Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C49/00—Ketones; Ketenes; Dimeric ketenes; Ketonic chelates
  • C07C49/587—Unsaturated compounds containing a keto groups being part of a ring
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
  • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
  • A61L27/54—Biologically active materials, e.g. therapeutic substances
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L29/00—Materials for catheters, medical tubing, cannulae, or endoscopes or for coating catheters
  • A61L29/14—Materials characterised by their function or physical properties, e.g. lubricating compositions
  • A61L29/16—Biologically active materials, e.g. therapeutic substances
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
  • A61L31/14—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
  • A61L31/16—Biologically active materials, e.g. therapeutic substances
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
• • 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
• • 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
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C49/00—Ketones; Ketenes; Dimeric ketenes; Ketonic chelates
  • C07C49/587—Unsaturated compounds containing a keto groups being part of a ring
  • C07C49/607—Unsaturated compounds containing a keto groups being part of a ring of a seven-to twelve-membered ring
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/20—Bacteria; Culture media therefor
  • C12N1/205—Bacterial isolates
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
  • C12P17/02—Oxygen as only ring hetero atoms
  • C12P17/08—Oxygen as only ring hetero atoms containing a hetero ring of at least seven ring members, e.g. zearalenone, macrolide aglycons
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
  • A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
  • A61L2300/404—Biocides, antimicrobial agents, antiseptic agents
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
  • C12R2001/07—Bacillus

Definitions

• the present application relates to a new class of terpene-derived compounds, having an antibiotic activity, of formula (I) or (II)
• A is selected from NR 1 , O, S, CR 2 R 3 or R- (CH 2 ) n -R 'wherein R and R' are, independently of one another, NH, O, S or CH 2 and R 1, R 2 and R 3 are substituents, and n is greater than or equal to 1, in particular 2.
• Staphylococci which are among the most resistant bacteria (eg methicillin-resistant Staphylococcus aureus), as well as
• Penicillin-resistant Streptococcus pneumoniae vancomycin-resistant enterococci, multidrug-resistant salmonella, and multidrug-resistant tuberculosis.
• imbalances in the intestinal flora may occur following antibiotic treatment (antibiotic therapy) intended for to cure an infection. These imbalances themselves cause digestive infections (diarrhea), including digestive infections related to Clost ⁇ dium difficile (ICD, or CDAD for Clostridium difficile-associated disease).
• ICD Clost ⁇ dium difficile
• Clostridium difficile is a gram-positive, anaerobic bacillus that is the leading cause of nosocomial infectious diarrhea in adults
• Antimicrobial impregnated catheters are the medical devices for which more data are available. There are essentially two combinations of molecules: chlorhexidine and silver sulfadiazine or minocycline and rifampicin. The in vivo efficacy of the former is controversial and would be limited in time ( ⁇ 10 days).
• rifampicin is one of the few antibiotics active on slow-growing bacteria, especially on coagulase-negative Staphylococci which are the most common cause of infection of catheters, stents and vein grafts. In large multicentre studies, it is estimated that they reduce the risk of infection by 80%, and that their use would be beneficial.
• the present application relates to a new class of compounds having antibiotic activity, more particularly broad-spectrum gram-positive antibacterial activity, on bacteria such as Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Bacillus anthracis and Clostdium difficile.
• the compounds of this class have a low molecular weight, are not toxic in vitro and in vivo and do not cross-react with known resistances.
• this class of antibiotic compounds there is preferentially margaucine, which can be isolated from a culture supernatant of a bacterial strain.
• the present invention relates to a compound of formula (I) or
• A is selected from NR 1, O, S, CR 2 R 3 or R- (CH 2 ) n -R 'wherein R and R' are, independently of one another, NH, O, S or CH 2 , and R 1, R 2 and R 3 are substituents, and n is greater than or equal to 1, in particular 2.
• constituent A atoms such as R 1, R 2 and R 3 ;
• the carbon in the 1-position is a CH 2
• the carbon in the 3-position is a CH
• the carbon in the terminal position is a CH 3 .
• A is selected from NH, O, S, CH 2 or R- (CH 2 ) 2 -R 'wherein
• R and R ' are, independently of one another, NH, O, S or CH 2 .
• the invention does not, as such, aim at the mixture chemically synthesized according to the method described by Schank et al. (Chemistry of free cyclic vicinal tricarbonyl compounds ('1,2,3-triones 1 ).) 2. Redox reactions of 1, 2,3-triones with ene-1,2-diols ('reductones'), 2-alkoxy -en-1-ols, ene-1, 2-diamines, and related species, Helvetica Chimica Acta (2002), 85 (5), 1295-1326), i.e.
• the invention relates to the compounds produced by a bacterium and mixtures thereof, according to the process described hereinafter by way of example, and concerns the use of these compounds and mixtures irrespective of their production method (in particular by synthesis or by a bacterium), in particular margaucine, in compositions or applications of these compounds or mixtures described in the present application.
• the present invention relates very particularly to a compound of formula (I) or (11):
• A is selected from NR-1, O, S, CR 2 R 3 or R- (CH 2 ) n -R 'wherein R and R' are, independently of one another, N, O, S or C, and R 1, R 2 and R 3 are substituents and n is greater than or equal to 1, in particular equal to 2.
• A is chosen from NH, O, S, CH 2 or R- (CH 2 ) 2 -R 'wherein R and R' are independently of each other, NH, O, S or CH 2.
• compound of the invention is meant both the compound in its formula (I) or in its formula (II), that is to say taken individually regardless of its method of preparation.
• the present invention relates to the compound of formula (I), as described in the context of the present application and in particular the compound of formula (I) produced by a bacterium, and its uses.
• the invention also relates to mixtures of these two isomeric compounds, irrespective of the proportion of compounds (I) and (II), with the exception of the mixture obtained by chemical synthesis according to the method of Schank et al.
• the mixtures can comprise the forms (I) and (II) even if the antibiotic activity of each of these two forms is not equivalent.
• the mixture comprises the forms (I) and (II), wherein optionally only one form has an antibiotic activity.
• the form (I) has an antibiotic activity.
• the present invention also relates to a process for preparing a compound of formula (I) or (II), and more particularly a process for preparing margaucine, comprising (a) culturing a bacterial strain, and b) recovering, in particular purifying, the compound of formula (I) or (II) from the culture supernatant.
• the bacterial strain used in the context of a process according to the invention is the strain JPL84 or JPL86, and the compound produced is the compound of formula
• culturing is meant the maintenance of the bacterial strain, particularly the strain JPL84, or any derivative strain as defined in the present application, under conditions of culture allowing its survival and its multiplication.
• the cultivation is also accompanied by the production of the antibiotic of formula (I) or (II) or of their mixture or promotes this production.
• the term “cultivation” will be used interchangeably and according to the same definition as the term “fermentation”.
• any liquid nutrient medium may be suitable for culturing the bacterial strain, strain JPL84, or derived strains, insofar as this medium contains a source of carbon, nitrogen and inorganic salts.
• this medium contains a source of carbon, nitrogen and inorganic salts.
• preference will be given to CYE (Casitone Yeast Extract) medium, an agar medium based on yeast extract, the composition of which is, for example, the following: peptone at 10 g / l, yeast extract at 1 g / l and CaCl 2 at 1 g / l.
• the bacterial strain is cultured at a temperature between 0 and 45 ° C, preferably between 20 and 37 0 C, and even more preferably at 28 0 C, under agitation and aeration (aerobic culture).
• the desired duration of the culture depends on the time at which the production of the desired antibiotic is optimal or maximum, and this duration is calculated with respect to the cultivation of the strain.
• the culture time (period between culturing and stopping this culture) is about 15 to 24 hours, preferably between 18 and 20 hours.
• purification is meant any technique for isolating the desired antibiotic from other constituents of the culture supernatant. For example, and without limitation, mention may be made of solvent extraction, precipitation, reverse phase chromatography (HPLC), ion exchange chromatography, or the combination of two or more of these techniques. .
• the method of the invention may also comprise an optional step between step (a) of culturing and step (b) of purification, consisting of the separation of the culture supernatant from the cells and other cellular debris, for example by centrifugation.
• the purified antibiotic may subsequently be converted to salts (salt forms), including pharmaceutically acceptable non-toxic salts, by standard reaction with organic or inorganic acids. These salts will be obtained, particularly when A is N.
• the purified antibiotic, or salts thereof may be obtained in anhydrous form, by lyophilization.
• the present invention also relates to a compound of formula (I) or (II) or a mixture thereof, as described above obtained by a method using a culture of bacteria, as described in this application.
• a mixture of isomers of formulas (I) and (II) as described above can also be obtained by chemical synthesis, as for example that disclosed in the publication of Schank et al. (Chemistry of free cyclic vicinal tricarbonyl compounds ('1,2,3-triones'). Part 2. Redox reactions of 1,2,3-triones with ene-1,2-diols ('reductones'), 2-alkoxy -en-1-ol, ene-1, 2-diamines, and related species, Helvetica Chimica Acta (2002), 85 (5), 1295-1326). This synthesis results in the production of a mixture from which it is possible to isolate, if appropriate, one of the compounds of formula (I) or (II).
• the compound of the invention When the compound of the invention is obtained by a method of culturing bacteria as described above, it retains a trace of its bacterial production and can be distinguished from the same compound produced by chemical synthesis, because of the difference observed in their respective ratios Ci 2 / C 13 .
• the compound of formula (I) or the compound of formula (II) produced chemically has a different C 12 / C- 13 ratio.
• the chemically produced margaucine has a C 12/3
• composition comprising at least one compound of formula (I) or (II) as described above, with the exception of the mixture of compounds of formulas (I) and (II), when it is obtained by chemical synthesis according to the method published by Schank et al.
• a composition according to the invention comprises at least one compound of formula (I) or (II) obtained by a method of production in a bacterium described above, particularly a composition comprising at least the compound of formula (I) obtained from the strain JPL84.
• the invention relates to a composition which comprises at least one carrier and / or a carrier, and a compound of formula (I)
• A is selected from NR-1, O, S, CR 2 R 3 or R- (Ch 1) n -R 'wherein R and R' are, independently of one another, NH, O, S or CH 2 , and
• the composition comprises at least one carrier and at least one compound of formula (I), in particular margaucine (compound of formula III).
• the compound is obtained by chemical synthesis or by a process comprising a culture of bacteria.
• vehicle is meant any substance that allows the formulation of the antibiotic within a composition.
• the vehicle is a physiologically acceptable substance or combination of substances, i.e., suitable for use of the composition in contact with a living being (e.g., a non-mammalian). -human, and preferably a human being), and is therefore preferentially nontoxic.
• compositions of the invention are pharmaceutical compositions, that is to say they are formulated to be administered or applied to a living being for therapeutic or prophylactic purposes.
• These pharmaceutical compositions contain pharmaceutically acceptable vehicles for systemic or local administration, particularly by injection, for application in contact with a living being, by ingestion, or use in a solid, gelatinous, liquid or aerosol form.
• the compositions of the invention are applied to materials that will be in contact with a living being as defined above.
• the composition comprises a pharmaceutically acceptable carrier and margaucine.
• the invention also relates to a composition which comprises at least one compound of formula (I) or (II) as defined in the present application, if appropriate associated with a carrier and / or a vehicle as defined in the present application, and which further comprises at least a second molecule, different, active against microorganisms.
• active molecule is meant a chemical or biological compound, especially a low molecular weight compound, for example a peptide, which has the capacity to destroy, neutralize the activity or prevent the proliferation of microorganisms.
• This active molecule may be, for example, an antibiotic or a bactericide.
• the active molecule is preferably chosen from a compound active against gram-negative bacteria and / or a fungicidal compound and / or the compounds listed in Table 3 below or the following compounds: silver salts, minocycline, chlorinexidine, sulfadiazine, rifampicin ....
• a composition of the invention comprises at least two compounds of formula (I) or (II) as defined above, but which differ from each other by their structure, namely their composition in atoms or by the position of their different substituents.
• the composition comprises at least two compounds of formula (I) which differ in the nature of element A, by the substituents on element A, and / or by the substituents at positions 1, 3 and / or 5.
• Another composition according to the invention comprises at least two compounds of formula (II) which differ in the nature of the element A, by substituents on element A, and / or substituents at positions 1, 3 and / or 5.
• the invention also relates to a composition which comprises at least one compound of formula (I) and a compound of formula (II), in which A is identical in the two compounds and is chosen from NH, S,
• R and R' are, independently of one another, NH, O, S or CH 2 , that is to say comprises two isomers of same compound as to the position of the double bonds.
• compositions according to the invention further comprise a vehicle or a transporter.
• the present invention also relates to the use of a compound of formula (I) or (II) taken individually or as a mixture or of a composition as defined above, as a medicament, and especially as an antibiotic (which destroys or prevents the proliferation of micro-organisms) and / or bactericidal (which destroys or prevents the proliferation of bacteria).
• the compound of formula (I) or (II) taken individually or as a mixture or the composition of the invention has a bactericidal activity against gram positive bacteria (gram stain) or bacteria having a wall a thin layer composed of several layers, one of which is lipidic, that is to say that the compound destroys the bacteria or slows down their growth.
• gram positive bacteria gram stain
• bacteria having a wall a thin layer composed of several layers, one of which is lipidic that is to say that the compound destroys the bacteria or slows down their growth.
• the compound of formula (I) or (II) taken individually or as a mixture or the composition of the invention is used to treat infections caused by bacteria of the genus Clostridium. More particularly, the compound of formula (I) or (II) taken individually or as a mixture or the composition of the invention is used to treat infections caused by Clostridium difficile, and preferably Clostridium difficile-related digestive infections (CDI). such as simple diarrhea, post-antibiotic, or colitis Pseudomembranous (CPM).
• CDI Clostridium difficile-related digestive infections
• CPM colitis Pseudomembranous
• the use as a medicament relates to the compound of formula (I) or that of formula (III).
• the invention relates to a compound, a mixture or a composition according to the invention, particularly margaucine or a composition comprising it, for use as a medicament, preferably as antibiotic or bactericidal.
• the ( s) compound (s), the mixture or composition (s) thereof may be in solid form (cachet, powder, capsule, pill, suppository, accelerated release tablet, enteric-coated tablet, sustained release tablet), gelatinous (gel, ointment, cream, ovum), liquid (syrup, solution for injection, eye drops) or aerosol (spray, steam, gas).
• the compound, the mixture or the composition of the invention may be administered orally, transmucosa-oral, nasal, ophthalmic, otological (in the ear), vaginal, rectal, parenteral (intravenous, intramuscular or subcutaneous), transcutaneous (or transdermal or percutaneous) or cutaneous.
• the doses administered are those conventionally used for other antibiotics or combinations of antibiotics.
• doses ranging from 30 mg / kg / day to 100 mg / kg / day, depending on the age or weight of the patient, the severity or stage of infection, the number of daily administrations or the mode of administration, may be administered; in adults mainly, doses between 1 g / day and 10 g / day are usual.
• the present invention also relates to the use of a compound of formula (I) or (II) taken individually or as a mixture, for the manufacture of a composition useful in the fight against bacterial infections, in the treatment or prophylaxis of bacterial infections, particularly antibiotic-resistant diseases administered to date.
• Resistant diseases means any infection whose origin is wholly or partially bacterial. More particularly, the term “resistant diseases” as used in the present application encompasses infections of which at least one causative agent is sensitive to an antibiotic compound of the invention, whether used alone or in combination with another compound of the invention or another active molecule. For example, multidrug-resistant diseases (resistant to several classes of antibiotics) or all infections caused by gram-positive bacteria.
• treatment includes both the curative effect (destruction of the microorganism), obtained with at least one compound of the invention or a composition of the invention, that the improvement of symptoms observed in the patient (and related to the presence of the microorganism (s)) or the improvement of the patient's condition.
• Treatment applies to the point of primary or secondary infection (s), as well as to the symptoms arising from the infection.
• the compounds, mixtures and compositions of the invention are particularly used in the treatment of nosocomial infections.
• prophylaxis is meant any use of at least one compound, a mixture or a composition of the invention, as a preventive measure, that is to say in order to avoid the appearance of symptoms. after contamination or suspicion of contamination, or to prevent infection by a microorganism or its consequences, particularly in the case of an infection caused by a bacterium, more particularly a gram-positive bacterium, and even more particularly an infection due to a bacterium of the genus Clostridium such as digestive infections related to Clost ⁇ dium difficile.
• the compounds, mixtures or compositions according to the invention are used in therapy and prophylaxis according to the galenic forms and modes of administration indicated above.
• the compound used is margaucine, or any composition as defined in the present application, comprising at least margaucine.
• the invention also relates to the use of compounds, mixtures or compositions of the invention in cosmetic, food or veterinary applications (domestic animals, poultry, pigs, sheep, cattle or pets).
• the compound of formula (I) or (II) of the invention taken individually or as a mixture or a composition according to the invention is particularly effective against bacteria of the genus Clost ⁇ dium, especially the toxinogenic forms, such as, for example , Clostridium difficile, Clostridium perfringens, Clostiridum botulinum, Clostridium tetani, Clostridium novyi, Clostridium histolyticum, Clostridium butyricum, Clostridium septicum, Clostridium sordellii, Clostridium ramosum, Clostridium bifermentans, Clostridium paraperfringens, Clostridium cadaveris, Clostridium clostridiiforme, Clostridium innocuum, Clostridium limosum, Clostridium Paraputrificum, Clostridium sporogenes, Clostridium subterminal, Clostdium tertium, Clostridium bar
• the invention aims to treat infections caused by toxigenic forms of C. difficile, such as the epidemic strain of PCR-ribotype 027. Due to its particularly effective action on bacteria of the genus Clostridium such as Clostridium difficile (MIC of 0.05 ⁇ g / ml) and its greater activity against Clostridium bacteria compared with other intestinal flora bacteria (30 time more active) on the other hand, the compound of formula (I) or (II) of the invention taken individually or as a mixture or a composition according to the invention, and in particular margaucine (III), is particularly advantageous for use for restoring the balance between different intestinal bacterial populations (microbial flora).
• the present invention also relates to the use of the compound of formula (I) or (II) of the invention taken individually or as a mixture or a composition according to the invention, and in particular margaucine (III), for treating infections due to Clostridium bacteria, particularly C. difficile infections, such as CDI.
• the compound of formula (I) or (II) of the invention taken individually or as a mixture or a composition according to the invention is used to treat DCIs consecutive to antibiotic therapy.
• this compound is margaucine or the composition comprises margaucine (compound of formula III).
• Particularly targeted by the therapeutic and prophylactic methods described in the present invention are patients with C. difficile infection, including (a) patients with CDI, (b) patients with severe CDI. (c) patients with recurrent CDI, recidivism being characterized by a new episode of CDI occurring within 8 weeks of the beginning of the previous episode, whether the strain of C. difficile is the same or different from that of the previous episode.
• Therapeutic and prophylactic methods apply to nosocomial CDIs (hospitalized or non-hospitalized patient who have been discharged from a health facility for less than 4 weeks) than to community DCIs.
• the invention also relates to the use of a compound of formula (I) or (II) taken individually or as a mixture or of a composition as defined above, for the impregnation of devices, among which medical devices (or biomaterial).
• impregnation is meant the application of at least one compound, a mixture or a composition of the invention, and possibly its penetration (deep or superficial) on the device.
• the invention also relates to devices impregnated with the compound (alone, in a mixture or composition) of the invention.
• the devices are biomedical devices, that is to say devices for medical use, such as for example, and without limitation, catheters, dressings, bone cements, brain shunts
• devices for medical use such as for example, and without limitation, catheters, dressings, bone cements, brain shunts
• devices for biomedical use devices made of a polymeric material, such as polyethylene, polypropylene, polyvinylchloride, polycarbonate, polyurethane, acrylate and methacrylate or polyamide or the materials obtained by weaving, will be preferred.
• these devices are biocompatible.
• the impregnated device is a fabric for domestic or industrial use, such as wipes.
• the invention also relates to bacterial strains that have the capacity to produce at least one compound of formula (I) or (II) according to the invention.
• the strains produce compounds according to the invention under the fermentation conditions set forth in point A below (material and methods). These strains can produce a compound of formula (I) or (II), taken individually or as a mixture and in particular margaucine.
• such a strain is the JPL84 strain deposited, for the purposes of the Budapest Treaty at the CNCM (National Collection of Cultures of Microorganisms, Institut Pasteur, 25, rue du Dondel Roux, 75724 Paris Cedex 15, France), the September 15, 2006, under number CNCM I-3669.
• the bacterial strains are Bacillus strains.
• the invention also relates to strains derived from the strains described above and in particular to any strain derived from the strain JPL84 or JPL86, characterized in that they retain the capacity to produce a compound of formula (I) or (II or a mixture, and more particularly in that they retain the ability to produce margaucine.
• “Derivative” means any strain that is not found in the natural state, and obtained from natural strains by modification of its genetic heritage, in particular a strain obtained by recombination (recombinant strain), provided that it continues to produce a compound of formula (I) or (II) according to the invention.
• the recombinant strain produces a greater quantity of compounds according to the invention than the natural strain.
• Modification of the genetic inheritance may consist, inter alia, in the modification of the enzymes and other proteins involved in the production and secretion of the compound of the invention.
• the invention also relates to a method for modulating the bacterial profile of a biological sample or a surface, comprising contacting this biological sample or this surface with at least one compound according to formula (I) or (II ) taken individually or as a mixture or a composition according to the invention, under conditions making it possible to modify the bacterial profile; the method may also comprise a second step of recognizing the modulation.
• the expression "modulate the bacterial profile” means modifying the relationship existing between the different strains. bacterial present in the biological sample or on the surface, before and after contacting with the compound or composition of the invention.
• the method of the invention is suitable for treating the surfaces of different objects, such as, for example, the surfaces of objects of the medical services (for example, in a hospital environment).
• the expression "Modulating the bacterial profile” includes the neutralization of bacterial activity, and in particular the destruction of all bacteria, that is to say the elimination of any bacterial profile.
• the expression "modulate the bacterial profile""Means to cause a modification of the ratio between the strains resistant to the compound of the invention and the most sensitive strains By way of example, the addition of a compound according to the invention results in a decrease in gram positive strains, which modifies the ratio of gram-positive strains to gram-negative strains.
• the carbon in position 1 is a CH 2
• the carbon in position 3 is a CH
• the carbon in the terminal position is a CH 3
• CM. National Collection of Culture of Microorganisms, Institut Pasteur, Paris, France), under number CNCM I-3669. Sequencing of the gene coding for 16S RNA has been performed; strain JPL84 could be a strain Bacillus sp. The strain is maintained at 4 ° C. on CYE agar medium (CYE agar), containing 10 g of casitone, 1 g of yeast extract, 1 g of CaCl 2 and 14 g of agar in 1 liter of water.
• CYE agar medium containing 10 g of casitone, 1 g of yeast extract, 1 g of CaCl 2 and 14 g of agar in 1 liter of water.
• the fermentation of this strain is carried out in a CYE medium containing 10 g / l of peptone, 1 g / l of yeast extract and 1 g / l of CaCl 2 .
• the molecule was purified by reverse phase chromatography: acetonitrile (10% WV) and fixing beads (Amberlite XAD-16) were added to the culture. The whole is placed at 4 ° C., with stirring for 12 hours. The XAD-16 beads are separated from the culture, washed with water and a water / methanol mixture (50/50) and then eluted with methanol (100%). This eluate is concentrated by evaporation under vacuum. The margaucine is finally purified by reverse phase HPLC on a C18 preparative column using a linear gradient of H2 ⁇ / 0.1% TFA and acetonitrile / 0.1% TFA ranging from 20% to 80% acetonitrile / 0.1% TFA in 30 minutes. with a flow of 10 ml / min. After lyophilization, 30 mg of margaucine was obtained from 10L of culture.
• the molecular formula was determined by a combination of spectroscopic techniques: one- and two-dimensional NMR (s) and mass spectrometry.
• MIC minimal inhibitory concentrations
• M07-A6 NCCLS medium Mueller Hinton Broth
• MCF7 breast cancer cell line
• ATCC HTB-22 TM approximately 10,000 cells / well
• RPMI medium containing 10% fetal calf serum
• t + 1 in days, relative to seeding
• cytotoxicity is measured by incorporation of MTT 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide), according to Heeg K et al. (J. Immunol Methods, 1985 Mar. 18, 77 (2): 237-46.)
• the results of the cytotoxicity are shown in Table 4.
• the molecule was tested on a Staphylococcus aureus septicemia model, after infection of OF1 mice by intraperitoneal injection of bacteria.
• mice From a total of 11 female OF1 mice, 25g (Charles River), at TO, 3 mice received an intraperitoneal injection of 200 ⁇ l of LB + 5% mucin (control of rearing conditions), and 8 mice received intraperitoneal injection of 200 ⁇ l of a bacterial suspension of Staphylococcus aureus Smith (10 8 CFU / ml) in LB medium + 5% mucin (infected mice).
• mice were divided into two lots: at t + 3 hours, 5 mice received a 12.5 mg / ml solution of margaucine in 10% methanol and 90% H 2 O (ie a dose of 100 mg / kg), while the other 3 mice did not receive margaucine (control of infection).
• mice The survival of the mice was monitored at 24 and 48 hours.
• the 1 H NMR shows two signal groups (Table 1): - a first group with 3 1 H signals (at 2.66, 3.63 and 5.81 ppm) corresponding to carbon-bound protons with a visible resonance on the spectrum 13 C, all these signals are singlets, and a second group with 2 1 H signals with a chemical shift almost identical to the first group, linked to carbon atoms with a non-visible resonance on the 13 C spectrum. They correspond to an alcohol form of margaucine. An unprotected methylene group at 3.63 ppm can be attributed to the presence in the ⁇ -position of an oxygen atom and a carbonyl group.
• the COZY spectrum shows no correlation while the protons are in the ⁇ position of a quaternary carbon or a heteroatom.
• the presence of two quaternary carbons (170.2 and 105 ppm), one methine (95.6 ppm), one methylene (71.6 ppm) and two methyl equivalent groups (40.4 ppm) was deduced from the DEPT spectrum (Table 1).
• Table 1 1 H and 13 C NMR, DEPT, HMQC and HMBC data for margaucine in CD 3 OD
• the combination of NMR data and mass information suggests a symmetrical molecule.
• the MS / MS experiments carried out on the molecular ion at m / z 211 make it possible to obtain a set of fragments in perfect adequacy with the proposed structure figurai, (ions obtained: 193, 175, 169, 165, 153, 151, 147, 141, 133, 129,
• bacterial wall (composed of several layers including a lipid in gram-negative bacteria whereas it is composed of a single thick layer of murein or peptidoglycan in gram-positive bacteria).
• Table 2 shows that the molecule is effective against Gram + bacteria at concentrations as low as 3.125 ⁇ g / ml or even 0.05 ⁇ g / ml for Clostridium difficile. GRAM ORGANIZATION MIC ( ⁇ g / mi)
• MIC Minimum inhibitory concentration
• margaucine is also active against multiresistant gram-positive strains, such as various strains of Staphylococcus aureus (H1, H3, H4, H7, H9, H16 and H18) (Table 3).
• Staphylococcus aureus H1, H3, H4, H7, H9, H16 and H18
• Table 3 Susceptibility of different strains of Stapi hyloco ⁇ aureus to various antibiotics including margaucine.
• Table 4 Cytotoxicity of margaucine on an MCF7 cell culture.
• margaucine is not toxic to eukaryotic cells, both in vitro and in vivo, at concentrations as high as 100 mg / kg.
• the molecule was tested on a model of infection by
• Clostridium difficile after subcutaneous injection of clindamycin (10 mg / kg body weight) to induce colitis, in hamsters
• a new compound with antibiotic activity, margaucine, produced by a bacterial strain has been identified.
• This compound is structurally characterized and is of low molecular weight.
• margaucine has a broad Gram-positive spectrum, is nontoxic, effective on animal models of infection and does not cross with any known resistance.
• Other compounds of similar structure and having an antibiotic activity are also provided in the present application, to illustrate the interest of this class of compounds for antibiotic application purposes. Because of its particularly effective action on bacteria of the Clostridium genus such as Clostridium difficile, at low concentrations (MIC of the order of 0.05 ⁇ g / ml), the compounds or compositions of the invention, and more particularly the margaucine, represent a strong therapeutic potential against gastrointestinal infections related to C.
• the compound or the composition of the invention is 30 times more active on C. difficile bacteria than on the other bacteria of the intestinal flora.
• vancomycin used to treat the most severe cases (MIC of the order of 1 ⁇ g / ml on C. difficile); however, vancomycin is already used to treat other bacterial infections, including Staphylococcus, the most serious, and has led to the appearance of many strains resistance; its use is limited to the maximum by the medical community in order to avoid the spread of resistance and the emergence of new resistant strains; and
• metronidazole used as first-line therapy but is inactive for 25% of patients and for which there is frequent relapse (25% of cases); moreover, while metronidazole is less expensive than vancomycin, it has more side effects.
• Nitazoxanide Phase III Romark Laboratories LC which is a close molecule in its mechanism of action of metronidazole; however, this molecule is not recommended for pregnant or lactating women;
• rifaximin (Xifaxan) (Salix Pharmaceuticals Phase III), a molecule derived from rifampicin; its minimum inhibitory concentration (MIC) is of the order of 0.08 to 0.2 ⁇ g / ml on C. difficile, and this molecule generates many spontaneous resistors; and
• the compound of the invention or a composition comprising it, and particularly margaucine constitutes a seductive alternative proposal to therapeutic molecules already on the market or those in clinical trials, in the treatment of digestive tract infections.
• C. difficile (ICD) C. difficile

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