WO2009021600A1 - Détermination du facteur de croissance de l'endothélium vasculaire humain - Google Patents

Détermination du facteur de croissance de l'endothélium vasculaire humain Download PDF

Info

Publication number
WO2009021600A1
WO2009021600A1 PCT/EP2008/006004 EP2008006004W WO2009021600A1 WO 2009021600 A1 WO2009021600 A1 WO 2009021600A1 EP 2008006004 W EP2008006004 W EP 2008006004W WO 2009021600 A1 WO2009021600 A1 WO 2009021600A1
Authority
WO
WIPO (PCT)
Prior art keywords
fluorophore
inhibitor
growth factor
vascular endothelial
endothelial growth
Prior art date
Application number
PCT/EP2008/006004
Other languages
German (de)
English (en)
Inventor
Martin KÜHNER
Original Assignee
Carl Zeiss Meditec 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 Carl Zeiss Meditec Ag filed Critical Carl Zeiss Meditec Ag
Publication of WO2009021600A1 publication Critical patent/WO2009021600A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
    • A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
    • A61K49/0032—Methine dyes, e.g. cyanine dyes
    • A61K49/0034—Indocyanine green, i.e. ICG, cardiogreen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
    • A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
    • A61K49/0041—Xanthene dyes, used in vivo, e.g. administered to a mice, e.g. rhodamines, rose Bengal
    • A61K49/0043—Fluorescein, used in vivo
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
    • A61K49/0052—Small organic molecules
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
    • A61K49/0058—Antibodies
    • 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
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/475—Assays involving growth factors

Definitions

  • the invention relates to the detection of the human vascular endothelial growth factor, and more particularly to a means for detecting said messenger, a method of making such an agent, a use of an inhibitor of the vascular endothelial growth factor to produce an agent for detection, use of such an agent as well as methods for detecting human vascular endothelial growth factor.
  • the messenger vascular endothelial growth factor plays a major role; it is also sometimes referred to in the literature as the Vascular Epithelial Growth Factor.
  • the usual abbreviation for this messenger substance is VEGF; It will also be used here.
  • VEGF occurs in diseases always z. T. increased to when blood vessels are formed. This may for example be the case with eye diseases, z. As in the wet age macular degeneration, but also in certain tumors, eg. B. colon cancer.
  • VEGF can serve as an early indicator since it is present at a time when (unwanted) growth has not yet occurred. As an example, this will be explained using the example of the wet age generation of morbulas.
  • Macular degeneration occurs a loss of function of the retina in the area of the macula lutea
  • the formation of the vessels is stimulated by the VEGF, which is therefore present in the affected areas in increased concentration.
  • fluorescein angiography is currently standard. It allows to detect whether vessels have grown into the retina. The diagnosis is therefore only possible if the ingrowth of the vessels and thus the retinal damage has occurred. It is therefore of interest to undertake an early diagnosis.
  • WO 2006/073314 A1 detects fluorescently labeled markers as ligands which react with receptors from the vascularization chain.
  • the problem here is that the statement on the existence of such receptors with the activation of vascularization, ie the actual vascular formation, has little to do, since the detected receptors are also present in normal endothelial cells, ie without vascular growth.
  • US 6371615 B1 proposes to detect autofluorescence and to measure the specific autofluorescence signature of the fundus area-wide, with the aim of concluding the presence of some substances characterizing disease progression.
  • the signature of visible substances is very molecule-specific, an indelible threshold for the intensity or distribution of autofluorescence has not yet been determined. It is also not clear how he could be determined.
  • the metrological design is very expensive.
  • fluorescently labeled or “labeled” is understood to mean the binding of a fluorophore to a substance. Including a variety of binding options in question, in particular a covalent bond, a complex formation, etc.
  • Hetero Fluorescence Resonance Energy Transfer also referred to as hetero-FRET, described, for example, in Lackowicz, "Principles of fluorescence microscopy", Plenum Publishers, 1999
  • One dye is optically excited and, because of its close proximity to the second dye, transmits vibrational energy and thus excitation to the second dye, which then optically emits light in another spectral range.
  • Resonance Energy can only occur if the two dyes are very close to each other, ie both are bound as ligands to the marker Unbound dyes can not carry the energy transfer, and then there is no emission of fluorescence radiation through the second fluorescence molecule.
  • the homo-FRET ultimately measures the lifetime of fluorescence anistropy, as described in the previously cited Gautier et al. via a time-resolved detection.
  • a phase measurement is described by Clyton et al., Dynamic
  • a means for detecting human VEGF comprising an inhibitor of VEGF to which a fluorophore is bound.
  • the invention uses as a marker a combination of a pharmaceutically active substance which acts as an inhibitor of VEGF with a fluorophore and thereby obtains in a surprisingly simple manner a marker for VEGF.
  • the procedure according to the invention makes it possible to use preferably an active substance which has already been authorized for human use.
  • a fluorophore is now bound to this inhibitor, and it is very advantageous that fluorophores already approved for human use are also available here.
  • Inhibitors of VEGF are already used in active ingredients for the treatment of wet age macular degeneration.
  • Examples of such inhibitors are ranibizumab, pegaptanib or bevacizumab.
  • no authorization procedure has to be carried out at all, which is why their use is particularly preferred. This applies quite fundamentally, of course, for the use of already approved inhibitors.
  • fluorescent dyes such as fluorescein or indocyanine green are already known and approved as approved fluorophores. It is therefore preferred a development in which such approved substances are used or the fluorophore has these substances. , ,
  • fluorophores of different substance classes are used for the hetero-FRET. It is therefore preferred a mixture for the agent in which the indicator in each case a fluorophore of the first kind and a fluorophore of the second kind is bound.
  • the fluorophores may be, for example, fluorescein or indocynine green.
  • the finding according to the invention makes it possible in a simple manner to prepare a means for detecting the human VEGF, so that according to the invention it is also intended to use an inhibitor of VEGF for the preparation of such an agent, a fluorophore being bound to the inhibitor.
  • inhibitor and fluorophore may include the substances already mentioned above.
  • the described agent can preferably be used as a medical diagnostic agent.
  • a substance that was actually developed and preferably also approved as a pharmacologically effective therapeutic agent is now being used as a medical diagnostic agent.
  • the use may relate in particular to age-related macular degeneration or intestinal tumors.
  • the invention further allows to detect the human VEGF in a method in which an agent of the previously described kind is added to a sample previously taken and a reading for the binding of the agent to the VEGF by fluorescence resonance energy transfer is determined on the sample.
  • the method provides for the use of the agent for in vitro detection of VEGF.
  • an in vivo detection is possible, so that a patient of an agent of the type described is injected and in vivo of a tissue to be examined by fluorescence resonance energy transfer, a measurement for the binding of the agent is determined to the VEGF.
  • an in vivo application is not limited to the described embodiments of the eye, but may, for example, in the context of an endoscopic examination, for example in the diagnosis of colon cancer, made.
  • the procedure according to the invention has the advantage that, in addition to significantly simpler approval of the diagnostic agent for human use, it is also ensured that a therapeutic agent is also available for diseases detected using the diagnostic agent according to the invention, since the inhibitor used for the preparation of the diagnostic agent can be used.
  • the VEGF diagnosis according to the invention further allows early diagnosis and therapy at a time before physiological deficits appear, thus the effect of VEGF already has negative consequences.
  • the approaches described above for the detection of wet age macular degeneration have problems with creating a comparison value which makes it possible to determine whether VEGF is not present in the examined tissue.
  • the method according to the invention can now be developed simply if an agent of the type mentioned is used, which is obtained using a fluorophore or has a fluorophore whose excitation and emission spectrum overlap.
  • a comparison value for the binding of the inhibitor to VEGF with an excitation wavelength is determined, which is above the wavelength of the intersection of excitation and emission spectrum of the fluorophore and lying above the measured value comparison value on the absence of VEGF in examined tissue is closed.
  • FIG. 1 is a simplified representation of a means for detecting the VEGF
  • FIG. 4 shows rotations of dipole moments that may occur in the device of FIG. 1 during detection
  • 5 is a schematic representation of an excitation and an emission spectrum, which are used for a comparison measurement, , ,
  • FIG. 6 shows a detection device for detecting the VEGF using the means of FIG. 1 by utilizing the hetero-FRET, FIG.
  • FIG. 7 is a detection device similar to FIG. 6,
  • Fig. 8 is a detection device similar to FIG. 6, but to use the homo-FRET and
  • FIG. 9 shows a device similar to FIG. 8.
  • VEGF molecularly-specific dimeric signaling agents
  • the molecular-specific detection consists in the fact that the agent binds to the dimeric signal substance on the one hand and has fluorescence properties on the other hand, which enables a fluorescence measurement or even imaging in the bound state.
  • Two diagnostic agent molecules bind to the VEGF and then show (and only then) certain fluorescence properties.
  • FIG. 1 schematically shows this state.
  • reference numeral 1 denotes the dimeric signal substance VEGF, which has a weight of 40 kD.
  • the diagnostic agent was prepared by binding the fluorophore or fluorophores (this difference will be discussed below) to the inhibitor.
  • all substances which bind to them for inhibiting the activity of the dimeric signal substance are suitable as inhibitors.
  • known inhibitors are ranibzizumab, pegaptanib or bevacizumab.
  • Their basic mode of operation and their structure are described, for example, in the publication Kowanetz / Ferrara, "Vascular Endothelial Growth Factor Signaling Pathways: Therapeutic Perspective", Clin. Cancer Res. 2006, 12 (17), p5018-5022, because of the relatively high molecular weight typically 150 kD, the binding of the fluorophore to the inhibitor is not critical since the latter has many possible binding sites.
  • fluorophores are substances already approved for human use, such as fluorescein or indocyanine green. Whether different , ,
  • Dyes used essentially depends on the detection method with which you want to use VEGF using the diagnostic agent.
  • the diagnostic agent will comprise a mixture consisting of the first dye-bound inhibitor and the second dye-bound inhibitor. This situation is illustrated in Fig. 1, in which different fluorophores 4 and 4 "are used, The fluorophores then act as donor dye or acceptor dye in the case of the hetero-FRET The detection is therefore possible only in that shown in FIG state when two diagnostic agent molecules are attached to a VEGF molecule.
  • the curve 5 represents the excitation spectrum of the donor dye, ie the fluorophore 4.
  • a fluorophore 4 excited by optical illumination radiation in this spectral range emits in a spectrum that is plotted as an emission spectrum 6 in Fig. 2, which shows the intensity I of the radiation as a function of the wavelength ⁇ .
  • the emission spectrum 6 of the donor dye overlaps with an excitation spectrum 7 which has the acceptor dye acting as a fluorophore 4 '. This in turn emits in the emission spectrum 8, which is once again shifted to the red.
  • An excitation in the spectral region of the excitation spectrum 5 then leads (and only then) to an emission in the region of the emission spectrum 8 when donor dye 4 and acceptor dye 4 'are in close proximity to one another.
  • This small distance designated d in Fig. 1, exists only when the respective molecules, i. Compounds of inhibitor 3 with fluorophore 4 or inhibitor 3 with fluorophore 4 ', both docked to the dimeric signaling substance VEGF. Only then is the distance so small that the illumination radiation radiated in the excitation spectrum 5 leads to an emission in the emission spectrum 8. Any emission in this spectral range thus indicates that VEGF is present with two diagnostic agent molecules attached to it. Diagnostic agent molecules, however, are not bound to VEGF, have no fluorophores 4 and 4 'at a distance d, so that the transmission of the emission spectrum 6 in the excitation spectrum 7 does not work there. The proof is therefore highly specific.
  • the ratio of emission without binding e.g. Emission in the emission spectrum 6 and e.g. Emission with binding, ie emission in the emission spectrum 8 determined. If the ratio exceeds a certain minimum value, VEGF is present.
  • the binding probability of the diagnostic agent for a molecule having fluorophore 4 is substantially equal to that of a molecule having fluorophore 4 ', only half of the VEGF molecules to which two diagnostic agent molecules are bound contribute to signaling, as to the other half statistically, two diagnostic agent molecules , ,
  • the diagnostic agent then consists of the inhibitor with only one dye.
  • the fluorophores 4 and 4 1 of FIG. 1 are therefore identical.
  • a polarization anistropy can be exploited, which occurs when two diagnostic agent molecules bind to the VEGF. 4 shows by way of example the positions of the dipole moments. Fig. 11 is the dipole moment at excitation. Without FRET, the dipole moment of the dye due to molecular rotation, e.g. rotated into the dipole momentum layer 12; with FRET, however, much further into the dipole moment position 13.
  • FIG. 6 schematically shows the structure of a detection device 17 using the hetero-FRET on the eye 18 or on the retina of the eye 18.
  • the radiation source emits in a spectral range which lies in the aforementioned excitation spectrum 5. This spectral property of the illumination radiation is optionally ensured by a suitable excitation filter 20.
  • a suitable excitation filter 20 About an unspecified partially transparent deflection mirror, the illumination radiation is directed to the eye 18 and there to the retina. An image of the retina is obtained through the partially transmissive mirror, _ _
  • a dichroid 21 in two spectral ranges. These are the spectral regions of the emission spectra 6 and 8. Suitable emission filters 22 and 23 ensure that only the radiation from the respective spectral region falls on downstream receivers 24 and 25 during imaging.
  • a comparison of the signals of the receiver 24, which receives the radiation from the spectral region of the emission spectrum 6, as well as the receiver 25, which receives the radiation from the spectral region of the emission spectrum 8, allows a statement whether VEGF is present with bound diagnostic agent molecules in the sample.
  • the structure according to FIG. 6 is comparatively simple since only one excitation wavelength or only one spectral range is required for the illumination radiation.
  • Another advantage is that the image of the retina or of the area of interest on the eye 18 (or another object to be examined) already contains the required location information, so that in the image the fluorescent sites can be clearly assigned to the detection of VEGF.
  • Fig. 7 shows a construction similar to Fig. 6, but the detection device 32 shown here is designed for the utilization of homo-FRET. Components that correspond to those of the detection device 17 from FIG. 6 in terms of construction and / or function in the detection device 32 of FIG. 7 are given the same reference numerals and therefore may not be explained again.
  • the detection device 32 has a radiation source 33 which emits at at least one wavelength. This emitted illumination radiation is passed through a polarizer 26, which specifies a polarization direction. About the partially transparent mirror is the
  • a birefringence compensator 40 can be used which compensates for birefringence of the cornea and the retina.
  • Birefringence compensator 27 is described, for example, in US 50303709, which describes a fixed birefringence compensator.
  • Capacitor is known from US 2002051333 A2 and can also be used.
  • Birefringence capacitor whose structure, application, function and advantages are fully incorporated herein.
  • Fluorescence radiation at the eye is then transmitted via an optionally polarizing beam splitter 29, which has an emission filter 28 (with filter properties for the selection of the _ u _
  • Emission spectrum 6 of Figure 2 upstream, again shown on two receivers 24, 25, which corresponding analyzers 30 and 31 are connected upstream.
  • the detection device 32 thus evaluates a polarization anistropy via the ratio of dipole moment position 12 and dipole moment position 13 of FIG. 4. Depending on this ratio, the presence of VEGF in the imaged sample is determined.
  • FIG. 8 schematically shows a detection device 32 for detecting homo-FRET on the basis of a lifetime measurement.
  • the radiation source 33 is now designed as a pulsed source which conducts pulsed illumination radiation to the eye 18. If necessary, the radiation source 33 can emit in a desired spectral range. A portion of the pulsed radiation is passed via a beam splitter 34 to a receiver 35 whose signals are read out by a pulse correlator 36. The remaining part of the illumination radiation reaches the eye 18 and excites fluorescence there. The fluorescence radiation is conducted via an emission filter to a receiver 38 whose signals are likewise read out by the pulse correlator 36.
  • a downstream evaluation unit now measures the lifetime and determines whether homo-FRET occurred. Since only a single beam for excitation of the eye is provided in the embodiment described here for FIG. 8 in order to be able to realize a higher excitation energy in the illumination radiation, the eye 18 is preceded by a scanning unit 40 which scans the region of interest of the sample. in which the position of the illumination spot on the sample is adjusted. By using corresponding high-energy illumination sources and a corresponding imaging optics and a corresponding planar receiver 38 can of course also be used without scanning; the scanning unit 40 is then omitted.
  • Fig. 9 shows a construction for detecting homo-FRET by means of phase measurement.
  • Elements of the corresponding detection device 32 which correspond to elements of previously described detection devices, are again provided with the same reference number and may not be explained again.
  • the radiation of the light source 33 is then modulated, eg by means of a modulator 41, with a frequency, for example by the current of the light source being modulated by the modulator 41 accordingly.
  • the modulator 41 outputs a signal via the modulation to a frequency- and phase-sensitive signal filter, which can be implemented, for example, as a lock-in detector 42.
  • the lock-in detector 42 evaluates the signal from the sensor 38 and determines the phase shift that the fluorescence radiation in the imaged eye 18 with respect to , ,
  • the downstream evaluation unit receives a signal on correlation amplitude and phase, which ultimately takes place the phase measurement.
  • the measured quantity is therefore the polarization anistropy, which can be obtained, for example, by the difference between intensity in parallel and perpendicular polarization divided by the sum of the intensity of the parallel polarized radiation and the double intensity of the perpendicularly polarized radiation.
  • the measured variable is the lifetime, in the case of the detection device according to FIG. 9 the phase.
  • the binding of the diagnostic agent to VEGF as the cause of the recorded fluorescence radiation can be recognized by the fact that the measured variable is above a certain threshold. In preferred developments, however, it is also possible to obtain a zero value for referencing, which gives a measure of the fluorescence that arises without binding of the diagnostic agent to VEGF.
  • Weber redshift is used (also referred to in the English literature as Weber Red Shift). This shift means that the emitted fluorescence radiation is regularly longer-wave than the illumination radiation, which triggers the emission as excitation radiation.
  • the corresponding excitation spectrum 14 and the red-shifted emission spectrum 15 are excited. The excitation radiation emitted can all be exploited in the FRET process to generate emission radiation in the emission spectrum 15.
  • the emission spectrum 15 is zero below the wavelength ⁇ 1, so that for no portions of the emission spectrum 15, the excitation wavelength is already longer wavelength than the emission wavelength to be emitted (which would not be due to the unavoidable redshift). If, however, the excitation wavelength is lengthened, for example, to the wavelength ⁇ 2, there are noticeable portions of the emission spectrum 15 which are shorter than the excitation wavelength. In these areas, therefore, an emission of fluorescence radiation is not possible. The emission intensity is thus significantly reduced.
  • This effect makes use of a preferred embodiment of the detection of VEGF according to the invention, since at sufficiently high excitation wavelength 12 virtually no emission radiation in the spectrum 15 can occur any more. All fluorescence radiation which is then collected and appears in the image thus results from primary fluorescence of the dyes used, from other fluorescence effects or is based on a principally imperfect separation of the excitation radiation from the fluorescence image, but not on the FRET effect.
  • the radiation taken in the image can only originate from the FRET effect to a negligible extent, that is to say caused by diagnostic agent molecules bound to VEGF.
  • Illumination radiation at such wavelengths thus leads to a signal which is the
  • the described detection devices can therefore be designed so that they at two wavelengths
  • Irradiate illumination radiation The radiation sources or filters are therefore designed to fit.
  • Such a configuration is optional and not mandatory, since even for certain purposes, a simple threshold monitoring without referencing to a zero value can be sufficient.
  • the detection of the VEGF dimer in the blood or tissue takes place via a fluorescently labeled inhibitor for VEGF, preferably a pharmaceutical active substance, which binds the dimer at two sites.
  • the binding to the dimer is detected by energy transfer of the fluorophores, either in the form of the hetero-FRET or in the form of the homo-FRET.
  • the ratio of the intensities of the primary and secondary fluorescence emission can be determined in fluorescence excitation of the primary dye, so be used hetero-FRET.
  • a first method according to the invention can thus provide:
  • a VEGF inhibitor or drug with a fluorescent dye Labein a VEGF inhibitor or drug with a fluorescent dye and Labein the same inhibitor with another fluorescent dye, so that the diagnostic agent is a mixture of inhibitor molecules with attached first fluorophore and , ,
  • the diagnostic agent thus obtained is either brought in vivo to the appropriate site to be diagnosed, e.g. by injection (into the bloodstream intravenously or intravitrally) or the diagnostic agent is mixed with a previously sampled sample in vitro.
  • excitation occurs at a first wavelength, which is the excitation wavelength for the first dye.
  • the recording of the fluorescence radiation from the sample then takes place either by image acquisition or by scanning of an area / volume, if imaging evidence is desired. In the case of homogeneous in vitro samples, a non-imaging recording of the fluorescence radiation is generally sufficient. To analyze the image, the corresponding measured variable (either the previously described anisotropy quotient or lifetime or phase) is determined.
  • the data output can take place, for example, in the form of a gray value image or a false color image, with regions having a specific threshold value of the
  • Fluorescence intensity can exceed or fall below, can be marked.
  • an overlay with a classical image of the object ie a mapping that does not address the peculiarities of the VEGF bond with the
  • Diagnosis means is tuned.
  • the corresponding image can be integrated in the detection device.
  • This described first method uses the hetero-FRET for detection and uses in the detection device e.g. already described for Fig. 6
  • a second method uses homo-FRET for detection. Again, the inhibitor is reacted with a fluorescent dye such that the fluorophore is bound to the inhibitor. In contrast to the first method, however, only one fluorophore is required. So there is a diagnostic agent that has the inhibitor with attached fluorophore.
  • a mixture of the inhibitor with different fluorophores bound thereto is no longer required.
  • the steps of producing the diagnostic agent and introducing the diagnostic agent are the same as in the already described methods.
  • the diagnostic agent is brought back into contact with the sample, as already described for the first method.
  • the second method provides for the detection of the binding of the diagnostic agent to VEGF by means of evaluation of the polarization anistropy in homo-FRET.
  • An optionally provided birefringence compensator is therefore, as described in the cited US Patent 2002051333 A2, in the position for maximum compensation of birefringence of cornea and , ,
  • FRET In the presence of VEGF, FRET approximately doubles the lifetime and changes the anisotropy.
  • the anisotropy decreases if, due to the binding of the fluorescently labeled marker to VEGF-, the anisotropy reduction due to slowing down of the rotational diffusion due to the anisotropy reduction is overcompensated due to the extension of the lifetime by energy transfer. This is the case for markers that are much lighter than the VEGF. This fact is accounted for by the reference anisotropy value of the pure diagnostic agent (either in vivo on the normal eye or in vitro).
  • a third method of detection is based on the second method, but uses a reference value currently determined on the relevant sample, which is determined using the Weber redshift already described.
  • the anisotropy ratio is also determined for the second, longer wavelength and the quotient of the two anisotropy ratios is analyzed. If the quotient is close to 1 (or the difference close to zero) then no binding of the diagnostic agent with VEGF has taken place. If the quotient differs substantially from 1 (or the difference of zero), ie the anisotropy ratio is greater at the shorter wavelength, the diagnostic agent bound to VEGF is present in the sample.
  • a fourth method like the second and third methods, also uses homo-FRET for detection.
  • the steps of producing the diagnostic agent and introducing the diagnostic agent are the same as in the already described methods.
  • the polarization anistropy is not determined during image acquisition, but a phase-sensitive analysis is carried out.
  • the fluorescence excitation takes place with a periodically oscillating intensity of the illumination radiation.
  • a correlation amplitude between the periodically oscillating excitation and the fluorescence signal at a fixed phase shift is determined.
  • the phase shift is changed and the measurement is repeated until minimum and maximum intensity have been achieved in the fluorescence image for a given location. From the maximum of the correlation amplitude, one then determines the phase shift between illumination radiation and emission radiation for each location. Is this phase shift _ _
  • the approach of the fourth method may in turn be combined around normalization by utilizing the Weber redshift to determine the reference value as zero value.
  • the comparison therefore does not take place with a value determined on the pure diagnostic agent, but with a phase shift which is determined with longer-wavelength illumination radiation.
  • a sixth method uses the lifetime measurement for homo-FRET-based detection of the binding of the diagnostic agent to VEGF.
  • the steps of preparing the diagnostic agent and introducing the diagnostic agent are the same as in previously described methods 2-5.
  • fluorescence excitation illumination is now pulsed to determine the lifetime for each measurement point in the sample.
  • This sixth method can be modified to a seventh method in that the comparison value is obtained by utilizing the Weber redshift, that is to say by illuminating the sample at a longer wavelength, as already explained above.
  • the evaluation in the case of a spatially resolving image can be averaged over the image or else pixel-wise or ger ⁇ ittelt over certain image areas to identify in the image sections in which VEGF is present.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Biochemistry (AREA)
  • Organic Chemistry (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biotechnology (AREA)
  • Cell Biology (AREA)
  • Genetics & Genomics (AREA)
  • Microbiology (AREA)
  • Endocrinology (AREA)
  • Food Science & Technology (AREA)
  • General Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

L'invention concerne la détermination du facteur de croissance de l'endothélium vasculaire (VEGF) humain. Pour cela, elle propose un agent comprenant un inhibiteur du VEGF auquel un fluorophore est lié. L'invention concerne en outre des utilisations correspondantes de l'inhibiteur, des procédés de préparation, des applications médicales ainsi que des procédés de détermination utilisant l'agent en question.
PCT/EP2008/006004 2007-08-16 2008-07-22 Détermination du facteur de croissance de l'endothélium vasculaire humain WO2009021600A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102007038730.1 2007-08-16
DE200710038730 DE102007038730A1 (de) 2007-08-16 2007-08-16 Nachweis des menschlichen Vascular Endothelial Growth Factor

Publications (1)

Publication Number Publication Date
WO2009021600A1 true WO2009021600A1 (fr) 2009-02-19

Family

ID=39769209

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2008/006004 WO2009021600A1 (fr) 2007-08-16 2008-07-22 Détermination du facteur de croissance de l'endothélium vasculaire humain

Country Status (2)

Country Link
DE (1) DE102007038730A1 (fr)
WO (1) WO2009021600A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3211422A1 (fr) * 2016-02-23 2017-08-30 Justus-Liebig-Universität Gießen Procédé de mesure et de commande de concentration de vegf intraoculaire

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040133357A1 (en) * 2001-04-17 2004-07-08 Abmaxis, Inc. Humanized antibodies against vascular endothelial growth factor
AU2006201128A1 (en) * 2000-03-02 2006-04-13 Vegenics Limited Methods for treating, screening for, and detecting cancers expressing vascular endothelial growth factor D
DE102006030382A1 (de) * 2006-06-29 2008-01-03 Carl Zeiss Meditec Ag Verfahren und Vorrichtung zur optischen Detektion am Auge

Family Cites Families (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5303709A (en) 1991-12-16 1994-04-19 Dreher Andreas W Retinal eye disease diagnostic system
GB9812596D0 (en) * 1998-06-11 1998-08-12 Amersham Pharm Biotech Uk Ltd Energy transfer assay method
US6252756B1 (en) 1998-09-18 2001-06-26 Illinois Tool Works Inc. Low voltage modular room ionization system
CN1330555A (zh) * 1998-12-21 2002-01-09 路德维格癌症研究所 抗截短的vegf－d的抗体及其应用
DE19920158A1 (de) 1999-04-29 2000-11-02 Univ Schiller Jena Verfahren und Anordnung zur Bestimmung von Fluorophoren an Objekten, insbesondere am lebenden Augenhintergrund
ATE315789T1 (de) * 1999-11-16 2006-02-15 Genentech Inc Elisa für vegf
DE602004009169D1 (de) * 2004-07-22 2007-11-08 Bayer Schering Pharma Ag Verwendung von Cyanin-farbstoffen zur Diagnose von Krankheiten, welche mit Angiogenese assoziert sind
US8236283B2 (en) 2005-01-06 2012-08-07 Ge Healthcare As Optical imaging
WO2007038453A2 (fr) * 2005-09-26 2007-04-05 Advanced Ocular Systems Limited Administration d'un agent pour l'amelioration de l'inflammation
US20070111326A1 (en) * 2005-11-14 2007-05-17 Abbott Laboratories Diagnostic method for proteinaceous binding pairs, cardiovascular conditions and preeclampsia

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2006201128A1 (en) * 2000-03-02 2006-04-13 Vegenics Limited Methods for treating, screening for, and detecting cancers expressing vascular endothelial growth factor D
US20040133357A1 (en) * 2001-04-17 2004-07-08 Abmaxis, Inc. Humanized antibodies against vascular endothelial growth factor
DE102006030382A1 (de) * 2006-06-29 2008-01-03 Carl Zeiss Meditec Ag Verfahren und Vorrichtung zur optischen Detektion am Auge

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
GAUTIER I ET AL: "Homo-FRET microscopy in living cells to measure monomer-dimer transition of GFP-tagged proteins", BIOPHYSICAL JOURNAL, NEW YORK, US, US, vol. 80, no. 6, 1 June 2001 (2001-06-01), pages 3000 - 3008, XP002977865, ISSN: 0006-3495 *
HOPPE, A., ET AL: "Fluorescence Resonance Energy Transfer-Based Stoichiometry in Living Cells", BIOPHYSICAL JOURNAL, vol. 83, December 2002 (2002-12-01), pages 3652 - 3664, XP002498565 *
SORBERA L A ET AL: "RANIBIZUMAB: TREATMENT OF AGE-RELATED MACULAR DEGENERATION HUMANIZED MONOCLONAL ANTI-VEGF ANTIBODY ANGIOGENESIS INHIBITOR", DRUGS OF THE FUTURE, PROUS SCIENCE, ES, vol. 28, no. 6, 1 June 2003 (2003-06-01), pages 541 - 545, XP008077008, ISSN: 0377-8282 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3211422A1 (fr) * 2016-02-23 2017-08-30 Justus-Liebig-Universität Gießen Procédé de mesure et de commande de concentration de vegf intraoculaire
WO2017144416A1 (fr) * 2016-02-23 2017-08-31 Justus-Liebig-Universität Giessen Procédé de mesure et de contrôle de la concentration de vegf intraoculaire

Also Published As

Publication number Publication date
DE102007038730A1 (de) 2009-02-19

Similar Documents

Publication Publication Date Title
DE69534228T2 (de) Nachweis von bestandteilen mittels der lumineszenz-dauer
DE60119556T2 (de) Optischer sensor zur in-situ messung von analyten
Shen et al. An artery-specific fluorescent dye for studying neurovascular coupling
DE60128138T2 (de) In vivo multiphoton diagnostische detektion und bilddarstellung einer neurodegenerativen erkrankung
DE69821475T2 (de) Verwendung von eindimensionaler kernspinresonanz fur identifizierung von liganden fur ziel-biomolekule
EP2032017A2 (fr) Procédé et dispositif de détection optique de l'oeil
WO2011038892A1 (fr) Procédé et dispositif de détection de dépôts dans l'oeil
DE102005013044B4 (de) Fluoreszenz-Scanner
WO2007101706A1 (fr) Procédé pour analyser des molécules ou des parties de molécules dans des échantillons biologiques
Lukacs et al. Differential effects of group III metabotropic glutamate receptors on spontaneous inhibitory synaptic currents in spine-innervating double bouquet and parvalbumin-expressing dendrite-targeting GABAergic interneurons in human neocortex
WO2000062071A1 (fr) Utilisation a des fins diagnostiques et therapeutiques d'anticorps diriges contre le recepteur de l'urokinase
WO2000071025A1 (fr) Procede et dispositif pour mesurer le degre d'organisation de l'eau dans le corps d'un homme ou d'un animal
EP1782044B1 (fr) Procédé et dispositif pour la séparation et la détermination précise d'un fluorophore d'un objet agissant localement
WO2009021600A1 (fr) Détermination du facteur de croissance de l'endothélium vasculaire humain
EP1705485B1 (fr) Procédure de disposition de la concentration de glucose par de polarisation de la fluorescence
DE102013200256B3 (de) Verfahren zur Erkennung von Tumorzellen und Mittel zur Diagnose und Therapie von Tumoren
EP1273904A2 (fr) Procédé et appareil pour la détection d'une substance dans un tissu vivant
DE102007028659A1 (de) Diagnosesubstanz und Verfahren zur Diagnose von Prostataerkrankungen
DE102020114278A1 (de) Bestimmung krankheitsspezifischer Protein-Aggregate in Stuhlproben
WO2013041522A1 (fr) Procédés et dispositifs pour déceler des dépôts de protéines tau dans l'oeil
EP2598174A1 (fr) Procédé de mesure pour la détermination de la fonction d'un organe
EP2316025B1 (fr) Moyen de détection et de traitement de cellules prostatiques
DE10220955B4 (de) Anordnung zur in-vivo Diagnostik von Gelenkveränderungen und deren Verwendung
DE102007005147A1 (de) Verfahren und Vorrichtung zur Untersuchung der Anheftung oder Ablösung lebender oder toter Zellen oder zellähnlicher Partikel oder sonstiger Oberflächenbelegung an Oberflächen mittels Plasmonenresonanz sowie Verwendung dieses Verfahrens und dieser Vorrichtung
DE4323368C2 (de) Verwendung einer Markersubstanz zur Unterscheidung von verändertem Gewebe in einer Umgebung von unverändertem Gewebe

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 08774035

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 08774035

Country of ref document: EP

Kind code of ref document: A1