Fluorescent markers for neurofibrillary tangles and uses thereof

Abstract

The present invention relates to a novel fluorescent marker that selectively binds to tau protein, its use, a method for imaging neurofibrillary tangles of tau protein in the retina of a subject, as well as an apparatus allowing said method to be carried out.

Description

[Technical Field]

[0001] The present invention relates to novel fluorescent markers that selectively bind to tau neurofibrillary tangles, compositions comprising said markers, uses thereof, methods for imaging tau neurofibrillary tangles in the retina of a subject, and devices that enable said methods to be carried out. [Background technology]

[0002] Alzheimer's disease (AD) is a neurodegenerative disorder that accounts for over 80% of cases of senile dementia. Limited scientific evidence underlying the pathogenesis of AD has made it difficult to develop targeted, effective treatments and diagnostics. To date, no solutions have been found that guarantee effective prevention, which could improve the lives of high-risk patients or reduce healthcare costs [Graham VW et al., 2017]. Despite numerous studies and pharmaceutical industry efforts to elucidate the pathogenesis, there are still no effective therapies that treat AD or significantly halt its progression [Masters CL et al., 2015; Graham VW et al., 2017]. As a result, research has focused on accurate, preferably early, and specific diagnostic methods for this disease in order to develop therapeutic strategies that can prevent AD. Much progress has been made since the introduction of cerebrospinal fluid analysis (CSF) [Olsson B. et al., 2016] and diagnostic neuroimaging techniques [Pietrzak K. et al., 2018].

[0003] These techniques allow the study of the presence of functional changes in the brain and biomarkers associated with AD.

[0004] However, despite being relatively specific and selective, these techniques have many limitations (they are very expensive and invasive, and only provide clear positive results when cognitive impairment is already advanced) and therefore cannot be used as the sole diagnostic tool and must be associated with postmortem evaluation of pathophysiological traits.

[0005] Three major biomarkers have been identified for AD. Aβ42 peptide, the main component of beta-amyloid plaques, a binding unit of the highly phosphorylated protein (P-Tau), and Total tau monomers (t-tau), a key component of neurofibrillary tangles (NFTs).

[0006] In recent years, it has been shown that the presence and amount of beta-amyloid plaques in dementia patients may not be completely related to the progression and clinical symptoms of the disease itself. In contrast, many studies have shown a closer correlation between the number of tau protein neurofibrillary tangles and disease progression.

[0007] In this regard, several fluorescent probes for identifying AD biomarkers have been described and / or patented in the literature, but only a limited number of these are highly selective for tau protein aggregates in vivo, and to date, no tau-specific fluorophores are commercially available. Therefore, there is a strong need to identify a new class of fluorophores that can bind highly selectively to tau protein NFTs. Summary of the Invention [Means for solving the problem]

[0008] The present invention relates to a compound of formula I [ka] and providing a novel fluorescent marker that selectively binds to tau protein, wherein X and Y are carbon atoms joined by a double bond in the E or Z configuration, or by an aromatic or heteroaromatic para-substituted ring, or by an aromatic or heteroaromatic 1,4-disubstituted ring; R is hydrogen, halogen, NH(RA), N(RA)2, NHC(=O)RA, ORA, OC(=O)RA, SRA, SO2RA, SO3RA, OSO2RA, OSO3RA, C(RA)3, or C5-7 an aromatic or aliphatic heterocycle, and In each substituent, R A is a C group containing up to three independent heteroatoms selected from hydrogen, halogen, hydroxyl, CF 3 , nitrogen, oxygen, and sulfur. 1-7 Saturated or unsaturated chain, straight or branched chain, C 5-7 Cycloalkyl, phenyl, C 5-7 Heterocycle or n-ethylene glycol.

[0009] As is evident from the data reported in the experimental section herein, the inventors have found that the fluorescent markers of formula I, and in particular the fluorescent compounds defined herein as BT1-BT4 and BT6, have high binding affinity to a hexameric model of the PHF6 fragment, which resides in the R3 region of the microtubule-associated tau protein and is responsible for the protein's tendency to assemble into fibrils.

[0010] The results obtained by the in vitro assays presented below clearly demonstrate that the fluorescent compounds are novel and effective markers capable of selectively binding to neurofibrillary aggregates of tau protein.

[0011] The present invention further provides methods for preparing the fluorescent markers of Formula I, as well as imaging methods using the markers.

[0012] Advantageously, the fluorescent marker of the present invention demonstrates the ability to efficiently penetrate the blood-brain barrier and bind to the neurofibrillary tangles of tau protein in the retina.Since neurofibrillary tau tangles are associated with tauopathy, the detection of neurofibrillary tangles in the retina of a subject can be used as relevant information in or to assist in the diagnosis of tauopathy.

[0013] Currently, most conventional methods for detecting tau neurofibrillary tangles are based on postmortem analysis of brain tissue. One major challenge to the early diagnosis of tauopathies is the complexity of conventional diagnostic methods, which generally involve not only immunohistochemical demonstration of abnormal tau deposits in the brain, but also the detection of the presence and amount of other non-tau proteins in the brain, as well as the study of morphological features of tau in different brain regions.

[0014] Advantageously, the fluorescent markers of the present invention, thanks to their high affinity and specificity for neurofibrillary tangles of tau protein together with their ability to cross the blood-brain barrier, can be used to provide novel imaging methods, providing images that can be subsequently analyzed by a specialist to obtain information that allows for an effective, non-invasive early diagnosis of tauopathies, or that can be used to assess the effectiveness of medical treatments for tauopathies and / or to assess the progression of tauopathies.

[0015] The subject of the present invention is therefore Formula I [ka] fluorescent markers wherein X and Y are carbon atoms joined by a double bond in the E or Z configuration, or by an aromatic or heteroaromatic para-substituted ring, or by an aromatic or heteroaromatic 1,4-disubstituted ring; R is hydrogen, halogen, NH(RA), N(RA)2, NHC(=O)RA, ORA, OC(=O)RA, SRA, SO2RA, SO3RA, OSO2RA, OSO3RA, C(RA)3, or C 5-7 an aromatic or aliphatic heterocycle, and In each substituent, R A is a C group containing up to three independent heteroatoms selected from hydrogen, halogen, hydroxyl, CF 3 , nitrogen, oxygen, and sulfur. 1-7 Saturated or unsaturated chain, straight or branched chain, C 5-7 Cycloalkyl, phenyl, C 5-7heterocycle or n-ethylene glycol, which marker selectively binds to tau protein neurofibrillary tangles);

[0016] i. 4,4-difluoro-1,3-dimethyl-4-bora-3a,4a-diaza-s-indacene of formula II [ka] wherein X and Y are carbon atoms joined by a double bond in the E or Z configuration, or by an aromatic or heteroaromatic para-substituted ring, or by an aromatic or heteroaromatic 1,4-disubstituted ring; R is hydrogen, halogen, NH(RA), N(RA)2, NHC(=O)RA, ORA, OC(=O)RA, SRA, SO2RA, SO3RA, OSO2RA, OSO3RA, C(RA)3, or C 5-7 an aromatic or aliphatic heterocycle, and In each substituent, R A is a C group containing up to three independent heteroatoms selected from hydrogen, halogen, hydroxyl, CF 3 , nitrogen, oxygen, and sulfur. 1-7 Saturated or unsaturated chain, straight or branched chain, C 5-7 Cycloalkyl, phenyl, C 5-7 heterocycle or n-ethylene glycol) and subjecting the resulting mixture to a Knoevenagel condensation reaction with an aldehyde of ii. subjecting the solution obtained in step i to liquid-liquid extraction (LLE), followed by a purification step to obtain said fluorescent marker of formula I; A method for preparing a fluorescent marker of formula I, comprising:

[0017] a composition comprising a fluorescent marker of formula I and one or more additional excipients and / or carriers,

[0018] a fluorescent marker of formula I as defined herein or a composition comprising a marker of formula I for use in detecting neurofibrillary tangles of tau protein,

[0019] - contacting a fluorescent marker of formula I or a composition comprising a fluorescent marker of formula I with a biological sample under conditions in which the fluorescent marker binds to neurofibrillary tangles of tau protein; detecting the fluorescent marker of formula I bound to said biological sample; a method for detecting neurofibrillary tangles of tau protein, comprising:

[0020] - administering to a subject a fluorescent marker of formula I or a composition comprising a fluorescent marker of formula I as described herein; performing non-invasive fluorescence imaging of the subject's retina, wherein detection of fluorescence from the marker of Formula I indicates binding of the marker to the retina. an imaging method,

[0021] administering to a subject a fluorescent marker of Formula 1 or a composition comprising a fluorescent marker of Formula 1, and performing non-invasive quantitative fluorescence imaging on the subject's retina at multiple consecutive time points ti (i ranging from 0 to n) to obtain corresponding fluorescence values; determining a fluorescence graph using the obtained fluorescence values; a method for determining a fluorescence graph, comprising:

[0022] - a computer program for monitoring the progression of the amount of neurofibrillary tangles of tau protein in the retina of a subject, the computer program, when executed on an electronic computer, providing a first fluorescence value f0 at time t0 and one or more time points t n One or more fluorescence values ​​f at n (n is an integer greater than 0, and at each subsequent time t n It increases gradually with each t n corresponds to a time point after t0, the value being obtained by any of the methods defined herein), Each of these values ​​is f0 to f n and each f n If the fluorescence value is lower than f0 and the n detecting a decrease in the neurofibrillary tangle load if the neurofibrillary tangle load is lower than the threshold; each fn The fluorescence value is higher than f0 and the above f n detecting an increase in the amount of neurofibrillary tangles when the amount is higher than the normal range; each f n The fluorescence value is f0 and the above f n detecting a substantial absence of variation in the amount of neurofibrillary tangles when the variation is equal to or substantially equal to a computer program for implementing the

[0023] - a device for the automated measurement of the fluorescence level of a marker of formula I of the present invention in the retina of a subject, the device comprising at least (i) a light source configured to emit light to illuminate the retina of the subject, the light having a wavelength comprised between 350 nm and 650 nm, and (ii) an optical unit configured to detect and / or quantify the fluorescence emitted by the fluorescent marker when the retina is illuminated with the light source, the device being configured to perform any of the methods described herein;

[0024] - use of the device according to the invention for carrying out any of the methods described in the present specification and claims, is.

[0025] Further advantages, as well as features and modes of use of the present invention will become apparent from the following detailed description of some preferred embodiments, given purely by way of example.

[0026] Glossary BODIPY in the present invention has the meaning commonly understood in the art, i.e., the technical generic name of a chemical compound whose molecule has the formula CHBN F consisting of a boron difluoride group BF bonded to a dipyrromethene group CHN, specifically the compound 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene in the IUPAC nomenclature. The generic name is an abbreviation for "boron-dipyrromethene."

[0027] The terms aromatic and non-aromatic are as generally understood in the state of the art and can therefore denote any aromatic heterocycle and any non-aromatic heterocycle, respectively.

[0028] As used herein, the term "ocular tissue" may include any tissue of the mammalian eye and / or optic nerve, such as the retina or substructures within the retinal periphery, such as the macula and fovea. The retina may include one or more of the inner nuclear layer of the retina and retinal ganglion cells of the retina.

[0029] The tau 1 probe has the structure described in the paper by Verwilst P et al., "Rational Design of in Vivo Tau Tangle-Selective Near-Infrared Fluorophores: Expanding the BODIPY Universe." J Am Chem Soc. 2017 Sep 27;139(38):13393-13403. [Brief explanation of the drawings]

[0030] [Figure 1] General formula for compounds of formula I. [Figure 2] Binding pose of BT1 compounds to the tau fibril lumen. [Figure 3](A) Representative binarized fluorescence image showing human iPSC-derived cortical neurons at day 30 in vitro after incubation with BT1 (100 μM) for 30 min at 37 °C and staining with anti-T22 and anti-AT8 antibodies. (B) Representative binarized fluorescence image showing human iPSC-derived cortical neurons at day 30 in vitro after incubation with TAU1 (100 μM) for 30 min at 37 °C and staining with anti-T22 and anti-AT8 antibodies. (C) Fluorescence composite of the above representative images. Fluorescence images of BT1 were obtained using excitation at 520 nm with a 555 / 15 nm filter for detection, and TAU1 were obtained using excitation at 470 nm with a 510 / 5 nm filter for detection. Both probes are shown. Cell nuclei were stained with DAPI. (D) Probe specificity was calculated as the ratio of antibody intensity within the area covered by BT1 or TAU1 out of the total antibody intensity determined using Image J (n = 3). Images were acquired with an FV10i confocal microscope using 60x magnification. [Figure 4] (A) Representative binarized fluorescence image showing human iPSC-derived retinal ganglion cells at day 30 in vitro after incubation with BT1 (100 μM) for 30 minutes at 37°C and staining with anti-T22 and anti-AT8 antibodies. (B) Representative binarized fluorescence image showing human iPSC-derived retinal ganglion cells at day 30 in vitro after incubation with TAU1 (100 μM) for 30 minutes at 37°C and staining with anti-T22 and anti-AT8 antibodies. (C) Fluorescence composite of the above representative images. Fluorescence images of BT1 were obtained using excitation at 520 nm with a 555 / 15 nm filter for detection, and TAU1 were obtained using excitation at 470 nm with a 510 / 5 nm filter for detection. Both probes are shown. Cell nuclei were stained with DAPI. (D) Probe specificity is calculated as the ratio of antibody intensity within the area covered by BT1 or TAU1 to the total antibody intensity determined using Image J (n = 3). Images were acquired with an FV10i confocal microscope using 60x magnification. DETAILED DESCRIPTION OF THE INVENTION

[0031] Among the BIODIPY-derived molecules, the present inventors have identified a compound of formula I [ka] The present invention identifies a novel fluorescent marker that selectively binds to tau protein, wherein X and Y are carbon atoms joined by a double bond in the E or Z configuration, or by a heteroaromatic or aromatic para-substituted ring, or by an aromatic or heteroaromatic 1,4 di-substituted ring; R is hydrogen, halogen, NH(RA), N(RA)2, NHC(=O)RA, ORA, OC(=O)RA, SRA, SO2RA, SO3RA, OSO2RA, OSO3RA, C(RA)3, or C 5-7 an aromatic or aliphatic heterocycle, and In each substituent, R A is a C group containing up to three independent heteroatoms selected from hydrogen, halogen, hydroxyl, CF 3 , nitrogen, oxygen, and sulfur. 1-7 Saturated or unsaturated chain, straight or branched chain, C 5-7 Cycloalkyl, phenyl, C 5-7 Heterocycle or n-ethylene glycol.

[0032] The compounds of Formula I are fluorescent molecules that have high affinity for tau protein neurofibrillary tangles (NFTs).

[0033] The fluorescent markers of formula I of the present invention are lipophilic compounds that exhibit efficient penetration of the blood-brain barrier.

[0034] One embodiment of the present invention relates to a fluorescent marker of formula I as defined above, wherein X and Y are carbon atoms linked by an aromatic para-substituted ring or an aromatic 1,4-disubstituted ring. R is H, halogen, NH(RA), N(RA)2, NHC(=O)RA, ORA, OC(=O)RA, SRA, SO2RA, SO3RA, OSO2RA, OSO3RA, C(RA)3, or C 5-7 an aromatic or aliphatic heterocycle, and In each substituent, R A is a C group containing up to three independent heteroatoms selected from hydrogen, halogen, hydroxyl, CF 3 , nitrogen, oxygen, and sulfur. 1-7 Saturated or unsaturated chain, straight or branched chain, C 5-7 Cycloalkyl, phenyl, C 5-7 Heterocycle or n-ethylene glycol.

[0035] According to the invention, preferred aromatic heterocycles are selected from among pyrrole, imidazole and piperidine.

[0036] According to the invention, preferred non-aromatic heterocycles are selected from among morpholine, piperazole and pyrrolidine.

[0037] In one preferred embodiment, R is selected from NH2, NH(CH3), N(CH3)2, N(Ph)2, imidazole, morpholine, piperazine.

[0038] Non-limiting examples of fluorescent markers of Formula I include 3-((E)-4-((E)-4-(dimethylamino)styryl)styryl)-5,5-difluoro-1-methyl-5H-dipyrrolo[1,2-c:2′,1′-f][1,3,2]diazaborinin-4-ium-5-uide (also referred to as BT1 in the specification, drawings and schemes); 3-((E)-4-((E)-4-(diphenylamino)styryl)styryl)-5,5-difluoro-1-methyl-5H-dipyrrolo[1,2-c:2′,1′-f][1,3,2]diazaborinin-4-ium-5-uide (also referred to herein and in the schemes as BT2), 3-((E)-4-((E)-4-aminostyryl)styryl)-5,5-difluoro-1-methyl-5H-dipyrrolo[1,2-c:2′,1′-f][1,3,2]diazaborinin-4-ium-5-uide (also referred to herein and in the schemes as BT3), 5,5-difluoro-1-methyl-3-((E)-4-((E)-4-(methylamino)styryl)styryl)-5H-dipyrrolo[1,2-c:2′,1′-f][1,3,2]diazaborinin-4-ium-5-uide (also referred to herein and in the schemes as BT4), 5,5-difluoro-1-methyl-3-((1E,3E,5E)-6-(pyrrolidin-1-yl)hexa-1,3,5trien-1-yl)-5H-dipyrrolo[1,2-c:2′,1′-f][1,3,2]diazaborinin-4-ium-5-uide (also referred to herein and in the schemes as BT5), 5,5-difluoro-1-methyl-3-((E)-4-((E)-4-morpholinostyryl)styryl)-5H-dipyrrolo[1,2-c:2′,1′-f][1,3,2]diazaborinin-4-ium-5-uide (also referred to herein and in the schemes as BT6), 5,5-difluoro-1-methyl-3-((E)-4-((E)-4-(piperazin-1-yl)styryl)styryl)-5H-dipyrrolo[1,2-c:2′,1′-f][1,3,2]diazaborinin-4-ium-5-uide (also referred to herein and in the schemes as BT7); and 3-((E)-4-((E)-4-(1H-imidazol-1-yl)styryl)styryl)-5,5-difluoro-1-methyl-5H-dipyrrolo[1,2-c:2',1'-f] [1,3,2]diazaborinin-4-ium-5-uide (also referred to herein and in the schemes as BT8). Examples include:

[0039] Thus, the fluorescent marker of formula I is 3-((E)-4-((E)-4-(dimethylamino)styryl)styryl)-5,5-difluoro-1-methyl-5H-dipyrrolo[1,2-c:2',1'-f] [1,3,2]diazaborin-4-ium-5-uide (BT1), 3-((E)-4-((E)-4-(diphenylamino)styryl)styryl)-5,5-difluoro-1-methyl-5H-dipyrrolo[1,2-c:2',1'-f] [1,3,2]diazaborin-4-ium-5-uide (BT2), 3-((E)-4-((E)-4-aminostyryl)styryl)-5,5-difluoro-1-methyl-5H-dipyrrolo[1,2-c:2',1'-f] [1,3,2]diazaborinin-4-ium-5-uide (BT3), 5,5-difluoro-1-methyl-3-((E)-4-((E)-4-(methylamino)styryl)styryl)-5H-dipyrrolo[1,2-c:2',1'-f] [1,3,2]diazaborin-4-ium-5-uide (BT4), 5,5-difluoro-1-methyl-3-((1E,3E,5E)-6-(pyrrolidin-1-yl)hexa-1,3,5-trien-1-yl)-5H-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinin-4-ium-5-uide (BT5), 5,5-difluoro-1-methyl-3-((E)-4-((E)-4-morpholinostyryl)styryl)-5H-dipyrrolo[1,2-c:2',1'-f] [1,3,2]diazaborin-4-ium-5-uide (BT6), 5,5-difluoro-1-methyl-3-((E)-4-((E)-4-(piperazin-1-yl)styryl)styryl)-5H-dipyrrolo[1,2-c:2',1'-f] [1,3,2]diazaborin-4-ium-5-uide (BT7), 3-((E)-4-((E)-4-(1H-imidazol-1-yl)styryl)styryl)-5,5-difluoro-1-methyl-5H-dipyrrolo[1,2-c:2',1'-f] [1,3,2]diazaborin-4-ium-5-uide (BT8) It can be one of the following:

[0040] The chemical structures of the fluorescent markers BT1 to BT8 are shown in Scheme 1 below.

[0041] [ka] Scheme 1 - Structures of compounds BT1-BT8 In a preferred embodiment, the fluorescent marker is 3-((E)-4-((E)-4-(dimethylamino)styryl)styryl)-5,5-difluoro-1-methyl-5H-dipyrrolo[1,2-c:2′,1′-f][1,3,2]diazaborinin-4-ium-5-uide.

[0042] The fluorescent marker of the present invention is characterized by an excitation wavelength of 350 to 650 nm and an emission wavelength of 450 to 800 nm.

[0043] The present invention further provides a method for preparing a fluorescent marker of the given formula I, comprising: i. 4,4-Difluoro-1,3-dimethyl-4-bora-3a,4a-diaza-s-indacene of formula II [ka] wherein X and Y are carbon atoms joined by a double bond in the E or Z configuration, or by a heteroaromatic or aromatic para-substituted ring, or by an aromatic or heteroaromatic 1,4-disubstituted ring; R is hydrogen, halogen, NH(RA), N(RA)2, NHC(=O)RA, ORA, OC(=O)RA, SRA, SO2RA, SO3RA, OSO2RA, OSO3RA, C(RA)3, or C 5-7 an aromatic or aliphatic heterocycle, and In each substituent, R A is a C group containing up to three independent heteroatoms selected from hydrogen, halogen, hydroxyl, CF 3 , nitrogen, oxygen, and sulfur. 1-7 Saturated or unsaturated chain, straight or branched chain, C 5-7 Cycloalkyl, phenyl, C 5-7 heterocycle or n-ethylene glycol) and subjecting the resulting mixture to a Knoevenagel condensation reaction with an aldehyde of ii. subjecting the solution obtained in step i to liquid-liquid extraction (LLE), followed by a purification step to obtain said fluorescent marker of formula I; The present invention provides a method for preparing the compound comprising the steps of:

[0044] In one embodiment, the Knoevenagel reaction is carried out under Dean-Stark conditions in the presence of piperidine (or pyrrolidine) and acetic acid using toluene (or benzene or trifluoromethylbenzene) as a solvent. The Knoevenagel reaction can be carried out under reflux for 2 to 4 hours.

[0045] In a specific embodiment of the invention, step ii of the above method comprises the following steps: ii.a adding saturated aqueous ammonium chloride solution (NH4Cl) to the solution obtained in step i; ii.b. subjecting the mixture obtained in step ii.a to liquid-liquid extraction (LLE); ii.c separating the aqueous phase obtained in step ii.b; ii.d. The organic phase obtained in step ii.b is collected and dried over Na2SO4.

[0046] The liquid-liquid extraction (LLE) can be carried out using DCM (dichloromethane) as the organic solvent.

[0047] According to one embodiment of the present invention, the purification step to provide the fluorescent marker of formula I is carried out by chromatography. In a specific embodiment, the purification is carried out by flash chromatography using the following eluent mixture: hexane:ethyl acetate in a ratio of 9:1.

[0048] In one preferred embodiment of the above process, the aldehyde of formula II is trans-4-[2-(4-dimethylaminophenyl)vinyl]benzaldehyde.

[0049] The trans-4-[2-(4-dimethylaminophenyl)vinyl]benzaldehyde can be obtained by the Heck reaction of 4-bromobenzaldehyde with 4-dimethylaminostyrene in the presence of a suitable catalyst and potassium carbonate.

[0050] A suitable catalyst that can be used to prepare the trans-4-[2-(4-dimethylaminophenyl)vinyl]benzaldehyde starting from 4-bromobenzaldehyde and 4-dimethylaminostyrene is, for example, a catalyst prepared in situ by mixing palladium(II) acetate (Pd(CHCOO)) with triphenylphosphine (PPh) in dimethylformamide (DMF).

[0051] The present invention also relates to compositions comprising at least one fluorescent marker of the present invention and optionally one or more carriers and / or excipients.

[0052] Suitable carriers and / or excipients are, for example, solvents such as DMSO, preferably pharmaceutically acceptable solvents, provided that they allow the fluorescent marker of formula I of the present invention to be dissolved or stably dispersed therein.

[0053] The concentration of the fluorescent marker of Formula I in the composition can be adjusted depending on the type of fluorescent marker of the present invention. In one embodiment, the concentration of the fluorescent marker can range from 0.5 micromolar to 50 millimolar. The excipients contained in the composition can be adjusted to obtain the desired dosage of the fluorescent marker.

[0054] The compositions of the present invention are preferably in the form of an oral composition or an ophthalmic composition.

[0055] Oral compositions may be in the form of solutions, suspensions, gels, soft or hard gelatin, capsules, tablets, lozenges, powders, granules, pills, oleogel.

[0056] Any suitable carrier or excipient known in the art for preparing a composition suitable for oral administration may be used by a person skilled in the art.

[0057] The ophthalmic composition according to the present invention may be in the form of eye drops, eye ointment or eye lotion.

[0058] Any suitable carrier or excipient known in the art for preparing a composition suitable for ophthalmic use can be used by one skilled in the art.

[0059] In one aspect the present invention refers to a fluorescent marker of formula I or a composition comprising said fluorescent marker for use in the detection of neurofibrillary tangles of tau protein.

[0060] In one embodiment, the present invention refers to a fluorescent marker of formula I or a composition comprising said fluorescent marker for use in detecting neurofibrillary tangles of tau protein in a subject; in other words, the present invention also refers to a fluorescent marker of formula I or a composition comprising said fluorescent marker for use in detecting neurofibrillary tangles of tau protein in a subject in vivo.

[0061] According to one embodiment of the present invention, any of the fluorescent markers or compositions taught herein can be administered to a subject in need thereof for use in detecting neurofibrillary tangles of tau protein, which, as described above, can be performed in the subject and can be performed in vivo without invasive tools.

[0062] In a further embodiment of the invention, detection can be performed in vitro on a suitable sample comprising an organoid.

[0063] In particular, the present invention further comprises: - contacting a fluorescent marker of formula I or a composition comprising a predetermined fluorescent marker of formula I with a biological sample under conditions in which the fluorescent marker binds to neurofibrillary tangles of tau protein; detecting said fluorescent marker bound to the biological sample; The present invention provides a method for detecting neurofibrillary tangles of tau protein, comprising:

[0064] Non-limiting examples of biological samples that can be analyzed using the above methods include ocular tissue, brain tissue, or olfactory epithelium. In a preferred embodiment, the biological sample is ocular tissue.

[0065] In one embodiment, the contacting step of the method can be carried out at a temperature of 37° C. The contacting step has a duration comprised between 10 and 120 minutes, such that the fluorescent marker selectively binds to neurofibrillary tangles of tau protein present in the biological sample to be analyzed.

[0066] The period of incubation of the biological sample in the presence of the fluorescent marker depends on the amount of fluorescent marker applied, but is within the ranges described above. In one embodiment, the contact time is 30 minutes.

[0067] Preferably, in the contacting step, the fluorescent marker of the present invention is at a concentration ranging from 0.5 micromolar to 50 millimolar.

[0068] The above method may further comprise, after said contacting step, a washing step in which any excess of said fluorescent marker is removed from the biological sample.

[0069] In one embodiment, the presence and / or amount of any of the fluorescent markers of the present invention bound to neurofibrillary tangles of tau protein in a biological sample can be determined by fluorescence measurement, preferably by fluorescence imaging.

[0070] Fluorescence imaging can be performed according to any of the fluorescence imaging techniques known in the art. For example, qualitative and / or quantitative assessment of the binding of the fluorescent markers of the present invention to neurofibrillary tangles of tau protein in the biological sample can be achieved using microscopic techniques.

[0071] In some embodiments, the detection step of the above-described method of the present invention further comprises the following steps: illuminating the biological sample with a light source having a wavelength (λ) suitable for determining fluorescent emission from bound fluorescent markers; and Detecting and optionally quantifying the fluorescence emitted by said fluorescent marker, said emitted fluorescence having a wavelength in the range of about 450 nm to 800 nm.

[0072] The excitation light source preferably has a narrow emission range to avoid excitation of any other components of the biological sample being analyzed. In one embodiment, the light source has a wavelength in the range of about 350 to 650 nm, preferably equal to 559 nm.

[0073] Yet another embodiment of the present invention comprises: - administering to a subject a fluorescent marker of formula I as defined herein or a composition comprising said fluorescent marker; performing non-invasive fluorescence imaging of the subject's retina, wherein detecting fluorescence from the fluorescent marker indicates binding of the marker to the retina. The present invention relates to an imaging method, including:

[0074] In one embodiment, the fluorescent marker of formula I according to the present invention or a composition comprising said fluorescent marker of formula I can be administered to a subject by oral or ocular administration.

[0075] In a preferred embodiment, the fluorescent marker taught herein or any composition comprising the fluorescent marker is administered to the subject at least 30 minutes to 1 day before fluorescence measurement.For example, administration is at least 1 hour, 2 hours, at least 4 hours, at least 8 hours, preferably at least 12 hours, at least 16 hours before fluorescence measurement.Due to its ability to efficiently penetrate the blood-brain barrier, the fluorescent marker of formula I of the present invention can reach the retina after administration and selectively bind to the neurofibrillary tangles of tau protein present therein.

[0076] The time required for such binding will vary depending on the method selected for administration of the marker or composition: topical administration in the form of ocular administration will require a shorter time for subsequent fluorescence detection stem compared to oral administration.

[0077] In one embodiment, the fluorescent marker of the present invention is administered in an amount comprising 0.5 micromoles to 50 millimoles per unit dose.

[0078] The fluorescence imaging of the subject's retina according to the method of the present invention can be carried out by any fluorescence imaging technique known in the art, as long as it is non-invasive.Non-limiting examples of non-invasive fluorescence imaging techniques include scanning laser ophthalmoscopy (SLO), confocal scanning laser ophthalmoscopy (cSLO), or fluorescence lifetime imaging ophthalmoscopy (FLIO).Fluorescence imaging can be qualitative or quantitative, in other words, it can simply indicate the presence or absence of fluorescence, or it can quantify the amount of detected fluorescence.

[0079] By way of example, non-invasive fluorescence imaging of the retina can be performed by using a light source to illuminate the subject's retina, together with means for detecting and / or quantifying fluorescence emitted by a fluorescent marker of the present invention bound to neurofibrillary tangles of tau protein in the retina, the emitted fluorescence having an emission wavelength in the range of about 450-800 nm.

[0080] As used herein, a "light source" refers to any light source that can be configured to illuminate a subject's retina and has a wavelength suitable for determining fluorescent emissions from a bound fluorescent marker of the present invention within the subject's retina. In a preferred embodiment, the light source has a wavelength that is comprised between 350 and 650 nm, preferably corresponding to 559 nm.

[0081] According to one embodiment of the present invention, detecting and / or quantifying the fluorescence emitted by the bound fluorescent marker of the present invention in the retina can be performed by any suitable device configured for fluorescence detection and recording. An example of a suitable device is a device including a unit configured to receive the fluorescence generated as a result of irradiating the subject's retina and detect the fluorescence emitted by the fluorescent marker of Formula I bound to neurofibrillary tangles of tau protein in the retina, i.e., to identify the presence and / or quantify the amount of the fluorescent marker bound to the retina. The fluorescent marker of the present invention is detectable only when bound to the retina, since the amount captured by the tangles is sufficient to emit sufficient fluorescence for its detection. Unbound, freely diffusing marker is unlikely to contribute to the observed fluorescent signal due to its low water solubility.

[0082] In one embodiment, the device may include a camera configured to form a camera image of the fluorescence emitted by markers bound to the retina illuminated with a suitable light source.

[0083] Such devices can be programmed to analyze the collected fluorescence intensities and provide quantitative measurements of the fluorescent markers of the present invention in the retina, for example, by calculating the mean fluorescence intensity value of the bound markers using dedicated computer programs and / or any suitable publicly available software.

[0084] Therefore, in one embodiment of the present invention, the imaging method described above may include determining the peak intensity of fluorescence generated by the fluorescent marker of Formula I bound to the NFTs of tau protein in the retina, and the amount of the fluorescent marker bound to the NFTs of tau protein can be determined based on the peak intensity.

[0085] As an example, a fluorescent marker of formula I of the present invention bound to an NFT of tau protein in the retina can be excited by a picosecond laser pulse and the fluorescence emission can be detected using time-correlated single photon counting (TCSPC) technology.

[0086] Advantageously, images obtained using the imaging methods of the present invention to detect fluorescence in a subject's retina can be used to diagnose a tauopathy. Because neurofibrillary tau tangles are associated with tauopathies, detection of neurofibrillary tangles in the retina can be used to diagnose or aid in the diagnosis of a tauopathy.

[0087] As used herein, the term "tauopathy" encompasses a class of neurodegenerative diseases in which tau protein aggregates into neurofibrillary or glial fibrillary tangles (NFTs), such as Alzheimer's disease, Down's syndrome, amyotrophic lateral sclerosis, Pick's disease, Parkinson's disease, primary age-related tauopathy (PART), chronic traumatic encephalopathy (CTE), progressive supranuclear palsy (PSP), corticobasal degeneration (CDB), frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), Litiko-Bodig disease (Parkinson-dementia complex of Guam), ganglioglioma and gangliocytoma, meningiomatosis, retroencephalitic parkinsonism, subacute sclerosing panencephalitis (SSPE), as well as lead encephalopathy, tuberous sclerosis complex, pantothenate kinase-associated neurodegeneration, and lipofuscinosis.

[0088] In a preferred embodiment, the tauopathy is selected from Alzheimer's disease, Down's syndrome, amyotrophic lateral sclerosis, Pick's disease or Parkinson's disease, preferably Alzheimer's disease.

[0089] According to one embodiment of the present invention, the method for detecting neurofibrillary tangles of tau protein in a biological sample or any of the imaging methods described herein can be combined with results obtained by other imaging techniques, for example techniques for detecting amyloid proteins, to aid in the diagnosis of tauopathies.

[0090] Administering the fluorescent marker according to any one of claims 1 to 6 or a composition comprising the fluorescent marker according to claim 11 to a subject, and performing non-invasive quantitative fluorescence imaging on the subject's retina at multiple consecutive time points ti (i is 0 to n) to obtain corresponding fluorescence values; determining a graph of fluorescence as a function of time using the obtained fluorescence values; 10. A method for determining a fluorescence graph, comprising:

[0091] When carrying out the above method, at time t i represents the time at which monitoring begins. The successive time points t where i is an increasing integer value from 0+1 to n i are time points that follow each other, and their temporal succession is indicated by increasing i values.

[0092] Therefore, at time t i is the time when monitoring begins, and this t i The fluorescence value of the fluorescent marker of formula I in the retina at can be considered as the starting fluorescence value from which the temporal progression of said fluorescence within the retina can be assessed.

[0093] Noninvasive quantitative fluorescence imaging of the retina is performed over a period of time t where i=0, such that time t precedes time t, which precedes time t, etc. i It can be repeated at later time points and at time points that follow each other in the progression from i=1 to n.

[0094] Therefore, comparison of fluorescence values ​​within images obtained for each subsequent time according to the method described above can be detailed in a graph of fluorescence as a function of time.

[0095] Graphs such as those provided by the above methods can be advantageously used to assess the effectiveness of medical treatments for tauopathy or to assess the progression of a tauopathy over time.

[0096] In this case, time t icorresponds to the time at which the medical treatment outcome is monitored, which may advantageously be before the start of the treatment itself, but can be introduced at any time during the treatment, even after the treatment has already begun.

[0097] A gradual decrease or stability of fluorescence in the graph provided by the above method may indicate the effectiveness of medical treatment in ameliorating tauopathy. The meaning of the graph and the associated degree of effectiveness can be assessed by interpretation of the graph by a clinical expert.

[0098] A gradual increase in fluorescence in the graph provided by the above method may indicate ineffectiveness of medical treatment. The meaning of the graph and the associated degree of ineffectiveness can be assessed by interpretation of the graph by a clinical expert.

[0099] In some cases, dietary and lifestyle changes may be suggested to a subject before undergoing medical treatment. In these cases, it may be useful to monitor the progression of the disease over time, for example, to see whether the dietary and lifestyle changes have a positive effect on the disease.

[0100] The graphs provided by the above methods can be used to monitor the effect of such changes on the disease.

[0101] In any case, it may be of interest to a physician to monitor the progression of a subject's tauopathy, whether or not possible therapeutic effects are being evaluated.

[0102] Thus, the methods of determining a fluorescence graph described herein can be advantageously used to monitor the progression of a tauopathy in a subject.

[0103] What has been said previously regarding the mode and dosage of administration of the fluorescent marker of formula I or a composition comprising said fluorescent marker in the description of the general imaging method also applies to any of the other methods described above.

[0104] Non-invasive quantitative fluorescence imaging of the retina of the subject can be performed according to any one of the non-invasive fluorescence imaging techniques known in the art, including those previously described herein. In particular, according to a preferred embodiment of the present invention, in any of the above imaging methods, the fluorescence imaging is performed by irradiating the subject's retina with a light source having a wavelength (λ) comprised between 350 and 650 nm and detecting and / or quantifying the fluorescence emitted by the fluorescent marker.

[0105] Any of the above methods involving fluorescence imaging of the retina may further include "normalization," i.e., determining the amount or fluorescence level of the fluorescent marker of the present invention bound to the NFTs of tau protein based on background autofluorescence emitted from the subject's retina. As used herein, such "normalization" may include subtracting the amount of background autofluorescence from the amount of fluorescence emitted from the fluorescent marker of the present invention bound to the NFTs of tau protein, including determining the ratio of such amounts, and using such normalized result as a normalized measure of the amount of the fluorescent marker of Formula I of the present invention bound to the NFTs of tau protein.

[0106] Detection of tau protein neurofibrillary tangles in the retina allows for early and non-invasive diagnosis of tauopathies, and the high specificity of the fluorescent markers of the present invention makes detection of the tau changes more sensitive and accurate, thereby improving early diagnosis of the disease.

[0107] Thereby, the imaging method of the present invention can be used in a method for treating tauopathies, where early diagnosis of the disease is followed by appropriate treatment of the disease at a stage where diagnosis is usually not possible.

[0108] Furthermore, the present invention also encompasses methods for treating tauopathies, in which the effectiveness of medical treatment is monitored by analysis of graphs of fluorescence as a function of time provided by the present invention, and in which medical treatment is continued, varied or modified by the attending physician depending on the results of said monitoring.

[0109] The following experimental section is provided for purposes of illustration only, not limitation, and is not intended to limit the scope of the invention as defined in the appended claims, which are an integral part of the description. [Example]

[0110] example Example 1 - Design of selectable markers for tau protein neurofibrillary tangles (NFTs) and molecular docking As shown in Scheme 2 below, a series of fluorescent probes, designated BT1–BT8, have been designed, consisting of a BODIPY core functionalized at the 3-position with highly conjugated systems terminated with aliphatic amines, cyclic, and non- or aromatic groups, characterized by distances between the electron donor and acceptor moieties of 13–19 Å and different polarities.

[0111] [ka] Scheme 2 - Structures of compounds BT1-8. To assess selectivity for NFTs and rule out any divergent candidates, we screened the molecules computationally against the crystal structure of the PHF6 fragment responsible for the protein's tendency to assemble into fibrils. A hexameric model of the PHF6 fragment hexapeptide 306VQIVYK311, present in the R3 region of the microtubule-binding tau protein, was constructed using extension and symmetry operators according to a procedure reported in the literature [Verwilst P. et al., 2017].

[0112] The high-resolution crystal structure of the peptide encoded by PDB-ID 5K7N [de la Cruz MJ et al., 2017] was used as a model system to construct 6-Wed. Small molecule docking was performed within the conserved amphipathic tunnel formed by the peptide monomer. Molecular docking was performed using AutoDock 4.2 [Morris GM et al., 2009].

[0113] The ligands were drawn using Picto (OpenEye) and subsequently converted to a three-dimensional format using OMEGA (OpenEye). [Hawkins PCD et al., 2010] Note that OpenEye and AutoDock 4.2 software do not provide force field parameters for docking boron-containing compounds; for this reason, the boron atom was replaced with a hybridized sp3 carbon atom. The ionization state of the pH 7.4 molecule was evaluated using QUACPAC (OpenEye) (QUACPAC 2.0.2.2: OpenEye Scientific Software, Santa Fe, NM. http: / / www.eyesopen.com), and a specific format compatible with AutoDock, PDBQT, was generated using the AutoDockTool GUI [Morris GM et al., 2009].

[0114] For each ligand, 10 runs of the genetic algorithm were performed and the statistically most relevant docking pose was determined by combined score analysis and visual inspection.

[0115] The binding pose of the BT1 compound to the tau fibril cavity is shown in Figure 2.

[0116] Table 1 below summarizes the predicted binding affinities for compounds BT1-BT8. [Table 1] The BT1 compound was found to be the most promising compound as a selectable marker for tau protein NFTs in terms of in silico affinity, binding conformation and polarity.

[0117] Example 2 - Design and synthesis of BT1 compounds In summary, for the synthesis of the BT1 compound, a two-step synthetic strategy was developed, namely, the Knoevenagel condensation of a selected commercially available Bodipy core with trans -4-[2-(4-dimethylaminophenyl)vinyl]benzaldehyde, the latter of which was synthesized by the Heck reaction of 4-bromobenzaldehyde with 4-dimethylaminostyrene (both commercially available) in the presence of an appropriately chosen catalyst to promote the stereoselectivity of the reaction (as shown in Scheme 3 ).

[0118] Chemicals, Reagents and Analytical Methods All reagents and solvents were commercially available and used without further purification.

[0119] Silica gel (230-400 mesh) was used for purification by column flash chromatography. All reactions were monitored by thin-layer chromatography (TLC) using f254 fluorescent gel silica plates (Sigma-Aldrich 99569). Melting points were determined using a B-454 melting point instrument. H and C NMR spectra were recorded on a Bruker 400 Ultra Shield™ instrument (400 MHz for H NMR and 100 MHz for C NMR) using tetramethylsilane (TMS) as the standard. Chemical substitutions are reported in parts per million (ppm). Multiplicities are reported as follows: singlet (s), doublet (d), triplet (t), and multiplet (m). Mass spectrometry was performed using a Thermo Finnigan LXQ linear ion trap mass spectrometer equipped with electrospray ionization (ESI). High-resolution mass spectra (HR-MS) were recorded using a Bruker BioApex Fourier transform ion cyclotron resonance (FT-ICR).

[0120] Synthesis procedure The compound trans-4-[2-(4-dimethylaminophenyl)vinyl]benzaldehyde (2) was prepared by the Heck reaction shown in Scheme 3 below.

[0121] [ka] Scheme 3 - Synthesis of Compound 2 The catalyst was prepared in situ. Palladium(II) acetate (Pd(CHCOO)) (Merck Life Science 3375-31-3) (16.8 mg, 0.075 mmol) and triphenylphosphine (PPh) (Merck Life Science 603-35-0) (19.7 mg, 0.075 mmol) were dissolved in dimethylformamide (DMF) (Merck Life Science 6812-2). After 10 min, a solution of 4-bromobenzaldehyde 3 (Merck Life Science 1122-91-4) (202 mg, 1.5 mmol), 4-dimethylaminostyrene 4 (component, Merck Life Science 2039-80-70) (264.6 mg, 1.8 mmol), and potassium carbonate (KCO) (Merck Life Science 584-08-7) (414 mg, 3.00 mmol) in DMF (3 mL) was added to the catalyst solution. The reaction was left stirring at 80 °C for 4 h. The reaction was then extracted with CHCl (3 times), and the organic phases were combined, dried over anhydrous NaSO, and concentrated under reduced pressure. Cold hexane crystallization afforded trans-4-[2-(4-dimethylaminophenyl)vinyl]benzaldehyde 2 (1,074 mmol, 270 mg) in 72% yield.

[0122] Yellow solid (yield 72%). MP: 218.0-220.0°C. 1 H NMR(400 MHz,CDCl3)δ 9.96(s,1H),7.83(d,J=8.2 Hz,2H),7.60(d,J=8.2 Hz,2H),7.45(d,J=8.7 Hz,2H),7.21(d,J=16.2 Hz,1H),6.94(d,J=16.2 Hz,1H),6.72(d,J=8.7 Hz,2H),3.01(s,5H). 13C NMR(101 MHz,CDCl3)δ 191.75,150.77,144.69,134.62,132.66,130.40,128.33,126.38,124.86,122.82,112.40,40.47.ESI-MS(m / z):[M+H] + C 17 H 18 Calculated NO value: 252.13, measured value: 252.17.

[0123] Compound BT1 has been prepared by Knoevenagel condensation (Scheme 4 below).

[0124] [ka] Scheme 4 - Synthesis of Compound BT1 A 10 mL solution of 4,4-difluoro-1,3-dimethyl-4-bora-3a,4a-diaza-s-indicene (TCI Europe 154793-49-4) (100 mg, 0.45 mmol) and trans-4-[2-(4-dimethylaminophenyl)vinyl]benzaldehyde (112.95 mg, 0.45 mmol) in toluene (Merck Life Science 108-88-3) was distilled at 120 °C for 2–4 h in the presence of piperidine (Merck Life Science 110-89-4) (0.35 mL, 6.12 mmol) and acetic acid (Merck Life Science 64-19-7) (0.35 mL, 3.5 mmol). The Dean-Stark reaction mixture was then removed. The reaction mixture was brought to room temperature, and 50 mL of aqueous ammonium chloride (NH4Cl) was added. The aqueous phase was then extracted with CHCl (3 × 50 ml), and the combined organic phases were dried over anhydrous NaSO and concentrated under reduced pressure. The reaction material was purified by flash chromatography using a 9:1 hexane:ethyl acetate eluent mixture. The compound 3-((E)-4-((E)-4-(dimethylamino)styryl)styryl)-5,5-difluoro-1-methyl-5H-dipyrrole[1,2-c:2',1'-f][1,3,2]diazaborin-4-ium-5-widoBT1 (mmol, mg) was obtained in 33% yield.

[0125] Black solid (yield 33%). MP: 257-262°C. 1 H NMR(400 MHz,CD2Cl2)δ 7.64-7.56(m,4H),7.53(d,J=8.2 Hz,2H),7.47-7.41(m,J=8.2 Hz,3H),7.23(s,1H),7.16(d,J=16.2 Hz,1H),6.94(d,J=16.2 Hz,1H),6.83(s,1H),6.72(d,J=8.2 Hz,2H),6.49-6.46(m,1H),2.99(s,6H),2.34(s,3H). 13 C NMR(101 MHz,CDCl3)δ 159.68,140.93,140.73,138.75,134.63,133.69,131.02,130.30,129.00,128.41,126.96,126.12,125. 63,123.60,123.36,118.80,117.96,117.84,117.42,116.77,112.80,40.68,30.26.ESI-MS(m / z):[M+H] + C 18 H 26 The calculated value of BF2N3 is 453.22, and the measured value is 454.33.

[0126] Example 3 - In vitro testing Maintenance of human iPSCs Human induced pluripotent stem cells (hiPSCs) were maintained by clonal expansion in mTeSR Plus medium (STEMCELL Technologies) on growth factor-reduced Matrigel-coated plates (Corning, dilution 1:100) at 37°C and 5% CO .

[0127] Differentiation of human iPSCs into retinal ganglion cells The differentiation protocol was a revised version of Sluch V. et al. (2017) with minor modifications. Human iPSCs were dissociated into single cells using 1x Accutase (Merck Life Science) and seeded onto growth factor-reduced Matrigel-coated plates at a density of 1000 cells / mm2 in mTeSR Plus supplemented with 10 μM Rock inhibitor Y-27632 (Peprotech). The seeding date was designated day minus 2 (D-2).

[0128] The next day (D-1), the stem cell medium was completely replaced with neurogenic basal medium (N2B27) consisting of 50% DMEM / F12 [1:1], 50% Neurobasal with 1% GlutaMAX, 1% non-essential amino acids (NEAA), 1% N2 Supplement, and 2% B27 Supplement without vitamin A (all from ThermoFisher Scientific). After another 24 hours (D0), fresh N2B27 medium was supplemented with a mixture of small molecules consisting of 25 μM forskolin (Peprotech), 1 μM dorsomorphin (Peprotech), 2.5 μM IDE2 (Peprotech), and 10 mM nicotinamide (Peprotech). At this stage, the medium was changed daily to enhance stem cell commitment to the anterior ventral forebrain.

[0129] Approximately 1 week after seeding (D7), when uniform, confluent neuroepithelial-like sheets were visible, cells were dissociated with 1x Accutase and seeded onto poly-L-ornithine / laminin-coated (Merck Life Science) dishes at a density of 1000 / mm2 in N2B27 + 10 μM Rock Inhibitor. The next day, the medium was switched to N2B27 containing 25 μM forskolin and 10 mM nicotinamide and changed daily for the next 3–4 days. Thereafter, N2B27 was supplemented with only 25 μM forskolin, 10 ng / mL IGF1 (Peprotech), and 10 ng / mL FGF2 (Peprotech) and refreshed twice weekly to promote the proliferation and expansion of retinal progenitor cells.

[0130] Upon reaching high-density confluence (around D18–20), retinal progenitor cell sheets were dissociated with 1x Accutase and seeded onto poly-L-ornithine / laminin-coated dishes at a density of 700 cells / mm. To enhance RGC maturation and escape from the proliferative state, N2B27 medium was supplemented with 10 μM Rock inhibitor (seeding only), 10 μM DAPT (Peprotech), and 25 μM forskolin, and the medium was changed every 3 days from day 30 to day 35.

[0131] Differentiation of human iPSCs into cortical neurons Human iPSC-cortical neurons were differentiated using a two-step protocol based on doxycycline-induced overexpression of the human NGN2 gene. Briefly, human iPSCs were treated with 1x Accutase and seeded onto growth factor-reduced Matrigel-coated plates at a density of 1,000 cells / mm² in mTeSR Plus containing 10 µM Rock inhibitor Y-27632. The seeding date is designated as day minus 3 (D-3). One day after seeding (D-2), the medium was switched to N2 medium consisting of DMEM / F12 [1:1] supplemented with 2 µg / mL doxycycline (Merck Life Science), 1% N2 supplement, 1% NEAA, and 1% GlutaMAX to induce human NGN2 expression. The N2 medium was refreshed daily. Three days later (D0), newborn neurons were dissociated with Accutase and plated at a density of 500 cells / mm on PDL / laminin-coated dishes in maturation medium consisting of Neurobasal, 2% B27 with vitamin A, 1% GlutaMAX, 0.5 μg / mL laminin (Merck Life Science), 20 ng / mL BDNF (Peprotech), 20 ng / mL ascorbic acid (Peprotech), 2 μg / mL doxycycline, 10 μM Rock inhibitor Y-27632, and 10 μM GDNF (Peprotech) supplemented with 10 μM DAPT. After 24 h, Y-27632 was removed, and DAPT and doxycycline were maintained in the medium until day 5. Optionally, 5 μM Ara-C (Merck Life Science) was added to the medium from days 6 to 10 to remove non-neuronal proliferating cells. Thereafter, half of the medium was replaced every week until the experimental window reached around D30.

[0132] Staining with BODIPY-based probes Human iPSC-derived neuronal cultures were incubated with either 100 μM TAU1 probe or 100 μM BT1 probe at 37°C for 30 min and then fixed with freshly prepared 4% cold PFA at room temperature for 15 min.

[0133] The tau 1 probe has the structure described in the paper by Verwilst P et al., "Rational Design of in Vivo Tau Tangle-Selective Near-Infrared Fluorophores: Expanding the BODIPY Universe." J Am Chem Soc. 2017 Sep 27;139(38):13393-13403.

[0134] immunocytochemistry Fixed hiPSC-derived cortical neurons and RGCs were permeabilized with 1x TBS containing 0.2% Triton X-100 (Merck Life Science) and incubated for 1 hour in blocking solution containing 1x TBS, 0.2% Triton X-100, and 5% goat serum (Merck Life Science). Cells were then incubated overnight at 4°C in blocking solution containing primary antibodies. The primary antibodies used in this study were goat anti-PHF-tau Ser202 / Thr205 (AT8; dilution 1:200; Thermo Fisher Scientific) and mouse anti-oligomeric TAU (T22; dilution 1:200; Merck Life Science), followed by incubation with secondary antibodies (dilution 1:1000) at room temperature for 1 hour. Images were acquired with an FV10i confocal system (Olympus) equipped with a 60x water-immersion objective. Fluorescence intensity per field of view was determined using Image J software.

[0135] The ability of the BT1 probe to specifically stain intracellular TAU aggregates was determined as a function of the antibody fluorescent signal detected within the binarized probe signal.

[0136] Figures 3 and 4 show the binarized signals of T22 and AT8 antibodies and BT1 or TAU1 probes detected in monolayer cultures of iPSC-derived cortical and retinal neurons after 30 days in vitro. Interestingly, the BT1 probe showed higher colocalization with the AT8 signal relative to T22, indicating that the BT1 probe preferentially stains intracellular aggregates enriched in phosphorylated TAU isoforms rather than oligomeric forms of TAU.

[0137] Furthermore, the BT1 probe exhibits a higher ability to detect phosphorylated aggregates compared to the TAU1 probe. Although performance is different, the enhanced detection of AT8-positive aggregates exhibited by the BT1 probe is conserved between iPSC-derived cortical neurons and iPSC-derived retinal ganglion cells, whereas the performance of the TAU1 probe is comparable.

Claims

1. Formula I, which selectively binds to tau protein 【Chemistry 1】 A fluorescent compound of the formula: wherein X and Y are carbon atoms linked by an aromatic para-substituted ring or by an aromatic 1,4-disubstituted ring; R is NH(RA), N(RA) ２ ,NHC(=O)RA,ORA,OC(=O)RA,SRA,SO ２ RA, SO ３ RA, OSO ２ RA, OSO ３ R.A., C.(R.A.) ３ , or C ５－７ an aromatic or aliphatic heterocycle, and In each substituent, RA is hydrogen, halogen, hydroxyl, CF ３ 、 C containing up to three independent heteroatoms selected from nitrogen, oxygen and sulfur １－７ saturated or unsaturated chain, straight or branched chain, C ５－７ cycloalkyl, phenyl, C ５－７ heterocycle or n-ethylene glycol; The fluorescent compound.

2. R is NH ２ , NH(CH ３ ), N(CH ３ ) ２ , N(Ph) ２ , imidazole, morpholine, or piperazine.

3. 3-((E)-4-((E)-4-(dimethylamino)styryl)styryl)-5,5-difluoro-1-methyl-5H-dipyrrolo[1,2-c:2′,1′-f][1,3,2]diazaborin-4-ium-5-uido, 3-((E)-4-((E)-4-(diphenylamino)styryl)styryl)-5,5-difluoro-1-methyl-5H-dipyrrolo[1,2-c:2′,1′-f][1,3,2]diazaborin-4-ium-5-uido, 3-((E)-4-((E)-4-aminostyryl)styryl)-5,5-difluoro-1-methyl-5H-dipyrrolo[1,2-c:2′,1′-f][1,3,2]diazaborinin-4-ium-5-uidate, 5,5-difluoro-1-methyl-3-((E)-4-((E)-4-(methylamino)styryl)styryl)-5H-dipyrrolo[1,2-c:2′,1′-f][1,3,2]diazaborin-4-ium-5-uido, 5,5-difluoro-1-methyl-3-((E)-4-((E)-4-morpholinostyryl)styryl)-5H-dipyrrolo[1,2-c:2′,1′-f][1,3,2]diazaborinin-4-ium-5-uidate, 5,5-difluoro-1-methyl-3-((E)-4-((E)-4-(piperazin-1-yl)styryl)styryl)-5H-dipyrrolo[1,2-c:2′,1′-f][1,3,2]diazaborin-4-ium-5-uido, 3-((E)-4-((E)-4-(1H-imidazol-1-yl)styryl)styryl)-5,5-difluoro-1-methyl-5H-dipyrrolo[1,2-c:2',1'-f] [1,3,2]diazaborin-4-ium-5-ui The fluorescent compound according to claim 1 or 2, wherein the compound is selected from the group consisting of:

4. The fluorescent compound according to claim 3, which is 3-((E)-4-((E)-4-(dimethylamino)styryl)styryl)-5,5-difluoro-1-methyl-5H-dipyrrolo[1,2-c:2',1'-f] [1,3,2]diazaborinin-4-ium-5-uido.

5. 10. The fluorescent compound of claim 1, having an excitation wavelength of 350 to 650 nm and an emission wavelength of 450 to 800 nm.

6. i. 4,4-difluoro-1,3-dimethyl-4-bora-3a,4a-diaza-s-indacene of formula II 【Chemistry 2】 wherein X and Y are carbon atoms linked by an aromatic para-substituted ring or by an aromatic 1,4-disubstituted ring; R is NH(RA), N(RA) ２ ,NHC(=O)RA,ORA,OC(=O)RA,SRA,SO ２ RA, SO ３ RA, OSO ２ RA, OSO ３ R.A., C.(R.A.) ３ , or C ５－７ an aromatic or aliphatic heterocycle, and In each substituent, R A is hydrogen, halogen, hydroxyl, CF ３ 、 C containing up to three independent heteroatoms selected from nitrogen, oxygen and sulfur １－７ saturated or unsaturated chain, straight or branched chain, C ５－７ cycloalkyl, phenyl, C ５－７ heterocycle or n-ethylene glycol) and subjecting the resulting mixture to a Knoevenagel condensation reaction with an aldehyde of ii. subjecting the solution obtained in step i to liquid-liquid extraction (LLE), followed by a purification step, to obtain said fluorescent compound of formula I; 10. A method for preparing the fluorescent compound of claim 1, comprising:

7. 7. The method of claim 6, wherein step i. is carried out in the presence of piperidine and acetic acid.

8. 8. The method of claim 6 or 7, wherein the aldehyde of formula II is trans-4-[2-(4-dimethylaminophenyl)vinyl]benzaldehyde.

9. Step ii is ii. a. Add saturated aqueous ammonium chloride solution (NH ４ Cl) and ii.b. subjecting the mixture obtained in step ii.a. to liquid-liquid extraction (LLE); ii.c. separating the aqueous phase obtained in step ii.b.; ii.d. The organic phase obtained in step ii.b is collected and treated with Na ２ SO ４ and a dehydration stage.

8. The method of claim 6 or 7, comprising:

10. A fluorescent marker comprising the fluorescent compound of claim 1.

11. A composition comprising the fluorescent compound of claim 1 and one or more additional excipients and / or carriers.

12. The composition of claim 11 in the form of an oral or ophthalmic composition.

13. 13. The composition according to claim 11 or 12, for use in detecting neurofibrillary tangles of tau protein.

14. detecting the fluorescent compound bound to a biological sample contacted with the fluorescent compound of claim 1, the fluorescent marker of claim 10, or the composition of claim 11 under conditions in which the fluorescent compound binds to neurofibrillary tangles of tau protein; 1. A method for detecting neurofibrillary tangles of tau protein, comprising:

15. performing non-invasive quantitative fluorescence imaging of the retina of a subject administered the fluorescent compound of claim 1, wherein detection of fluorescence from the fluorescent compound indicates binding of the compound to the retina; An imaging method comprising:

16. performing non-invasive quantitative fluorescence imaging on the retina of a subject administered with the fluorescent compound of claim 1 at a plurality of consecutive time points ti (i ranging from 0 to n) to obtain corresponding fluorescence values; using the obtained fluorescence values ​​to determine a graph of fluorescence as a function of time; 10. A method for determining a fluorescence graph, comprising:

17. 17. The method of claim 16, wherein the consecutive time points are separated by a period of one or more weeks, or one or more months, between each time point and the subsequent time point.

18. 18. The method of any one of claims 15 to 17, wherein the non-invasive quantitative fluorescence imaging is performed 30 minutes to 1 day after administration.

19. 18. The method of any one of claims 15 to 17, wherein the fluorescence imaging is performed by illuminating the subject's retina with a light source having a wavelength (λ) comprised between 350 and 650 nm and detecting and / or quantifying the fluorescence emitted by the fluorescent compound.

20. The method of any one of claims 15 to 17, wherein the fluorescent compound is administered orally or in the form of an eye ointment or eye drops.

Images