US20140303070A1 - Process for the manufacture of ajoene derivatives - Google Patents

Process for the manufacture of ajoene derivatives Download PDF

Info

Publication number
US20140303070A1
US20140303070A1 US13/992,052 US201113992052A US2014303070A1 US 20140303070 A1 US20140303070 A1 US 20140303070A1 US 201113992052 A US201113992052 A US 201113992052A US 2014303070 A1 US2014303070 A1 US 2014303070A1
Authority
US
United States
Prior art keywords
ajoene
bacteria
formula
acid
subject
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US13/992,052
Other languages
English (en)
Inventor
Thomas Bjarnsholt
Niels Høiby
Peter Østrup Jensen
Richard Phipps
Meenakshi Sundaram Shanmugham
Maria Alhede
Louise Dahl Hultqvist
Tim Holm Jakobsen
David Tanner
Thomas Ostenfeld Larsen
Michael Givskov
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Danmarks Tekniskie Universitet
Original Assignee
Danmarks Tekniskie Universitet
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Danmarks Tekniskie Universitet filed Critical Danmarks Tekniskie Universitet
Priority to US13/992,052 priority Critical patent/US20140303070A1/en
Assigned to DANMARKS TEKNISKE UNIVERSITET reassignment DANMARKS TEKNISKE UNIVERSITET ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: ALHEDE, MARIA, BJARNSHOLT, THOMAS, HOIBY, NIELS, HULTQVIST, LOUISE DAHL, JAKOBSEN, TIM HOLM, LARSEN, THOMAS OSTENFELD, PHIPPS, RICHARD, SHANMUGHAM, MEENAKSHI SUNDARAM, TANNER, DAVID, GIVSKOV, MICHAEL, JENSEN, PETER OSTRUP
Publication of US20140303070A1 publication Critical patent/US20140303070A1/en
Abandoned legal-status Critical Current

Links

Images

Classifications

    • 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
    • A61K31/095—Sulfur, selenium, or tellurium compounds, e.g. thiols
    • A61K31/105—Persulfides
    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/7036—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin having at least one amino group directly attached to the carbocyclic ring, e.g. streptomycin, gentamycin, amikacin, validamycin, fortimicins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • 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
    • C07C319/00—Preparation of thiols, sulfides, hydropolysulfides or polysulfides
    • C07C319/22—Preparation of thiols, sulfides, hydropolysulfides or polysulfides of hydropolysulfides or polysulfides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M16/00—Devices for influencing the respiratory system of patients by gas treatment, e.g. mouth-to-mouth respiration; Tracheal tubes
    • A61M16/06—Respiratory or anaesthetic masks
    • A61M16/0683—Holding devices therefor
    • 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 chemical process the end product of which exhibits direct medical applications.
  • the product of the chemical process known as ajoene has been found highly useful for the treatment of bacterial infections comprising biofilm forming bacteria by blocking expression of important quorum sensing controlled virulence factors.
  • the invention also relates to a product obtainable by the above chemical process which shows synergistic antimicrobial effects with antibiotics.
  • the present invention relates to the synthesis of ajoene derivatives via an oxidation of diallyldisulphide to allicin which is subsequently treated with an acid to obtain ajoene E,Z isomers in a given ratio.
  • Ajoene may be subjected to oxidation to obtain oxidized variations.
  • Further aspects of the invention involve a composition of ajoene derivatives with an antibiotic and the use of ajoene derivatives for the treatment of bacterial infections comprising biofilm forming bacteria.
  • Bacterial infections are an increasing problem worldwide.
  • the widespread use of conventional antibiotics has provoked the development of increasingly resistant bacterial strains along with a depleting arsenal of sufficiently effective antibiotics.
  • QS enables bacteria to keep track of their numbers, and is considered to afford them a mechanism for minimizing host response by delaying the production of virulence factors until sufficient bacteria have been amassed to overwhelm host-defence mechanisms. Blocking of QS (either completely or partly) reinstates proper action of the host-defence system which subsequently eliminates bacterial intruders. Therefore, inhibition of QS presents an alternative therapeutic approach to the traditional antibiotic-mediated bacterial killing or growth inhibition.
  • Ajoene [(E,Z)-4,5,9-trithiadodeca-1,6,11-triene-9-oxide] has previously been shown to be an active ingredient in the antimicrobial garlic extracts along with allicin and other organosulfur derivatives.
  • an object of the present invention relates to providing a synthetic procedure for the effective production of ajoene derivatives or specific mixtures thereof which are applicable in the treatment of bacterial infections using QS inhibition.
  • one aspect of the present invention is (E,Z)-ajoene of formula (1) for use in treatment of bacterial infections.
  • Another aspect of the present invention is a composition comprising (E,Z)-ajoene of formula (1) and at least one antibiotic.
  • Yet another aspect of the invention relates to a method for manufacturing (E,Z) ajoene of formula (1)
  • conformation of the internal —C ⁇ C— bond can be either E or Z or a mixture thereof, said method comprising reacting allicin of formula (3)
  • FIG. 1 shows an exemplary scheme depicting the synthetic route towards (E,Z)-ajoene derivatives, including control of E:Z ratio of isomers.
  • the reaction conditions conditions were i) Dimethyldioxirane (DMDO), acetone, ⁇ 50° C. to ⁇ 20° C., 30 min, 96% yield; ii) either A) 20% AcOH, 40% aqueous acetone, 64° C., 4 h, 36% yield, (Z:E ⁇ 4:1) or B) 20% AcOH, 40% toluene, 40° C., 48 h, 32% yield, (Z:E ⁇ 1:10).
  • DMDO Dimethyldioxirane
  • acetone ⁇ 50° C. to ⁇ 20° C., 30 min, 96% yield
  • FIG. 2 shows an exemplary scheme depicting the synthetic route towards further oxidized derivatives of ajoene.
  • the reaction conditions were iii) 4 equivalents of peracetic acid in dichloromethane at 0° C. to room temperature for 12 h, 38% yield or 4 equivalents of DMDO in acetone at ⁇ 10° C. for 6 h at 82% yield.
  • FIG. 3 shows a comparison of fold change ( ⁇ indicates down regulation) in gene expression of rhIA and lasB as measured by RT-PCR (dark grey bars) and DNA microarray (light grey bars). Data represent the average of tree individual experiments. A star (*) indicates P ⁇ 0.05, Student's t-test. Error bars are mean ⁇ SD.
  • FIG. 4 shows total rhamnolipid concentration in untreated (“no add”) and ajoene treated planktonic grown P. aeruginosa .
  • Data represent the average of tree individual experiments. Error bars are mean ⁇ SD.
  • FIG. 5 Shows combined fluorescence and light microscopic investigations of biofilms of P. aeruginosa exposed to PMNs (a single PMN pointed out by the arrow) at day four for 180 min at 37° C. and then subsequently stained with the DNA stain propidium iodide (PI).
  • Red fluorescence indicates necrotic PMN's leaking out their content of DNA (as stained by PI).
  • Green fluorescence indicates top areas of the P. aeruginosa biofilm.
  • FIG. 6 shows biofilms of P. aeruginosa PAO1 (light grey) (A) and a clinical P. aeruginosa isolate CF438 (light grey, stained with syto9) (B) at day four after 24 hours with 10 ⁇ g/ml tobramycin treatment. Dead cells are stained with the DNA stain PI (Dark grey/black). The biofilm were visualized with CSLM.
  • FIG. 7 shows combined results of three separate experiments of ajoene treatment versus no treatment (placebo) using the pulmonary infectious mouse model.
  • Mice were sacrificed on day one or day three post-infection and the contents of bacteria in the lungs were determined. Open squares indicate bacterial lung content per lung for each individual mouse. The median values are indicated with a filled black square. The statistic significance of difference in clearance was tested by a Mann-Whitney U test (analysis of nonparametric data) and p-values for the difference at day one and day three were 0.9 and 0.002 respectively.
  • FIG. 8 shows results of experiments of treatment with HPLC purified ajoene (top graph, ajoene purity >99.5%) and ajoene which have not been HPLC purified (bottom graph, purity 95-99.5%) versus no treatment (placebo) using the pulmonary infectious mouse model. Mice were sacrificed on day three post-infection and the contents of bacteria in the lungs were determined.
  • (E,Z)-ajoene derivatives or (E, Z)-isomers of ajoene derivatives refers to derivatives of ajoene which are either isolated as substantially pure E-ajoene derivatives, substantially pure Z-ajoene derivatives or as mixtures of E- and Z-ajoene derivatives.
  • the (E,Z) designations refers to the internal —C ⁇ C— double bond of ajoene as shown in the structures by a wavy line where the configuration may vary.
  • derivatives refers to both the two isomers (E) and (Z) and mixtures thereof and also to oxidized and/or reduced derivatives of (E,Z) ajoene and mixtures thereof.
  • An oxidizing agent is any chemical reactant capable of changing the oxidation state of a molecule from its original oxidation state to a higher oxidation state.
  • a reducing agent is any chemical reactant capable of changing the oxidation state of a molecule from its original oxidation state to a lower oxidation state.
  • an acid comprises both inorganic and organic acids.
  • a pro-acid is any compound capable of converting into an acid under certain conditions. Such conditions could include heating or subjection to an aqueous environment. Any mixture of acids is also inferred.
  • biofilm refers to any aggregated, often but not necessarily a surface bound (sessile) community of bacteria embedded in a biopolymeric matrix showing increased resistance e.g. to antibiotics and important parts of the innate immune system, as compared to their planktonic counterparts.
  • quorum sensing refers to a bacterial behavioural coordination mechanism operating by means of cell-to-cell communication transmitted by low molecular weight, diffusible signal molecules.
  • QS systems play an important role during the initial event of an infection.
  • three QS systems denoted Las, Rhl and PQS
  • virulence factors many of which are antigenic determinants
  • certain bacteria such as P. aeruginosa is able to operate in a stealthy manner until a certain cell density is reached where the QS system is activated and virulence and immune protection is switched on.
  • a coordinated release of tissue damaging and immune defence degrading virulence factors takes place.
  • N-acyl-homoserine lactone producing bacteria is defined as bacteria utilising N-acyl-homoserine lactones as messenger molecules in their cell-to-cell communication during quorum sensing.
  • a quorum sensing inhibitor or QS-inhibitor is a substance or mixture of substances capable of inhibiting bacterial QS based communication. Blockage of QS processes either by mutation in regulatory genes or by a QS inhibitor makes said bacteria more vulnerable to both conventional antibiotics, in particular aminoglycosides such as tobramycin and important components of the cellular host-defence such as PMN-leukocytes (PolyMorphoNuclear-leukocytes).
  • the first aspect of the present invention provides a method for making compounds of general formula (1)
  • the method comprises providing an intermediate compound of formula (3) also referred to as allicin
  • the concentration of the solution containing compound (3) may be 0.06-6.0 M, preferably 0.12-3.0 M, 0.24-1.5 M, 0.48-0.8 M, such as 0.6 M.
  • the acid can be added in an excess or stoichiometric amount, but may typically be added in a catalytic amount such as 5-50 mol %, 10-40 mol %, 15-30 mol %, such as 20 mol %.
  • the acid added in the conversion of intermediate (3) to compounds of formula (1) may be a a number of acids, such as carboxylic acids including acetic acid, propionic acid, butyric acid, pentanoic acid, hexanoic acid, or trifluoroacetic acid (TFA), or p-toluenesulfonic acid (TsOH), methanesulfonic acid (MsOH), camphorsulphonic acid (CSA).
  • the preferred acid is acetic acid.
  • the choice of acid has impact on the obtained ratio of E:Z isomers, i.e.
  • the solvent used is 10-70 V/V % aqueous acetone, preferably 20-60 V/V %, 30-50 V/V %, such as 40 V/V % aqueous acetone (i.e. 60% acetone in 40% water).
  • the reaction temperature may be adjusted to be in the range of 0-100° C., preferably 20-80° C., such as 40-70° C., preferably 50-65° C., such as 64° C.
  • the reaction time can be from 1-24 h, preferably 2-10 h, such as 3-5 h, preferably 4 h.
  • the use of aqueous acetone as solvent provides the compounds of formula (1) in an E:Z ratio of 1:2-1:6, preferably 1:3-1:5, such as about 1:4, when using acetic acid in the acid treatment.
  • the solvent used during the acid treatment of intermediate (3) is toluene or aqueous benzene.
  • the aqueous benzene can be 1-50 V/V % aqueous benzene, preferably 5-30 V/V %, such as 7-20 V/V %, preferably 10 V/V % aqueous benzene (i.e. 90% benzene in 10% water).
  • the reaction temperature may be adjusted to be in the range of 0-100° C., preferably 10-80° C., such as 20-60° C., preferably 30-50° C., such as 40° C.
  • the reaction time for the acid treatment in toluene or aqueous benzene can be from 2-96 h, preferably 24-72 h, such as 36-60 h, preferably 48 h.
  • the use of toluene or aqueous benzene as solvent provides the compounds of formula (1) in an E:Z ratio of 6:1-20:1, such as about 10:1, when using acetic acid in the acid treatment.
  • the purification of the obtained products may typically be performed using standard solvent extraction techniques followed by column chromatographic purification, but other methods may also be used such as distillation, preparative thin layer chromatography or crystallization for solid compounds.
  • the reaction mixture is cooled to room temperature and diluted with 3 volumes of a 1:1 mixture of water and methanol followed by an extraction with an apolar solvent such as pentane.
  • the aqueous fraction may be saturated with ammonium sulphate and further extracted with a more polar solvent than pentane such as dichloromethane.
  • (E,Z)-ajoene (1) can be isolated by concentrating the combined organic extracts and subjecting the crude product to silica column chromatography using an eluent mobile phase comprising 40-99 V/V % ethyl acetate in pentane, preferably 50-90 V/V %, 60-80 V/V %, such as 70 V/V %.
  • the purity of (E,Z)-ajoene after the above procedure may be 95-99.5%, such as 97-99.5%, 98-99.5%, 99-99.5% such as about 99.5%. It was surprisingly found by the inventors, that upon further purification of the product obtained after column chromatography, e.g. using reverse phase HPLC chromatography, or other purification steps, said (E,Z)-ajoene product lost all or parts of its biological activity, in e.g. the assays as described in the examples. This was also found when isolating pure (E)-ajoene and/or pure (Z)-ajoene, e.g. using HPLC.
  • ajoene derived chemistry i.e. compounds related to ajoene, such as less than 5%, 3% , 2%, 1%, such as less than 0.5% may be highly important in the stabilisation of (E,Z)-ajoene and derivatives, towards degradation, e.g. oxidation, reduction and or cleavage, both in vitro but importantly also in vivo.
  • One embodiment of the invention is a method as described above wherein the (E,Z)-ajoene of formula (1) is subjected to no more than one chromatographic purification step, said chromatographic purification step not comprising reversed phase HPLC.
  • the (E,Z)-ajoene derivatives can be isolated as mixtures of isomers in yields based on compound (3) of at least 10 mol %, preferably 15 mol %, 20 mol %, 25 mol %, such as at least 30 mol %.
  • the allicin of formula (3) may be manufactured by treating a compound of formula (2), also referred to as diallyldisulphide (DADS)
  • DADS diallyldisulphide
  • the oxidizing agent used to oxidize compounds of formula (2) to compounds of formula (3) may be selected from the group consisting of DMDO, MCPBA, peracetic acid, potassium permanganate, magnesium oxide and Swern oxidants (e.g. oxalyl chloride, dimethyl sulfoxide (DMSO) and an organic base, such as triethylamine)
  • DMDO is a preferred oxidant as it provides the highest yields.
  • the oxidizing agent may be added in catalytic or stoichiometric amount relative to compound (2) but preferably in an excess amount such as 1.01-10.0 mol equivalents, preferably 1.05-5.0 mol equivalents, such as 1.07-2.0 mol equivalents, preferably 1.10 mol equivalents.
  • the compound of formula (2) may be advantageously added to a solution of the oxidizing agent at low temperature over a period of several minutes.
  • the concentration of oxidizing agent in the solution can be 0.001-1.0 M, preferably 0.005-0.5 M, 0.01-0.1 M, such as 0.07 M.
  • the solution may be in a solvent capable of staying fluid at low temperatures, such as acetone, dichloromethane, chloroform, tetrachloromethane, diethylether, ethyl acetate, or tetrahydrofurane.
  • the solution temperature while adding compound (2) may be ⁇ 100° C. to 10° C., preferably ⁇ 80° C. to ⁇ 10° C., ⁇ 60° C. to ⁇ 40° C., such as ⁇ 50° C.
  • Compound (2) may be added over a period of 1-120 min, preferably 5-90 min, 7-60 min, 10-30 min, 12-20 min, such as 15 min.
  • this may advantageously be allowed to warm up slowly by 10-60° C., preferably 20-40° C., such as 30° C., over a period of 10-120 min, preferably 15-90 min, 20-60 min, 25-40 min, such as 30 min.
  • Concentration via evaporation of solvents provides the intermediate compound (3) in a crude form with a purity of at least 80 mol %, preferably at least 85 mol %, at least 90 mol %, such as at least 95 mol %.
  • the crude allicin (3) may be used without further purification, and still provide good yields in the following step. This could be due to the lack of acid present in the oxidation procedure, which seems to contribute to degradation of allicin during oxidation and subsequent storage.
  • allicin (3) may advantageously be purified in order to isolate a substantially allicin (3) prior to the subsequent acid treatment. This was found to provide an even cleaner reaction and higher yields in the subsequent acid treatment step to provide compounds of formula (1).
  • the purification can be performed using any conventional purification methods, preferably silica column chromatography.
  • Chromatography can be performed using a mobile phase eluent consisting of a mixture of diethyl ether in pentane, preferably 0.5-20 V/V % diethylelether in pentane, such as 1-10 V/V %, 2-8 V/V %, 3-7 V/V %, 4-6 V/V %, preferably 5 V/V %.
  • a compound of formula (3) is obtained in a yield based on compound (2) of at least 80 mol %, preferably at least 85 mol %, at least 90 mol %, such as at least 95 mol %.
  • Y is chosen from —S—, —S(O), or —S(O 2 )—
  • R is chosen from allyl, phenyl, methyl, ethyl, propyl, butyl, pentyl, hexyl, benzyl, p-tolouyl, m-tolouyl, o-tolouyl, and the conformation of the internal —C ⁇ C— bond can be either E or Z or a mixture thereof.
  • This process comprises the method as defined above to make compound of formula (1) followed by an additional reaction step involving reacting (E,Z) ajoene of formula (1) with at least one additional oxidizing reagent to provide a compound of formula (4) wherein R is allyl, and optionally reacting the compound of formula (4) with an R substituted lithiated sulphide (R-SLi) to provide compounds of formula (4) wherein R is phenyl, methyl, ethyl, propyl, butyl, pentyl, hexyl, benzyl, p-tolouyl, m-tolouyl, or o-tolouyl.
  • the second oxidizing agent used to oxidize compounds of formula (1) to compounds of formula (4) may be selected from the group consisting of DMDO, MCPBA, peracetic acid, potassium permanganate, Hydrogen peroxide, Bis TMS Peroxide, or any mild peroxides.
  • the reaction with R-substituted lithiated sulphide may be performed in tetrahydrofurane solvent at ⁇ 78° C. up to 0° C. Reaction time may be 30 min to 24 hours, preferably 30 min to 2 hours.
  • the compounds obtained of general formula (4) can be isolated as mixtures of (E,Z)-isomers, or these isomers can be separated to provide substantially pure (E)-isomers or substantially pure (Z)-isomers. Also, the compounds of formula (4) having different oxidation states of Y, i.e. Y equal to —S—, S(O), or —S(O 2 )— can be either separated or isolated as mixtures.
  • Another embodiment of the present invention is the compounds of formula (1) obtained by the methods of described above or alternatively the compounds of formula (1) obtainable by the methods of described above.
  • the above method leads to good crude purities, which leads to the use of only one chromatographic purification step, which has proved to be vital to obtain a more biologically active (E,Z)-ajoene derivative (1).
  • active ajoene derivatives may be said to have a purity in the range of 95-99.5%, such as about 97-99.5%, 98-99.5%, 99-99.5% such as about 99.5%.
  • the E:Z ratio of the (E,Z) ajoene obtained may preferably be from 20:1 to 1:10, such as from 15:1 to 1:6, 12:1 to 1:5, preferably from about 10:1 to about 1:4.
  • the E:Z ratio may preferably be from 1:1 to 1:10, such as from 1:2 to 1:8, 1:3 to 1:5, preferably about 1:4.
  • compositions comprising a (E,Z)-ajoene having formula (1) and at least one antibiotic.
  • the compound i.e. (E,Z)-ajoene having an obtainable E:Z ratio including pure E or pure Z
  • Such compositions may be used in the formulation of medicaments and thus may be formulated as a medicament or dosage form comprising a (E,Z)-ajoene having formula (1), at least one antibiotic and pharmaceutically acceptable carriers and/or binders.
  • the medicament may be formulated as liquid or solid dosage forms, where liquid compositions may include liquid compositions for topical administration, liquid compositions for intravenous injection, intramuscular or subcutaneous injection, or a liquid composition for inhalation as an aerosol.
  • Solid dosage forms may include tablets, capsules, powders, including powders for inhalation.
  • Embodiments wherein the (E,Z)-ajoene is administered in one of the above ways, while the antibiotic is administered in any other of the above ways are also inferred.
  • the ajoene may be administered by inhalation, via powder or aerosol, while the antibiotic is administered intravenously.
  • the E:Z ratio of the (E,Z) ajoene in the composition may preferably be from 20:1 to 1:10, such as from 15:1 to 1:6, 12:1 to 1:5, preferably from about 10:1 to about 1:4.
  • the E:Z ratio may preferably be from 1:1 to 1:10, such as from 1:2 to 1:8, 1:3 to 1:5, preferably about 1:4.
  • One embodiment is thus a composition as described above comprising (E,Z)-ajoene having formula (1) and at least one antibiotic for use in the treatment of infections, preferably bacterial infections comprising biofilm forming bacteria.
  • an advantage of such compositions is that a synergistic effect is achieved when using a QS-inhibitor in a composition with an antibiotic, as demonstrated herein.
  • an antibiotic is defined as any antibacterial substance where the antibacterial effect is not based on QS inhibition.
  • the antibiotic used in combination with a QS-inhibitor can be classified as either bactericidal or bacteriostatic. Bactericidals kill bacteria directly where bacteriostatics prevent bacteria from dividing.
  • Classes of antibiotics include aminoglycosides, ansamycins, carbacephem, carbapenems, cephalosporins, glycopeptides, macrolides, monobactams, penicillins, polypeptides, quinolones, sulfonamides, and tetracyclines.
  • the antibiotic may be selected from any of these classes.
  • the antibiotic may be selected from the group consisting of Ampicillin, Bacampicillin, Carbenicillin Indanyl, Mezlocillin, Piperacillin, Ticarcillin, Amoxicillin-Clavulanic Acid, Ampicillin-Sulbactam, Benzylpenicillin, Cloxacillin, Dicloxacillin, Methicillin, Oxacillin, Penicillin G, Penicillin V, Piperacillin Tazobactam, Ticarcillin Clavulanic Acid, Nafcillin, Cefadroxil, Cefazolin, Cephalexin, Cephalothin, Cephapirin, Cephradine, Cefaclor, Cefamandol, Cefonicid, Cefotetan, Cefoxitin, Cefprozil, Ceftmetazole, Cefuroxime, Loracarbef, Cefdinir, Ceftibuten, Cefoperazone, Cefixime, Cefotaxime, Cefpodoxime prox
  • One aspect of the present invention is (E,Z)-ajoene of formula (1) for use in treatment of bacterial infections, preferably bacterial infections comprising biofilm forming bacteria.
  • Another aspect of the invention is (E,Z)-ajoene of formula (1) as obtained by the herein described method for use in treatment of bacterial infections, preferably bacterial infections comprising biofilm forming bacteria.
  • the E:Z ratio of the (E,Z) ajoene obtained may preferably be from 20:1 to 1:10, such as from 15:1 to 1:6, 12:1 to 1:5, preferably from about 10:1 to about 1:4.
  • the E:Z ratio may preferably be from 1:1 to 1:10, such as from 1:2 to 1:8, 1:3 to 1:5, preferably about 1:4.
  • the treatment of bacterial infections may be prophylactic.
  • the compounds used may be in a composition with an antibiotic and/or a pharmaceutically acceptable carrier.
  • the compounds or compositions may be administered by intravenous injections, intramuscular or subcutaneous injections, orally, topically or they may be inhaled, preferably using a suitable inhalation device. They may be inhaled as solid compositions, such as powders, or as liquid compositions in the form of aerosols.
  • the bacterial infection treated is in a mammal with an immune deficiency.
  • a mammal with immune deficiency is a mammal that for any reason is immuno-compromised, meaning that the performance of some or all aspects of the natural defence is lower than normal. Typically, this may be due to an immuno-compromising disease or due to therapy using immuno-suppressant medication.
  • Such mammals are at higher risk of infections with biofilm forming bacteria such as Pseudomonas aeruginosa and would therefore potentially benefit from both prophylactic and acute treatment with a QS inhibitor which may be in a composition with an antibiotic.
  • Mammals may comprise humans, pets and livestock, or mammals may be selected from the group consisting of humans, pets and livestock.
  • Immuno-compromising diseases include diseases selected from the group consisting of cystic fibrosis, diabetes mellitus, COPD, malignant haematological disease, cancer, HIV, AIDS, chronic wounds, burn wounds.
  • Patients may include patients with indwelling catheters, patients treated with medical equipment, implants, stents and patients in ICU's.
  • the bacteria, particularly the biofilm forming bacteria causing the infections described above may be selected from the group of bacteria capable of QS controlled virulence such as gram negative bacteria, particularly the following groups of Gram negative bacteria: Vibrio fischeri, Aeromonas hydrophila, Aeromonas salmonicida, Agrobacterium tumefaciens, Burkholderia cepacia, Chromobacterium violaceum, Enterobacter agglomerans, Erwinia carotovora, Erwinia chrysanthemi, Erwinia Stewartii, Escherichia coli, Pseudomonas aureofaciens, Pseudomonas aeruginosa, Ralstonia solanacearum, Rhizobium etli, Rhizobium leguminosarum, Rhodobacter sphaeroides, Salmonella typhimurium, Serratia liquefaciens, Sinorh
  • (E,Z)-ajoene of formula (1) for use in the treatment of bacterial infections where the infected area is a wound, such as a chronic wound and/or non-healing wound.
  • the compounds may advantageously be administered topically.
  • Another useful embodiment is (E,Z)-ajoene of formula (1) for use in the treatment of bacterial infections where the infected area is the lungs.
  • This treatment is especially relevant for cystic fibrosis patients who, due to their reduced immune response, often suffer from persistent lung infections comprising biofilm forming bacteria, such as Pseudomonas aeruginosa . Therefore, a preferred embodiment is (E,Z)-ajoene of formula (1) for use in the treatment of bacterial infections where the infected area is the lungs of a cystic fibrosis patient.
  • the compounds may advantageously be administered via inhalation or by intravenous injections.
  • the infected area is an implant or the area around an implant.
  • an implant is a medical device made to replace or act as a missing biological structure in a mammal.
  • Such implants may include implants containing electronics such as artificial pacemakers and cochlear implants, subcutaneous drug delivery devices in the form of implantable pills or drug-eluting stents, a prosthetic device such as bone-replacement and support devices or dental implants.
  • the infected area may be the part of the digestive system, such as the stomach and/or intestines.
  • infections may include food poisoning or infections arising from pancreatic or biliary stents.
  • Oxidized sulphone derivatives of (E,Z) ajoene has been shown to have QS inhibitory effects. Therefore such derivatives, i.e. compounds of formula (4) were synthesized using various routes as described below (see also FIG. 2 ):
  • DNA microarray analysis was used to identify genes regulated by ajoene treatment.
  • QS regulon previously identified by Hentzer et al., [M. Hentzer et al., EMBO J., 2003, 22, p. 3803-3815] and these data has been used to validate target specificity of putative quorum sensing inhibiting (QSI) compounds.
  • Exponential growing P. aeruginosa cultures were treated with the following four concentrations of ajoene; 10 ⁇ g/ml (42.7 ⁇ M), 20 ⁇ g/ml (85.4 ⁇ M), 40 ⁇ g/ml (170.8 ⁇ M) and 80 ⁇ g/ml (341.6 ⁇ M).
  • RT-PCR was performed with two of the key QS genes; lasB and rhIA (see FIG. 3 ).
  • lasB and rhIA the key QS genes
  • rhIA the repression of both the lasB and rhIA gene expression follow the same trend, with a general slightly increase in fold reduction observed with the RT-PCR based method.
  • the RT-PCR data shows that a concentration of 80 ⁇ g/ml ajoene lowers the expression of rhIA almost 12 fold and lasB almost 5 fold.
  • rhIA The expression of rhIA is primarily controlled by the Rhl QS system and the signal molecule BHL whereas the expression of lasB is affected by both the Las and the Rhl QS systems and the concentration of the signal molecules OdDHL and BHL.
  • the genes listed in table 3 are (except prpL which regulation is entirely governed by the Las QS system) subject to regulation by both the Las and Rhl QS systems.
  • rhamnolipid concentration present in the cultures grown for DNA array and RT-PCR where directly quantified by LC-MS.
  • the production of rhamnolipids is initiated early in stationary phase co-ordinately regulated by the Rhl QS system encoded by the rhIA, rh/B and rh/C genes (PA3479, PA3478 and PA1131) [V. E. Wagner et al., J. Bacteriol., 2003, 185, p. 2080-2095 and R. Rahim et al., Mol. Microbiol., 2001, 40, p. 708-718].
  • Biofilms exposed to PMN leukocytes produce rhamnolipids that function as a shield and protects the biofilm bacteria from phagocytosis.
  • the influence of ajoene on the lysis of PMNs was demonstrated by using an in vitro continuous-culture once through flow chamber biofilm system [B. B. Christensen et al., Methods Enzymol., 1999, 310, p. 20-42].
  • the P. aeruginosa biofilms were grown for four days either in the presence or absence of 100 ⁇ g/ml ajoene.
  • mice were intracheally challenged (at day 0) with alginate beads containing 1.5 ⁇ 10 8 CFU/ml P. aeruginosa .
  • the two groups of mice were either untreated (placebo) or treated with ajoene 25 ⁇ g/g mouse once a day.
  • the mice were given two days of prophylactic treatment or placebo prior to bacterial challenge. Treatment was continued for three days.
  • the present study offers a convincing indication of ajoene and close derivatives being the major active components in garlic able to reduce a P. aeruginosa infection.
  • rhamnolipid lyses the PMNs that subsequently spill out their content of DNA, hydrolytic enzymes and oxygen radicals. This creates an “evil circle” particularly with respect to tissue damage, increasing inflammation and induction of mutations in P. aeruginosa of which appearance of the mucoid phenotype significantly contributes to exacerbations.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Organic Chemistry (AREA)
  • Epidemiology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Pulmonology (AREA)
  • Dermatology (AREA)
  • Communicable Diseases (AREA)
  • Oncology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
  • Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
US13/992,052 2010-12-08 2011-12-07 Process for the manufacture of ajoene derivatives Abandoned US20140303070A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US13/992,052 US20140303070A1 (en) 2010-12-08 2011-12-07 Process for the manufacture of ajoene derivatives

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
US42092210P 2010-12-08 2010-12-08
EP10194154.0 2010-12-08
EP10194154 2010-12-08
US13/992,052 US20140303070A1 (en) 2010-12-08 2011-12-07 Process for the manufacture of ajoene derivatives
PCT/DK2011/050467 WO2012076016A1 (en) 2010-12-08 2011-12-07 Process for the manufacture of ajoene derivatives

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
PCT/DK2011/050467 A-371-Of-International WO2012076016A1 (en) 2010-12-08 2011-12-07 Process for the manufacture of ajoene derivatives

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US15/980,471 Continuation US10603289B2 (en) 2010-12-08 2018-05-15 Process for the manufacture of ajoene derivatives

Publications (1)

Publication Number Publication Date
US20140303070A1 true US20140303070A1 (en) 2014-10-09

Family

ID=43903893

Family Applications (2)

Application Number Title Priority Date Filing Date
US13/992,052 Abandoned US20140303070A1 (en) 2010-12-08 2011-12-07 Process for the manufacture of ajoene derivatives
US15/980,471 Expired - Fee Related US10603289B2 (en) 2010-12-08 2018-05-15 Process for the manufacture of ajoene derivatives

Family Applications After (1)

Application Number Title Priority Date Filing Date
US15/980,471 Expired - Fee Related US10603289B2 (en) 2010-12-08 2018-05-15 Process for the manufacture of ajoene derivatives

Country Status (9)

Country Link
US (2) US20140303070A1 (de)
EP (2) EP3020397B1 (de)
JP (2) JP6088433B2 (de)
CN (1) CN103328441B (de)
AU (1) AU2016203651B2 (de)
CA (1) CA2820393A1 (de)
ES (1) ES2571998T3 (de)
HK (1) HK1223013A1 (de)
WO (1) WO2012076016A1 (de)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111166732A (zh) * 2020-01-14 2020-05-19 广东省微生物研究所（广东省微生物分析检测中心） 二烯丙基二硫醚在制备抑制铜绿假单胞菌双组分系统药物中的应用
WO2020157463A1 (en) * 2019-01-30 2020-08-06 Neem Biotech Limited Disulphide compounds
US10751495B2 (en) * 2013-12-20 2020-08-25 Koninklijke Philips N.V. Patient interface
WO2021182958A1 (en) * 2020-03-13 2021-09-16 Ahv International Compositions for disrupting biofilm formation and for treating biofilm-related disorders

Families Citing this family (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2014001170A (ja) * 2012-06-19 2014-01-09 Nagoya Seiraku Kk 分泌型ＩｇＡ誘導剤、及びその分泌型ＩｇＡ誘導剤を含む飲食品
GB201420902D0 (en) * 2014-11-25 2015-01-07 Neem Biotech Ltd A process for producing Ajoene
GB2533975A (en) * 2014-12-30 2016-07-13 Trio Healthcare Ltd Antibiofilm compositions
WO2017044044A1 (en) * 2015-09-08 2017-03-16 Nanyang Technological University Method of inhibiting quorum sensing in pseudomonas aeruginosa
CN106311734B (zh) * 2016-08-18 2019-06-25 浙江工商大学 用于土壤抗生素抗性基因污染消减的药剂及其制备和应用
CN106518739B (zh) * 2016-10-31 2018-12-04 中山大学 一种从黑大蒜中提取分离阿霍烯的方法
CA3051598A1 (en) 2017-03-30 2018-10-04 Mootral Sa Antimicrobial garlic compositions
WO2018190352A1 (ja) * 2017-04-11 2018-10-18 三井化学アグロ株式会社 ピリドン化合物およびそれを有効成分とする農園芸用殺菌剤
GB201818029D0 (en) * 2018-11-05 2018-12-19 Neem Biotech Ltd Organosulfur compounds
WO2023038522A1 (en) * 2021-09-10 2023-03-16 AHV International B.V. Compositions and methods for treating biofilm disorders and infection

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003024437A1 (en) * 2001-09-21 2003-03-27 Stone Island Holdings Ltd Allicin

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4665088A (en) 1984-11-14 1987-05-12 The Research Foundation Of State University Of New York (E-Z)-4,5,9-trithiadodeca-1,6,11-triene 9-oxides
US4643994A (en) 1984-12-17 1987-02-17 The Research Foundation Of State University Of New York Novel organic trithio oxides and method for the preparation thereof
AU1118299A (en) * 1997-10-23 1999-05-10 Pharmaprint, Inc. Pharmaceutical grade garlic
GB0120887D0 (en) * 2001-08-29 2001-10-17 Sahajanand Biotech Private Ltd Local drug delivery system in coronary stents
WO2005089571A1 (es) * 2004-03-03 2005-09-29 Mousala, S., L. Utilización de extractos y compuestos de plantas del género allium como conservantes en las industrias alimentaria y agroalimentaria
GB0903869D0 (en) * 2009-03-05 2009-04-22 Neem Biotech Ltd Process for the preparation of ajoene

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003024437A1 (en) * 2001-09-21 2003-03-27 Stone Island Holdings Ltd Allicin

Non-Patent Citations (13)

* Cited by examiner, † Cited by third party
Title
Anderson, Gregory G. et al, "In vitor analysis of tobramycin treated pseudomonas aeruginosa biofilms on cystic fibrosis derived airway epithelial cells." Infect. Immun. (2008) 76(4) p1423-1433) *
Ankri, Serge and Mirelman, David; "Antimicrobial properties of allicin from garlic." Microbes and Infection (1999) 2 p125-129 *
Apitz-Castro, Rafael et al; "Ajoene, the antiplatelet principle of garlic, synergistically potentiates the antiaggregatory action of prostacyclin, forskolin, indomethacin, and dypirdamole on human platelets." Thromb. Res. (1986) 42 p303-311 *
Bjarnsholt, Thomas et al; "Garlic blocks quorum sensing and promotes rapid clearing of pulmonary pseudomonas aeruginosa infections." Microbiology (2005) 151(12) p3873-3880 *
Hudault, S. et al, "Escherichia coli strains colonising the gastrointestinal tract protect germ free mice against salmonella typhimurium infection." Gut (2001) 49 p47-55 *
Kalia, Vipin Chandra, "Quorum sensing inhibitors: an overview." Biotech. Adv. (2013) 31 p224-245) *
Katzen, Mollie; The Moosewood Cookbook (1977) ISBN 0-89815-490-1 *
Lawson, Larry D. et al, "Identification and hplc quantitation of the sulfides and dialk(en)yl thiosulfinates in commercial garlic products." Planta Med. (1991) 57 p363-370) *
Naganawa, Rie et al; "Inhibition of microbial growth by ajoene, a sulfer containing compound derived from garlic." App. Environ. Microbiol (1996) 62(11) p4238-4242 *
Rasmussen, Thomas Bovbjerg et al; "Screening for quorum-sensing inhibitors (qsi) by use of a novel genetic system, the qsi selector." J. Bacteriol. (2005) 187(5) p1799-1814 *
Ratjen, Felix et al, "Aminoglycoside therapy against pseudomonas aeruginosa in cystic fibrosis: a review." J. Cystic Fibrosis (2009) 8 p361-369 *
USPTO. 35 USC 101 Myriad Guideline. March 4, 2014. *
Walker, Travis S. et al; "Pseudomonas aeruginosa-plant root interactions. Pathogenicity, biofilm formation, and root exudation." Plant. Physiol. (2004) 134 p320-331 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10751495B2 (en) * 2013-12-20 2020-08-25 Koninklijke Philips N.V. Patient interface
WO2020157463A1 (en) * 2019-01-30 2020-08-06 Neem Biotech Limited Disulphide compounds
CN111166732A (zh) * 2020-01-14 2020-05-19 广东省微生物研究所（广东省微生物分析检测中心） 二烯丙基二硫醚在制备抑制铜绿假单胞菌双组分系统药物中的应用
WO2021182958A1 (en) * 2020-03-13 2021-09-16 Ahv International Compositions for disrupting biofilm formation and for treating biofilm-related disorders

Also Published As

Publication number Publication date
AU2016203651A1 (en) 2016-06-16
AU2016203651B2 (en) 2017-06-01
EP3020397B1 (de) 2019-04-17
US10603289B2 (en) 2020-03-31
HK1223013A1 (zh) 2017-07-21
JP6088433B2 (ja) 2017-03-01
JP2017061525A (ja) 2017-03-30
JP6253749B2 (ja) 2017-12-27
EP3020397A3 (de) 2016-08-17
EP2649047B1 (de) 2016-02-24
ES2571998T3 (es) 2016-05-27
EP3020397A2 (de) 2016-05-18
AU2011341155A1 (en) 2013-06-27
CN103328441A (zh) 2013-09-25
EP2649047A1 (de) 2013-10-16
WO2012076016A1 (en) 2012-06-14
CN103328441B (zh) 2016-08-31
CA2820393A1 (en) 2012-06-14
JP2014508719A (ja) 2014-04-10
US20180360773A1 (en) 2018-12-20

Similar Documents

Publication Publication Date Title
US10603289B2 (en) Process for the manufacture of ajoene derivatives
CN101232884B (zh) 作为抗菌剂的喹啉衍生物
CN110662746B (zh) 作为β-内酰胺酶抑制剂的经口给药的前药的化合物、其药物组合物和治疗细菌感染的用途
DK1879655T3 (en) Pharmaceutical composition comprising an antibacterial agent and an active substance selected from carveol, carvacrol, alpha-ionone, beta-ionone, and thymol
CN101277696B (zh) 作为抗菌药物的喹啉衍生物
JP6050763B2 (ja) 抗菌性の化合物／アジュバント化合物および方法
EA013244B1 (ru) 8-МЕТОКСИ-9Н-ИЗОТИАЗОЛО[5,4-b]ХИНОЛИН-3,4-ДИОНЫ И РОДСТВЕННЫЕ СОЕДИНЕНИЯ В КАЧЕСТВЕ ПРОТИВОИНФЕКЦИОННЫХ СРЕДСТВ
JP7443361B2 (ja) 加齢黄斑変性症の治療方法
KR20200032115A (ko) 세균 감염의 치료를 위한 색소 생산에 영향을 미치는 화합물 및 이의 용도
KR101329587B1 (ko) 항균제로서의 퀴놀린 유도체
CA3090833A1 (en) Compound acting as antibiotics
AU2011341155B2 (en) Process for the manufacture of ajoene derivatives
TWI392494B (zh) 作為抗細菌劑之喹啉衍生物（一）
WO2003063927A2 (en) Compositions and methods to treat gastrointestinal disorders
ES2733474T3 (es) Ajoeno para su uso en el tratamiento de infecciones bacterianas
US20100137388A1 (en) Therapeutic Pro-Antibiotic Agents and Methods of Use Thereof
WO2018213378A1 (en) Inhibitors of nucleotidyltransferase superfamily enzymes as antibiotics
CN104981238A (zh) 抗细菌化合物
JP7437847B2 (ja) レレバクタムの誘導体およびその使用
CN109121411B (zh) 嘧啶并-异喹啉-醌衍生的化合物、含有它们的药物组合物和它们在细菌性疾病治疗中的用途
JP2024511165A (ja) バンコマイシンと組み合わせたリファマイシン類似体およびその使用
WO2023193101A1 (en) Antibacterial 4,5 diaryl-thiophen-2-yl hexafluoropropan-2-ols
FR2941955A1 (fr) Nouvelles molecules hybrides vancomycine-aminoquinoleine denommees " vancomyquines", leur preparation et leur application en therapeutique

Legal Events

Date Code Title Description
AS Assignment

Owner name: DANMARKS TEKNISKE UNIVERSITET, DENMARK

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:BJARNSHOLT, THOMAS;HOIBY, NIELS;JENSEN, PETER OSTRUP;AND OTHERS;SIGNING DATES FROM 20130627 TO 20130904;REEL/FRAME:031527/0827

STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION