as well as their toxicity, safety and most appropriate route of administration, so the number of clinical trials carried out to determine these parameters would be fewer. This allows reducing costs and time in research, resulting in the introduction of molecules already known for the treatment of a new disease relatively quickly (Oprea TI, Mestres, J. (2012). Drug repurposing: far beyond new targets for old drugs.The AAPS Journal 14: 759-763.
- the present invention relates to a compound selected from:
- the invention refers to the compound or a pharmaceutically acceptable salt thereof for the use defined above, selected from:
- the invention refers to the compound or a pharmaceutically acceptable salt thereof for the use defined above, selected from:
- Another aspect of the present invention relates to a pharmaceutical composition
- a pharmaceutical composition comprising a compound selected from:
- Another aspect of the present invention refers to a method for the treatment of microbial diseases caused by Streptococcus, preferably for the treatment of diseases caused by Streptococcus pneumoniae (pneumococcus), Streptococcus oralis, Streptococcus mitis, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus viridans o Streptococcus mutans, even more preferably for the treatment of diseases caused by Streptococcus pneumoniae (pneumococcus), even more preferably for the treatment of pneumonia, meningitis, bacteremia, sinusitis or otitis, and even more preferably for the treatment of community-acquired pneumonia, bacterial meningitis or otitis media, in a subject in need thereof, especially in humans, comprising administering to said subject an effective amount of a compound selected from:
- GBR 12909 (Vanoxerin) 1-(2-[bis(4-fluorophenyl)methoxy)ethyl)-4- (3-phenylpropyl)piperazine
- Another aspect of the present invention relates to the use of a compound selected from:
- the compounds of the invention have bactericidal properties which therefore also make them useful for non-therapeutic uses such as antimicrobials for disinfection of eg surfaces.
- Examples of application of the compounds of the invention include, among others, the cleaning or coating of medical, veterinary, dental and surgical material, implants in general such as dental, cardiac or cochlear, as well as catheters, ventilation tubes, ventilators, endotracheal tubes and in general any other surface on which a biofilm of the pathogen can grow.
- another aspect of the invention refers to the non-therapeutic use as an antimicrobial against pathogens of the genus Streptococcus, preferably against Streptococcus pneumoniae (pneumococcus), Streptococcus oralis, Streptococcus mitis, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus viridans or Streptococcus mutans, more preferably against Streptococcus pneumoniae (pneumococcus), and even more preferably for its non-therapeutic use as an antimicrobial for the disinfection of the surface of a material, of a compound selected from:
- GBR 12909 (Vanoxerin) 1-(2-[bis(4-fluorophenyl)methoxy)ethyl)-4- (3-phenylpropyl)piperazine
- the compounds of the invention have been selected by screening compounds approved by the Food and Drug Administration (FDA) of the United States and by the European Medicines Agency (EMA) included in the library Prestwick® commercial chemistry. These compounds have demonstrated in vitro bactericidal effect against S. pneumoniae at therapeutic concentrations, with minimum inhibitory concentrations (MIC) between 26 and 50 pg mL -1 in capsuiated strain (infective), causing a decrease in bacterial viability of between 80 and 99.9%. In all cases, the addition of these compounds induces a disturbance of the bacterial membrane that gives rise to an increase in its permeability, so it is likely that they act through similar mechanisms.
- FDA Food and Drug Administration
- EMA European Medicines Agency
- the present invention also relates to a pharmaceutical composition
- a pharmaceutical composition comprising a compound of the invention (or a pharmaceutically acceptable salt or solvate thereof) 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 being harmful to the user of said composition.
- the compounds of the present invention can be administered in the form of any pharmaceutical formulation, the nature of which, as is well known, will depend on the nature of the active ingredient and its route of administration. In principle any route of administration can be used, eg oral, parenteral, nasal, ocular, rectal, and topical.
- Solid compositions for oral administration include tablets, granules, and capsules.
- the manufacturing method is based on a simple mixture, dry granulation or wet granulation of the active ingredient with excipients.
- excipients can be, for example, diluents such as lactose, microcrystalline cellulose, mannitol or calcium hydrogen phosphate; binding agents such as starch, gelatin or polyvinylpyrrolidone; disintegrants such as sodium carboxymethyl starch or croscarmellose sodium; and lubricating agents such as magnesium stearate, stearic acid or talc.
- the tablets can also be coated with suitable excipients and using known techniques in order to delay their disintegration and absorption in the gastrointestinal tract and thus achieve a sustained action for a longer period of time, or simply to improve their organoleptic properties or their stability.
- the active principle can also be incorporated by coating on inert pellets through the use of natural or synthetic film-forming polymers. It is also possible to make soft gelatin capsules, in which the active ingredient is mixed with water or with oily medium, for example coconut oil, liquid paraffin or olive oil.
- Powders and granules for the preparation of oral suspensions can be obtained by adding water, mixing the active ingredient with dispersing or wetting agents; suspensive and preservatives.
- Other excipients can also be added, for example sweeteners, flavors and colorants.
- Liquid forms for oral administration may include emulsions, solutions, suspensions, syrups, and elixirs containing commonly used inert diluents, such as distilled water, ethanol, sorbitol, glycerol, polyethylene glycols (macrogols), and propylene glycol.
- Said compositions may also contain adjuvants such as wetting agents, suspending agents, sweeteners, flavoring agents, preservatives and pH regulators.
- Injectable preparations for parenteral administration, comprise sterile solutions, suspensions or emulsions, in an aqueous or non-aqueous solvent such as propylene glycol, polyethylene glycol or vegetable oils. These compositions may also contain adjuvants, such as wetting agents, emulsifiers, dispersants, and preservatives. They may be sterilized by any of the known methods or prepared as sterile solid compositions to be dissolved in water or any other sterile injectable medium immediately before use. It is also possible to start from sterile raw materials and keep them in these conditions throughout the manufacturing process.
- the active ingredient may preferably be formulated as a suppository in an oily base, such as vegetable oils or solid semi-synthetic glycerides, or in a hydrophilic base such as polyethylene glycols (macrogol).
- an oily base such as vegetable oils or solid semi-synthetic glycerides
- a hydrophilic base such as polyethylene glycols (macrogol).
- the compounds of the invention can also be formulated for topical application for the treatment of pathologies in areas or organs accessible by this route, such as the eyes, skin and intestinal tract.
- Formulations include creams, lotions, gels, powders, solutions, and patches in which the compound is dispersed or dissolved in suitable carriers.
- the compound can be formulated in aerosol form from which it is conveniently released with the use of suitable propellants.
- the dosage and frequency of doses will vary depending on the nature and severity of the disease to be treated, the age, general condition and weight of the patient, as well as the particular compound administered and the route of administration, among other factors. .
- the compounds of the invention can be administered alone or in combination.
- carrier media for such compounds in one or multiple copies for example through the use of nanoparticles, should also be envisaged for their administration, since it has been shown that the antimicrobial dose of a given compound can be reduced by several orders of magnitude after their multiple arrangement on the surface of such particles, as has been shown, for example, with dendrimeric particles containing several copies of choline against Streptococcus pneumoniae (Hernández-Rocamora V, Maestro B, Waal B, Morales M, Garc ⁇ a P, Meijer EW, Merkx M, Sanz JM (2009).
- Multivalent choline dendrimers as potent inhibitors of pneumococcal cell-wall hydrolysis.
- Fig. 1 shows the growth curves of S. pneumoniae R6CIB17 in the presence of 25 pM of the compound to be tested, except perhexiline maieate (50 pM) and mianserin hydrochloride (100 pM).
- Fig. 2 shows the variation in the permeability of pneumococcal S. pneumoniae R6CIB17 cells incubated with each compound at a concentration of 25 pM. Positive control: Triton X100 at 1%.
- Prestwick Chemical A chemical library of 1,280 compounds authorized by both the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA) and marketed by the company Prestwick Chemical (http://www.prestwickchemical.com/libraries) has been screened. -screening-lib-pcl.html) called Prestwick® (Prestwick Chemical Library®).
- the assays were first performed blindly on the non-capsulated pneumococcus strain R6CIB17 ((GenBank accession number: CP038808), a non-flocculant variant of the usual laboratory strain R6), and the screening was performed sequentially (100, 50 and 25 pM) in planktonic cultures (liquid culture medium (Roig-Molina E, Sánchez-Angulo M, Seele, J, Garc ⁇ a-Asencio F, Nau R, Sanz JM, Maestro B. (2020). Searching for antipneumococcal targets : choline-binding modules as phagocytosis enhancers. ACS Inf. Dis. 6(5): 954-974. https://dx.doi.org/10.1021/acsinfecdis.9b00344), adding the compound to an O.D.550nm of 0, 1 .
- Bactericidal activity (plate viability assays) of selected compounds was carried out at 25 pM, except mianserin hydrochloride (100 pM) and perhexiline maleate (50 pM). Those that decrease viability by more than 90% (viable count ⁇ 3.6 x 10 7 colony-forming units per milliliter) were selected from this assay, with the exception of mianserin hydrochloride.
- Table 1 shows the cell viability of R6CIB17 after 140 min in the presence of the compound.
- La F ⁇ g. 2 shows that all compounds, to a greater or lesser degree, permeabilized the membrane, which seems to indicate that they all exert their antimicrobial activity through a similar molecular mechanism.
- Table 1 shows the data of the permeability of the membrane at 95 min, with respect to the control of 100% permeability in the presence of triton X100 at 1%. In any case, it is necessary to emphasize that many other compounds in the library that have similar chemical characteristics (amines bound to a hydrophobic region), do not have any antimicrobial activity, so the compounds that are the subject of this patent have a high degree of specificity. additional.
- MIC minimum inhibitory concentration of the compounds was determined using the criteria of the Clinical and Laboratory Standards Institute (CLSI). (2009). Performance standards for antimicrobial susceptibility testing; 19th informational supplement.
- CLSI documents M100-S19. CLSI, Wayne, PA.) using the microdilution system, following the indications described in International Standard ISO 20776 and using pneumococcal strains R6 (non-capsulated) and D39 (capsulated strain, and both infective).
- the MIC was calculated as the lowest concentration of the agent at which growth is visibly inhibited.
- growth controls were performed for each tested strain, and a positive control (type strain ATC49619 + ampicillin) and negative (sterility: culture medium + same volume of drug added to the experiment) (Table 1).
- Table 1 Bactericidal activity of selected compounds against S. pneumoniae R6CIB17. The cultures were grown in C+Y medium until an O.D.ssonm of 0.1, at which point the compound to be studied was added at a concentration of 25 pM except * (50 pM) and ** (100 pM), taking the sample for viable study at 140 min after addition. The count control of colony forming units (CFU) of S. pneumoniae and the same in the presence of DMSO at the final concentration in which the compounds in the test are found (0.5%) is shown.
- CFU colony forming units
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Abstract
Description
DESCRIPCIÓN DESCRIPTION
Antimicrobianos frente a patógenos del género Streptococcus Antimicrobials against pathogens of the Streptococcus genus
La presente invención se refiere a una serie de compuestos para su uso antimicrobiano frente a patógenos del género Streptococcus. The present invention relates to a series of compounds for antimicrobial use against pathogens of the Streptococcus genus.
ANTECEDENTES DE LA INVENCIÓN BACKGROUND OF THE INVENTION
Streptococcus pneumoniae, también conocido como neumococo, es una bacteria cocoide Gram-positiva, anaerobia facultativa, no esporulada e inmóvil cuyo hábitat principal son las vías respiratorias superiores de humanos (Brooks L, Mias G. (2018) Streptococcus pneumoniae’s virulence and host immunity: aging, diagnostics, and prevention. Front. Immunol. 9(1136): 1-29. https://doi.org/10.3389/fimmu.2018.01366). Streptococcus pneumoniae, also known as pneumococcus, is a Gram-positive, facultatively anaerobic, non-sporulating, nonmotile coccoid bacterium whose main habitat is the upper respiratory tract of humans (Brooks L, Mias G. (2018) Streptococcus pneumoniae's virulence and host immunity: aging, diagnostics, and prevention. Front. Immunol. 9(1136): 1-29. https://doi.org/10.3389/fimmu.2018.01366).
Neumococo es un importante patógeno humano responsable tanto de patologías invasivas como no invasivas, tales como la meningitis y la otitis media, siendo el principal agente causal de la neumonía adquirida en comunidad (Loughran AJ, Orihuela CJ, Tuomanen El. (2019). Streptococcus pneumoniae: Invasion and Inflammation. Microbiol Spectr. 7(2):GPP3-0004-2018. https://dx.doi.org/10.1128%2Fmicrobiolspec.GPP3- 0004-2018)). Además, se ha descrito recientemente como el mayor agente co-infectante con el virus SARS-CoV-2 (Zhu X, Ge Y, Wu T, Zhao K, Chen Y, Wu B, Zhu F, Zhu B, Cui L. (2020). Co-infection with respiratory pathogens among COVID-2019 cases. Virus Research 285: 198005. https://doi.Org/10.1016/j.virusres.2020.198005; y Garcia-Vidal C, Sanjuan G, Moreno-García E, Puerta-Alcalde P, Garcia-Pouton N, Chumbita M, Fernandez-Pittol M, Pitart e, Inciarte A, Bodro M, Morata L, Ambrosioni J, Grafía I, Meira F, Macaya I, Cardozo C, Casals C, Tellez A, Castro P, Marco F, García F, Mensa J, Martinez JA, Soriano A for the COVID-19 Researchers Group. (2021). Incidence of coinfections and superinfections in hospitalized patients with COVID-19: a retrospective cohort study. Clin. Microbiol. Infect 27(1):83-88. doi: 10.1016/j.cmi.2020.07.041), y en aquellos casos de co-infección se relaciona con un peor pronóstico (Pal C, Przydzial P, Chika-Nwosuh O, Shah O, Patel P, Madan N. (2020). Streptococcus pneumoniae coinfection in COVID-19: a series of three cases. Hindawi Case Reports in Pulmonology, article ID 8849068. https://doi.org/10.1155/2020/8849068; y Nieto-Moro M, Giuseppe Ecclesia F, Tomé-Masa I, De Lama Caro-Patón G, Leoz-Gordillo I, Cabrero-Hernández M, García-Salido A. (2020) SARS-CoV-2 and Streptococcus pneumoniae coinfection as a cause of severe pneumonia in an infant. Pediatric Pulmonology 2020; 1-3. http://doi.org/10.1002/ppul.24916). Pneumococcus is an important human pathogen responsible for both invasive and non-invasive pathologies, such as meningitis and otitis media, being the main causal agent of community-acquired pneumonia (Loughran AJ, Orihuela CJ, Tuomanen El. (2019). Streptococcus pneumoniae: Invasion and Inflammation. Microbiol Spectr. 7(2):GPP3-0004-2018. https://dx.doi.org/10.1128%2Fmicrobiolspec.GPP3-0004-2018). Furthermore, it has recently been described as the major co-infecting agent with the SARS-CoV-2 virus (Zhu X, Ge Y, Wu T, Zhao K, Chen Y, Wu B, Zhu F, Zhu B, Cui L. ( 2020). Co-infection with respiratory pathogens among COVID-2019 cases. Virus Research 285: 198005. https://doi.Org/10.1016/j.virusres.2020.198005; and Garcia-Vidal C, Sanjuan G, Moreno-García E , Puerta-Alcalde P, Garcia-Pouton N, Chumbita M, Fernandez-Pittol M, Pitart e, Inciarte A, Bodro M, Morata L, Ambrosioni J, Grafía I, Meira F, Macaya I, Cardozo C, Casals C, Tellez A, Castro P, Marco F, García F, Mensa J, Martinez JA, Soriano A for the COVID-19 Researchers Group.(2021).Incidence of coinfections and superinfections in hospitalized patients with COVID-19: a retrospective cohort study.Clin . Microbiol. Infect 27(1):83-88. doi: 10.1016/j.cmi.2020.07.041), and in those cases of co-infection it is related to a worse prognosis (Pal C, Przydzial P, Chika-Nwosuh O, Shah O, Patel P, Madan N. (2020).Streptococcus pneumoniae coinfe action in COVID-19: a series of three cases. Hindawi Case Reports in Pulmonology, article ID 8849068. https://doi.org/10.1155/2020/8849068; and Nieto-Moro M, Giuseppe Ecclesia F, Tomé-Masa I, De Lama Caro-Patón G, Leoz-Gordillo I, Cabrero-Hernández M, García-Salido A. (2020) SARS-CoV-2 and Streptococcus pneumoniae coinfection as a cause of severe pneumonia in an infant. Pediatric Pulmonology 2020; 1-3. http://doi.org/10.1002/ppul.24916).
La insuficiente cobertura procurada por las vacunas actuales, así como la falta de antimicrobianos eficaces para el tratamiento de ciertas cepas, lo ha convertido en un patógeno prioritario para la investigación de nuevas estrategias terapéuticas (World Health Organization. (2017). List of bacteria for which new antibiotics are urgently needed. Available from: htps://www.who.int/news/item/27-02-2017-who-publishes-list- of-bacteria-for-which-new-antibiotics-are-urgentiy-needed; y Centers for Disease Control and Prevention (CDC). (2019). Antibiotic resistance threats in the United States. Atlanta, GA: U.S. Department of Health and Human Services, CDC. Available from: https://www.cdc.gov/drugresistance/biggest-threats.html). The insufficient coverage provided by current vaccines, as well as the lack of effective antimicrobials for the treatment of certain strains, has made it a priority pathogen for the investigation of new therapeutic strategies (World Health Organization. (2017). List of bacteria for Available from: htps://www.who.int/news/item/27-02-2017-who-publishes-list-of-bacteria-for-which-new-antibiotics-are urgentiy-needed and Centers for Disease Control and Prevention (CDC) (2019). Antibiotic resistance threats in the United States. Atlanta, GA: U.S. Department of Health and Human Services, CDC. Available from: https://www. cdc.gov/drugresistance/biggest-threats.html).
Una de estas estrategias es el reposicionamiento de drogas, basada en emplear fármacos ya autorizados por las agencias reguladoras en el tratamiento de nuevas patologías, o al menos identificar moléculas bioactivas que permitan su investigación y desarrollo. Al ser fármacos ya autorizados por las agencias reguladoras, se conocen sus propiedades farmacocinéticas y farmacodinámicas, así como su toxicidad, seguridad y vía de administración más adecuada, por lo que el número de ensayos clínicos realizados para determinar estos parámetros sería menor. Esto permite reducir costes y tiempo en la investigación, dando como resultado la introducción de moléculas ya conocidas para el tratamiento de una nueva enfermedad de manera relativamente rápida (Oprea TI, Mestres, J. (2012). Drug repurposing: far beyond new targets for old drugs. The AAPS Journal 14: 759-763. http://doi.Org/10.1208/s 12248-012-9390- 1^Xue H, Li J, Xie H, Wang Y. (2018). Review of drug repositioning approaches and resources. Int. J. Biol. Sci. 14: 1232-1244. https://doi.org/10.7150/ijbs.24612; y Corsello SM, Bittker JA, Liu Z, Gould J, McCarren P, Hirschman JE, Johnston SE, Vrcic A, Wong B, Khan M, Asiedu J, Narayan R, Mader CC, Subramanian A, Golub TR. (2017). The drug repurposing hub: a next generation drug library and information resource. Nat. Med. 23: 405-408. https://doi.org/10.1038/nm.4306). One of these strategies is the repositioning of drugs, based on using drugs already authorized by regulatory agencies in the treatment of new pathologies, or at least identifying bioactive molecules that allow their research and development. Being drugs already authorized by regulatory agencies, their pharmacokinetic and pharmacodynamic properties are known, as well as their toxicity, safety and most appropriate route of administration, so the number of clinical trials carried out to determine these parameters would be fewer. This allows reducing costs and time in research, resulting in the introduction of molecules already known for the treatment of a new disease relatively quickly (Oprea TI, Mestres, J. (2012). Drug repurposing: far beyond new targets for old drugs.The AAPS Journal 14: 759-763. http://doi.Org/10.1208/s 12248-012-9390-1^Xue H, Li J, Xie H, Wang Y. (2018).Review of drug repositioning approaches and resources Int J Biol Sci 14: 1232-1244 https://doi.org/10.7150/ijbs.24612 and Corsello SM, Bittker JA, Liu Z, Gould J, McCarren P, Hirschman JE, Johnston SE, Vrcic A, Wong B, Khan M, Asiedu J, Narayan R, Mader CC, Subramanian A, Golub TR. (2017). The drug repurposing hub: a next generation drug library and information resource. Nat. Med 23: 405-408. https://doi.org/10.1038/nm.4306).
Por tanto, debido a los niveles preocupantes de resistencia a antimicrobianos y a la insuficiente cobertura de las vacunas actuales, sería deseable disponer de nuevos antimicrobianos frente a Streptococcus. DESCRIPCIÓN DE LA INVENCIÓN Therefore, due to the worrying levels of antimicrobial resistance and the insufficient coverage of current vaccines, it would be desirable to have new antimicrobials against Streptococcus. DESCRIPTION OF THE INVENTION
En un primer aspecto, la presente invención se refiere a un compuesto seleccionado de: In a first aspect, the present invention relates to a compound selected from:
GBR 12909 (Vanoxerina)GBR 12909 (Vanoxerine)
1-(2-[bis(4-fluorofenil)metoxi)etil)-4- (3-fenilpropil)piperazina 1-(2-[bis(4-fluorophenyl)methoxy)ethyl)-4- (3-phenylpropyl)piperazine
Clomifeno (Z, E) (E,Z)-2-(4-(2-cloro-1 ,2- difeniletenil)fenoxi) -N, N- dietiletanoamina Clomiphene (Z, E) (E,Z)-2-(4-(2-chloro-1,2-diphenylethenyl)phenoxy)-N,N- diethylethanolamine
MianserinaMianserin
(±)-2-metil-1 ,2,3,4, 10, 14 b- hexahidrodibenzofc, f]pirazino[ 1 , 2- a]azepína(±)-2-methyl-1 ,2,3,4, 10, 14 b- hexahydrodibenzofc, f]pyrazino[ 1 , 2- a] azepine
ClofiiiumClofiiium
4-(4-clorofenil)butil-dietil- heptilazanio 4-(4-chlorophenyl)butyl-diethyl- heptylazanium
AmiodaronaAmiodarone
(2-butil- 1 -benzofuran-3-il)-[4-[2- (dietilamino)etoxi]-3,5- diyodofenil]metanone (2-butyl- 1 -benzofuran-3-yl)-[4-[2- (diethylamino)ethoxy]-3,5-diiodophenyl]methanone
PerhexilinaPerhexiline
2-(2,2-diciclohexiletil)pipendina 2-(2,2-dicyclohexylethyl)pipendine
TerfenadinaTerfenadine
1 -(4-tert-butilfenil)-4-[4- [hidroxi(difen¡l)met¡l]piperidin- 1 - il]butan-1-ol
En otra realización la invención se refiere al compuesto o a una sal farmacéuticamente aceptable del mismo para el uso definido anteriormente, seleccionado de:
Clofilium
(2-butil- 1 -benzofuran-3-il)-[4-[2-(2-butyl- 1 -benzofuran-3-yl)-[4-[2-
(dietilamino)etoxi]-3,5- diyodofenil]metanone
En otra realización la invención se refiere al compuesto o a una sal farmacéuticamente aceptable del mismo para el uso definido anteriormente, seleccionado de: In another embodiment, the invention refers to the compound or a pharmaceutically acceptable salt thereof for the use defined above, selected from:
GBR 12909 (Vanoxerina)GBR 12909 (Vanoxerine)
1-(2-[bis(4-fluorofenil)metoxi)etil)-4-1-(2-[bis(4-fluorophenyl)methoxy)ethyl)-4-
(3-fenilpropil)piperazina
Clomifeno (Z, E)Clomiphene (Z, E)
(E,Z)-2-(4-(2-cloro-1 ,2- difeniletenil)fenoxi) -N, N- dietiletanoamina
GBR 12909 (Vanoxerina)GBR 12909 (Vanoxerine)
1-(2-[bis(4-fluorofenil)metoxi)etil)-4-1-(2-[bis(4-fluorophenyl)methoxy)ethyl)-4-
(3-fenilpropil)piperazina
GBR 12909 (Vanoxerina)GBR 12909 (Vanoxerine)
1-(2-[bis(4-fluorofenil)metoxi)etil)-4- (3-fenilpropil)piperazina 1-(2-[bis(4-fluorophenyl)methoxy)ethyl)-4- (3-phenylpropyl)piperazine
Ciomifeno (Z, E) (E,Z)-2-(4-(2-cloro-1 ,2- difeniletenil)fenoxi) -N, N- dietiietanoamma Cyomiphene (Z, E) (E,Z)-2-(4-(2-chloro-1 ,2- diphenylethenyl)phenoxy) -N, N- diethietanoamma
MianserinaMianserin
(±)-2-metil-1 ,2,3,4, 10, 14b- hexahidrodibenzo[c, f]pirazino[ 1 , 2- ajazepina(±)-2-methyl-1 ,2,3,4, 10, 14b- hexahydrodibenzo[c, f]pyrazino[ 1 , 2- ajazepine
ClofiliumClophylium
4-(4-clorofenil)butil-dietil- heptilazanio 4-(4-chlorophenyl)butyl-diethyl- heptylazanium
AmiodaronaAmiodarone
(2-butil- 1 -benzofuran-3-il)-[4-[2- (dietilamino)etoxi]-3,5- diyodofenil]metanone (2-butyl- 1 -benzofuran-3-yl)-[4-[2- (diethylamino)ethoxy]-3,5-diiodophenyl]methanone
PerhexilinaPerhexiline
2-(2,2-diciclohexiletil)piperidina 2-(2,2-dicyclohexylethyl)piperidine
Terfenadina 1-(4-tert-butilfenil)-4-[4- [hidroxi(difenil)metil]piperidin- 1 - il]butan-1 -ol
Otro aspecto de la presente invención se refiere a un método para el tratamiento de enfermedades microbianas causadas por Streptococcus, preferiblemente para el tratamiento de enfermedades causadas por Streptococcus pneumoniae (neumococo), Streptococcus oralis, Streptococcus mitis, Streptococcus, pyogenes, Streptococcus agalactiae, Streptococcus viridans o Streptococcus mutans, aún más preferiblemente para el tratamiento de enfermedades causadas por Streptococcus pneumoniae (neumococo), todavía más preferiblemente para el tratamiento de neumonía, meningitis, bacteriemia, sinusitis u otitis, y aún más preferiblemente para el tratamiento de neumonía adquirida en comunidad, meningitis bacteriana u otitis media, en un sujeto que lo necesite, especialmente en humanos, que comprende administrar a dicho sujeto una cantidad efectiva de un compuesto seleccionado de: Another aspect of the present invention refers to a method for the treatment of microbial diseases caused by Streptococcus, preferably for the treatment of diseases caused by Streptococcus pneumoniae (pneumococcus), Streptococcus oralis, Streptococcus mitis, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus viridans o Streptococcus mutans, even more preferably for the treatment of diseases caused by Streptococcus pneumoniae (pneumococcus), even more preferably for the treatment of pneumonia, meningitis, bacteremia, sinusitis or otitis, and even more preferably for the treatment of community-acquired pneumonia, bacterial meningitis or otitis media, in a subject in need thereof, especially in humans, comprising administering to said subject an effective amount of a compound selected from:
GBR 12909 (Vanoxerina) 1-(2-[bis(4-fluorofenil)metoxi)etil)-4- (3-fenilpropil)piperazina GBR 12909 (Vanoxerin) 1-(2-[bis(4-fluorophenyl)methoxy)ethyl)-4- (3-phenylpropyl)piperazine
Clomifeno (Z, E)Clomiphene (Z, E)
( E,Z) -2- (4- (2-cloro -1,2- difeniletenil)fenox¡) -N, N- dietiletanoamina (E,Z)-2-(4-(2-chloro-1,2-diphenylethenyl)phenoxy)-N,N- diethylethanolamine
MianserinaMianserin
(±)-2-metil- 1 ,2,3,4, 10, 14b- hexahidrodibenzo[c,f]pirazino[1,2- ajazepina(±)-2-methyl- 1 ,2,3,4, 10, 14b- hexahydrodibenzo[c,f]pyrazino[1,2- ajazepine
ClofiliumClophylium
4-(4-dorofenil)butil-díetíl- heptilazanio
(2-butil- 1 -benzofuran-3-il)-[4-[2- (dieti!amino)etoxi]-3,5- diyodofeniljmetanone (2-butyl- 1 -benzofuran-3-yl)-[4-[2- (diethyl!amino)ethoxy]-3,5- diiodophenyljmethanone
PerhexilinaPerhexiline
2-(2,2-diciclohexiletil)pipendina 2-(2,2-dicyclohexylethyl)pipendine
TerfenadinaTerfenadine
1 -(4- tert-butilfenil) - 4-[4 - [hidroxi(difenil)metil]piperidin- 1 - il]butan-1-ol
Otro aspecto de la presente invención se refiere al uso de un compuesto seleccionado de: Another aspect of the present invention relates to the use of a compound selected from:
GBR 12909 (Vanoxerina)GBR 12909 (Vanoxerine)
1-(2-[bis(4-fluorofenil)metox¡)et¡l)-4-1-(2-[bis(4-fluorophenyl)methoxy¡)ethyl)-4-
(3-femlpropil)piperazina (3-femlpropyl)piperazine
Clomifeno (Z, E) (E,Z)-2-(4-(2-cloro-1 ,2- difeni1eteni1)fenoxi) -N, N- dietiletanoamina Clomiphene (Z, E) (E,Z)-2-(4-(2-chloro-1,2-diphenyletheni1)phenoxy)-N,N- diethylethanolamine
MianserinaMianserin
(±)-2-metil-1,2,3,4, 10, 14b- hexahidrodibenzo[c, f]pirazino[ 1 ,2- a]azepina(±)-2-methyl-1,2,3,4, 10, 14b- hexahydrodibenzo[c, f]pyrazino[ 1 ,2- a]azepine
Clofíliumclophylium
4-(4-clorofenil)butil-dietil- heptilazanio
(2-butil- 1 -benzofuran-3-il)-[4-[2- (dietiiamino)etoxi]-3,5- diyodofeniljmetanone (2-butyl- 1 -benzofuran-3-yl)-[4-[2- (diethylamino)ethoxy]-3,5- diiodophenyljmethanone
PerhexilinaPerhexiline
2-(2,2-diciclohexiletil)piperidina 2-(2,2-dicyclohexylethyl)piperidine
TerfenadinaTerfenadine
1 -(4- tert-butilfenil) - 4-[4 - [hidroxi(difenil)metil]piperidin- 1 - il]butan-1-ol
Los compuestos de la invención tienen propiedades bactericidas que, por tante, también los hacen útiles para usos no terapéuticos tales como antimicrobianos para la desinfección de, por ejemplo, superficies. Ejemplos de aplicación de los compuestos de la invención incluyen, entre otros la limpieza o recubrimiento de material médico, veterinario, dental y quirúrgico, implantes en general tales como dentales, cardiacos o cocleares, asi como catéteres, tubos de ventilación, ventiladores, tubos endotraqueales y en general cualquier otra superficie en la que pueda crecer una biopelícula del patógeno. The compounds of the invention have bactericidal properties which therefore also make them useful for non-therapeutic uses such as antimicrobials for disinfection of eg surfaces. Examples of application of the compounds of the invention include, among others, the cleaning or coating of medical, veterinary, dental and surgical material, implants in general such as dental, cardiac or cochlear, as well as catheters, ventilation tubes, ventilators, endotracheal tubes and in general any other surface on which a biofilm of the pathogen can grow.
Así, otro aspecto de la invención se refiere al uso no terapéutico como antimicrobiano frente a patógenos del género Streptococcus, preferiblemente frente aStreptococcus pneumoniae (neumococo), Streptococcus oralis, Streptococcus mitis, Streptococcus, pyogenes, Streptococcus agalactiae, Streptococcus viridans o Streptococcus mutans, más preferiblemente frente a Streptococcus pneumoniae (neumococo), y aún más preferiblemente para su uso no terapéutico como antimicrobiano para la desinfección de la superficie de un material, de un compuesto seleccionado de: Thus, another aspect of the invention refers to the non-therapeutic use as an antimicrobial against pathogens of the genus Streptococcus, preferably against Streptococcus pneumoniae (pneumococcus), Streptococcus oralis, Streptococcus mitis, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus viridans or Streptococcus mutans, more preferably against Streptococcus pneumoniae (pneumococcus), and even more preferably for its non-therapeutic use as an antimicrobial for the disinfection of the surface of a material, of a compound selected from:
GBR 12909 (Vanoxerina) 1-(2-[bis(4-fluorofenil)metoxi)etil)-4- (3-fenilpropil)piperaz¡na GBR 12909 (Vanoxerin) 1-(2-[bis(4-fluorophenyl)methoxy)ethyl)-4- (3-phenylpropyl)piperazine
Clomifeno (Z, E) (E,Z)-2-(4-(2-cloro-1 ,2- difeniletenil)fenoxi) -N, N- dietiletanoamina Clomiphene (Z, E) (E,Z)-2-(4-(2-chloro-1,2-diphenylethenyl)phenoxy)-N,N- diethylethanolamine
MianserinaMianserin
(±)-2-metil-1 ,2,3,4, 10, 14b- hexahidrodibenzo[c, f]pirazino[ 1,2- ajazepina(±)-2-methyl-1 ,2,3,4, 10, 14b- hexahydrodibenzo[c, f]pyrazino[ 1,2- ajazepine
ClofiliumClophylium
4-(4-clorofenil)butil-dietil- heptilazanio 4-(4-chlorophenyl)butyl-diethyl- heptylazanium
AmiodaronaAmiodarone
(2-butil- 1 -benzofuran-3-il)-[4-[2-(2-butyl- 1 -benzofuran-3-yl)-[4-[2-
(dietilamino)etox¡]-3,5- diyodofenil]metanone
Los compuestos de la invención han sido seleccionados por cribado de compuestos aprobados por la Administración de Alimentos y Medicamentos (Food and Drug Administration’, FDA) de Estados Unidos y por la Agencia Europea del Medicamento (European Medicines Agency; EMA) incluidos en la librería química comercial Prestwick®. Estos compuestos han demostrado efecto bactericida in vitro frente a S. pneumoniae a concentraciones terapéuticas, con concentraciones mínimas inhibitorias (CMI) entre 26 y 50 pg mL-1 en cepa capsuiada (infectiva), ocasionando una disminución de viabilidad bacteriana de entre un 80 y un 99,9 %. En todos los casos la adición de estos compuestos induce una perturbación de la membrana bacteriana que da lugar a un incremento de su permeabilidad, por lo que es probable que actúen mediante mecanismos similares. The compounds of the invention have been selected by screening compounds approved by the Food and Drug Administration (FDA) of the United States and by the European Medicines Agency (EMA) included in the library Prestwick® commercial chemistry. These compounds have demonstrated in vitro bactericidal effect against S. pneumoniae at therapeutic concentrations, with minimum inhibitory concentrations (MIC) between 26 and 50 pg mL -1 in capsuiated strain (infective), causing a decrease in bacterial viability of between 80 and 99.9%. In all cases, the addition of these compounds induces a disturbance of the bacterial membrane that gives rise to an increase in its permeability, so it is likely that they act through similar mechanisms.
La presente invención también se refiere a una composición farmacéutica que comprende un compuesto de la invención (o una sal o solvato farmacéuticamente aceptable del mismo) y uno o más excipientes farmacéuticamente aceptables. Los excipientes deben ser “aceptables” en el sentido de ser compatibles con los demás ingredientes de la composición y de no ser perjudiciales para quién tome dicha composición. The present invention also relates to a pharmaceutical composition comprising a compound of the invention (or a pharmaceutically acceptable salt or solvate thereof) 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 being harmful to the user of said composition.
Los compuestos de la presente invención pueden ser administrados en forma de cualquier formulación farmacéutica, la naturaleza de la cual, como es bien sabido, dependerá de la naturaleza del principio activo y de su via de administración. En principio se puede utilizar cualquier vía de administración, por ejemplo, oral, parenteral, nasal, ocular, rectal, y tópica. The compounds of the present invention can be administered in the form of any pharmaceutical formulation, the nature of which, as is well known, will depend on the nature of the active ingredient and its route of administration. In principle any route of administration can be used, eg oral, parenteral, nasal, ocular, rectal, and topical.
Las composiciones sólidas para la administración oral incluyen comprimidos, granulados y cápsulas. En cualquier caso el método de fabricación está basado en una mezcla simple, granulación seca o granulación húmeda del principio activo con excipientes. Estos excipientes pueden ser, por ejemplo, diluyentes tales como lactosa, celulosa microcristalina, manitol o hidrogenofosfato calcico; agentes aglutinantes como por ejemplo almidón, gelatina o polivinilpirrolidona; disgregantes como carboximetilalmidón sódico o croscarmelosa sódica; y agentes lubricantes como por ejemplo estearato magnésico, ácido esteárico o talco. Los comprimidos pueden ser además recubiertos con excipientes adecuados y mediante técnicas conocidas con el objeto de retrasar su disgregación y absorción en el tracto gastrointestinal y así conseguir una acción sostenida durante un mayor período de tiempo, o simplemente para mejorar sus propiedades organolépticas o su estabilidad. El principio activo puede también ser incorporado por recubrimiento sobre pellets inertes mediante el uso de polímeros filmógenos naturales o sintéticos. También es posible la realización de cápsulas de gelatina blanda, en las que el principio activo se mezcla con agua o con medio oleoso, por ejemplo aceite de coco, parafina líquida o aceite de oliva. Solid compositions for oral administration include tablets, granules, and capsules. In any case, the manufacturing method is based on a simple mixture, dry granulation or wet granulation of the active ingredient with excipients. These excipients can be, for example, diluents such as lactose, microcrystalline cellulose, mannitol or calcium hydrogen phosphate; binding agents such as starch, gelatin or polyvinylpyrrolidone; disintegrants such as sodium carboxymethyl starch or croscarmellose sodium; and lubricating agents such as magnesium stearate, stearic acid or talc. The tablets can also be coated with suitable excipients and using known techniques in order to delay their disintegration and absorption in the gastrointestinal tract and thus achieve a sustained action for a longer period of time, or simply to improve their organoleptic properties or their stability. The active principle can also be incorporated by coating on inert pellets through the use of natural or synthetic film-forming polymers. It is also possible to make soft gelatin capsules, in which the active ingredient is mixed with water or with oily medium, for example coconut oil, liquid paraffin or olive oil.
Se pueden obtener polvos y granulados para la preparación de suspensiones orales mediante la adición de agua, mezclando el principio activo con agentes dispersantes o humectantes; suspensantes y conservantes. También pueden añadirse otros excipientes, por ejemplo edulcorantes, aromatizantes y colorantes. Powders and granules for the preparation of oral suspensions can be obtained by adding water, mixing the active ingredient with dispersing or wetting agents; suspensive and preservatives. Other excipients can also be added, for example sweeteners, flavors and colorants.
Como formas líquidas para la administración oral se pueden incluir emulsiones, soluciones, suspensiones, jarabes y elixires que contienen diluyentes inertes comúnmente utilizados, tales como agua destilada, etanol, sorbitol, glicerol, polietilenglicoles (macrogoles) y propilénglicol. Dichas composiciones pueden también contener coadyuvantes como agentes humectantes, suspensantes, edulcorantes, aromatizantes, conservantes y reguladores de pH. Liquid forms for oral administration may include emulsions, solutions, suspensions, syrups, and elixirs containing commonly used inert diluents, such as distilled water, ethanol, sorbitol, glycerol, polyethylene glycols (macrogols), and propylene glycol. Said compositions may also contain adjuvants such as wetting agents, suspending agents, sweeteners, flavoring agents, preservatives and pH regulators.
Preparaciones inyectables, de acuerdo con la presente invención, para la administración parenteral, comprenden soluciones, suspensiones o emulsiones estériles, en un solvente acuoso o no acuoso como propilénglicol, polietilénglicol o aceites vegetales. Estas composiciones pueden también contener coadyuvantes, como humectantes, emulsionantes, dispersantes y conservantes. Podrían ser esterilizadas por cualquiera de los métodos conocidos o preparadas como composiciones sólidas estériles que serán disueltas en agua o cualquier otro medio inyectable estéril inmediatamente antes de uso. También es posible partir de materias primas estériles y mantenerlas en estas condiciones durante todo el proceso de fabricación. Injectable preparations, according to the present invention, for parenteral administration, comprise sterile solutions, suspensions or emulsions, in an aqueous or non-aqueous solvent such as propylene glycol, polyethylene glycol or vegetable oils. These compositions may also contain adjuvants, such as wetting agents, emulsifiers, dispersants, and preservatives. They may be sterilized by any of the known methods or prepared as sterile solid compositions to be dissolved in water or any other sterile injectable medium immediately before use. It is also possible to start from sterile raw materials and keep them in these conditions throughout the manufacturing process.
Para la administración rectal, el principio activo puede ser formulado preferentemente como supositorio en una base oleosa, como por ejemplo aceites vegetales o glicéridos semisintéticos sólidos, o en una base hidrófila como polietilénglicoles (macrogol). For rectal administration, the active ingredient may preferably be formulated as a suppository in an oily base, such as vegetable oils or solid semi-synthetic glycerides, or in a hydrophilic base such as polyethylene glycols (macrogol).
Los compuestos de la invención pueden también ser formulados para su aplicación tópica para el tratamiento de patologías en zonas o órganos accesibles por esta vía, como ojos, piel y tracto intestinal. Formulaciones incluyen cremas, lociones, geles, polvos, soluciones y parches en las que el compuesto se encuentra dispersado o disuelto en excipientes adecuados. The compounds of the invention can also be formulated for topical application for the treatment of pathologies in areas or organs accessible by this route, such as the eyes, skin and intestinal tract. Formulations include creams, lotions, gels, powders, solutions, and patches in which the compound is dispersed or dissolved in suitable carriers.
Para la administración nasal o por inhalación, el compuesto puede presentarse formulado en forma de aerosol de dónde es convenientemente liberado con el empleo de propelentes adecuados. For nasal or inhalation administration, the compound can be formulated in aerosol form from which it is conveniently released with the use of suitable propellants.
La dosificación y ia frecuencia de las dosis variarán en función de la naturaleza y gravedad de la enfermedad a tratar, la edad, la condición general y el peso del paciente, así como también del compuesto particular administrado y la vía de administración, entre otros factores. The dosage and frequency of doses will vary depending on the nature and severity of the disease to be treated, the age, general condition and weight of the patient, as well as the particular compound administered and the route of administration, among other factors. .
Asimismo, los compuestos de la invención se pueden administrar solos o en combinación. También se ha de prever, para su administración, la utilización de soportes transportadores de tales compuestos en una o en múltiples copias, por ejemplo mediante el uso de nanopartículas, puesto que se ha demostrado que la dosis antimicrobiana de un determinado compuesto puede reducirse en varios órdenes de magnitud tras su disposición múltiple en la superficie de tales partículas, como por ejemplo se ha demostrado con partículas dendriméricas conteniendo varias copias de colina frente a Streptococcus pneumoniae (Hernández- Rocamora V, Maestro B, Waal B, Morales M, García P, Meijer EW, Merkx M, Sanz JM. (2009). Multivalent choline dendrimers as potent inhibitors of pneumococcal cell-wall hydrolysis. Angew. Chem. Int. Ed Engl. 48(5): 948-951. https://doi.org/10.1002/anie.200803664: y de Gracia Retamosa M, Diez-Martinez R, Maestro B, García-Fernández E, de Waal B, Meijer EW, Sanz JM. (2015). Aromatic esters of bicyclic amines as antimicrobials against Streptococcus pneumoniae. Angew. Chem. Int. Ed Engl. 54(46): 13673-13677. https://doi.org/10.1002/anie.201505700). Likewise, the compounds of the invention can be administered alone or in combination. The use of carrier media for such compounds in one or multiple copies, for example through the use of nanoparticles, should also be envisaged for their administration, since it has been shown that the antimicrobial dose of a given compound can be reduced by several orders of magnitude after their multiple arrangement on the surface of such particles, as has been shown, for example, with dendrimeric particles containing several copies of choline against Streptococcus pneumoniae (Hernández-Rocamora V, Maestro B, Waal B, Morales M, García P, Meijer EW, Merkx M, Sanz JM (2009). Multivalent choline dendrimers as potent inhibitors of pneumococcal cell-wall hydrolysis. Angew. Chem. Int. Ed Engl. 48(5): 948-951. https://doi. org/10.1002/anie.200803664: and de Gracia Retamosa M, Diez-Martinez R, Maestro B, García-Fernández E, de Waal B, Meijer EW, Sanz JM (2015).Aromatic esters of bicyclic amines as antimicrobials against Streptococcus pneumoniae.Angew.Ch em. Int. Ed Engl. 54(46): 13673-13677. https://doi.org/10.1002/anie.201505700).
A Io largo de la descripción y las reivindicaciones la palabra "comprende" y sus variantes no pretenden excluir otras características técnicas, aditivos, componentes o pasos. Para los expertos en la materia, otros objetos, ventajas y características de la invención se desprenderán en parte de la descripción y en parte de la práctica de la invención. Los siguientes ejemplos y figuras se proporcionan a modo de ilustración, y no se pretende que sean limitativos de la presente invención. Throughout the description and claims, the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. Other objects, advantages and features of the invention will be apparent to those skilled in the art in part from the description and in part 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.
BREVE DESCRIPCIÓN DE LAS FIGURAS BRIEF DESCRIPTION OF THE FIGURES
Fig. 1 Muestra las curvas de crecimiento de S. pneumoniae R6CIB17 en presencia de 25 pM del compuesto a testar, excepto maieato de perhexilina (50 pM) y clorhidrato de mianserina (100 pM). Fig. 2 Muestra la variación de la permeabilidad de las células neumocócicas S. pneumoniae R6CIB17 incubadas con cada compuesto a una concentración de 25 pM. Control positivo: Tritón X100 al 1 %. Fig. 1 shows the growth curves of S. pneumoniae R6CIB17 in the presence of 25 pM of the compound to be tested, except perhexiline maieate (50 pM) and mianserin hydrochloride (100 pM). Fig. 2 shows the variation in the permeability of pneumococcal S. pneumoniae R6CIB17 cells incubated with each compound at a concentration of 25 pM. Positive control: Triton X100 at 1%.
EJEMPLOS EXAMPLES
A continuación, se ilustrará la invención mediante unos ensayos realizados por los inventores, que ponen de manifiesto la efectividad del producto de la invención. Next, the invention will be illustrated by tests carried out by the inventors, which show the effectiveness of the product of the invention.
Procedimiento: Se ha cribado una quimioteca de 1280 compuestos autorizados tanto por la U.S Food and Drug Administration (FDA) como por la European Medicines Agency (EMA) y comercializada por la empresa Prestwick Chemical (http://www. prestwickchemical.com/libraries-screening-lib-pcl.html) denominada Prestwick® (Prestwick Chemical Library®). Procedure: A chemical library of 1,280 compounds authorized by both the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA) and marketed by the company Prestwick Chemical (http://www.prestwickchemical.com/libraries) has been screened. -screening-lib-pcl.html) called Prestwick® (Prestwick Chemical Library®).
Los compuestos se obtuvieron en una concentración de 10 mM en dimetil sulfóxido (DMSO). Compounds were obtained at a concentration of 10 mM in dimethyl sulfoxide (DMSO).
Los ensayos se realizaron en primera instancia de forma ciega sobre la cepa no capsulada R6CIB17 de neumococo ((GenBank accession number: CP038808), variante no floculante de la cepa usual de laboratorio R6), y el cribado se realizó de manera secuencial (100, 50 y 25 pM) en cultivos planctónicos (medio de cultivo líquido (Roig- Molina E, Sánchez-Angulo M, Seele, J, García-Asencio F, Nau R, Sanz JM, Maestro B. (2020). Searching for antipneumococcal targets: choline-binding modules as phagocytosis enhancers. ACS Inf. Dis. 6(5): 954-974. https://dx.doi.org/10.1021/acsinfecdis.9b00344), añadiendo el compuesto a una D.O.550nm de 0,1 . The assays were first performed blindly on the non-capsulated pneumococcus strain R6CIB17 ((GenBank accession number: CP038808), a non-flocculant variant of the usual laboratory strain R6), and the screening was performed sequentially (100, 50 and 25 pM) in planktonic cultures (liquid culture medium (Roig-Molina E, Sánchez-Angulo M, Seele, J, García-Asencio F, Nau R, Sanz JM, Maestro B. (2020). Searching for antipneumococcal targets : choline-binding modules as phagocytosis enhancers. ACS Inf. Dis. 6(5): 954-974. https://dx.doi.org/10.1021/acsinfecdis.9b00344), adding the compound to an O.D.550nm of 0, 1 .
Ejemplo 1.- Efecto sobre la curva de crecimiento Example 1.- Effect on the growth curve
Tras el cribado a 25 pM, se seleccionaron los compuestos más eficaces que afectaban a la curva de crecimiento con respecto al control, y que no han sido descritos para su uso frente a S. pneumoniae. En la Fig. 1 se muestran las curvas de crecimiento, mientras que en la Tabla 1 se muestra el descenso en densidad óptica en el crecimiento de los compuestos definitivamente seleccionados, en % con respecto al control sin compuesto. After screening at 25 pM, the most effective compounds that affected the growth curve with respect to the control, and that have not been described for use against S. pneumoniae, were selected. In Fig. 1 the growth curves are shown, while in Table 1 the decrease in optical density in the growth of the definitively selected compounds is shown, in % with respect to the control without compound.
En segundo lugar, los más efectivos se testaron de nuevo sobre curva de crecimiento a concentración de 0,1 , 1 y 5 pM, pero ninguno de ellos tuvo efecto sobre la misma. Second, the most effective were tested again on a growth curve at concentration of 0.1, 1 and 5 pM, but none of them had an effect on it.
Ejemplo 2.- Efecto sobre la viabilidad Example 2.- Effect on viability
La actividad bactericida (ensayos de viabilidad en placa) de los compuestos seleccionados se llevó a cabo a 25 pM, excepto clorhidrato de mianserina (100 pM) y maléate de perhexilina (50 pM). De este ensayo se seleccionaron aquellos que disminuyen la viabilidad en más de un 90 % (recuento de viables < 3,6 x 107 unidades formadoras de colonia por mililitro), a excepción del clorhidrato de mianserina. Bactericidal activity (plate viability assays) of selected compounds was carried out at 25 pM, except mianserin hydrochloride (100 pM) and perhexiline maleate (50 pM). Those that decrease viability by more than 90% (viable count < 3.6 x 10 7 colony-forming units per milliliter) were selected from this assay, with the exception of mianserin hydrochloride.
En la Tabla 1 se muestra la viabilidad celular de R6CIB17 tras 140 min en presencia del compuesto. Table 1 shows the cell viability of R6CIB17 after 140 min in the presence of the compound.
Ejemplo 3.- Efecto sobre la permeabilidad Example 3.- Effect on permeability
Para investigar el posible mecanismo bactericida de estos compuestos, y dado que todos ellos tienen una estructura química general comúnmente implicada en mecanismos de perturbación de la membrana celular (aminas terciarias o cuaternarias unidas a una parte hidrofóbica, casi siempre con grupos aromáticos) (Alkhalifa S, Jennings MC, Granata D, Klein M, Wuest WM, Minbiole KPC, Carnevale V. (2020). Analysis of the destabilization of bacterial membranes by quaternary ammonium compounds: a combined experimental and computational study. Chem.Bio.Chem. 21(10):1510-1516. https://doi.org/10.1002/cbic.201900698), se realizaron ensayos de permeabilidad de la membrana bacteriana (Roig-Molina E, Domenech M, Retamosa MG, Nácher-Vázquez M, Rivas L, Maestro B, García P, García E, Sanz JM. (2019). Widening the antimicrobial spectrum of esters of bicyclic amines: In vitro effect on grampositive Streptococcus pneumoniae and gram-negative non-typeable Haemophilus influenzae biofilms. BBA-General Subjects 1863(1). 96-104. https://doi.Org/10.1016/j.bbagen.2O18.10.001). To investigate the possible bactericidal mechanism of these compounds, and given that they all have a general chemical structure commonly involved in cell membrane disruption mechanisms (tertiary or quaternary amines attached to a hydrophobic part, almost always with aromatic groups) (Alkhalifa S , Jennings MC, Granata D, Klein M, Wuest WM, Minbiole KPC, Carnevale V. (2020).Analysis of the destabilization of bacterial membranes by quaternary ammonium compounds: a combined experimental and computational study.Chem.Bio.Chem.21( 10):1510-1516.https://doi.org/10.1002/cbic.201900698), bacterial membrane permeability assays were performed (Roig-Molina E, Domenech M, Retamosa MG, Nácher-Vázquez M, Rivas L , Maestro B, García P, García E, Sanz JM.(2019).Widening the antimicrobial spectrum of esters of bicyclic amines: In vitro effect on gram-positive Streptococcus pneumoniae and gram-negative non-typeable Haemophilus influenzae biofilms.BBA-General Subjects 1863(1). 96-104. https://doi.org/10.1016/j.bbagen.2O18.10.001).
El estudio de la permeabilización de la membrana celular se realizó sobre S. pneumoniae R6 con los compuestos mostrados en la Tabla 1 , y se llevó a cabo utilizando como sonda Sytox Green. Un aumento en la permeabilidad de la membrana conlleva un incremento en la emisión de fluorescencia, causado por la entrada del colorante a través de la membrana y su unión subsiguiente a los ácidos nucleicos (Roig-Molina E, Domenech M, Retamosa MG, Nácher-Vázquez M, Rivas L, Maestro B, García P, García E, Sanz JM. (2019). Widening the antimicrobial spectrum of esters of bicyclic amines: In vitro effect on gram-positive Streptococcus pneumoniae and gram-negative non- typeable Haemophilus influenzae biofilms. BBA-Generai Subjects 1863(1). 96-104. https://doi.Org/10.1016/j.bbagen.2018.10.001). The study of cell membrane permeabilization was performed on S. pneumoniae R6 with the compounds shown in Table 1, and was carried out using Sytox Green as probe. An increase in membrane permeability leads to an increase in fluorescence emission, caused by the entry of the dye through the membrane and its subsequent binding to nucleic acids (Roig-Molina E, Domenech M, Retamosa MG, Nácher- Vazquez M, Rivas L, Maestro B, Garcia P, Garcia E, Sanz JM. (2019). Widening the antimicrobial spectrum of esters of bicyclic amines: In vitro effect on gram-positive Streptococcus pneumoniae and gram-negative non-typeable Haemophilus influenzae biofilms. BBA-Generai Subjects 1863(1). 96-104. https://doi.org/10.1016/j.bbagen.2018.10.001).
La Fí g. 2 muestra que todos los compuestos, en mayor o menor grado, permeabilizaron la membrana, lo que parece indicar que todos ejercen su actividad antimicrobiana mediante un mecanismo molecular similar. En la Tabla 1 se muestran los datos de la permeabilidad de la membrana a los 95 min, respecto al control del 100 % de permeabilidad en presencia de tritón X100 al 1 %. En cualquier caso, es necesario recalcar que muchos otros compuestos de la librería que poseen similares características químicas (aminas unidas a una región hidrofóbica), no presentan actividad antimicrobiana alguna, por lo que luego los compuestos objeto de esta patente presentan un alto grado de especificidad adicional. La Fí g. 2 shows that all compounds, to a greater or lesser degree, permeabilized the membrane, which seems to indicate that they all exert their antimicrobial activity through a similar molecular mechanism. Table 1 shows the data of the permeability of the membrane at 95 min, with respect to the control of 100% permeability in the presence of triton X100 at 1%. In any case, it is necessary to emphasize that many other compounds in the library that have similar chemical characteristics (amines bound to a hydrophobic region), do not have any antimicrobial activity, so the compounds that are the subject of this patent have a high degree of specificity. additional.
Ejemplo 4.- Concentración mínima inhibitoria (CMI) Example 4.- Minimum inhibitory concentration (MIC)
Finalmente, se determinó la concentración mínima inhibitoria (CMI) de los compuestos utilizando los criterios del Instituto de Estándares del Laboratorio Clínico (CLSI) (Clinical and Laboratory Standards Institute (CLSI). (2009). Performance standards for antimicrobial susceptibility testing; 19th informational supplement. CLSI documents M100-S19. CLSI, Wayne, PA.) mediante el sistema de microdilución, siguiendo las indicaciones descritas en la Norma Internacional ISO 20776 y empleando las cepas de neumococo R6 (no capsulada) y D39 (cepa capsulada, y por tanto infectiva). La CMI se calculó como la concentración más baja del agente a la cual se inhibe el crecimiento de forma visible. En paralelo se realizaron controles de crecimiento para cada cepa ensayada, y un control positivo (cepa tipo ATC49619+ ampicilína) y negativo (de esterilidad: medio de cultivo + mismo volumen del fármaco añadido al experimento) (Tabla 1).
Tabla 1 : (a): Actividad bactericida de compuestos seleccionados frente a S. pneumoniae R6CIB17. Los cultivos se crecieron en medio C+Y hasta una O.D.ssonm de 0,1 , momento en el que se adicionó el compuesto a estudiar a la concentración de 25 pM excepto * (50 pM) y ** (100 pM), tomando la muestra para el estudio de viables a los 140 min tras la adición. Se muestra el control de recuento de unidades formadoras de colonia (UFC) de S. pneumoniae y del mismo en presencia de DMSO a la concentración final en la que se encuentran los compuestos en el ensayo (0,5 %). Table 1: (a): Bactericidal activity of selected compounds against S. pneumoniae R6CIB17. The cultures were grown in C+Y medium until an O.D.ssonm of 0.1, at which point the compound to be studied was added at a concentration of 25 pM except * (50 pM) and ** (100 pM), taking the sample for viable study at 140 min after addition. The count control of colony forming units (CFU) of S. pneumoniae and the same in the presence of DMSO at the final concentration in which the compounds in the test are found (0.5%) is shown.
(b): Incremento de la entrada intracelular del colorante SyTOX Green desde el exterior, tras 90 min de incubación. Los cultivos de R6CIB17 se crecieron en medio C+Y hasta una O.D.550nm de 0,5-0, 6, posteriormente se centrifugaron y resuspendieron en tampón fosfato sódico 5 mM, pH 7,0 conteniendo sorbitol 280 nM y SyTOX Green 1 pM y se determinó la línea base. A continuación, se añadió el compuesto, a una concentración de 25 pM, y se midió el incremento de fluorescencia a 520 nm tras excitación a 485 nm en función del tiempo. (b): Increased intracellular influx of SyTOX Green dye from the outside, after 90 min of incubation. The R6CIB17 cultures were grown in C+Y medium to an O.D.550nm of 0.5-0.6, subsequently centrifuged and resuspended in 5 mM sodium phosphate buffer, pH 7.0 containing 280 nM sorbitol and 1 pM SyTOX Green and baseline was determined. The compound was then added, at a concentration of 25 pM, and the increase in fluorescence at 520 nm after excitation at 485 nm was measured as a function of time.
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| GROSSMAN, S. ET AL.: "Tackling antimicrobial resistance through two novel methods: repurposing vanoxerine as a novel anti-tubercular drug; and silencing virulence in Pseudomonas aeruginosa through inhibition of PqsR", MRES THESIS, 4 November 2019 (2019-11-04), University of Nottingham, Retrieved from the Internet <URL:http://eprints.nottingham.ac.uk/55397/> * |
| KANVATIRTH PANCHALI, JEEVES ROSE E., BACON JOANNA, BESRA GURDYAL S., ALDERWICK LUKE J.: "Utilisation of the Prestwick Chemical Library to identify drugs that inhibit the growth of mycobacteria", PLOS ONE, vol. 14, no. 3, 12 March 2019 (2019-03-12), pages e0213713, XP055960774, DOI: 10.1371/journal.pone.0213713 * |
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