WO2004015416A2 - Procedes pour identifier des substances a action anti-microbienne - Google Patents

Procedes pour identifier des substances a action anti-microbienne Download PDF

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Publication number
WO2004015416A2
WO2004015416A2 PCT/EP2003/008145 EP0308145W WO2004015416A2 WO 2004015416 A2 WO2004015416 A2 WO 2004015416A2 EP 0308145 W EP0308145 W EP 0308145W WO 2004015416 A2 WO2004015416 A2 WO 2004015416A2
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WO
WIPO (PCT)
Prior art keywords
microbes
eukaryotic cells
cells
test
concentration
Prior art date
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PCT/EP2003/008145
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German (de)
English (en)
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WO2004015416A3 (fr
Inventor
Gerald Kleymann
Hans-Otto Werling
Original Assignee
Bayer Healthcare Ag
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 Bayer Healthcare Ag filed Critical Bayer Healthcare Ag
Priority to AU2003255282A priority Critical patent/AU2003255282A1/en
Publication of WO2004015416A2 publication Critical patent/WO2004015416A2/fr
Publication of WO2004015416A3 publication Critical patent/WO2004015416A3/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/18—Testing for antimicrobial activity of a material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5014—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing toxicity

Definitions

  • a goal of inventions in the pharmaceutical industry is to provide the healthcare system with compatible medication and therapy options for treating patients.
  • This invention describes a method for the identification of anti-infectively active substances for the treatment of intra- or extracellular, non-viral microbial infections.
  • the method is based on a combined analysis of the anti-microbial effect and the tolerance of the agent in viti-o.
  • this assay allows the characterization of an anti-microbial agent with respect to the anti-microbial effect, the inhibition of the cytopatic effect which the pathogen exerts on the infected culture, a possible cytotoxicity and protective effects on the infected cell culture.
  • Targets for drug discovery have skyrocketed, but even though these targets and assay systems are well characterized, the identified compounds must continue to meet the criteria listed above that are essential for successful medication.
  • the NCCLS committee (National Committee for Clinical Laboratory Standards, USA) publishes standardized guidelines for the MIC test (minimum inhibitory concentration, or MIC test, minimum inhibitory concentration), which is based on solid or liquid media.
  • the aim of this invention is to provide a method which makes it possible to find alternative or more active non-viral, anti-microbial compounds which have a better selectivity index and / or tolerance and / or protective effects on the infected eukaryotic cell culture.
  • Another object of this invention is to provide a robust, inexpensive, sensitive and efficient method by which alternative, new or more potent anti-microbial substances can be identified.
  • Another object of this invention is to provide a method that is compatible with high throughput test methods.
  • Another aspect of the present invention is to provide a method by which substances can be identified which have a broad-spectrum activity or an activity against therapy-resistant pathogens.
  • the present invention solves the problems listed above by providing a method for finding active substances against non-viral microbes, characterized in that, in a test batch, potential active substances are simultaneously incubated together with eukaryotic cells and with non-viral microbes, the vitality of the eukaryotic cells are determined and then those active substances are selected in which the signal of the corresponding test batch is significantly larger than the mean value of the infection control at at least one concentration of the active substance.
  • this method allows a combined assessment of both the antimicrobial activity of the compound and, at the same time, its tolerance (selectivity index) and its cytoprotective effects on an infected cell culture. From a different perspective, it is precisely this in vitro assay
  • the infectiological assay according to the invention allows an efficient screening of substance libraries and the subsequent optimization of the identified anti-microbial active substances, because a reproducible and
  • Dose-effect-dependent test which identifies anti-microbial activities of substances with predominantly suitable tolerability, ultimately represents the simplest stage in vitro of a sensible evaluation of substances in relation to their anti-microbial potential.
  • a further advantage of this invention is that this infectiological in vitro method not only simultaneously tests all of the pathogen's targets that are essential for microbial growth and infection, but also the targets of the eukaryotic host cells that are relevant for possible therapeutic intervention in the almost natural infection process without, for example like with
  • the infectiological assay therefore provides answers to the question of whether there are structures that can be optimized, are tolerable and that are anti-microbial and that inhibit the occurrence of infection.
  • Once a suitable substance has been identified the underlying mechanism of action or the target can be quickly clarified using the available methods. With this procedure, the previously necessary identification, evaluation and testing of numerous individual targets can become redundant sequentially or in parallel.
  • the assay clearly differs from all publications published to date. Numerous assays have been described in various modifications to identify bacteriostatic or bactericidal compounds, but these test systems are basically all based on the growth inhibition of the pathogen in the presence of the active ingredient or the test substance.
  • these tests identify not only the desired anti-microbial compounds with respect to the development of a drug, but to a large extent cytotoxic and cytostatic active substances, which require numerous downstream test systems in order to filter out compatible active substances from the undesired cell-toxic substances.
  • microbe is a collective term and stands for a wide variety of microorganisms including bacteria, viruses, protozoa and fungi etc.
  • the term signal describes any form of tapping information from the test batch which is suitable for evaluating the anti-microbial activity and tolerance of the potential active compounds, e.g. a signal can be referred to as a signal, which corresponds to the vitality of the cells and thus indicates the anti-microbial activity of the test substance and its tolerance.
  • prokaryote describes an organism that, unlike eukaryotic cells, has no nucleus. In this context, it also includes genetically engineered prokaryotes, such as those that produce a reporter gene product.
  • eukaryotic cell describes an organism that, unlike prokaryotes, has a nucleus. In this context, it also includes genetically engineered eukaryotes, such as those that produce a reporter gene product.
  • the abbreviation MIC is a synonym for the German name MHK (Minimum Inhibitory Concentration) stands for “Minimum Inhibition Concentration” and describes the lowest concentration of a compound that is still able to completely inhibit bacterial growth, for example, with reference to visual turbidity the
  • Gram-positive and Gram-negative describe the result of the so-called Gram staining, which is only one of a large number of published staining methods for microorganisms to examine samples from organisms. (Principles and Practice of Infectious Diseases, GL Mandall, RG Douglas, JE Bennett ed., Churchill Livingstone Inc., 1995)
  • SI selectivity index
  • simultaneous with reference to the infectious assay refers to a situation in which the active substance, microbe and eukaryotic cells are simultaneously incubated in one solution at a time; however, the sequence of adding the components is variable.
  • TI therapeutic index
  • LD lethal dose
  • ED effective dose
  • vital dyes refers to dyes that allow a distinction to be made between vital and non-vital cells, e.g. by only penetrate into non-vital cells (tryp blue) or are only converted by intact cells (e.g. fluorescein diacetate). Fluorescent dyes are preferred.
  • the method is characterized in that
  • Active substances are selected for which the signal of the corresponding test rate is greater than 2 times, particularly greater than 4 times, very particularly greater than 10 times the mean value of the infection control and in particular greater than 50% of the cell control.
  • the method is characterized in that the non-viral microbes are prokaryotic cells.
  • the prokaryotic cells are preferably bacteria, in particular intracellular and or therapy-resistant microbes.
  • Fluorescent dyes in particular fluorescein diacetate, fluorescein dibutyrate, 5-carboxyfluorescein diacetate, acetoxymethyl ester (5-CFDA, AM) and 4,5,6,7-tetrafluorofluorescein diacetate (TFFDA) are preferred.
  • a preferred embodiment of the invention is characterized in that eukaryotic cells are used which have a so-called reporter gene for generating the test signal.
  • a preferred embodiment of the invention is characterized in that the non-viral microbes, such as genetically engineered organisms, have non-naturally occurring properties.
  • the non-viral microbes such as genetically engineered organisms
  • microbes can be used which have a changed adhesion behavior on cell surfaces or which produce special toxins.
  • a preferred embodiment of the invention is characterized in that the eukaryotic cells are Vero (african green monkey kidney cells) or CHO cells (chinese hamster ovary cells).
  • Another preferred embodiment of the invention is characterized in that a mixture of different microbial strains is used. So can Test substances are tested directly for possible anti-microbial broadband activity.
  • the method is characterized in that a mixture of various eukaryotic cell lines is used.
  • the invention relates to compounds or active ingredients which have been identified by means of the above-mentioned method, to pharmaceutical compositions which contain a compound found in this way, which
  • the method is carried out as high-throughput screening (HTS) at a concentration of the active substance in the concentration range from 100 nM to 100 ⁇ M.
  • HTS high-throughput screening
  • compounds are preferred which have a so-called broad-spectrum spectrum anti-microbial effect.
  • active substances whose mechanism of action is based on a target of the pathogen or pathogen.
  • the method is characterized in that those active substances are selected which have the property of inhibiting microbial growth in cell culture at a concentration of approximately 100 ⁇ M and less by at least 50%.
  • the present invention therefore relates to a method for identifying active substances, characterized in that
  • test substances or control antibiotics are presented at a suitable substance concentration; b) appropriate eukaryotic cells are added; c) microbes are added; d) the assay is incubated; e) a read out or a so-called test signal is generated.
  • any concentration is suitable at which there is anti-microbial activity and a certain tolerance to the eukaryotic cells.
  • concentrations of 250 ⁇ M to less than 1/10 (one tenth) of the MIC concentration or less are usually used.
  • Eukaryotic cells which optionally carry a reporter gene or mixtures of the corresponding cells or cell lines can be used. Any one
  • the number of cells per area can be sown provided that a reasonable quotient of the signals from the cell control and the infection control is achieved.
  • the number of cells sown should not exceed an amount which leads to so-called contact inhibition of the cells during the incubation period; furthermore, cell vitality should not be impaired by the resulting cell metabolism products or nutrient deficiency. Suitable cell numbers allow a clear differentiation between possible cytostatic or cytotoxic effects of the test substances and the contact inhibition or nutrient deficiency as described above.
  • 20,000 eukaryotic cells per well of a 96-well MTP are preferred.
  • Microbes that may have been genetically manipulated, as well as intracellularly or extra-cellularly growing microbial cells or mixtures thereof, are added to the test in the desired multiplicity of infection (moi). It is necessary to have a microbial cell count that exerts a cytopathic effect on the eukaryotic cells directly or indirectly during the incubation period, so that a suitable measurement signal is generated after the corresponding incubation period. A moi of 10 to 0.001 is preferred. Step d)
  • a microtiter plate which consists of a mixture of the assay components, namely the test substance, the eukaryotic cells and the microbes, is incubated under the optimal conditions for the growth of eukaryotic cells for a corresponding period of time in order to generate a suitable measurement signal.
  • the cell culture supernatant is optionally removed, the remaining eukaryotic
  • cells are washed and a suitable dye or the appropriate reagent for generating the signal in the case of the reporter gene-expressing eukaryotic cells is added to generate the measurement signal, which is recorded with the aid of a suitable measuring device or camera system at the corresponding wavelengths.
  • step e) without first removing the cell culture supernatant and or carrying out additional washing steps, for example this is possible if defined cell culture media are used which do not interfere with the generation of the measurement signal.
  • this can also be achieved by using reporter gene-expressing, eukaryotic cells that allow a suitable measurement signal to be recorded after the corresponding incubation time.
  • the vitality of the cells can be measured continuously (on-line) from the time of incubation by providing the cells with a suitable reporter system (eg GFP).
  • a suitable reporter system eg GFP
  • any eukaryotic cell including those that express a so-called reporter gene
  • a mixture of the named eukaryotic cells that are able to generate a suitable test signal and the measurement. of the corresponding selectivity index allow to be used in this assay.
  • the eukaryotic cells can be sown long before the test incubation; preference is given to adding the eukaryotic cells to a microtiter plate suitable for cell culture, which already contains an active ingredient or test substance of a suitable concentration.
  • these eukaryotic cells are then infected with the corresponding inoculum (these are microbes, including those that carry a so-called reporter gene, or hybrids of the microbes that have a cytophatic effect on the infected eukaryotic cells), with a suitable ratio of the multiplicity of infection (moi) allows a significant difference in the test signal (read out) between the eukaryotic cell control or the therapy control (antibiotic control) compared to the infection control.
  • moi multiplicity of infection
  • the test substances can be present in any concentration, ideally in an area that is not toxic to the eukaryotic cells, so that an anti-microbial effect as a result generates a positive test signal.
  • the microbes whether they are Gram-positive or Gram-negative bacteria or intra- or extracellularly growing microbes, are added in an infection dose of (moi about 0.001 to 10). It is possible to vary the moi over a wide range and yet a reasonable test signal can be generated, but a change in the infection dose or the moi can also affect the IC 50 and or the maximum test Increase signal accordingly or shift to lower values.
  • the assay is then incubated for a suitable period of time under the conditions necessary for cell culture, such as temperature, atmosphere, etc.
  • test signal or read out is generated.
  • a large number of fluorescent dyes can be used for various cell lines, while cell lines with reporter genes such as luciferase, green fluorescent protein (GFP), alkaline phosphatase etc. have to be generated or acquired in individual cases; However, such embodiments can provide better test signal / background ratios or make various steps such as washing steps superfluous.
  • the assay is for Vero cells (growth in medium 199 with Earle's salts supplemented with 5% fetal calf serum (FCS) and 2 mM L-glutamine) and CHO cells (growth in RPMI-1640 medium supplemented with 2 mM L-glutamine and 10%
  • FCS FCS
  • the cells are available from ATCC (American Tissue Cell Culture) or the DSMZ (German Collection for Microorganisms and Cell Cultures). The cells constitutively expressing the reporter gene were produced using standard methods.
  • the reporter-bearing plasmids for luciferase, aequorin and eGFP (green fluorescent protein) as well as the transfection and detection reagents of the reporter genes are available from various commercial suppliers (e.g. Clontech).
  • test substance is placed in an MTP, so that the final concentration for a high-throughput test is 10 ⁇ M or for a gradient
  • the gradient test is particularly suitable for determining the selectivity index. If the substances are dissolved in dimethyl sulfoxide, the solvent should not exceed final concentrations of 1% or better than 0.5%. Then the eukaryotic cells in the corresponding
  • Medium was added (e.g. 20,000 cells per well of a 96 well MTP, or 5,000 cells per well of a 384 well MTP and so on and so on for 1,536 well MTP or other cell culture vessels).
  • the microbes are added at a moi of 0.025.
  • the assay with a total volume of 200 ⁇ l is then carried out for 2 to 3 days, in the case of Vero or CHO cells at 37 ° C. and 5%
  • the MTPs are washed once with 200 ⁇ l PBS (phosphate buffer saline) and the read out or test signal is generated by adding 200 ⁇ l 10 ⁇ g / ml fluorescein diacetate in PBS or the corresponding detection reagents for luciferase or aequorin.
  • the MTP are incubated for 15-45 min at room temperature until a visually visible discoloration of the cell control and the resulting fluorescence signal is measured at an excitation wavelength of 485 nm and an emission wavelength of 538 nm in a suitable measuring device (eg Fluoroskan Ascent from Labsystems).
  • the other test signals are generated according to the manufacturer's instructions for the detection reagents.
  • Table 1 shows typical standardized data of the test described above.
  • Table 2 shows the evaluation of the standardized data of a 96-well microtiter plate from a high-throughput screening. Two hits are marked in bold (B8 and C4), the connections of which meet the selection criteria and are examined further.
  • the percentage value of the test batch is significantly larger than the mean value of the infection control. It is preferred if the significance value is at least 99.95%.
  • Tables 3 and 4 are examples of a simultaneous dose effect and tolerance test over a broad concentration gradient for determining the IC 50 values and the SI.
  • Normalized data of cell control 100%, bacterial control, infection control, and therapy control are listed above.
  • the percentage signal (signal of the therapy or drug test approach) / ' (signal of the cell control) * 100

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
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  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
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  • Urology & Nephrology (AREA)
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Abstract

La présente invention concerne un nouveau procédé et son application pour identifier de nouvelles substances à action anti-microbienne. L'invention se caractérise en ce que, dans une préparation d'essai, des substances potentiellement actives sont mises en incubation simultanément avec des cellules eucaryotes et des microbes non viraux, la viabilité des cellules eucaryotes est déterminée, puis une sélection s'effectue pour choisir les substances actives pour lesquelles le signal de la préparation d'essai correspondante est significativement supérieur à la valeur moyenne des témoins d'infection pour au moins une concentration de la substance active.
PCT/EP2003/008145 2002-08-06 2003-07-24 Procedes pour identifier des substances a action anti-microbienne WO2004015416A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2003255282A AU2003255282A1 (en) 2002-08-06 2003-07-24 Methods for the identification of agents with anti-microbial action

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE2002135967 DE10235967B4 (de) 2002-08-06 2002-08-06 Methode zum Identifizieren von Substanzen mit antimikrobieller Wirkung
DE10235967.9 2002-08-06

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WO2004015416A2 true WO2004015416A2 (fr) 2004-02-19
WO2004015416A3 WO2004015416A3 (fr) 2004-05-06

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010064141A1 (fr) * 2008-12-05 2010-06-10 Christoph Merten Essai
WO2019068817A1 (fr) 2017-10-05 2019-04-11 Innovative Molecules Gmbh Énantiomères de thiazoles substitués utilisés comme composés antiviraux
US10590094B2 (en) 2016-04-06 2020-03-17 Innovative Molecules Gmbh Aminothiazole derivatives useful as antiviral agents

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US5369095A (en) * 1990-02-14 1994-11-29 Alcon Laboratories, Inc. Compositions and method comprising substituted glycosides as mucus membrane permeation enhancers
US6046021A (en) * 1995-04-12 2000-04-04 Biolog, Inc. Comparative phenotype analysis of two or more microorganisms using a plurality of substrates within a multiwell testing device
WO2001000209A1 (fr) * 1999-06-29 2001-01-04 Smithkline Beecham Corporation Methodes d'utilisation de composes fluoroquinolone pour lutter contre des bacteries
WO2001081920A2 (fr) * 2000-04-20 2001-11-01 Biolog, Inc. Analyse comparative de phenotypes pour l'evaluation de composes actifs biologiquement tels que des produits antimicrobiens

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010064141A1 (fr) * 2008-12-05 2010-06-10 Christoph Merten Essai
US10590094B2 (en) 2016-04-06 2020-03-17 Innovative Molecules Gmbh Aminothiazole derivatives useful as antiviral agents
WO2019068817A1 (fr) 2017-10-05 2019-04-11 Innovative Molecules Gmbh Énantiomères de thiazoles substitués utilisés comme composés antiviraux
US11278534B2 (en) 2017-10-05 2022-03-22 Innovative Molecules GmbG Enantiomers of substituted thiazoles as antiviral compounds
EP4209491A1 (fr) 2017-10-05 2023-07-12 Innovative Molecules GmbH Enantiometres d'une serie de composes antiviraux

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WO2004015416A3 (fr) 2004-05-06
DE10235967A1 (de) 2004-02-26
DE10235967B4 (de) 2005-09-08
AU2003255282A1 (en) 2004-02-25

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