WO2013041522A1 - Procédés et dispositifs pour déceler des dépôts de protéines tau dans l'oeil - Google Patents

Procédés et dispositifs pour déceler des dépôts de protéines tau dans l'oeil Download PDF

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WO2013041522A1
WO2013041522A1 PCT/EP2012/068343 EP2012068343W WO2013041522A1 WO 2013041522 A1 WO2013041522 A1 WO 2013041522A1 EP 2012068343 W EP2012068343 W EP 2012068343W WO 2013041522 A1 WO2013041522 A1 WO 2013041522A1
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Prior art keywords
light emission
probes
eye
retina
light
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PCT/EP2012/068343
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German (de)
English (en)
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Michael Kempe
Tobias Schmitt-Manderbach
Jochen Herms
Christian Schön
Boris Schmidt
Daniel Kieser
Alexander BOLÄNDER
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Carl Zeiss Ag
Technische Universität Darmstadt
Ludwig-Maximilians-Universität München
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Publication of WO2013041522A1 publication Critical patent/WO2013041522A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/0059—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence
    • A61B5/0071—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence by measuring fluorescence emission
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B3/00—Apparatus for testing the eyes; Instruments for examining the eyes
    • A61B3/10—Objective types, i.e. instruments for examining the eyes independent of the patients' perceptions or reactions
    • A61B3/1025—Objective types, i.e. instruments for examining the eyes independent of the patients' perceptions or reactions for confocal scanning
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B3/00—Apparatus for testing the eyes; Instruments for examining the eyes
    • A61B3/10—Objective types, i.e. instruments for examining the eyes independent of the patients' perceptions or reactions
    • A61B3/12—Objective types, i.e. instruments for examining the eyes independent of the patients' perceptions or reactions for looking at the eye fundus, e.g. ophthalmoscopes
    • A61B3/1225—Objective types, i.e. instruments for examining the eyes independent of the patients' perceptions or reactions for looking at the eye fundus, e.g. ophthalmoscopes using coherent radiation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/0059—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence
    • A61B5/0062—Arrangements for scanning
    • A61B5/0066—Optical coherence imaging
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/0059—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence
    • A61B5/0075—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence by spectroscopy, i.e. measuring spectra, e.g. Raman spectroscopy, infrared absorption spectroscopy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/40—Detecting, measuring or recording for evaluating the nervous system
    • A61B5/4076—Diagnosing or monitoring particular conditions of the nervous system
    • A61B5/4088—Diagnosing of monitoring cognitive diseases, e.g. Alzheimer, prion diseases or dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/48—Other medical applications
    • A61B5/4842—Monitoring progression or stage of a disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6408—Fluorescence; Phosphorescence with measurement of decay time, time resolved fluorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/0004—Microscopes specially adapted for specific applications
    • G02B21/0012—Surgical microscopes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B3/00—Apparatus for testing the eyes; Instruments for examining the eyes
    • A61B3/10—Objective types, i.e. instruments for examining the eyes independent of the patients' perceptions or reactions
    • A61B3/102—Objective types, i.e. instruments for examining the eyes independent of the patients' perceptions or reactions for optical coherence tomography [OCT]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/0059—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence
    • A61B5/0062—Arrangements for scanning
    • A61B5/0068—Confocal scanning

Definitions

  • the present invention relates to methods and devices for the detection of deposits of the ⁇ protein in the eye for the diagnosis of diseases from the group of so-called tauopathies, for example on the retina of a human eye.
  • tauopathies belong to a group of neurodegenerative diseases that share a pathological deposit of ⁇ -proteins in the brain.
  • the group of tauopathies includes, for example, Alzheimer's disease, Pick's disease, cortico-basal degeneration, frontotemporal dementia or progressive supranuclear palsy.
  • Alzheimer's disease i. Alzheimer's disease, is by far the most common and also the most intensively researched disease of this group and therefore serves as an application example below.
  • Alzheimer's disease the most common form of progressive neurodegenerative disease in the elderly, is about 40% -50% in the 85-90 age group. 60% of people affected by dementia worldwide have Alzheimer's disease. Currently, Alzheimer's can only be diagnosed with certainty after the death of the patient by autopsy of the brain.
  • PHF paired helical filaments
  • the formation of neurofibrillary bundles of x-proteins proceeds approximately as follows: In the course of their formation and accumulation, paired helical filaments of the x-protein first assemble within the cytoplasm, probably from x-oligomers that are truncated during or before PHF formation. This results in classic intracellular neurofibrillary tangles. In this state, the x-PHFs consist of a core of truncated x-proteins and a frayed outer region with complete x-proteins. It is believed that these aggregates result in functional impairment of the affected neurons.
  • MRI magnetic resonance imaging
  • FDG-PET positron emission tomography using fluorodeoxyglucose
  • positron emission tomograms of the brain have been performed using contrast agents (eg, [ 11 C] PIB, [ 18 F] FDDNP) which selectively bind to amyloid plaques and / or x-PHF give direct indication of pathological changes of the nerve tissue.
  • contrast agents eg, [ 11 C] PIB, [ 18 F] FDDNP
  • Such examinations are hardly suitable for large-scale preventive examinations or for continuous monitoring of a therapy.
  • such examinations can not be repeated as often as desired due to the radiation exposure to a patient.
  • x-PHF paired helical x-filaments
  • an efficient examination can be carried out.
  • the areas of interest can be accurately examined by means of laser beam scanning, without a total duration of the investigation becoming unnecessarily long.
  • the regions of interest may in particular be regions in which light emission from the probes can be identified on the overview image.
  • the light emission may in particular comprise a fluorescence emission or an emission of inelastically scattered scattered light, for example by Raman scattering.
  • the method further comprises carrying out supplementary diagnostics, such as polarization-based or optical coherence tomography-based morphological characterization of the retinal neuronal tissue, comprising, for example, a layer thickness measurement of the retinal nerve fiber cell layer or the entire retina.
  • supplementary diagnostics such as polarization-based or optical coherence tomography-based morphological characterization of the retinal neuronal tissue, comprising, for example, a layer thickness measurement of the retinal nerve fiber cell layer or the entire retina.
  • different tauopathies ie the presence of x-PHF, associated diseases can be distinguished, for example eye diseases such as glaucoma of neurodegenerative diseases.
  • the detection comprises a lifetime measurement of the fluorescence, whereby probes bound to paired helical x-filaments can be distinguished from unbound probes.
  • Morphological information can also be obtained via optical coherence tomography, which can be coupled with information from the detection of the fluorescence in order to enable the fluorescence of cellular structures in different layers of the eye.
  • the resolution of the scanning is preferably 20 ⁇ or less, more preferably 10 ⁇ or less.
  • one or more substances are preferably selected from the group comprising arylaminothiazoles, 4,6-divinylpyrimidines, 2,5- and 3,6-divinylpyrazines, [4- (1, 3-benzothiazol-2-yl) phenyl] hydrazones , Diaryl ureas, tetracycline, 4,4 '- (1l, 1' £) -2,2'- (1-aryl-1 / - / - pyrazole-3,5-diyl) bis (ethene-2,1 - diyl) bis-2-methoxyphenols, 2,5-bis (4-hydroxystyryl) benzonitrile and 2,5-bis (4-hydroxystyryl) - / V-benzamides.
  • other probes sensitive to paired helical x-filaments may also be used.
  • FIG. 1 shows a block diagram of an embodiment of a device according to the invention
  • FIG. 2 shows a diagram of a further embodiment of a device according to the invention
  • FIGS. 3 (a) to (j) show various probes usable in embodiments of the invention
  • FIG. 4 shows a flow chart for illustrating an embodiment of a method according to the invention
  • Figure 5 histological sections of a retina of patients with tauopathies
  • Figure 6 in vivo recordings of ⁇ -PHF in retinal ganglion cells of a mouse model of Alzheimer's disease.
  • FIG. 1 shows a block diagram of a device according to the invention for detecting paired helical x-filaments (x-PHT) 107 in an eye 100, for example a human eye.
  • x-PHT paired helical x-filaments
  • probes 108, 109 which have characteristic fluorescence properties and which are sensitive to paired helical x-filaments, i. bind to these administered.
  • the probes 108 are already bound to paired helical x-filaments 107 while a probe 109 is unbound.
  • the apparatus of the exemplary embodiment of FIG. 1 comprises a laser scanner device 102 and a camera device 103, which in the embodiment of FIG. 1 are optically coupled to the eye 100 via a common ophthalmoscope lens 101, in particular a retina of the eye 100 in which x-PHT 107 is embedded, to be able to illuminate and to be able to absorb optical radiation emanating from the retina.
  • the laser scanner device 102 and the camera device 103 are controlled by a controller 104.
  • the controller 104 may be implemented by means of a computer, for example, and in the illustrated embodiment comprises a screen 106 and an input device 105, wherein the input device 105 may comprise, for example, a keyboard and / or a mouse.
  • the controller 104 is set up to control the camera device 103 to record an overview image of the retina of the eye 100.
  • the camera device 103 can have illumination for illuminating the retina, which is adapted to an excitation wavelength of the probes 108, 109 in order to excite the probes 108, 109 for fluorescence.
  • a color filter can be provided for recording the overview image, which essentially allows only light with wavelengths corresponding to the fluorescent light of the probes 108, 109 to pass through the illumination, so that an overview image of the fluorescence of the retina is taken.
  • other types of images for example, multicolor images for identifying structures or infrared images may be included.
  • the controller 104 is further set up to evaluate the acquired overview image and to define one or more regions of interest (ROI), the regions of interest in particular being regions in which fluorescence is displayed on the overview image from the probes 108, 109.
  • ROI regions of interest
  • Such an evaluation can be carried out fully automatically by means of corresponding automatic image processing
  • the selection of regions of interest can also be semi-automatic
  • the overview image can be displayed on the screen 106, with regions having fluorescence from the probes 108, 109 may be highlighted or highlighted, and a user may then select the regions of interest by means of the input device 105.
  • the controller 104 then controls the laser scanner device 102 to scan the regions of interest with a laser beam and from which the ret Ina in response to the laser beam emitted light, in particular fluorescent light to detect.
  • the spatial resolution in such a scanning of the regions of interest is preferably less than 20 ⁇ , more preferably less than 10 ⁇ .
  • the detection can be carried out in particular time-resolved, for example by means of FLI (Fluorescence Lifetime Imaging), and / or spectrally resolved, in order to be able to distinguish bound probes 108 from unbound probes 109, since typically by binding to paired helical ⁇ filaments changes a lifetime and / or a spectrum of fluorescence.
  • FLI Fluorescence Lifetime Imaging
  • the laser scanner device can work in particular as a confocal laser scanning ophthalmoscope (cSLO, Confocal Scanning Laser Ophthalmoscope).
  • cSLO Confocal Scanning Laser Ophthalmoscope
  • optical coherence tomography measurements or polarization-dependent measurements can also be carried out with the laser scanner device 102 in some embodiments in order to obtain morphological information about the retina in addition to a preferably depth-resolved fluorescence distribution.
  • the morphological information and the information obtained from the fluorescence can then be linked by the controller 104 and, for example, displayed on the screen 106 for review, for example by a physician.
  • fluorescence signals from the probes can be assigned in this way to cellular structures in the inner nuclear layer (INL) or the ganglion cell layer (GCL, or "ganglion cell layer”) or other layers.
  • FIG. 2 shows a more detailed diagram of a further exemplary embodiment of the present invention, wherein in particular a laser scanner device and a camera device are shown in detail.
  • a controller such as the controller 104 of FIG. 1 is not explicitly shown, but may be provided as described in FIG.
  • the embodiment of FIG. 2 comprises a confocal laser scanning ophthalmoscope (cSLO), which comprises one or more laser light sources 1, a housing head 41 and a detection device 15.
  • cSLO confocal laser scanning ophthalmoscope
  • the embodiment of FIG. 2 includes a camera device 40.
  • the number and type of the laser light sources 1 can be selected depending on the probes 50 to be detected in an eye 10, such that the one or more laser light sources emit corresponding excitation wavelengths around the probe 50 to stimulate fluorescence.
  • the one or more laser light sources 1 are coupled to the housing head 41 via light guides 2, preferably singlemodige fibers.
  • the laser light emitted by the light guides 2 is collimated by means of collimator lenses 3.
  • the laser light of the different laser light sources in the example of FIG. 2 two laser light sources, is combined via a deflection mirror 4 and a dichroic beam splitter 29 or another optical device into a single illumination beam, which via another deflection mirror 4 to another dichroic beam splitter 30 is directed.
  • the dichroic beam splitter 30 is used to separate an illumination beam path, via which the laser light emitted by the laser light sources 1 is finally directed into the eye 10, from a detection beam path with which light emerging from the eye 10 is directed to the detector device 15. From the dichroic beam splitter 30, the laser light of the illumination beam reaches a scanner mirror 5, which is preferably arranged in a conjugate pupil plane of the optical arrangement.
  • the scanner mirror 5 can, for example, have a biaxial scanner mirror or two uniaxial scanner mirrors connected one after another in order to be able to scan a surface in the eye 10, in particular on a retina of the eye 10, by moving the scanner mirror 5.
  • the scanning movement of the scanner mirror 5 can be controlled by the controller 104 of FIG. 1, for example.
  • the laser light is then focused on a desired layer, in particular the retina or a layer of the retina, in the eye 10.
  • the objective 6 can also be referred to as a scanning objective
  • the objective 7 can also be referred to as a field objective.
  • the beam splitter 8 can, for example, be pivoted in and out in order to be able to selectively use the laser scanner device or the camera device 40.
  • the laser scanner device and the camera device use different wavelengths, and the beam splitter 8 may be configured as wavelength-selective, for example dichroic, beam splitters.
  • the pinhole 12 preferably has a variable size, so that confocality and signal strength are adaptable to the detection device 15, wherein a further opened pinhole causes a poorer confocality at the same time higher signal strength.
  • the light passing through the pinhole 12 is then coupled into a light guide 14, preferably a multimode fiber, which guides the light to the detection device 15.
  • the detection device 15 is used for spectrally resolved detection and / or for time-resolved detection or fluorescence lifetime measurement (fluorescence lifetime measurement).
  • Time Imaging, FLI is set up in order to be able to distinguish bound from unbound probes as already explained.
  • the camera device 40 of the exemplary embodiment of FIG. 2 has an illumination 16, which may comprise one or more light sources, in particular broadband emitting light sources, for example a flash lamp, one or more white light LEDs or combinations of light emitting diodes emitting in different spectral ranges.
  • an illumination 16 which may comprise one or more light sources, in particular broadband emitting light sources, for example a flash lamp, one or more white light LEDs or combinations of light emitting diodes emitting in different spectral ranges.
  • a collimator 17 a lens 18 (field objective), a lens 20 (ring diaphragm lens) and a lens 22, which may be configured in particular as an anti-reflection lens and this may have appropriate coatings gene, and a beam splitter 23, the light passes through the
  • a retinal aperture 19 and a field stop 21 are provided.
  • a spectral range for illuminating the eye 10 can be selected, for example a spectral range in which a fluorescence of the probe 50 can be excited.
  • the beam splitter 23 is designed in the illustrated embodiment as a perforated mirror, so that in the illustrated example, the detection beam path of the camera device as shown "inside" the illumination beam path runs.
  • light passing through the beam splitter 28 is directed via a camera lens 25 to a further camera 26.
  • the further camera 26 may, for example, be an infrared camera, while the camera 27 may be, for example, a color camera. However, it can also be provided two color cameras or two infrared cameras or a black and white camera.
  • a preferably exchangeable color filter 32 is arranged, which can be designed, for example, to place only light in the wavelength range of fluorescence of the probe 50, so that the camera 27 produces an overview image of the fluorescence in the eye 10 can be based on which then as already explained with reference to Fig. 1, the laser scanner device, in particular the scanner mirror 5 is driven can scrape out areas of interest.
  • the further camera 26 can be used for example for tracking movements of the eye 10 or for other settings.
  • the objectives 7 and 24 have a variable focus, so that by means of these lenses on a region of interest of the eye 10, in particular the retina of the eye 10, can be focused.
  • probes for detecting paired helical ⁇ filaments in particular aggregates thereof, it is possible to use, for example, the substances shown in FIG. 3, for example arylaminothiazoles (FIG. 3 (a)), 4,6-divinylpyrimidines (FIG. b)), 2,5-divinylpyrazines ( Figure 3 (c)), benzothiazolylphenylhydrazones (eg, the substance shown in Figure 3 (d)), 3,6-divinylpyridanzines ( Figure 3 (e)), diaryl ureas (Fig .. 3 (f)), tetracyclines (Fig.
  • radicals shown can be, for example, C 1 -C 6 -alkyl radicals.
  • the compounds mentioned preferably act as fluorescent probes. They have a (preferably high) affinity for the Aß protein, ⁇ -synuclein and / or for tau-PHF aggregates and bind - preferably specifically - to these.
  • the binding of the compounds of the invention to one or more of the above target proteins is optically detectable.
  • probes used have sufficient solubility, low lipophilicity (logP ⁇ 5) and selective binding to ⁇ -PHF even in the presence of other ⁇ -sheet aggregates such as ⁇ -amyloid.
  • a dissociation constant of probes used is in an in vitro displacement assay against thioflavin-S or thioflavine-T or auto-displacement assay against radiolabeled analogs at IC / EC 50 ⁇ 200 nM.
  • One-photon fluorescence excitation of the probes bound to ⁇ -PHF is preferably between 390 and 550 nm, although other wavelengths are also possible then the corresponding excitation wavelength, for example by the laser light sources 1 or the illumination 6, are adjusted accordingly.
  • substances used as probes preferably have 3 or more of the following properties a) -f): a) a greater than five-fold, preferably greater than ten-fold, increase in absorbance at a detection wavelength after binding to ⁇ -PHF aggregates (or also to ⁇ -amyloid ) response in comparison with the unbound state, b) a Stokes shift of> 10 nm, preferably> 20 nm, c) an extinction coefficient e> 5000 L-mo 1-cm "1, preferably greater than 10,000 L-rmol" 1 -cm "1 , d) EC50 ⁇ 400 nM, preferably ⁇ 200 nM in a displacement assay against fluorescent reference probe or in the auto-displacement assay against the radiolabeled compound e) solubility / lipophilicity with logP ⁇ 5, preferably between 1 and 2.8, and f) a topological polar surface area (TPSA) ⁇ 200 ⁇ 2 , more preferably ⁇ 100 ⁇
  • the abovementioned increase in absorbance upon binding to x-PHF indicates an improvement in the signal-to-noise ratio and can be experimentally determined, for example, by changing, for example reducing, background noise.
  • the determination of the extinction coefficient E can be measured for example at 25 ° C, a pH of 7, at a respective absorption maximum of the respective substance with DMSO (dimethyl sulfoxide) as a solvent.
  • the Stokes shift denotes a difference between excitation (excitation) maximum and emission maximum. A larger Stokes shift facilitates separation of excitation light and fluorescence, for example by the dichroic beam splitters or color filters mentioned above.
  • the abovementioned EC50 value characterizes the affinity of the respective probe for the substance to be detected, for example ⁇ -PHF, and can be determined indirectly, for example by displacing fluorescent or radioactive reference ligands, for example displacement of thioflavin S, thioflavin T or 1 1 C-PIB.
  • a suitable measuring method is described, for example, in Lockhart, et al., The Journal of Biological Chemistry, 280, 7677-7684 of March 4, 2005, under Material and Methods.
  • probes for example the abovementioned arylamino-notyazoles, 4,6-divinylpyrimidines, 3,6-divinylpyridazines, 2,5-divinylpyrazines, [4- (1,3-benzothiazol-2-yl) phenyl] hydrazones and / or diaryl ureas 2, preferably at least 3 aromatic rings, which are bonded together directly via a vinyl bridge or a urea bridge.
  • used materials 3 may have aromatic rings, which are connected to each other via vinyl bridges, resulting in extended p-electron systems.
  • compounds of the abovementioned classes with delocalized electrons are used in particular via at least 15 atoms involved, for example at least 20 atoms, in particular 22 or more atoms.
  • probes whose conformation in the excited state are stabilized by binding to the target protein.
  • This stabilization may be by hydrogen bonding or van der Waals interactions of the aryl moieties with the target protein.
  • Such twisting can be induced by 1,3-allyl stress or by 1,4-butadiene voltage alkylated vinyl aromatic compounds.
  • Particular preference is furthermore given to compounds which have a half-life in vivo of> 60 min.
  • residence time and excretion rate of correspondingly labeled probes eg 3H, 11C, 18F
  • Preference is given to compounds which have an increased potential brain activity with reduced binding to white brain mass and have reduced plasma protein binding.
  • the diffusivity of a compound through the endothelium of the blood-brain barrier is largely determined by its lipid solubility (lipophilicity) and size.
  • the compounds of the invention have a molecular weight ⁇ 500 g / mol.
  • the log P value and the log D value are model measures of the ratio between lipophilicity (fat solubility) and hydrophilicity (water solubility) of a substance.
  • the expectation is to be able to estimate the distribution coefficients of this substance in other systems with an aqueous and a lipophilic phase with the help of the octanol-water partition coefficient.
  • the log P value is greater than one if a substance is more soluble in fat-like solvents such as n-octanol, less than one if it is better soluble in water. Accordingly, log P value is positive for lipophilic and negative for hydrophilic substances. Preference is given to compounds which have a log P value of from 1 to 2.8.
  • compounds with a log D ⁇ 5 are preferred.
  • the log P value or the log D value are measured by means of an octanol / water two-phase system and UVA / IS spectroscopy at 25 ° C. and pH 7. Since not all of the chemicals have the log P value and / or the log D value can be measured, there are other models for the prediction, eg by Quantitative Structure-Activity Relationships (QSAR) or by Linear Free Energy Relationships (LFER).
  • QSAR Quantitative Structure-Activity Relationships
  • LFER Linear Free Energy Relationships
  • TPSA topological polar surface area
  • the compounds of the invention are still characterized by good photostability (low photobleaching) and short-lived singlet excitation versus long-lived triplet excitation.
  • FIG. 4 shows a flowchart for illustrating an embodiment of a method according to the invention.
  • the method of Fig. 4 may be performed using the devices of Figs. 1 or 2, but may be implemented independently thereof.
  • a step 140 for example, sensitive probes are supplied to a human patient or to an animal to be examined, for example one of the probes explained with reference to FIG. 3. These probes attach themselves after a certain time to ⁇ -PHF in the eye.
  • step 141 an overview image of an ocular fundus is then created with a corresponding camera device such as the camera devices explained with reference to FIGS. 1 and 2.
  • a wavelength of illumination and a detection by means of a camera for example by means of color filters such as the color filters 31 and 32 of FIG. 2, can be spectrally adjusted to the excitation wavelength or emission wavelength of the corresponding probe.
  • the fundus of the eye When creating the overview image, the fundus of the eye is generally excited to fluorescence over a large area.
  • various measures can be taken.
  • the fundus may be illuminated in a structured manner, resulting in lighter and darker areas on the ocular fundus. From intensity measurements in the darker areas, a spatially resolved background signal can be determined as a false light distribution via the camera, which is subtracted from a picture of the fundus taken later and evenly illuminated. Thus, a picture of the fundus essentially free of false light can be generated.
  • the spatial resolution corresponds to the resolution of the camera used as the camera 27 of FIG.
  • two different light patterns are preferably generated in such a way that the bright areas of the two light patterns complement each other to form a complete gapless image (light image) and the darker areas of the two light patterns also complement each other without gaps to form an image (dark image).
  • scattered light distribution scattering centers in an entire cross section of the beam path of the camera device can be taken into account.
  • such a scattered light distribution can be determined with high accuracy. Details on corresponding methods of scattered light determination can be found, for example, in DE 103 30 716, DE 103 47 389, DE 10 2004 053 730 and DE 10 2006 031 177.
  • regions of interest are then defined, in particular regions in which fluorescence can be detected on the overview image. This can be done fully automatically by image analysis or semi-automatically involving user input.
  • a fluorescence lifetime and / or a spectral fluorescence distribution are already determined when the overview image is generated in order to be able to distinguish bound from unbound probes, since binding of the probes to ⁇ -PHF is particularly slow Component of an exponential decay of fluorescence decay behavior of the fluorescence influenced.
  • a lifetime measurement can be accomplished, in particular, by combining the illumination at at least two frequencies, preferably in the megahertz range, and using a runtime camera as the camera.
  • fluorescence from the probes of interest can also be distinguished from other fluorescence sources, for example autofluorescence of the ocular fundus.
  • the regions of interest can then be created in step 142 based solely on fluorescence bound to x-PHF.
  • step 143 the regions of interest defined in step 142 are then scanned with a laser and fluorescence detected.
  • a con- focal laser scanning ophthalmoscope as shown in Fig. 2 are used.
  • pixel-by-pixel spectral detection can take place on at least two channels during this image recording.
  • structures can then be segmented, ie identified, which have features which resemble or correspond to features of paired helical ⁇ -filaments.
  • a lifetime measurement may be performed, in which case a number of data points in some embodiments are limited to one or a few data points in the regions of interest and adjacent regions or in the structures mentioned above having the x-PHF-like features and adjacent regions.
  • the presence and binding of the probe to helical x-filaments can be detected analogously to the molecule by a microspectroscopic examination, eg Raman spectroscopy.
  • Such measurements of scattered light from inelastic scattering, eg, Raman scattered light may be used in some embodiments as an alternative or in addition to fluorescence measurements to detect the probes.
  • an optical coherence tomography image can be taken with a corresponding laser scanner device.
  • areas of interest which may be relatively small, and restricting the lifespan, spectroscopic measurements to only a few data points, a relatively short examination time can be achieved, which is more comfortable for a patient and at the same time a closer examination of the actual areas of interest.
  • step 143 Various possibilities and variants of the implementation of step 143, in particular the measures mentioned above, will now be explained in more detail.
  • a serial point-like excitation of the fluorescent objects takes place, wherein the point-shaped excited field is confocally imaged in a field stop, which is preferably arranged in front of a detector, preferably a single detector. From successively detected light from each point, a structure of a two-dimensional image of the fluorescent object takes place after passing through a notch filter. Due to the confocality, which can be influenced in the embodiment of FIG. 2, for example, by the size of the pinhole 12 is the effect of fluorescence or scattered light from layers that are not in the focal plane of the excitation or imaging system is greatly reduced.
  • the laser wavelength is chosen in particular to match the probe used, i. a laser wavelength is selected in which the probe has an increased absorption and / or scattering and / or excitation for fluorescence or bioluminescence.
  • the laser wavelength can be in the visible or infrared spectral range.
  • a spectrally resolved detection for example for the abovementioned "spectral unmixing" to distinguish bound probes from unbound probes or other fluorescence sources, can for example be spectral splitting of the light and simultaneous detection in several channels, for example in the detection device 15 of FIG. 2.
  • To determine the fluorescence lifetime imaging it is possible, for example, to use a time-correlated single-photon count, as described, for example, in DE 199 20 158 or DE 101 45 823.
  • a reflection image can be recorded simultaneously with fluorescence measurements, on the basis of which, for example, a compensation of eye movements between the individual measurements, for example measurements at different points or between spectrally resolved measurement and life-time measurement, can take place.
  • a supplementary diagnostic can be performed in step 144, whereby this can also be carried out in parallel to the preceding steps.
  • an autofluorescence detection can take place, or other types of measurements can be carried out, such as optical coherence tomography measurements, for example in order to additionally detect eye diseases, especially eye diseases associated with neurodegenerative diseases.
  • the information obtained by the method according to the invention of FIG. 4 can then be used for diagnosis.
  • the data can be stored, for example, with data from previous examinations on the same patient or data from a data Bank can be compared.
  • Steps 141 and 143 of FIG. 4 a movement of the patient and thus of the eye to be examined can be detected and taken into account, whereby such a movement can be detected by the infrared camera 26 of FIG. 2, for example.
  • an ante-mortem diagnosis of tauopathies can be carried out by the illustrated methods and devices according to the invention, a course of the disease can be monitored and / or a corresponding therapy can be controlled.
  • FIG. 1 histological sections of the retina of patients suffering from two different tauopathies are shown in FIG.
  • AD Alzheimer's disease
  • PSP progressive supranuclear palsy
  • FIG. 6 shows in vivo images of ⁇ -PHF in retinal ganglion cells of a mouse model of Alzheimer's disease. On the left, a photograph is shown during a preliminary examination. On the right, a photograph is shown 48 hours after the administration of a specific probe (FSB). The illustrated laser scanning ophthalmoscope image shows brightly marked ⁇ -PHF deposits. From this it can be seen that the described devices and methods are suitable for the detection of ⁇ -PHF in a retina.
  • FSB specific probe

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Abstract

L'invention concerne des procédés et des dispositifs pour déceler la présence de paires de filaments τ en hélice τ-PHF dans l'œil (100), procédés selon lesquels une vue d'ensemble est réalisée par exemple au moyen d'un appareil de prise de vues (103) et des zones identifiées à examiner sont balayées au moyen d'un scanner à laser sur la base de la vue d'ensemble pour détecter la fluorescence de sondes (108) liées aux paires de filaments τ en hélice
PCT/EP2012/068343 2011-09-20 2012-09-18 Procédés et dispositifs pour déceler des dépôts de protéines tau dans l'oeil WO2013041522A1 (fr)

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DE102016125524A1 (de) * 2016-12-22 2018-06-28 Arnold & Richter Cine Technik Gmbh & Co. Betriebs Kg Elektronisches Mikroskop
DE102022131803A1 (de) 2022-11-30 2024-06-06 Rheinische Friedrich-Wilhelms-Universität Bonn, Körperschaft des öffentlichen Rechts Verfahren und Bildgebungseinrichtung zur in-vivo Erfassung von Autofluoreszenz eines Augenhintergrundes

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