Mutant bestrophin 1 modulators for the treatment of BEST1-associated retinopathy

Abstract

The present invention relates to a biochanin A derivative or a salt thereof as defined in formula (II), which is used in a method for treating or preventing BEST1-related retinal diseases such as autosomal dominant Best yolk-shaped macular dystrophy. The present invention also relates to a pharmaceutical composition comprising a biochanin A derivative or a salt thereof as defined in formula (II) and a pharmaceutically acceptable excipient and / or carrier, which is used in a method for treating or preventing BEST1-related retinal diseases such as autosomal dominant Best yolk-shaped macular dystrophy. In addition, the present invention relates to a pharmaceutical package comprising one or more compartments, wherein at least one compartment comprises a biochanin A derivative or a salt thereof as defined in formula (II) of the present invention used according to the present invention, or a pharmaceutical composition or eye drops, eye ointments or implantable devices, in particular a microdrug delivery system, which comprises (i) a biochanin A derivative or (ii) a pharmaceutical composition as defined in formula (II) of the present invention used according to the present invention.

Description

[0001] The present invention relates to a biochanin A derivative or a salt thereof as defined in formula (II) for use in a method for treating or preventing BEST1-related retinopathy such as autosomal dominant Best yolk-shaped macular dystrophy. The present invention also relates to a pharmaceutical composition comprising a biochanin A derivative or a salt thereof as defined in formula (II) and a pharmaceutically acceptable excipient and / or carrier for use in a method for treating or preventing BEST1-related retinopathy such as autosomal dominant Best yolk-shaped macular dystrophy. In addition, the present invention relates to a pharmaceutical package comprising one or more compartments, wherein at least one compartment comprises a biochanin A derivative or a salt thereof or a pharmaceutical composition as defined in formula (II) of the present invention, or an eye drop, an eye ointment or an implantable device, in particular a microdrug delivery system, comprising (i) a biochanin A derivative as defined in formula (II) or (ii) a pharmaceutical composition according to the present invention used according to the present invention.

[0002] BEST1 (MIM 607854) belongs to the bestrophin family (BEST1-4) of four evolutionarily related genes, each encoding an integral membrane protein. In humans, they function as calcium-activated anion channels, but each channel is specific in terms of gene regulation and tissue distribution. BEST1 mutations are most prominently localized to the basolateral plasma membrane of the human retinal pigment epithelium (RPE) at the back of the eye, which is associated with at least four different retinal diseases, the so-called bestrophin diseases (bestrophinopathies), including autosomal dominant Best vitelloin macular dystrophy (BVMD; MIM 153700) (Marquardt et al., 1998; Petrukhin et al., 1998), autosomal dominant vitreoretinal choroidopathy (ADVIRC; MIM 193220) (Yardley et al., 2004) and autosomal recessive bestrophin disease (ARB; MIM 611809) (Burgess et al., 2008). Finally, a phenotype called graphic dystrophy can be segregated from typical BVMD and inherited as an autosomal dominant trait with a specific single mutation in BEST1,p.(Ala243Val) (Boon et al. 2009; Khan et al. 2018).

[0003] BVMD is the most common pathology of bestrophin disease, with an estimated prevalence of between 1:5000 and approximately 1:50000. It is a typically early-onset progressive disease. The disease is characterized by the accumulation of a lipofuscin-like material resembling an "egg yolk (= yolk-like)" in the macular region at the posterior pole. Later, disintegration of the lesions leads to atrophy of the RPE / photoreceptor complex, resulting in visual impairment. An important diagnostic feature is the abnormal Arden ratio (light peak / dark trough ratio) in the electrooculogram (EOG) response. Patients with Best macular dystrophy show a reduction in the slow light peak rise compared to healthy subjects, which is partly thought to reflect an increase in chloride conductance on the basolateral membrane of the RPE.

[0004] To date, more than 250 independent pathogenic mutations in the BEST1 gene have been reported in human gene mutation databases. These mutations affect BEST1 localization, protein stability, and ion gating properties. Therefore, these dysfunctions lead to the loss of BEST1 chloride transport function.

[0005] The X-ray structure of the chicken BEST1-Fab complex shows that the eukaryotic BEST1 channel is a pentameric structure composed of five homomeric BEST1 subunits, forming a The chicken BEST1 structure has a long pore that is just wide enough to allow the passage of dehydrated chloride ions. Two constrictions in the pore, the so-called "neck" and "aperture," are responsible for ion gating and ion selectivity, respectively. Cryo-electron microscopy and electrophysiological recordings revealed that introducing various mutations into the chicken BEST1 structure leads to (i) changes in the relative permeability between anions, (ii) the generation of channels with significantly altered gating properties and (iii) reduced channel inactivation.

[0006] So far, there is no treatment for BVMD or any BEST1-related retinopathies. Surprisingly, it was found that biochanin A diacetate and several other biochanin A derivatives can be used to specifically improve impaired BEST1 channel function. They are able to restore (BEST1-mediated) anion transport to address the main defect in BEST1-related bestrophin diseases (such as BEST1-related BVMD), namely impaired chloride conductance.

[0007] Therefore, the problem addressed by the present invention is to provide compounds capable of restoring (BEST1-mediated) anion transport in order to address the major defect in BEST1-related bestrophin diseases such as BEST1-related BVMD, in particular autosomal dominant BVMD, namely impaired chloride conductance.

[0008] Biochanin A diacetate is a biologically active substance belonging to the class of isoflavones. Isoflavones belong to the class of polyphenols and have estrogen agonist and estrogen antagonist properties. They are present in high concentrations in certain plants such as soybeans or red clover. According to WO2008 / 011083, biochanin A diacetate is known to be used in a method for treating ischemic, hypoxic or neurodegenerative diseases.

[0009] The problem underlying the invention is solved by the subject matter defined in the claims.

[0010] The following drawings are used to illustrate the present invention.

[0011] Figure 1 The generation of MDCKII stable cell lines expressing wild-type and mutant BEST1 is described. (A) Figure 1 As shown in A, seven disease-associated mutations representing hotspot mutation regions of the protein were selected. The wild-type BEST1 sequence was cloned into pcDNA3 and mutants were generated by site-directed mutagenesis. (B) Wild-type (WT) and mutant (p.(T6P), p.(L21V), p.(W93C), p.(R218C), p.(L224M), p.(Y227N), p.(F305S)) BEST1 constructs were stably transfected in MDCKII cells. Single clones were isolated in selection medium containing G418. Stable cell lines were grown on coverslips for 7 days to obtain cell polarity, and BEST1 localization was determined by immunocytochemistry.

[0012] Figure 2 Analysis of BEST1-mediated halide transport in MDCKII cell lines stably co-expressing YFP and wild-type or mutant BEST1 is shown. (A) Representative fluorescence images of MDCKII cell lines stably co-expressing wild-type BEST1 and YFP. (B) Schematic illustration of the principle behind the halide transport assay. When iodide (I - ) When YFP stably expressed in MDCKII cells enters the cell through an open anion channel, the fluorescence decreases. (C) Time course of cell fluorescence signal in MDCKII cells expressing wild-type BEST1 and untransfected control (as indicated). Cells were incubated in 100mM Cl- (=100%) and then Cl- was replaced by I- (n=6). (D) Same experiment as in C.MDCKII cells not expressing BEST1, expressing wild-type or mutant BEST1 (as indicated) (n=6).

[0013] Figure 3Schematic diagram of the drug screening method is shown. Stably transfected MDCKII cells co-expressing YFP and wild-type BEST1 (BEST1-WT; white circles) or mutant p.(R218C) (BEST1-R218C; gray circles) were cultured in black 96-well plates for 6 days. After adding the test compound, the cells were screened by adding I - The solution induced I - Each of the eight rows of grey wells is loaded with a different compound and plotted against I - YFP quenching values ​​after 90 seconds after application (boxes).

[0014] Figure 4 The effect of BioA(C2H3O2)2 on the anion transport function of BEST1 in MDCKII cells was revealed. (A) Chemical structures of the chickpea protein A derivatives. Top: The structure of BioA(C2H3O2)2, which has the greatest effect on the anion transport activity of BEST1: Bottom: Two biosimilars showed no effect. (BD) Addition of the compound BioA(C2H3O2)2 ( Figure 4 B and 4C) or BioA( Figure 4 D) After Figure 3 Functional assay of BEST1 in .

[0015] Figure 5 Examples of the effects of BioA(C2H3O2)2 on BEST1 chloride conductance in hiPSC-RPE cells are provided. (A) Immunostaining in + / + and R218C / +hiPSC-RPEs (B) IV graphs of selected recordings from -60 to 120 mV after Ca2+ stimulation (0.38 μM Ca2+ free) in + / + (black), untreated (light gray), and BioA(C2H3O2)2-treated (gray) R218C / +hiPSC-RPEs. (C) Statistical analysis of maximal Ca2+-activated currents at -60 and 120 mV. Data points are corrected for cell volume. The values ​​are given as mean ± SEM. n = 8-13; unpaired Student's T test: *P < 0.05; **P < 0.01

[0016] Figure 6 revealed that compared with untreated MDCKII cells, biochanin A ( Figure 6 A) and several biochanin A derivatives (i.e., trimethoxyisoflavones ( Figure 6 B), 5,4-dimethoxy-7-hydroxyisoflavone ( Figure 6 C), Biochanin A-7-methyl ether ( Figure 6 D), biochanin A-7-acetate ( Figure 6 E), biochanin A diacetate ( Figure 6F), biochanin A-7-valerate ( Figure 6 G) and biochanin A divalerate ( Figure 6 H)) on the anion transport function of BEST1 in MDCKII cells. Although compared with untreated cells, biochanin A ( Figure 6 A), trimethoxy isoflavones ( Figure 6 B), 5,4-dimethoxy-7-hydroxyisoflavone ( Figure 6 C), Biochanin A-7-methyl ether ( Figure 6 D), biochanin A-7-acetate ( Figure 6 E) and biochanin A-7-valerate ( Figure 6 G) showed no or only minimal effect when the corresponding compound was added, but showed no or only minimal effect when biochanin A diacetate ( Figure 6 F) and biochanin A divalerate ( Figure 6 H), the anion transport activity of the mutant BEST1-R218C was significantly increased.

[0017] Figure 7 revealed that several other biochanin A derivatives (i.e., biochanin A-7-pivalate ( Figure 7 A), biochanin A-dipipentanoate ( Figure 7 B), biochanin A-7-acrylate ( Figure 7 C), biochanin A diacrylate ( Figure 7 D), biochanin A-7-methyl fumarate ( Figure 7 E), biochanin A-di(methyl fumarate) Figure 7 F), biochanin A-7-butylcarbamate ( Figure 7 G) and biochanin A di(butylcarbamate) ( Figure 7 H)) on the anion transport function of BEST1 in MDCKII cells. Compared with untreated cells, although biochanin A-7-pivalate ( Figure 7 A), biochanin A-7-methyl fumarate ( Figure 7 E) and biochanin A-7-butylcarbamate ( Figure 7 G) showed no or only minimal effects after the addition of the respective compound delivery, and after the addition of biochanin A-divalerate ( Figure 7 B) and biochanin A-7-acrylate ( Figure 7 C), the anion transport activity of mutant BEST1-R218C was significantly increased, and after addition of biochanin A diacrylate ( Figure 7 D), biochanin A-di(methyl fumarate) ( Figure 7F) and biochanin A di(butylcarbamate) ( Figure 7 H), the anion transport activity of the mutant BEST1-R218C was significantly increased.

[0018] Figure 8 Several additional biochanin A derivatives (i.e., biochanin A-7-phosphate disodium salt ( Figure 8 A), biochanin A-7-isopropyl ester ( Figure 8 B), 7-neopentyloxymethyl-biochanin A ( Figure 8 C), biochanin A diisopropyl ester ( Figure 8 D), Biochanin A-7-isopropyl carbonate ( Figure 8 E), biochanin A isopropyl dicarbonate ( Figure 8 F) and isopropoxy-carbonylmethoxy-biochanin A ( Figure 8 G)) on the anion transport function of BEST1 in MDCKII cells. Although compared with untreated cells, biochanin A-7-isopropyl ester ( Figure 8 B) showed no or only minimal effects after addition of the corresponding compound delivery, addition of biochanin A-7-phosphate disodium salt ( Figure 8 A), 7-neopentyloxymethyl-biochanin A ( Figure 8 C) and isopropyloxycarbonylmethoxy-biochanin A ( Figure 8 G) after the addition of biochanin A diisopropyl ester ( Figure 8 D), Biochanin A-7-isopropyl carbonate ( Figure 8 E) and biochanin A diisopropyl carbonate ( Figure 8 F) After PCR, the anion transport activity of the mutant BEST1-R218C was significantly increased.

[0019] As used herein, the term "biochanin A diacetate" refers in particular to a compound of formula (I):

[0020]

[0021] As used herein, the term "biochanin A diacetate" may be used synonymously with the term "BioA(C2H3O2)2" or "3-(4-methoxyphenyl)-4-oxo-4H-chromene-5,7-diyl diacetate".

[0022] The term "BEST1 associated retinopathy" relates to the retinopathy (MIM607854) caused by BEST1 gene mutation. BEST1 is most prominently positioned at the basolateral plasma membrane of the RPE at the back of the eye. The mutation in BEST1 can affect BEST1 positioning, protein stability and ion gating properties. As a result, these functional impairments cause the loss of BEST1 channel function, particularly anion transport function, more preferably chloride transport function, which causes the damage of the eye retina.

[0023] As used herein, the term "BEST1" is an abbreviation for bestrophin-1. It can be used synonymously with the term "VMD2". BEST1 belongs to the bestrophin family of four evolutionarily related genes (BEST1-4), which encode complete membrane proteins. In humans, they play a role as calcium-activated anion channels, although each channel is specific in terms of gene regulation and protein tissue distribution. Wild-type BEST1 preferably has the amino acid sequence shown in SEQ ID NO:1. Wild-type BEST1 is preferably encoded by the nucleic acid sequence shown in SEQ ID NO:2.

[0024] The term "bestrophin disease" refers to a group of degenerative retinal disease phenotypes caused by one or more mutations in the BEST1 gene, particularly one mutation (in the case of autosomal dominant inheritance) or two mutations (in the case of autosomal recessive inheritance) in the BEST1 gene.

[0025] As used herein, the term "Best's vitelliform macular dystrophy (BVMD)" may also be referred to as "Best's macular dystrophy," "vitelliform macular dystrophy-2 (VMD2)," or simply "Best's disease." It is an inherited retinal dystrophy involving the RPE / photoreceptor complex that manifests as yellow "egg yolk"-like lesions in the macula in the early stages of the disease. It is the most common phenotype of bestrophin disease. Subjects with autosomal dominant Best's vitelliform macular dystrophy have a mutation in the BEST1 gene, resulting in loss of channel function and ultimately retinal degeneration.

[0026] The term "autosomal dominant vitreoretinal choroidopathy (ADVIRC)" refers to a chorioretinal pigment disorder affecting the peripheral retina. According to the prior art, in particular five BEST1 gene mutations p.(Gly83Asp), p.(Val86Met), p.(Val235Ala), p.(Tyr236Cys) and p.(Val239Met) are the cause of ADVIRC. The disease is typically characterized by a peripheral hyperpigmented band of the peripheral retina with a clear posterior demarcation and may be associated with developmental ocular abnormalities such as microcornea, microphthalmia, angle-closure glaucoma and cataracts. The term "autosomal recessive bestrophin disease (ARB)" refers to a disease caused by homozygous or compound heterozygous BEST1 gene mutations. The main features of ARB are multifocal subretinal deposits outside the macular area, abnormal autofluorescence and accumulation of subretinal fluid or macular edema. Heterozygous parents usually have no retinal symptoms.

[0027] As used herein, the term "pattern dystrophy" refers to a specific form of autosomal dominant Best macular dystrophy, which is particularly associated with the p.(Ala243Val) mutation. In this form, the yolk-like lesions disappear and patterned pigmentary changes form instead. The course of the disease is usually mild. Preferably, the term "pattern dystrophy" as used herein is associated only with the p.(Ala243Val) mutation.

[0028] Any mutation described in the present invention refers to wild-type BEST1, as shown in SEQ ID NO:1.

[0029] The term "increase chloride conductivity" refers to an increase in the level of chloride conductivity measurable in a given assay in the presence of a candidate compound relative to the measurable level of chloride conductivity in the absence of the candidate compound when tested under the same conditions. Chloride conductivity is improved according to the present invention if the chloride conductivity is increased by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 150%, 200%, 250%, 300% or more compared to the absence of the candidate compound. Improved chloride conductivity indicates improved anion transport activity. Improvements in chloride conductance and anion transport activity, respectively, can be tested, for example, as described in Example 9 or 10.

[0030] The term "recovering BEST1 channel function" refers in particular to repairing BEST1 anion transport function, particularly chloride transport function, if BEST1 channel function is impaired, for example due to one or more sudden changes in the BEST1 gene. When tested under the same conditions and compared with the BEST1 channel function of the control without sudden change in the BEST1 gene, can be in a given test in the presence of candidate compound, relative to the measurable level of BEST1 channel function when there is no candidate compound, determine the repair of BEST1 channel function. If in the presence of candidate compound, can recover at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more BEST1 channel function, then according to the present invention, recovered BEST1 channel function. If in the presence of candidate compound, BEST1 channel function improves at least about 100%, 150%, 200%, 250%, 300% or more, also the term "recovering BEST1 channel function" can be used.

[0031] The term "comprising" as used herein should not be interpreted as being limited to the meaning of "consisting of" (i.e., excluding the presence of additional other materials). Rather, "comprising" means that optional additional materials may be present. The term "comprising" includes within its scope the specifically envisioned embodiments of "consisting of" (i.e., excluding the presence of additional other materials) and "comprising but not consisting of" (i.e., requiring the presence of additional other materials), with the former being more preferred.

[0032] In the first object of the present invention, it is envisaged to provide a modulator of a BEST1 chloride channel that can restore BEST1 channel function. Specifically, the present invention provides a chickpea derivative or a salt thereof for use in a method for treating or preventing BEST1-related retinopathy such as BVMD, particularly autosomal dominant BVMD. Preventing BEST1-related retinopathy such as BVMD preferably includes delaying the onset and / or development of BEST1-related retinopathy such as BVMD. Treatment of BEST1-related retinopathy such as BVMD may also include delaying the progression of BEST1-related retinopathy such as BVMD, or preventing the recurrence of disease symptoms after the disease has been treated before. The chickpea derivative according to the present invention can regulate the BEST1 transport channel, particularly by improving chloride conductivity across the basolateral membrane of the subject's RPE, thereby restoring the BEST1 channel function. Specifically, the chickpea derivative used according to the present invention can compensate for the reduced BEST1 anion transport activity by enhancing the remaining BEST1 channel function in the subject's RPE or activating other undetermined anion channels.

[0033] The biochanin A derivative that solves the potential problems of the present invention is a biochanin A derivative according to formula (II):

[0034]

[0035] Where R1 is a proton or any of the following residues:

[0036]

[0037] and R2 is one of the following residues:

[0038]

[0039] Provided that when R1 is a proton, R2 is selected from any of the following residues:

[0040]

[0041]

[0042] Therefore, R1 in formula (I) is preferably a proton, acetate, valerate, isopropyl, acrylate, methyl fumarate, butylcarbamate, isopropyl carbonate, isopropyloxycarbonylmethoxy, disodium phosphate or neopentyloxymethyl, and R2 in formula I is preferably acetate, valerate, isopropyl, acrylate, methyl fumarate, butylcarbamate, isopropyl carbonate, isopropyloxycarbonylmethoxy, disodium phosphate or neopentyloxymethyl, provided that when R1 is a proton, R2 is acrylate, isopropyl carbonate, isopropyloxycarbonylmethoxy, disodium phosphate or neopentyloxymethyl.

[0043] In a particularly preferred embodiment of the present invention, R1 and R2 in formula (I) are identical, or R1 is a proton and R2 is isopropyl carbonate.

[0044] In a preferred embodiment, the biochanin A derivative used according to the present invention is selected from the group shown in Table 1. The compounds marked with an asterisk in the circle represent particularly preferred biochanin A derivatives.

[0045] Table 1:

[0046]

[0047] In contrast, it has been found that the following biochanin A compounds have no or only a minimal effect on anion transport activity. Therefore, the following biochanin A compounds do not represent a preferred embodiment for use according to the present invention.

[0048]

[0049] In a preferred embodiment, the biochanin A derivative used according to the present invention is selected from: biochanin A diacetate, biochanin A divalerate, biochanin A diacrylate, biochanin A di(methyl fumarate), biochanin A-7-isopropyl carbonate, biochanin A di(butylcarbamate), biochanin A diisopropyl carbonate, biochanin A diphosphate tetrasodium salt, dineopentyloxymethyl-biochanin A, biochanin A diisopropyl ester, and diisopropyloxycarbonylmethoxy-biochanin A.

[0050] In a further particularly preferred embodiment, the biochanin A derivative is selected from the group shown in Table 2.

[0051] Table 2:

[0052]

[0053]

[0054] In a further particularly preferred embodiment, the biochanin A derivative is selected from the group consisting of: biochanin A diacetate, biochanin A divalerate, biochanin A diacrylate, biochanin A di(methyl fumarate), biochanin A-7-isopropyl carbonate, biochanin A di(butylcarbamate), biochanin A dicarbonate isopropyl, biochanin A diisopropyl. Biochanin A diacetate represents a particularly preferred embodiment of the present invention.

[0055] The method for treating, preventing and / or delaying the onset or development of BEST1-related retinopathy such as BVMD preferably comprises administering to a subject a preferred chickpea A derivative or a salt thereof as defined above. Preferably, the chickpea A derivative or a salt thereof is administered in an amount sufficient to improve the chloride conductivity across the basolateral membrane of the subject's RPE. Preferably, it is administered in an amount sufficient to restore the BEST1 channel function. Preferably, it is administered in an amount sufficient to compensate for the reduced BEST1 anion transport activity by enhancing the remaining BEST1 channel function in the subject's RPE or activating other undetermined anion channels. In a preferred embodiment, the method further comprises a molecular genetics testing step of BEST1. In a particularly preferred embodiment, the method comprises the following steps

[0056] (i) Assess the subject's vision before treatment;

[0057] (ii) molecular genetic testing of the BEST1 gene;

[0058] (iii) administering to the subject a biochanin A derivative or a salt thereof as defined above; and

[0059] (iv) assessing the subject's visual acuity after step (iii).

[0060] Molecular genetic testing of the BEST1 gene can be performed from DNA extracted from a peripheral blood sample of a subject. Mutation analysis can be performed by the Sanger chain termination method, a DNA sequencing technique based on the selective incorporation of chain-terminating dideoxynucleotides (ddNTPs) by DNA polymerase during in vitro DNA replication. Specifically, the nucleotide sequences of 11 coding sequences (exons) and the corresponding flanking intronic regions (about 20 base pairs each) will be sequenced from the subject's BEST1 gene.

[0061] The present invention also relates to a pharmaceutical composition comprising the preferred biochanin A derivative or a salt thereof as defined above and a pharmaceutically acceptable excipient and / or carrier, for use in a method for treating, preventing and / or delaying the onset or progression of BEST1-related retinopathy as defined above.

[0062] The pharmaceutical composition according to the present invention may additionally contain one or more conventional additives. Some examples of such additives include solubilizers, such as glycerol; antioxidants, such as benzalkonium chloride, benzyl alcohol, chloretone or chlorobutanol; and / or isotonic agents. As a further precaution against oxidation or other deterioration, the pharmaceutical composition may be stored under a suitable gas (e.g., nitrogen) in a vial sealed with a sealing stopper.

[0063] Preferably, the preferred biochanin A derivatives or salts thereof as defined above and / or the pharmaceutical compositions of the present invention are used in a method for treating or preventing BEST1-related retinopathy, in particular the so-called bestrophin disease. In a preferred embodiment of the present invention, the BEST1-related retinopathy or bestrophin disease is caused by a change in the BEST1 protein, which may be caused by a mutation in the BEST1 gene. Preferably, such BEST1-related retinopathy or bestrophin disease is caused or characterized by an impaired or reduced anion transport activity of BEST1. BEST1-related retinopathy or bestrophin disease is selected from: autosomal dominant Best macular dystrophy, vitelliform macular dystrophy-2 (VMD2), autosomal dominant vitreoretinal choroidopathy (ADVIRC), autosomal recessive bestrophin disease or pattern dystrophy, more preferably from autosomal dominant Best macular dystrophy, vitelliform macular dystrophy-2 (VMD2)), autosomal recessive bestrophin disease or pattern dystrophy. The treatment and prevention of autosomal dominant BVMD is particularly preferred.

[0064] The subject to be treated is preferably a human or an animal, in particular a mammal, most preferably a human. The preferred biochanin A derivatives or salts thereof as defined above and / or the pharmaceutical composition of the present invention can be administered to a subject in need in an effective amount. Those skilled in the art can easily determine the effective amount of the compound to be administered during preclinical trials and clinical trials by methods familiar to physicians and clinicians.

[0065] According to all embodiments of the present invention, an effective amount of a preferred chickpea A derivative or a salt thereof as defined above, preferably in the form of a pharmaceutical composition, for use in a method for treating or preventing BEST1-related retinopathy can be administered to a subject in need by any of a number of known methods of administering pharmaceutical compounds. The compound can be administered topically or systemically. The route of administration can be oral, topical, ocular, intraocular, by eye drops, or by intravitreal injection, or any other suitable route of administration.

[0066] In another aspect, the present invention relates to a method for preparing a pharmaceutical composition, which comprises mixing a biochanin A derivative or a salt thereof and / or a pharmaceutical composition of the present invention with a pharmaceutically acceptable diluent, excipient or carrier.

[0067] The present invention also relates to a pharmaceutical pack comprising one or more compartments, wherein at least one compartment comprises a preferred biochanin A derivative or a salt thereof as defined above or a pharmaceutical composition according to the present invention.

[0068] In a preferred embodiment, the chickpea derivative A derivative or its salt or pharmaceutical composition of the present invention is formulated for topical administration, in particular for intraocular administration. It is preferably formulated in the form of eye drops or eye ointments. Alternatively, the chickpea derivative A derivative or its salt or pharmaceutical composition of the present invention may be formulated for oral administration, for example in the form of tablets, capsules, dragees or pills, but may also be in the form of injectable solutions or any other medically reasonable galenic formulations. Preferably, the galenic formulation comprises a suitable carrier, stabilizer, flavoring agent, buffer or other suitable agent.

[0069] Therefore, the present invention also relates to eye drops or eye ointments comprising a preferred biochanin A derivative or its salt as defined above and a pharmaceutically acceptable excipient and / or carrier. Eye drops or eye ointments or preparations in the form of eye drops or eye ointments can be prepared as known to those skilled in the art. For example, eye drops can be prepared using a tonicity agent, such as concentrated glycerol or sodium chloride, a buffer, such as sodium phosphate or sodium acetate, a surfactant, such as polyoxyethylene sorbitan monooleate or polyoxyethylene 40 stearate, a stabilizer, such as sodium citrate or sodium edetate and / or a preservative such as benzalkonium chloride or parabens. The pH of the eye drops is preferably within the acceptable range of ophthalmic preparations. For example, it can be within the range of pH 4 to 8. Eye ointments can be prepared using commonly used bases such as white soft paraffin or liquid paraffin.

[0070] In a preferred embodiment, the biochanin A derivative or its salt or pharmaceutical composition according to the present invention is formulated for intraocular administration, in particular in the form of an implantable device, in particular a micro drug reservoir, for effective and controlled ocular drug delivery, in particular intraocular drug delivery. When the reservoir is implanted, the doctor can replenish the drug without removing the reservoir. The implantable device can be prepared as known to those skilled in the art. For example, a port delivery system currently being tested in a phase III clinical trial (ClinicalTrials.gov identifier: NCT03677934) can be implanted.

[0071] Therefore, the present invention also relates to an implantable device, in particular an implantable micro drug delivery system, comprising the preferred biochanin A derivative or salt thereof as defined above and a pharmaceutically acceptable excipient and / or carrier.

[0072] In a preferred embodiment, the pharmaceutical pack, eye drops, eye ointment and / or implantable device as described above is used for the use according to the invention as described herein.

[0073] The preferred salts of the biochanin A derivatives as defined above of all embodiments of the present invention are preferably pharmaceutically acceptable salts of the respective biochanin A derivatives.

[0074] In a specific embodiment of the present invention, the preferred biochanin A derivatives as defined above or their salts and / or the pharmaceutical compositions of the present invention are used as treatment for BEST1-related retinopathies, in particular so-called bestrophin diseases, which are selected from: autosomal dominant Best macular dystrophy, vitelliform macular dystrophy-2 (VMD2), autosomal dominant vitreoretinal choroidopathy (ADVIRC), autosomal recessive bestrophin diseases, or pattern dystrophy, more preferably from autosomal dominant Best macular dystrophy, vitelliform macular dystrophy-2 (VMD2), autosomal recessive bestrophin diseases or pattern dystrophy. The treatment of autosomal dominant BVMD is particularly preferred.

[0075] In another specific embodiment of the present invention, the biochanin A derivative or its salt and / or pharmaceutical composition of the present invention is used for the preparation of a medicament for the treatment of BEST1-related retinopathies, in particular the so-called bestrophin disease, selected from the group consisting of autosomal dominant Best macular dystrophy, vitelliform macular dystrophy-2 (VMD2), autosomal dominant vitreoretinal choroidopathy (ADVIRC), autosomal recessive bestrophin disease, or pattern dystrophy, more preferably from autosomal dominant Best macular dystrophy, vitelliform macular dystrophy-2 (VMD2), autosomal recessive bestrophin disease or pattern dystrophy. The treatment of autosomal dominant BVMD is particularly preferred.

[0076] Another aspect of the present invention is a method for treating the above-mentioned medical conditions by administering or applying to a subject, in particular to a human or an animal, a biochanin A derivative or a salt thereof as defined above or a pharmaceutical composition comprising such a biochanin A derivative or a salt thereof.

[0077] In yet another aspect, the present invention provides a method for improving chloride conduction across the basolateral membrane of the RPE in a subject in need thereof. The method comprises administering to the subject an effective amount of a preferred biochanin A derivative or a salt thereof as defined above or a pharmaceutical composition comprising such a biochanin A derivative or a salt thereof.

[0078] In another aspect, the present invention provides a method for restoring BEST1 channel function, which comprises administering to a subject an effective amount of a preferred biochanin A derivative or a salt thereof as defined above, or a pharmaceutical composition comprising such a biochanin A derivative or a salt thereof.

[0079] Finally, the present invention provides a method for activating other channels besides BEST1 to compensate for the reduced anion transport activity of BEST1. The method comprises administering to a subject an effective amount of a preferred biochanin A derivative or a salt thereof as defined above or a pharmaceutical composition comprising such a biochanin A derivative or a salt thereof.

[0080] The following examples illustrate the present invention but are not to be considered as limiting. It should be understood that the detailed description and specific examples disclosed herein, which indicate specific embodiments of the present invention, are given by way of illustration only, as various changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art based on this description.

[0081] Example 1 - Generation of a stable MDCKII cell line

[0082] According to the instructions of the manufacturer (Thermofisher Scientific, Waltham, USA), MDCKII cells were transfected with Lipofectamine3000 transfection reagent. MDCKII cells expressing wild-type and mutant BEST1 were cultured for 2 weeks in a selection medium containing 500 mg / ml G418, and then single cells were inoculated into 96-well plates. 1-5 monoclones of each cell line were selected.

[0083] Example 2 - Generation of hiPSC-RPE cell lines from Best macular dystrophy patients

[0084] Skin biopsies were collected from Best macular dystrophy patients and healthy controls carrying BEST1 gene mutation-related diseases. Adult dermal fibroblasts were established and reprogrammed into human induced pluripotent stem cells (hiPSC) (Takahashi et al., 2007) by overexpression of transcription factors OCT3 / 4, Sox2, Klf4 and c-Myc. As described in Brandl et al. (2014), the cells were subsequently differentiated into hiPSC-derived RPE cells.

[0085] Example 3 - Immunofluorescence labeling

[0086] Cell monolayers were fixed in 4% paraformaldehyde (PFA) / PBS for 10 min and blocked with PBS containing 0.3% TritonX-100 and 10% goat serum for 25 min. Incubation with BEST1 primary antibody and fluorescently conjugated secondary antibodies was performed at 4°C ON. Immunolabeled hiPSC-RPE or MDCKII cells were imaged on a Zeiss confocal microscope LSM 510 (Zeiss, Germany).

[0087] Example 4 - YFP-halide transport assay

[0088] Human hiPSC-RPE or MDCKII cells were transduced with lentiviral particles using Ca 2+ The phosphate transfection method was used to co-transfect HEK293T cells with yellow fluorescent protein (YFPH148Q / I152L)-pLJM1 and the helper plasmids pMD2.G and psPAX2. After culturing for six weeks in 96-well black microtiter plates, the cells were incubated with 100 mM Cl - The cells were then incubated with an equimolar solution of I - Replace Cl - The decrease in YFP fluorescence intensity was monitored for an additional 2 minutes at 10-second intervals.

[0089] Example 5 - Whole-cell patch clamp recording

[0090] Human iPSC-RPE cells were trypsinized and seeded on fibronectin-coated glass coverslips for electrophysiological characterization. Whole-cell patch clamp recordings were performed using an EPC10 patch clamp amplifier (HEKAElectronics) controlled by PatchMaster v2x90.2 software. Capacitance and series resistance were adjusted using built-in compensation algorithms. Patch pipettes had a tip resistance of 3–5 MΩ and were loaded with an internal solution containing (in mM): 40 mM CsCl, 100 mM Cs methanesulfonate, 1 mM MgCl2, 5 mM EGTA, 4 mM ATP-Na2 (freshly added), and 10 mM HEPES (pH 7.4). 0.38 μM free Ca was obtained by adding CaCl2 2+ Concentration (Ca-EGTA Calculator TS v1.3). Voltage-driven inward and outward currents were induced by applying voltage-ramps from -60 to +120 mV at 2-second intervals (500 ms duration). The formula of the external solution was (in mM): 150 NaCl, 6 CsCl, 1.5 CaCl2, 1 MgCl2, 10 glucose and 10 HEPES (pH 7.4).

[0091] Example 6 - Generation of MDCKII stable cell lines expressing wild-type and mutant BEST1

[0092] Initially, stable MDCKII cell lines were established that constitutively expressed wild-type BEST1 and disease-associated BEST1 mutants (i.e., p.(T6P), p.(L21V), p.(W93C), p.(R218C), p.(L224M), p.(Y227N), p.(F305S)). The mutants were located in four mutational hotspots of BEST1 (White et al., 2000) ( Figure 1 A). The results show that polarized MDCKII cell lines are very suitable for studying protein trafficking and anion conductance in epithelial cells (Milenkovic et al., 2011; Milenkovic et al., 2018). Immunostaining of BEST1 with human polyclonal antibodies showed that the proteins of wild-type BEST1 and mutant BEST1-R218C were localized to the plasma membrane, while the remaining six mutant proteins mainly accumulated in the cytoplasm ( Figure 1 B).

[0093] Example 7 - Analysis of BEST1-mediated anion transport in cell lines

[0094] To analyze BEST1-mediated anion transport in the cell lines generated in the previous examples, a well-established halide transport assay was used (Galietta et al., 2001). All cell lines were virally transduced with the yellow YFP-based halide sensor YFP (H148Q / I152L) and seeded in black 96-well plates, showing bright and uniform cell fluorescence that is highly sensitive to iodide ions (I-) (Jayaraman et al., 1999) ( Figure 2 A). The addition of I- results in a specific quenching of YFP over time, and the change in fluorescence intensity can be monitored on a plate reader. MDCKII cells were incubated with a solution containing 100 mM Cl, and the steady-state YFP fluorescence signal intensity was measured after 6 minutes. Subsequently, Cl- was replaced by equimolar I-, and anion permeability was monitored over a period of up to 2 minutes by a decrease in YFP intensity due to BEST1-mediated I- influx ( Figure 2 B). Rapid kinetics of fluorescence intensity reduction to approximately 30 to 40% were observed for several independent BEST1 wild-type cell lines, whereas the untransfected control cell lines remained flat ( Figure 2 C and D). To demonstrate the utility of this assay for characterizing BEST1 channel function, all BEST macular dystrophy-related cell lines were analyzed under identical conditions. There was no significant difference in fluorescence intensity between mutant cell lines compared to untransfected controls, indicating that BEST1 mutants have significantly reduced channel activity, whereas cell lines expressing wild-type BEST1 exhibited rapid kinetics of reduced fluorescence signal due to BEST1-mediated ion flux ( Figure 2C). Taken together, these data suggest that this assay is a suitable system to test whether compounds act on the BEST1 ion channel by either enhancing residual BEST1 channel function (potentiators) or activating channels other than BEST1 to compensate for its reduced anion transport activity.

[0095] Example 8 - Evaluation of BioA(C2H3O2)2 activity in MDCKII cells

[0096] MDCKII cells co-expressing YFP and BEST1 wild type or mutant p.(R218C) are inoculated on black 96-well plates and cultured for 6 days to achieve polarity, which is the requirement for the correct positioning of BEST1 in epithelial cells. Compounds are tested at 10 μM concentrations in 96-well format, respectively. After adding the compound for 24 hours, cells are subjected to an inwardly directed I- gradient to drive I-influx and produce a decreasing (quenching) fluorescent signal. Each assay includes recording baseline fluorescence for 10 seconds (=100% value), and then recording 120 seconds of fluorescence reduction after adding I- solution. Figure 3 A schematic diagram of the screening method is shown. The effect of BioA(C2H3O2)2 on the I- influx kinetics was tested ( Figure 4 AD). Both the BEST1 wild-type cell line and the untransfected control had little or no effect on the delivery of (C2H3O2)2 ( Figure 4 B), while the anion transport activities of mutants BEST1-R218C and BEST-Y227N were strongly and moderately increased at various concentrations (from 2 μM to 20 μM), respectively ( Figure 4 C). Notably, BiochaninA (BioA) had no effect on BEST1-mediated I-influx ( Figure 4 D).

[0097] Example 9 - Evaluation of the biological activity of BioA(C2H3O2)2 in hiPSC-RPE cells generated from patients with BEST macular dystrophy

[0098] Next, we tested whether the results of the fluorescence-based assay in the MDCKII cell line could be validated in a cell line-based disease-relevant model system. To this end, we generated iPSC-RPE heterozygous for the BEST1 mutation p.(R218C) (hiPSC-RPE-R218C / +) from skin fibroblasts of healthy donors (hiPSC-RPE+ / +) and patients with BEST macular dystrophy. Consistent with the immunostaining results for BEST1 in the MDCKII test series, correct localization of BEST1 was confirmed on the plasma membrane of mutant and control hiPSC-RPE treated with BioA(C2H3O2)2 ( Figure 5A). Subsequently, whole-cell patch clamp recordings were performed to analyze calcium-activated chloride currents in treated and untreated hiPSC-RPE cells and controls. Figure 5 As shown, hiPSC-RPE+ / + produced significant Cl- current amplitudes, which were greatly reduced in hiPSC-RPE-R218C / + cells. However, the addition of 10 μM BioA(C2H3O2)2 resulted in a significant increase in Cl- currents in hiPSC-RPER218C / + cells, confirming the efficacy of BioA(C2H3O2)2 in enhancing BEST1 channel function in RPE ( Figure 5 B and C).

[0099] Example 10 - Evaluation of the activity of several biochanin A compounds / derivatives in MDCKII cells

[0100] MDCKII cells co-expressing yellow fluorescent protein (YFP) and mutant BEST1 R218C were seeded on black 96-well plates and cultured for 6 days to achieve polarity, a key requirement for the correct localization of BEST1 in epithelial cells. Compounds were tested at a concentration of 5 μM in a 96-well format. 24 hours after the addition of the compound, the cells were subjected to an outwardly directed iodine (I-)-gradient to drive BEST1-mediated I-efflux and produce an increased fluorescence signal. Each assay consisted of recording YFP quenching after 6 minutes of I-preincubation (=100% value), followed by recording a 7-minute fluorescence increase after adding a chloride (Cl-)-containing solution. Changes in fluorescence intensity were monitored on a plate reader. Biochanin A ( Figure 6 A), trimethoxy isoflavones ( Figure 6 B), 5,4-dimethoxy-7-hydroxyisoflavone ( Figure 6 C), Biochanin A-7-methyl ether ( Figure 6 D), biochanin A-7-acetate ( Figure 6 E), biochanin A diacetate ( Figure 6 F), biochanin A-7-valerate ( Figure 6 G), biochanin A divalerate ( Figure 6 H), biochanin A-7-pivalate ( Figure 7 A), biochanin A-dipipentanoate ( Figure 7 B), biochanin A-7-acrylate ( Figure 7 C), biochanin A diacrylate ( Figure 7 D), biochanin A-7-methyl fumarate ( Figure 7 E), biochanin A-di(methyl fumarate) Figure 7 F), biochanin A-7-butylcarbamate ( Figure 7 G) and biochanin A di(butylcarbamate) ( Figure 7 H), biochanin A-7-phosphate disodium salt ( Figure 8 A), biochanin A-7-isopropyl ester ( Figure 8 B), 7-neopentyloxymethyl-biochanin A ( Figure 8 C), biochanin A diisopropyl ester ( Figure 8 D), Biochanin A-7-isopropyl carbonate ( Figure 8 E), biochanin A isopropyl dicarbonate ( Figure 8 F) and isopropoxy-carbonylmethoxy-biochanin A ( Figure 8 G) Effects of biochanin A ( Figure 6 A), trimethoxy isoflavones ( Figure 6 B), 5,4-dimethoxy-7-hydroxyisoflavone ( Figure 6 C), Biochanin A-7-methyl ether ( Figure 6 D), biochanin A-7-acetate ( Figure 6 E), biochanin A-7-valerate ( Figure 6 G), biochanin A-7-pivalate ( Figure 7 A), biochanin A-7-methyl fumarate ( Figure 7 E), biochanin A-7-butylcarbamate ( Figure 7 G), biochanin A-7-isopropanol ester ( Figure 8 B) After the addition of the corresponding compound delivery, no or only minimal effect was shown, and the addition of biochanin A-dipivalate ( Figure 7 B), biochanin A-7-acrylate ( Figure 7 C), biochanin A-7-phosphate disodium salt ( Figure 8 A), 7-neopentyloxymethyl-biochanin A ( Figure 8 C), isopropyloxycarbonylmethoxy-biochanin A ( Figure 8 G), the anion transport activity of mutant BEST1-R218C was significantly increased, and the addition of biochanin A diacetate ( Figure 6 F), biochanin A divalerate ( Figure 6 H), biochanin A diacrylate ( Figure 7 D), biochanin A-di(methyl fumarate) Figure 7 F), biochanin A di(butylcarbamate) Figure 7 H), biochanin A diisopropyl ester ( Figure 8 D), biochanin A-7-isopropyl carbonate ( Figure 8 E), biochanin A isopropyl dicarbonate ( Figure 8 F), the anion transport activity increased significantly.

[0101] References

[0102] Boon, C. J., Theelen, T., Hoefsloot, E. H. et al. (2009) Clinical and molecular genetic analysis of best vitelliform macular dystrophy. Retina 29: 835 - 847.

[0103] Brandl, C., Zimmermann, S. J., Milenkovic, V. M., Rosendahl, S. M., Grassmann, F., Milenkovic, A., Hehr, U., Federlin, M., Wetzel, C. H., Helbig, H. et al. (2014). In - Depth Characterisation of Retinal Pigment Epithelium (RPE) Cells Derived from Human Induced Pluripotent Stem Cells (hiPSC). Neuromolecular Med.

[0104] Burgess, R., Millar, I. D., Leroy, B. P., Urquhart, J. E., Fearon, I. M., De Baere, E., Brown, P. D., Robson, A. G., Wright, G. A., Kestelyn, P. et al. (2008). Biallelic mutation of BEST1 causes a distinct retinopathy in humans. Am J Hum Genet 82, 19 - 31.

[0105] Galietta, L. V., Jayaraman, S. and Verkman, A. S. (2001). Cell - based assay for high - throughput quantitative screening of CFTR chloride transport agonists. Am J Physiol Cell Physiol 281, C1734 - 42.

[0106] Jayaraman,S.,Teitler,L.,Skalski,B.and Verkman,A.S.(1999).Long-wavelength iodide-sensitive fluorescent indicators for measurement offunctional CFTR expression in cells.Am J Physiol 277,C1008-18.

[0107] Khan,K.N.,Islam,F.,Moore,A.T.et al.(2018)The fundus phenotypeassociated with the p.Ala243Val BEST1 mutation.Retina 38:606-613.

[0108] Marquardt,A.,Stohr,H.,Passmore,L.A.,Kramer,F.,Rivera,A.and Weber,B.H.(1998).Mutations in a novel gene,VMD2,encoding a protein of unknownproperties cause juvenile-onset vitelliform macular dystrophy(Best'sdisease).Hum Mol Genet 7,1517-25.

[0109] Milenkovic,A.,Milenkovic,V.M.,Wetzel,C.H.and Weber,B.H.F.(2018).BEST1protein stability and degradation pathways differ between autosomal dominantBest disease and autosomal recessive bestrophinopathy accounting for thedistinct retinal phenotypes.Hum Mol Genet 27,1630-1641.

[0110] Milenkovic,V.M.,Rohrl,E.,Weber,B.H.and Strauss,O.(2011).Disease-associated missense mutations in bestrophin-1 affect cellular trafficking andanion conductance.J Cell Sci 124,2988-96.

[0111] Petrukhin,K.,Koisti,M.J.,Bakall,B.,Li,W.,Xie,G.,Marknell,T.,Sandgren,O.,Forsman,K.,Holmgren,G.,Andreasson,S.et al.(1998).Identification of thegene responsible for Best macular dystrophy.Nat Genet 19,241-7.

[0112] Takahashi,K.,Tanabe,K.,Ohnuki,M.,Narita,M.,Ichisaka,T.,Tomoda,K.andYamanaka,S.(2007).Induction of pluripotent stem cells from adult humanfibroblasts by defined factors.Cell 131,861-72.

[0113] White,K.,Marquardt,A.and Weber,B.H.(2000).VMD2 mutations invitelliform macular dystrophy(Best disease)and other maculopathies.Hum Mutat15,301-8.

[0114] Yardley,J.,Leroy,BP,Hart-Holden,N.,Lafaut,BA,Loeys,B.,Messiaen,LM,Perveen,R.,Reddy,MA,Bhattacharya,SS,Traboulsi,E.et al.(2004).Mutations of VMD2 splicing regulators cause nanophthalmos and autosomaldominant vitreoretinochoroidopathy(ADVIRC).Invest Ophthalmol Vis Sci 45,3683-9. SEQUENCE LISTING <110> University of Regensburg <120> Mutant bestrophin 1 modulators for the treatment of BEST1-associated retinopathy <130> REG-001 PCT <140> unknown <141> 2021-03-30 <150> EP20166646.8 <151> 2020-03-30 <160> 2 <170> PatentIn Version 3.5 <210> 1 <211> 585 <212> PRT <213> Homo sapiens bestrophin 1 (BEST1) <400> 1 Met Thr Ile Thr Tyr Thr Ser Gln Val Ala Asn Ala Arg Leu Gly Ser 1 5 10 15 Phe Ser Arg Leu Leu Leu Cys Trp Arg Gly Ser Ile Tyr Lys Leu Leu 20 25 30 Tyr Gly Glu Phe Leu Ile Phe Leu Leu Cys Tyr Tyr Ile Ile Arg Phe 35 40 45 Ile Tyr Arg Leu Ala Leu Thr Glu Glu Gln Gln Leu Met Phe Glu Lys 50 55 60 Leu Thr Leu Tyr Cys Asp Ser Tyr Ile Gln Leu Ile Pro Ile Ser Phe 65 70 75 80 Val Leu Gly Phe Tyr Val Thr Leu Val Val Thr Arg Trp Trp Asn Gln 85 90 95 Tyr Glu Asn Leu Pro Trp Pro Asp Arg Leu Met Ser Leu Val Ser Gly 100 105 110 Phe Val Glu Gly Lys Asp Glu Gln Gly Arg Leu Leu Arg Arg Thr Leu 115 120 125 Ile Arg Tyr Ala Asn Leu Gly Asn Val Leu Ile Leu Arg Ser Val Ser 130 135 140 Thr Ala Val Tyr Lys Arg Phe Pro Ser Ala Gln His Leu Val Gln Ala 145 150 155 160 Gly Phe Met Thr Pro Ala Glu His Lys Gln Leu Glu Lys Leu Ser Leu 165 170 175 Pro His Asn Met Phe Trp Val Pro Trp Val Trp Phe Ala Asn Leu Ser 180 185 190 Met Lys Ala Trp Leu Gly Gly Arg Ile Arg Asp Pro Ile Leu Leu Gln 195 200 205 Ser Leu Leu Asn Glu Met Asn Thr Leu Arg Thr Gln Cys Gly His Leu 210 215 220 Tyr Ala Tyr Asp Trp Ile Ser Ile Pro Leu Val Tyr Thr Gln Val Val 225 230 235 240 Thr Val Ala Val Tyr Ser Phe Phe Leu Thr Cys Leu Val Gly Arg Gln 245 250 255 Phe Leu Asn Pro Ala Lys Ala Tyr Pro Gly His Glu Leu Asp Leu Val 260 265 270 Val Pro Val Phe Thr Phe Leu Gln Phe Phe Phe Tyr Val Gly Trp Leu 275 280 285 Lys Val Ala Glu Gln Leu Ile Asn Pro Phe Gly Glu Asp Asp Asp Asp 290 295 300 Phe Glu Thr Asn Trp Ile Val Asp Arg Asn Leu Gln Val Ser Leu Leu 305 310 315 320 Ala Val Asp Glu Met His Gln Asp Leu Pro Arg Met Glu Pro Asp Met 325 330 335 Tyr Trp Asn Lys Pro Glu Pro Gln Pro Pro Tyr Thr Ala Ala Ser Ala 340 345 350 Gln Phe Arg Arg Ala Ser Phe Met Gly Ser Thr Phe Asn Ile Ser Leu 355 360 365 Asn Lys Glu Glu Met Glu Phe Gln Pro Asn Gln Glu Asp Glu Glu Asp 370 375 380 Ala His Ala Gly Ile Ile Gly Arg Phe Leu Gly Leu Gln Ser His Asp 385 390 395 400 His His Pro Pro Arg Ala Asn Ser Arg Thr Lys Leu Leu Trp Pro Lys 405 410 415 Arg Glu Ser Leu Leu His Glu Gly Leu Pro Lys Asn His Lys Ala Ala 420 425 430 Lys Gln Asn Val Arg Gly Gln Glu Asp Asn Lys Ala Trp Lys Leu Lys 435 440 445 Ala Val Asp Ala Phe Lys Ser Ala Pro Leu Tyr Gln Arg Pro Gly Tyr 450 455 460 Tyr Ser Ala Pro Gln Thr Pro Leu Ser Pro Thr Pro Met Phe Phe Pro 465 470 475 480 Leu Glu Pro Ser Ala Pro Ser Lys Leu His Ser Val Thr Gly Ile Asp 485 490 495 Thr Lys Asp Lys Ser Leu Lys Thr Val Ser Ser Gly Ala Lys Lys Ser 500 505 510 Phe Glu Leu Leu Ser Glu Ser Asp Gly Ala Leu Met Glu His Pro Glu 515 520 525 Val Ser Gln Val Arg Arg Lys Thr Val Glu Phe Asn Leu Thr Asp Met 530 535 540 Pro Glu Ile Pro Glu Asn His Leu Lys Glu Pro Leu Glu Gln Ser Pro 545 550 555 560 Thr Asn Ile His Thr Thr Leu Lys Asp His Met Asp Pro Tyr Trp Ala 565 570 575 Leu Glu Asn Arg Asp Glu Ala His Ser 580 585 <210> 2 <211> 1758 <212> DNA <213> Homo sapiens bestrophin 1 (BEST1) <400> 2 atgaccatca cttacacaag ccaagtggct aatgcccgct taggctcctt ctcccgcctg 60 ctgctgtgct ggcggggcag catctacaag ctgctatatg gcgagttctt aatcttcctg 120 ctctgctact acatcatccg ctttatttat aggctggccc tcacggaaga acaacagctg 180 atgtttgaga aactgactct gtattgcgac agctacatcc agctcatccc catttccttc 240 gtgctgggct tctacgtgac gctggtcgtg acccgctggt ggaaccagta cgagaacctg 300 ccgtggcccg accgcctcat gagcctggtg tcgggcttcg tcgaaggcaa ggacgagcaa 360 ggccggctgc tgcggcgcac gctcatccgc tacgccaacc tgggcaacgt gctcatcctg 420 cgcagcgtca gcaccgcagt ctacaagcgc ttccccagcg cccagcacct ggtgcaagca 480 ggctttatga ctccggcaga acacaagcag ttggagaaac tgagcctacc acacaacatg ttctgggtgc cctgggtgtg gtttgccaac ctgtcaatga aggcgtggct tggaggtcga atccgggacc ctatcctgct ccagagcctg ctgaacgaga tgaacacctt gcgtactcag 660 tgtggacacc tgtatgccta cgactggatt agtatcccac tggtgtatac acaggtggtg 720 actgtggcgg tgtacagctt cttcctgact tgtctagttg ggcggcagtt tctgaaccca 780 gccaaggcct accctggcca tgagctggac ctcgttgtgc ccgtcttcac gttcctgcag 840 ttcttcttct atgttggctg gctgaaggtg gcagagcagc tcatcaaccc ctttggagag900 960. gatgatgatg attttgagac caactggatt gtcgacagga atttgcaggt gtccctgttg gctgtggatg agatgcacca ggacctgcct cggatggagc cggacatgta ctggaataag cccgagccac agccccccta cacagctgct tccgcccagt tccgtcgagc ctcctttatg 1080 ggctccacct tcaacatcag cctgaacaaa gaggagatgg agttccagcc caatcaggag 1140 gacgaggagg atgctcacgc tggcatcatt ggccgcttcc taggcctgca gtcccatgat 1200 caccatcctc ccagggcaaa ctcaaggacc aaactactgt ggcccaagag ggaatccctt 1260 ctccacgagg gcctgcccaa aaccacaag ccagccaaac agaacgttag gggccaggaa 1320 gacaacaagg cctggaagct taaggctgtg gacgcttca agtctgcccc actgtatcag 1380 aggccaggct actacagtgc cccacagcg cccctcagcc ccactcccat gttcttcccc 1440 ctagaaccat cagcgccgtc aaagcttcac agtgtcacag catagacac caaagacaaa 1500 agcttaaaga ctgtgagttc tggggccaag aaaagttttg aattgctctc agagagcgat 1560 ggggccttga tggagcaccc agaagtatct caagtgagga ggaaaactgt ggagtttaac 1620 ctgacggata tgccagagat ccccgaaaat cacctcaaag aacctttgga acaatcacca 1680 accaacatac acactacact caaagatcac atggatcctt attgggcctt ggaaaacagg 1740 gatgaagcac attcctaa 1758

Claims

1. Use of a biochanin A derivative or a salt thereof in the preparation of a medicament for treating or preventing Best macular dystrophy, wherein the biochanin A derivative has formula (II): in R1 is a proton, or OR1 is acetate, valerate, isopropionate, acrylate, methyl fumarate, butylcarbamate, isopropyl carbonate, isopropoxycarbonylmethoxy, disodium phosphate or neopentyloxymethyl, and OR2 is acetate, valerate, isopropionate, acrylate, methyl fumarate, butylcarbamate, isopropyl carbonate, isopropoxycarbonylmethoxy, disodium phosphate or neopentyloxymethyl, Provided that when R1 is a proton, OR2 is acrylate, isopropyl carbonate, isopropoxycarbonylmethoxy, disodium phosphate or neopentyloxymethyl, and The biochanin A derivative is selected from the group consisting of: biochanin A diacetate, biochanin A divalerate, biochanin A diacrylate, biochanin A di(methyl fumarate), biochanin A-7-isopropyl carbonate, biochanin A di(butylcarbamate), biochanin A diisopropyl carbonate, biochanin A diisopropionate, biochanin A-7-acrylate, biochanin A-7-phosphate disodium salt, 7-neopentyloxymethyl-biochanin A, and isopropoxy-carbonylmethoxy-biochanin A.

2. The use according to claim 1, wherein OR1 and OR2 are the same or R1 is a proton and OR2 is isopropyl carbonate.

3. The use according to claim 1 or 2, wherein preventing Best macular dystrophy comprises delaying the onset or progression of BEST macular dystrophy.

4. The use according to claim 1 or 2, wherein the biochanin A derivative or a salt thereof is administered in an amount sufficient to (i) improve chloride conduction across the basolateral membrane of retinal pigment epithelial cells and / or (ii) restore BEST1 channel function. 5 . The use according to claim 1 or 2 , wherein the biochanin A derivative or a salt thereof is administered in an amount sufficient to activate channels other than BEST1 to compensate for the impaired anion transport activity of BEST1.

6. The method according to claim 1 or 2, comprising the following steps (i) Assess the subject's vision before treatment; (ii) molecular genetic testing of the BEST1 gene; (iii) administering to the subject a biochanin A derivative according to formula (II) or a salt thereof as defined in any one of the preceding claims; and (iv) assessing the subject's visual acuity after step (iii).

7. The use according to claim 1 or 2, wherein the biochanin A derivative or its salt is formulated for topical, oral, ocular and / or intraocular administration.

8. The use according to claim 1 or 2, wherein the biochanin A derivative or its salt is formulated into the form of eye drops, eye ointments, tablets, capsules, pills, injections or implants.

9. The use according to claim 8, wherein the pellets are dragees.

10. Use of a pharmaceutical composition comprising a biochanin A derivative according to formula (II) or a salt thereof as defined in claim 1 or 2 and a pharmaceutically acceptable excipient and / or carrier in the preparation of a medicament for treating or preventing Best macular dystrophy.

11. The use according to claim 1 or 2, wherein the Best macular dystrophy is characterized by impaired anion transport activity of BEST1.

12. The use according to claim 1 or 2, wherein the BEST macular dystrophy is autosomal dominant Best macular dystrophy.

13. The use according to claim 12, wherein the autosomal dominant Best macular dystrophy is a pattern dystrophy caused by the BEST1 mutation p.(Ala243Val).

14. Use of (i) a biochanin A derivative as defined in claim 1 or 2 or (ii) a pharmaceutical composition as defined in claim 10 for the preparation of a medicament in the form of eye drops, eye ointment or implantable device for the treatment or prevention of Best macular dystrophy.

15. The use according to claim 14, wherein the implantable device is a micro drug delivery system.

16. Use of (i) a biochanin A derivative as defined in claim 1 or 2 or (ii) a pharmaceutical composition as defined in claim 10 for the preparation of a medicament in the form of a pharmaceutical pack for treating or preventing Best macular dystrophy, the pharmaceutical pack comprising one or more compartments.

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