Pharmaceutical composition and treatment methods for genetic diseases associated with splice anomalies

Description

Technical Field

[0001] The present disclosure relates to a pharmaceutical composition for genetic diseases caused by aberrant splicing events, and a method for, using the pharmaceutical composition preventing, ameliorating, suppressing progression of, and / or treating genetic diseases caused by an aberrant splicing regulation, particularly Fabry disease.Background Art

[0002] Patent Document 1 discloses a reporter system capable of detecting alternative splicing, and a method for identifying a compound that affects alternative splicing, using the reporter system.Prior Art DocumentsPatent Document

[0003] [Patent Document 1] WO 2011 / 152043 A1 (US9273364B2)Disclosure of InventionProblem to be Solved by the Invention

[0004] Various genetic diseases resulting from aberrant splicing are known. Fabry disease is known as one of such genetic diseases. Fabry disease is a disease, to which a genetic mutation in alternative splicing or the like resulting from a splicing mutation is attributed. In recent years, although enzyme replacement therapy by a recombinant α-galactosidase A (GLA) enzyme protein has been developed for Fabry disease, Fabry disease patients still rely on many symptomatic therapies. Thus, in general, development of curative therapeutics with a novel strategy has been unmet clinical needs for various genetic diseases caused by aberrant splicing events.

[0005] The present disclosure is concerned with a pharmaceutical composition capable of preventing, ameliorating, suppressing progression of, and / or treating genetic diseases caused by aberrant splicing events, and a method for, using the pharmaceutical composition, preventing, ameliorating, suppressing progression of, and / or treating genetic diseases caused by aberrant splicing events. The invention is concerned with Fabry disease.Means for Solving Problem

[0006] In one or more embodiments, the present disclosure relates to a pharmaceutical composition for use in preventing, ameliorating, suppressing progression of, and / or treating Fabry disease, the pharmaceutical composition containing an active ingredient capable of suppressing a splicing abnormality that contributes to the development or progression of the Fabry disease, wherein the compound is: wherein Y represents a halogen atom, or a pharmaceutically acceptable salt thereof.Brief Description of Drawings

[0007] FIG. 1 is a conceptual diagram illustrating splicing mutations found in genetic diseases. FIG. 2 is a diagram illustrating a GLA gene pseudo exon skipping evaluation system vector. pAM1 is constituted by a normal IVS4 sequence, and pAM2 has the IVS4+919G>A mutation. A precursor mRNA (pre-mRNA) including exon 4 to exon 5 is transcribed, and splicing alteration in patient cells with the identical mutation is recapitulated. pAM1 and pAM2 share sequences other than the sequence of the IVS4+919G>A point mutation. FIGS. 3A and 3B show one example of the effect of administration of Compound 1 on inhibiting the GLA gene pseudo exon in Fabry disease caused by the IVS4+919G>A mutation. FIG. 3A shows one example in which, with regard to splicing of the normal GLA and the IVS4+919G>A mutant GLA, the fact that the production of a normal isoform (pseudo exon skipping) is restored through treatment with Compound 1 was confirmed by RT-PCR. The control, for which intracellular Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is used, is shown in a lower panel, showing that the amounts of RNA used in analysis are equal to each other. FIG. 3B is a schematic diagram, illustrating GLA splice switching and recovery of an active enzyme using Compound 1. FIGS. 4A, 4B and 4C show one example of the effect of administration of Compound 1 on inhibiting the GLA gene pseudo exon in Fabry disease caused by the IVS4+919G>A mutation. FIG. 5 is a diagram illustrating one example of the configuration of a SPREADD reporter system for a splicing mutation in the IKBKAP gene in familial dysautonomia. FIGS. 6A and 6B show one example of the effect of administration of Compound III-1 on inhibiting a CFTR gene pseudo exon in cystic fibrosis. FIGS. 7A and 7B show one example of a functional isoform induction effect of administration of Compound III-1 on the COL4A5 gene in Alport syndrome and the TSC2 gene in tuberous sclerosis. Description of the Invention

[0008] Various genetic diseases caused by an aberrant splicing regulation are known. Splicing mutations found in genetic diseases are classified into 1) an exon skipping type, 2) a splice site selection type, 3) an intron retention type, and 4) a pseudo exon type (FIG. 1).

[0009] An exon skipping mutation refers to a splicing mutation in which an exon that is normally recognized cannot be recognized (skipping occurs) due to a mutation within the exon or a peripheral intron sequence. An exon skipping mutation results in suppression or loss of a 5' splice site, suppression or loss of a 3' splice site, suppression or loss of an enhancer element, or formation of a silencer element. Out of exon skipping mutations, mutations other than mutations in GU located at the 5' splice site +1 or +2 and AG located at the 3' splice site -1 or -2, which are essential for splicing, are considered as targets for splicing therapeutic agents.

[0010] A splice site selection mutation refers to a splicing mutation in which a plurality of 5' splice sites or 3' splice sites occur due to a mutation in a splicing regulatory sequence in an exon region or an intron region. Similarly to exon skipping mutations, mutations other than mutations in GU located at the 5' splice site +1 or +2 and AG located at the 3' splice site -1 or -2, which are essential for splicing, are considered as targets for splicing therapeutic agents.

[0011] An intron-retention mutation refers to a splicing mutation in which recognition of an intron region (intron definition) is incomplete due to a mutation in an exon or intron region near the 5' splice site or the 3' splice site, and intron retention is induced. Similarly to exon skipping mutations, mutations other than mutations in GU located at the 5' splice site +1 or +2 and AG located at the 3' splice site -1 or -2, which are essential for splicing, are considered as targets for splicing therapeutic agents.

[0012] A pseudo exon mutation refers to a splicing mutation in which a sequence that is originally a sequence of an intron region is recognized as an exon due to a mutation. A pseudo exon mutation occurs due to a newly created 5' splice site, 3' splice site, or enhancer element, or suppression or loss of a silencer element occurring due to a mutation occurring within an intronic sequence. As for pseudo exon mutations, any pseudo exon mutations are considered as targets for splicing therapeutic agents.

[0013] Inventors of the present invention found a compound capable of enhancing exon recognition in splicing in which exon recognition is incomplete due to a splicing abnormality, and a compound capable of suppressing exon recognition in the splicing. Also, the inventors found that a compound capable of enhancing exon recognition in splicing in which exon recognition is incomplete due to a splicing abnormality exhibits therapeutic effects on both exon skipping mutations and pseudo exon mutations. Also, the inventors found that a compound capable of suppressing exon recognition in splicing in which exon recognition is incomplete due to a splicing abnormality exhibits therapeutic effects on pseudo exon mutations. The inventors found that a compound capable of suppressing exon recognition in splicing in which exon recognition is incomplete due to a splicing abnormality exhibits the effects of inducing a functional splicing isoform in an exon (a PTC exon) into which a premature termination codon (PTC) is introduced, and being capable of avoiding PTC.

[0014] Examples of genetic diseases caused by an aberrant splicing regulation resulting from exon skipping mutations include Pompe disease, mucopolysaccharidoses, congenital long QT syndrome, Fukuyama congenital muscular dystrophy, progeria syndrome, amyotrophic lateral sclerosis, atypical adenofibrosis, autism, autism spectrum disorder, Charcot-Marie-Tooth disease, CHARGE syndrome, dementia, epilepsy, epileptic encephalopathies, familial dysautonomia (IKBKAP), familial isolated growth hormone deficiency type II, Frasier syndrome, frontotemporal dementia, Parkinson's disease, Huntington's disease, Marfan syndrome, mental retardation, Menkes disease, muscular dystrophy, myopathy, myotonic dystrophy type I, myotonic dystrophy type 2, neurofibromatosis type 1, von Recklinghausen NF, peripheral NF, occipital horn syndrome, retinoblastoma, schizophrenia, and tuberous sclerosis.

[0015] Examples of genetic diseases caused by an aberrant splicing regulation resulting from pseudo exon mutations include Fabry disease (GLA), cystic fibrosis (CFTR), homocystinuria (MTRR), hereditary breast / ovarian cancer syndrome (BRCA 1, BRCA2), ataxia-telangiectasia / Louis-Bar syndrome (ATM), Lynch syndrome (MSH2), neurofibromatosis type 1 (NF1), tuberous sclerosis (TSC2), atypical pyridoxine-dependent epilepsy (ALDH7A1), Leber congenital amaurosis (CEP290), Alport syndrome (COL4A3), chronic granulomatous disease (CYBB), 17α-hydroxylase deficiency (CYP17A1), Marfan syndrome (FBN1), X-linked hypophosphatemia (PHEX), and polycystic kidney disease (PKHD1) (responsible genes with pseudo exon are indicated in parentheses).

[0016] Examples of genetic diseases in which it is expected that a PTC can be avoided in a similar manner through induction of a splicing isoform include Alport syndrome (COL4A5), Bartter syndrome (CLCNKA), Becker muscular dystrophy (DMD), hereditary ovarian cancer and breast cancer (BRCA1, BRCA2, PALB2), colon cancer / T-cell acute lymphoblastic leukemia (BAX), arrhythmia (KCNH2), cardiomyopathy (TNNT2), Carney complex (PRKAR1A), CHARGE syndrome (CHD7), chronic granulomatous disease (CYBB), ciliary dyskinesia syndrome (ZMYND10), Cockayne syndrome (ERCC8), congenital disorders of glycosylation type I (SSR4), Cornelia de Lange syndrome (NIPBL), cystic fibrosis (CFTR), hearing impairment (RDX, OTOF, SMPX), dilated cardiomyopathy (DSP), Duchenne muscular dystrophy (TTN, DMD), familial adenomatous polyposis (APC), hypertrophic cardiomyopathy (MYBPC3), fibrochondrogenesis (COL11A1), Finnish congenital nephrotic syndrome (NPHS1), β-galactosidase deficiency (GALC), glycogen storage disease type III (AGL), hereditary neoplastic syndrome (CDH1, STK11), Hermansky-Pudlak syndrome (HPS5), hypogonadotropic hypogonadism (TACR3), I-cell disease (GNPTAB), juvenile polyposis syndrome (SMAD4), limb-girdle muscular dystrophy (CAPN3, ANO5), lissencephaly (PAFAH1B1), Lynch syndrome (MLH1, PMS2), Marfan syndrome (FBN1), meconium ileus (GUCY2C), merosin-deficient muscular dystrophy (LAMA2), congenital mirror movement disorder (DCC), Miyoshi muscular dystrophy, mucolipidosis type III (GNPTG), myopathy, early-onset-areflexia-respiratory-distress-dysphagia (MEGF10), nemaline myopathy, nonimmunologic hydrops fetalis (NEB), neutral lipid storage disease with myopathy (PNPLA2), nonketotic hyperglycinemia (GLDC), Hurler syndrome (IDUA), maple syrup urine disease (BCKDHA), oligodontia-colorectal cancer syndrome (AXIN2), orofaciodigital syndrome (OFDI), gyrate atrophy (OAT), Nance-Sweeney syndrome (COL11A2), palmoplantar keratoderma (SERPINB7), Parkinson's disease (LRRK2), phenylketonuria (PAH), pituitary hormone deficiency (POU1F1), pyridoxine-dependent epilepsy (ALDH7A1), severe combined immunodeficiency (JAK3), severe myoclonic epilepsy of infancy (SCN1A), myotubular myopathy (MTM1), Sotos syndrome (NSD1), spinal muscular atrophy (SMN1), spinocerebellar ataxia (ANO10), tuberous sclerosis (TSC2), and familial tumoral calcinosis (GALNT3) (responsible genes with PTC are indicated in parentheses).

[0017] The invention is concerned with treating, preventing, ameliorating or suppressing the progression of Fabry disease.Pharmaceutical composition for genetic diseases resulting from an aberrant splicing regulation

[0018] The present disclosure provides a pharmaceutical composition for use in preventing, ameliorating, suppressing progression of, and / or treating genetic diseases caused by an aberrant splicing regulation, in particular Fabry disease, the pharmaceutical composition containing, as an active ingredient, a compound capable of suppressing a splicing abnormality that contributes to the development or progression of the genetic diseases. In accordance with the invention, the compound is: wherein Y represents a halogen atom, or a pharmaceutically acceptable salt thereof.

[0019] The compound in the pharmaceutical composition of the present disclosure may enhance exon recognition in splicing in which exon recognition is incomplete due to a splicing mutation, and suppress recognition of an exon created by a splicing mutation.

[0020] The above compounds provide examples of a compound represented by Formulae (II) and (II').

[0021] In Formulae (II) and (II'), R 1a< and R 2a< each independently represent a hydrogen atom, a substituted or unsubstituted C 1-6 alkyl group, a substituted or unsubstituted benzyl group, a substituted or unsubstituted heteroarylmethyl group, a substituted or unsubstituted heteroarylethyl group, a substituted or unsubstituted aryloxy group, a substituted or unsubstituted heteroaryloxy group, a substituted or unsubstituted alkoxyamidoalkyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted heteroaryl group, or alternatively R 1a< and R 2a< bind to each other to form a ring together with N, and the ring is a substituted or unsubstituted monocyclic heterocyclic ring, or a substituted or unsubstituted bicyclic heterocyclic ring; R 5< represents a hydrogen atom, a halogen atom, or a substituted or unsubstituted C 1 -C 6 alkoxy group; X 1< represents N or CH; X 2< represents -N(R 3< )-, S, or O; R 3< represents a hydrogen atom, a C 1 -C 6 alkyl group, a benzyl or heteroarylmethyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, or CH 2 OC(O)R 4< -; R 4< represents a C 1 -C 6 alkyl group, a benzyl or heteroarylmethyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted heteroaryl group; and X represents a hydrogen atom, a halogen atom, an amino group, an R 1a< - and R 2a< -substituted amino group, an azido group, a cyano group, a nitro group, a hydroxy group, a C 1 -C 6 alkyl group, a substituted or unsubstituted aryloxy group, a substituted or unsubstituted heteroaryloxy group, a mercapto group, a linear, branched, or cyclic alkylthio group having 1 to 6 carbon atoms, a substituted or unsubstituted arylthio group, a substituted or unsubstituted heteroarylthio group, a benzyl or heteroarylmethyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted heteroaryl group.

[0022] In Formula (II) or (II'), if X 1< and X 2< respectively represent N and NH, Formulae (II) and (II') above are tautomers. Although only one tautomer is illustrated in the above-described specific examples, disclosure of one tautomer also discloses the other tautomer in the present disclosure. If a compound represented by Formula (II) or (II') includes an asymmetric carbon atom, and / or if a stereoisomer thereof is present, the compound may be a mixture of isomers or an isolated isomer, in one or more embodiments. The tautomers of the compounds for use in accordance with the invention are shown below:

[0023] In the present disclosure, the number of substituents of a "substituted or unsubstituted group" may be one or more and the substituents may be the same as or different from each other, and in one or more embodiments, examples thereof include a halogen atom, a cyano group, a trifluoromethyl group, a nitro group, a hydroxy group, a methylenedioxy group, a lower alkyl group, a lower alkoxy group, a benzyloxy group, a lower alkanoyloxy group, an amino group, a mono-lower alkylamino group, a di-lower alkylamino group, a carbamoyl group, a lower alkylaminocarbonyl group, a di-lower alkylaminocarbonyl group, a carboxyl group, a lower alkoxycarbonyl group, a lower alkylthio group, a lower alkylsulfinyl group, a lower alkylsulfonyl group, a lower alkanoylamino group, and a lower alkylsulfonamide group. In one or more embodiments, examples of the halogen atom include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom.

[0024] Examples of the compound for use according to the invention, which fall within Formula (II) or (II') include the compounds below, which show the tautomer forms of two different compounds.

[0025] In one or more embodiments, the pharmaceutical composition for use according to the invention contains, as an active ingredient, a compound as set forth above, and may further contain a medicinally acceptable carrier, an antiseptic, a diluent, an excipient, or other medicinally acceptable component.Method for preventing, ameliorating, suppressing progression of, and / or treating genetic diseases resulting from an aberrant splicing regulation

[0026] The present invention relates to a pharmaceutical composition as described hereinbefore for use in preventing, ameliorating, suppressing progression of, and / or treating genetic diseases caused by aberrant splicing events, in particular Fabry disease. The use may be achieved by administering a compound as described herein which is capable of suppressing a splicing abnormality that contributes to the development or progression of the genetic diseases to a subject that requires the compound. The compound is capable of enhancing exon recognition in splicing in which exon recognition is incomplete due to a splicing abnormality or capable of suppressing exon recognition.

[0027] Fabry disease is a genetic disease resulting from an aberrant splicing regulation. Fabry disease is a disease, in which glycolipids such as globotriaosylceramide (Gb3) accumulate in lysosomes due to a deficiency of the GLA enzyme, which is a lysosomal hydrolase, resulting in various symptoms relating to various organs such as circulatory organs (e.g., the heart) and kidney.

[0028] Fabry disease is classified into three types according to symptoms: classic, atypical, and heterozygous. With classic Fabry disease, normally, the GLA enzyme activity is low or barely detectable. On the other hand, with atypical Fabry disease, especially with a cardiac variant (cardiac Fabry disease), whose symptoms mainly appear in the cardiovascular system, the GLA enzyme activity can be detectable, and thus, the onset age of atypical Fabry disease is higher than in classic Fabry disease. Although heterozygous Fabry diseases have individual differences, such as due to effects of X-chromosome inactivation in female, the symptoms of Fabry disease are often recognized.

[0029] Fabry disease screening of cardiac hypertrophy patients was performed, and it was reported that 3.0% of Japanese male patients with left ventricular hypertrophy (Nakao S et al., NEJM 333, 288-293, 1995), 6.3% of British patients diagnosed with hypertrophic cardiomyopathy after age 40 (Sachdev B et al., Circulation 105, 1407-1411, 2002), and 12% of Italian female patients with hypertrophic cardiomyopathy (Chimenti C et al., Circulation 110, 1047-1053, 2004) were Fabry disease. Thus, it has been pointed out that there is a possibility that patients with cryptogenic left ventricular hypertrophy and hypertrophic cardiomyopathy are likely to be cardiac Fabry disease patients.

[0030] A single base substitution (IVS4+919G>A mutation) within the intron 4 of the GLA gene has been reported as etiology of a subset of cardiac Fabry disease patients. The IVS4+919G>A mutation results in alternative splicing in transcription of the GLA gene, and as a result, a GLA enzyme deficiency in lysosomes occurs. It has been reported that many cardiovascular abnormalities and the like are confirmed in adult Taiwanese people having the IVS4+919G>A mutation. Also, it has been reported that, when screening of Taiwanese newborns for the GLA enzyme activity was performed, about 70% to 80% of newborns with low plasma GLA enzyme activity and a causative mutation for Fabry disease had the IVS4+919G>A mutation (Lin H-Y, et al., Circ Cardiovasc Genet 2(5) 450-456 2009, Hwu W-L et al., Hum Mutat 30(10) 1397-1405 2009).Pharmaceutical composition for Fabry disease

[0031] As discussed above, the present invention relates to a pharmaceutical composition for use in preventing, ameliorating, suppressing progression of, and / or treating Fabry disease, the pharmaceutical composition containing an active ingredient capable of suppressing an aberrant splicing regulation that contributes to the development or progression of the Fabry disease (abnormal splicing that contributes to Fabry disease) which is a compound as described hereinbefore. In one or more embodiments, the pharmaceutical composition according to the present disclosure may be used to suppress an aberrant splicing regulation that contributes to Fabry disease.

[0032] In one or more embodiments, "An aberrant splicing regulation that contributes to Fabry disease" in the present disclosure results from a mutation in a gene to be spliced. In one or more non-limiting embodiments, an example of abnormal splicing that contributes to Fabry disease is splicing of a pre-mRNA of the mutant GLA gene having the IVS4+919G>A mutation (see the above description). In one or more embodiments, the pharmaceutical composition for use according to the present invention may be used to prevent, ameliorate, suppress progression of, and / or treat cardiac Fabry disease out of the Fabry diseases.

[0033] In one or more embodiments, the pharmaceutical composition for use according to the present invention may be used to alter abnormal splicing that contributes to Fabry disease in mammalian cells or mammalian individuals. In one or more embodiments, the abnormal splicing that contributes to Fabry disease may result from a mutation within a gene to be spliced. In another one or more embodiments, the abnormal splicing that contributes to Fabry disease may be splicing of a pre-mRNA of the mutant GLA gene with the IVS4+9 19G>A mutation.

[0034] In one or more embodiments, the pharmaceutical composition for use according to the present invention may be used to increase the ratio of normal splicing to abnormal splicing that contributes to Fabry disease in mammalian cells or mammalian individuals. In one or more embodiments, the abnormal splicing that contributes to Fabry disease may result from a mutation in a gene to be spliced. In another one or more embodiments, the abnormal splicing that contributes to Fabry disease may be splicing of a pre-mRNA of the mutant GLA gene having the IVS4+919G>A mutation.

[0035] In one or more embodiments, the pharmaceutical composition for use according to the present invention may be used to alter splicing of a pre-mRNA of the mutant GLA gene having the IVS4+9 19G>A mutation in human cells or human individuals. Also, in one or more embodiments, the pharmaceutical composition for use according to the present invention may be used to increase the ratio of normal splicing to splicing abnormality of a pre-mRNA of the mutant GLA gene having the IVS4+919G>A mutation in human cells or human individuals.

[0036] In one or more embodiments, mammalian cells or human cells of the present disclosure include in vivo cells, in vitro cells, or ex vivo cells. Also, in one or more embodiments, mammalian cells may be human cells or cells of a mammal other than a human.

[0037] In one or more embodiments, human cells and human individuals of the above-described embodiment may have the IVS4+9 19G>A mutation in the endogenous GLA gene. As described above, the IVS4+9 19G>A mutation of the present disclosure is a single base substitution (G → A) in intron 4 of the GLA gene. In one or more non-limiting embodiments, whether human cells and human individuals have an IVS4+9 19G>A mutation may be determined using a method for detecting a single base substitution. Alternatively, base sequence, array, and various gene amplification methods may be used.

[0038] In one or more embodiments, examples of the compound according to the present disclosure include the compounds below, where Y represents a halogen atom in the above compounds. In one or more embodiments, examples of the halogen atom represented by Y include a chlorine atom, a fluorine atom, and an iodine atom.

[0039] In one or more embodiments, examples of the compound for use according to the present disclosure include the compounds below.

[0040] The compounds disclosed herein can be synthesized by referring to the method disclosed in WO2010 / 118367 or the method disclosed in WO2016 / 115434.

[0041] In the present disclosure, a "pharmaceutically acceptable salt" refers to a pharmaceutically, pharmacologically, and / or medicinally acceptable salt, and examples thereof include inorganic acid salts, organic acid salts, inorganic base salts, organic base salts, and acidic or basic amino acid salts.

[0042] Preferred examples of the inorganic acid salts include hydrochloride, hydrobromide, sulfate, nitrate, and phosphate, and preferred examples of the organic acid salts include acetate, succinate, fumarate, maleate, tartrate, citrate, lactate, stearate, benzoate, methanesulfonate, and p-toluenesulfonate.

[0043] Preferred examples of the inorganic base salts include alkali metal salts such as sodium salts and potassium salts, alkaline earth salts such as calcium salts and magnesium salts, aluminum salts, and ammonium salts. Preferred examples of the organic base salts include diethylamine salts, diethanolamine salts, meglumine salts, and N', N-dibenzylethylenediamine salts.

[0044] Preferred examples of the acidic amino acid salts include aspartate and glutamate. Preferred examples of the basic amino acid salts include arginine salts, lysine salts, and ornithine salts.

[0045] In the present disclosure, a "salt of a compound" may include a hydrate that can be formed as a result of a compound being left in the air and absorbing moisture. Also, in the present disclosure, a "salt of a compound" may include a solvate that can be formed as a result of a compound absorbing a certain type of solvent.

[0046] In one or more embodiments, a known drug preparation technique may be applied to the pharmaceutical composition for use according to the present invention to have a dosage form that is suitable for an administration form. An example of the administration form is, but not limited to, oral administration via dosage forms such as tablets, capsules, granules, powders, pills, troches, syrups, and liquid formulations. Alternatively, an example of the administration form is parenteral administration via dosage forms such as injections, liquid formulations, aerosols, suppositories, plasters and pressure sensitive adhesives, cataplasms, lotions, liniments, ointments, and eye drops. Although these pharmaceutical preparations are not limited thereto, they may be manufactured using a known method using additives such as excipients, lubricants, binders, disintegrants, stabilizing agents, corrigents, and diluents.

[0047] In one or more embodiments, the pharmaceutical composition for use according to the present invention does not contain other active ingredients having a therapeutic effect, or contains another one or more active ingredients.

[0048] Examples of the excipient include, but not limited to, starches such as starch, potato starch, and corn starch, lactose, crystalline cellulose, and calcium hydrogen phosphate. Examples of the coating agent include, but not limited to, ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, shellac, talc, carnauba wax, and paraffin.

[0049] Examples of the binder include, but not limited to, polyvinylpyrrolidone, macrogol, and compounds that are similar to the above-described excipients.

[0050] Examples of the disintegrant include, but not limited to, compounds that are similar to those given as examples of the excipient, chemically-modified starches and celluloses such as croscarmellose sodium, sodium carboxymethyl starch, and crosslinked polyvinylpyrrolidone.

[0051] Examples of the stabilizing agent include, but not limited to, para-hydroxybenzoic acid esters such as methylparaben and propylparaben; alcohols such as chlorobutanol, benzyl alcohol, and phenylethyl alcohol; benzalkonium chloride; phenols such as phenol and cresol; thimerosal; dehydroacetic acid; and sorbic acid.

[0052] Examples of the corrigent include, but not limited to, sweeteners, acidulants, and flavors that are usually used.

[0053] Although there is no limitation on a solvent, ethanol, phenol, chlorocresol, purified water, distilled water, and the like can be used as a solvent to manufacture liquid formulations, and a surfactant, an emulsifying agent, or the like can also be used as needed. Examples of the surfactant or emulsifying agent include, but not limited to, polysorbate 80, polyoxyl 40 stearate, and lauromacrogol.

[0054] Methods for using a pharmaceutical composition for use according to the present invention may change depending on the symptoms, age, administration method, and the like. Although there is no limitation on the usage method, a pharmaceutical composition can be intermittently or continuously administered orally, percutaneously, submucosally, subcutaneously, intramuscularly, intravascularly, intracerebrally, or intraperitoneally such that the concentration of the compound in the body that is an active ingredient and is as described hereinbefore is in a range of 100 nM to 1 mM. In a non-limiting embodiment, in the case of oral administration, the pharmaceutical composition may be administered to a subject (an adult human if the subject is a human) in a dosage of 0.01 mg (preferably 0.1 mg) to 2000 mg (preferably 500 mg and more preferably 100 mg), which is expressed in terms of the compound as described hereinbefore, once or over several times in a day according to a symptom. In a non-limiting embodiment, in the case of intravenous administration, the pharmaceutical composition may be administered to a subject (an adult human if the subject is a human) in a dosage of 0.001 mg (preferably 0.01 mg) to 500 mg (preferably 50 mg) once or over several times in a day according to a symptom.Method and use

[0055] Although not an aspect of the invention, the present disclosure also provides the following methods: a method for altering splicing of a pre-mRNA of a mutant GLA gene having the IVS4+919G>A mutation in human cells or human individuals; and a method for increasing a ratio of normal splicing to splicing abnormality of a pre-mRNA of a mutant GLA gene having the IVS4+9 19G>A mutation in human cells or human individuals.

[0056] These methods may be performed by bringing the compound described herein or the pharmaceutical composition described herein into contact with the human cells or the human individuals.

[0057] The compound described herein or the pharmaceutical composition described herein may be brought into contact with in vitro or ex vivo human cells through addition of the compound, a salt thereof, or the pharmaceutical composition to a cell culture medium. The addition may be performed so that the concentration of the compound is in a range of 100 nM to 1 mM. The compound or the pharmaceutical composition may be brought into contact with in vivo human cells and human individuals according to the method for use of the pharmaceutical composition as described above.

[0058] In preferred aspects, the pharmaceutical composition as described herein for use in preventing, ameliorating, suppressing progression of, and / or treating Fabry disease, contains an active ingredient capable of suppressing a splicing abnormality that contributes to development or progression of the Fabry disease, preferably in which the splicing abnormality is a splicing abnormality that contributes to at least one of a GLA enzyme deficiency and a decrease in activity of the GLA enzyme. The splicing abnormality is preferably a splicing abnormality caused by the IVS4+9 19G>A mutation in the GLA gene.

[0059] The pharmaceutical composition for use according to the invention may be used to alter splicing of a pre-mRNA of a mutant GLA gene having an IVS4+919G>A mutation in human cells or human individuals; or to increase the ratio of normal splicing to splicing abnormality of a pre-mRNA of a mutant GLA gene having the IVS4+919G>A mutation in human cells or human individuals.

[0060] The pharmaceutical composition for use according to the invention may also be used in a method for altering splicing of a pre-mRNA of a mutant GLA gene having an IVS4+919G>A mutation in human cells or human individuals, or a method for increasing a ratio of normal splicing to splicing abnormality of a pre-mRNA of a mutant GLA gene having the IVS4+919G>A mutation in human cells or human individuals, the method including bringing the pharmaceutical composition into contact with the cells.

[0061] The pharmaceutical composition for use according to the invention is also capable of preventing or ameliorating GLA deficiency or a decrease in GLA activity.

[0062] The pharmaceutical composition for use according to the invention may be used for diseases in which GLA deficiency contributes to the development or progression thereof, the pharmaceutical composition containing an active ingredient capable of suppressing the splicing abnormality.

[0063] The pharmaceutical composition for use according to the invention may also be used for diseases in which GLA deficiency contributes to the development or progression thereof.Examples

[0064] Hereinafter, although the present disclosure will be described in more detail by way of examples, these are illustrative, and the present disclosure is not limited to these examples.Manufacturing Example 1: Manufacture of Compound 1

[0065]

[0066] Compound 1 was synthesized in the following manner with reference to the method disclosed in WO2010 / 118367.

[0067] Triethylamine (0.15 mL, 1.08 mmol) was added at room temperature to an acetonitrile (20 mL) solution containing 2,6-dichloro-1H-purine (189 mg, 1.00 mmol, commercial product) and furfurylamine (97.0 mg, 1.00 mmol, commercial product). The mixture was stirred at room temperature for 6 hours, and then stirred at 60°C for 3 hours. After this mixed solution was concentrated under reduced pressure, water was added to the solution to form white precipitates, and the white precipitates were removed through filtration. The resultant solid was washed with water and subsequently with diethyl ether, and 2-chloro-N-(2-furanylmethyl)-7H-purin-6-amine (Compound 1) (19.8 mg, 0.0795 mmol, 8.0%) was obtained as a white solid. TLC Rf 0.22 (ethyl acetate); 1< H NMR (DMSO-d 6 , 400 MHz) δ 4.56-4.67 (br, 2H), 6.24-6.28 (br, 1H), 6.35-6.40 (br, 1H), 7.54-7.57 (br, 1H), 8.11-8.15 (br, 1H), 8.54-8.64 (br, 1H), 13.05-13.17 (br, 1H).Manufacturing Example 2

[0068] Compounds shown in Table 1 below were synthesized with reference to Manufacturing Example 1 and the method disclosed in WO2010 / 118367. Some, but not all, of these compounds are according to the invention. Manufacturing Example 3

[0069] Compounds shown in Table 2 below were synthesized with reference to the method disclosed in WO2010 / 010797. These compounds are not according to the invention. [Table 2] R 11< CH 3 CH 3 CH 3 CH 3 CH 3 CH 3 R 12< CH 3 CH 3 CH 3 CH 3 CH 3 CH 3 R 13< HCH 3 CH 3 OFC 2 H 5 O R 11< CH 3 CH 3 CH 3 CH 3 CH 3 CH 3 CH 3 R 12< CH 3 CH 3 CH 3 R 13< CH 3 OHCH 3 OCH 3 OCH 3 O Manufacturing Example 4

[0070] Compounds VIII-A to VIII-O below were synthesized with reference to the method disclosed in WO2017 / 175842. These compounds are not according to the invention. System for evaluating splicing abnormality caused by the GLA IVS4+919G>A mutation in Fabry disease

[0071] A region starting from GLA exon 4 to exon5 (nt 7272-9215 in a GLA gene sequence) having a normal IVS4 or the IVS4+919G>A mutation downstream of a cytomegalovirus (CMV) early gene promoter was cloned, and vectors pAM1 (wild-type IVS4) and pAM2 (the IVS4+919 G>A mutant) that serve as evaluations systems were produced (FIG. 2), for investigating the effects of a splicing operation compound on a splicing abnormality caused by the GLA IVS4+919G>A mutation. Splicing abnormalities (inclusion of the pseudo exon) in Fabry disease patient cells were demonstrated by introducing these vectors into culture cells, and therapeutic effects of the compounds were determined.

[0072] The GLA gene pseudo exon skipping evaluation vector is shown in FIG. 2.

[0073] As shown in FIG. 2, pAM1 is constituted by a normal IVS4 sequence, and pAM2 has the IVS4+919G>A mutation. A precursor mRNA including exon 4 to exon 5 is transcribed, and splicing alteration recapitulating patient cells with the same mutation is demonstrated. Sequences of pAM1 and pAM2 other than the IVS4+919G>A point mutation were the same.Confirmation of effect of Compound 1 in suppression of the pseudo exon splicing of GLA gene caused by the IVS4+919G>A

[0074] HeLa cells of human epithelial origin were cultured on 6 cm-plates (0.5x10 6< cells), and the vectors shown in FIG. 2 were introduced into cells using lipofection reagents. Compound 1 was added at a concentration of 0 pM, 5 pM, or 10 pM, 4 hours after the vector transfection (final concentration of DMSO was 0.1%). Cellular RNA was collected 24 hours after the Compound 1 treatment, and treated with DNase to be applied for RT-PCR for evaluation of the alternative splicing of GLA gene. Results thereof are shown in FIGS. 3A and 3B.

[0075] FIGS. 3A and 3B show the effect of Compound 1 through administration on inhibition of the GLA gene pseudo exon caused by the IVS4+919G>A mutation in Fabry disease. FIG. 3A shows results of RT-PCR for GLA gene with wild-type or IVS4+919G>A mutant IVS4. The production of a normal isoform (pseudo exon skipping) was restored following Compound 1 treatment. The lower panel of FIG. 3A shows Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as an internal control for RNA applied to RT-PCR and the amounts of RNA used in analysis are equal to each other. FIG. 3B is a schematic diagram illustrating GLA splicing control and recovery of the active enzyme using Compound 1.

[0076] As shown in FIG. 3A, Compound 1 suppressed aberrant splicing (incorporation of the pseudo exon) of GLA gene at a concentration as low as 5 µM. In addition, splice correcting activity of Compound 1 was observed in a concentration-dependent manner.

[0077] Then, a SPREADD reporter system, which is a system such that shown in FIG. 4A, that is capable of visualization and quantitative analysis of splicing alteration in living cells for GLA gene splicing, was newly constructed. In this reporter system, a red fluorescent protein (RFP) is expressed if a pseudo exon is incorporated, and a green fluorescent protein (GFP) is expressed if a pseudo exon is skipped.

[0078] HeLa cells were transfected with the SPREADD reporter vectors, and as shown in FIG. 4B, pseudo exon skipping products (4 / 5 / GFP) were dominantly expressed in the cells transfected with the SPREADD vector with wild-type IVS4 (pAM17 (WT)), while pseudo exon inclusion products (4 / ps / 5 / RFP) were dominant when cells were transfected with the SPREADD vector with the IVS4+919 G>A mutation (pAM18 (IVS4+919 G>A)).

[0079] HeLa cells transfected with the SPREADD reporter pAM18 (IVS4+919 G>A mutant) were treated with Compound 1 (concentrations: 10 pM, 20 µM, and 50 µM) for 24 hours, and analyzed for fluorescent intensities of GFP and RFP for the GLA splicing. Results thereof are shown in FIG. 4C. The graph shown in FIG. 4C shows the relationship between the administration concentration and the fluorescence intensity ratio (GFP / RFP) of the control (solvent alone) and Compound 1. As shown in FIG. 4C, GFP / RFP of Compound 1 increased in a concentration-dependent manner. Thus, Compound 1 suppressed the pseudo exon of GLA gene, caused by the IVS4+9 19 G>A splicing mutation in a concentration-dependent manner.

[0080] Inclusion of the pseudo exon is a direct cause of a decrease in the GLA activity in cardiac Fabry disease. Thus, the above-described results indicate evidence of expected recovery of the GLA enzyme activity and therapeutic potential for cardiac Fabry disease by Compound 1.

[0081] Confirmation of the suppressing activity of the aberrant splicing (exon skipping) caused by the IVS20+6T>C splicing mutation of IKBKAPgene. This example does not concern a disease encompassed by the appended claims

[0082] The SPREADD reporter system as shown in FIG. 5 for a splicing mutation in the IKBKAP gene in familial dysautonomia was produced. In the reporter system, GFP is expressed if the transcription product from the reporter vector is subjected to a normal splicing (exon 19 / 20 / 21), whereas RFP is expressed if the transcription product from the reporter vector subjected to an abnormal splicing (exon 19 / 21).

[0083] HeLa cells transfected with the SPREADD reporter construct were brought into contact with the compounds shown in Table 3 and cultured (concentration: 10 µM or 50 pM), and cellular fluorescence was measured after 24 hours. As a result, with regard to the compounds shown in Table 3 below, the relative GFP intensities over RFP (GFP / RFP) was higher than control (DMSO), confirming suppression effect of the exon 20 skipping of IKBKAP gene with the IVS20+6T>C mutation. Thus, the compounds represented by Formula (II), such as Compound 1, was capable of suppressing abnormal splicing resulting from the IVS20+6T>C mutation. [Table 3] Y XClClClFClClClHR 5< HHHHHHHHGFP / RFP4.01.9 (50 uM)1.5 (50 uM)3.03.02.51.61.4* control (DMSO) : GFP / RFP 1.0

[0084] Confirmation of the suppressive effect on the pseudo exon-type mis-splicing caused by the c.3849+10kbC>T splicing mutation of CFTR gene. This example uses a compound which is not encompassed by the appended claims.

[0085] The SPREADD reporter system as shown in FIG. 6A that is a system capable of visualization and quantitative analysis of the splicing alteration in living cells for CFTR gene splicing in cystic fibrosis was produced. In the reporter system, RFP is expressed if the pseudo exon within the intron 22, caused by the c.3849+10kb C>T is included in the mRNA, whereas GFP is expressed if the pseudo exon is skipped.

[0086] HEK293 cells transfected with the SPREADD reporter construct were brought into contact with Compound III-1 and cultured (concentrations: 10 µM and 30 pM). Fluorescence observation was performed after 6 hours. Results thereof are shown in FIG. 6B.

[0087] The graph shown in FIG. 6B shows the relationship between the administration concentration and the fluorescence intensity ratio (GFP / (GFP+RFP)) of the control (solvent alone) and Compound III-1. The results of HEK293 cells transfected with the normal reporter construct are also shown. As shown in FIG. 6B, GFP / (GFP+RFP) of Compound III-1 increased in a concentration-dependent manner. Also, if the concentration was 30 µM, the fluorescence intensity ratio was at about the same level as that of the normal type. Thus, the pseudo exon caused by the 3849+10kbC>T mutation in the CFTR gene was suppressed by Compound III-1 in a concentration-dependent manner, and as a result, the normal CFTR splicing products was restored.

[0088] Similar examinations were performed using the compounds shown in the following table, instead of Compound III-1 (concentration: 10 pM). Results thereof are shown in Table 4 below.

[0089] As shown in the Table 4 above, Compounds A to Q exhibited activity that is about the same as Compound III-1 or higher than that of Compound III-1. Compounds H, P, and Q exhibited activity with a statistically significant differences in the recovery rate (splicing recovery effects of compound treatment where GFP / (GFP+RFP) of the solvent treated CFTRc. 3849+10kb mutant vector was set to 0% and GFP / (GFP+RFP) of the wild-type CFTRvector was 100%), and, in particular, Compounds H and Q exhibited higher activity values, compared to Compound III-1. Thus, the pseudo exon caused by the c.3849+10kb C>T mutation in the CFTR gene was suppressed by the above described compounds, and as a result, the normal CFTR splicing products was restored.

[0090] If a pseudo exon is produced within the intronic sequence of the CFTR gene, and the pseudo exon is included into mRNA, a premature stop codon is introduced for CFTR, resulting in functional and quantitative loss of CFTR. Thus, the above described compounds are expected to show therapeutic effect for cystic fibrosis by restoring normal splicing and functional production of CFTR.

[0091] Confirmation of effect of suppressing PTC exon recognition. This example uses a compound which is not encompassed by the appended claims.

[0092] A vector obtained by cloning target exon regions (COL4A5 gene exons 40, 41, and 42, and TSC2 gene exons 15, 16, and 17) for the COL4A5 gene (c.3710_3761del52 deletion mutation, PTC mutation) in Alport syndrome and the TSC2 gene (c. 1783C>T, PTC mutation) in tuberous sclerosis was expressed in HeLa cells. Then the cells were treated with Compound III-1 (0 µM, 5 µM, 10 pM, 20 µM, 30 pM, and 50 µM) and induction of splicing alteration was verified through RT-PCR. Results thereof are shown in FIG. 7.

[0093] FIGS. 7A and 7B show examples of the results of RT-PCR on HeLa cells subjected to minigene transfection with the COL4A5 gene (FIG. 7A) in Alport syndrome, and the TSC2 gene (FIG. 7B) in tuberous sclerosis. As shown in FIGS. 7A and 7B, Compound III-1 had a concentration-dependent splicing induction effect on the COL4A5 gene and the TSC2 gene.

Claims

1. A pharmaceutical composition for use in preventing, ameliorating, suppressing progression of, and / or treating Fabry disease, wherein the pharmaceutical composition comprises, as an active ingredient, the compound: wherein Y represents a halogen atom, or a pharmaceutically acceptable salt thereof.

2. The pharmaceutical composition for the use of claim 1, wherein the compound is: or a pharmaceutically acceptable salt thereof.