US20200113907A1 - Pharmaceutical composition and treatment method for genetic disease associated with splicing abnormalities - Google Patents

Pharmaceutical composition and treatment method for genetic disease associated with splicing abnormalities Download PDF

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US20200113907A1
US20200113907A1 US16/486,980 US201816486980A US2020113907A1 US 20200113907 A1 US20200113907 A1 US 20200113907A1 US 201816486980 A US201816486980 A US 201816486980A US 2020113907 A1 US2020113907 A1 US 2020113907A1
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Masatoshi Hagiwara
Masahiko Ajiro
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Kyoto University NUC
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Definitions

  • 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.
  • 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.
  • Patent Document 1 WO 2011/152043 A1 (U.S. Pat. No. 9,273,364B2)
  • 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.
  • enzyme replacement therapy by a recombinant ⁇ -galactosidase A (GLA) enzyme protein has been developed for Fabry disease
  • Fabry disease patients still rely on many symptomatic therapies.
  • GLA ⁇ -galactosidase A
  • the present disclosure provides 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 present disclosure relates to a pharmaceutical composition for preventing, ameliorating, suppressing progression of, and/or treating genetic diseases caused by aberrant splicing events, the pharmaceutical composition containing, as an active ingredient, a compound capable of suppressing an aberrant splicing that contributes to the development or progression of genetic diseases.
  • the present disclosure relates to a method for preventing, ameliorating, suppressing progression of, and/or treating genetic diseases caused by aberrant splicing events, and the method includes administering a compound 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 present disclosure relates to a pharmaceutical composition for 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.
  • the present disclosure relates to a pharmaceutical composition for preventing, ameliorating, suppressing progression of, and/or treating Fabry disease, the pharmaceutical composition containing, as an active ingredient, a compound represented by Formula (I) or (I′) below, a prodrug thereof, or a pharmaceutically acceptable salt thereof,
  • R 1 and R 2 each independently represent a hydrogen atom, a linear, branched, or cyclic alkyl group having 1 to 6 carbon atoms, a benzyl or heteroarylmethyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted heteroaryl group;
  • R 3 represents a hydrogen atom, a linear, branched, or cyclic alkyl group having 1 to 6 carbon atoms, 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 linear, branched, or cyclic alkyl group having 1 to 6 carbon atoms, a benzyl or heteroarylmethyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted heteroaryl group;
  • X represents a hydrogen atom, a halogen atom, an amino group, an R 1 - and R 2 -substituted amino group, an azide group, a cyano group, a nitro group, a hydroxy group, a linear, branched, or cyclic alkyloxy group having 1 to 6 carbon atoms, 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 linear, branched, or cyclic alkyl group having 1 to 6 carbon atoms, a benzyl or heteroarylmethyl group, a substituted or unsubstituted aryl group, or a substituted or
  • the present disclosure relates to a pharmaceutical composition for preventing, ameliorating, suppressing progression of, and/or treating Fabry disease, the pharmaceutical composition containing, as an active ingredient, a compound represented by Formula (IX) or (IX′) below, a prodrug thereof, or a pharmaceutically acceptable salt thereof,
  • R 1 and R 2 each independently represent a hydrogen atom, a linear, branched, or cyclic alkyl group having 1 to 6 carbon atoms, a benzyl or heteroarylmethyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted heteroaryl group;
  • R 5 represents a hydrogen atom, a halogen atom, a substituted or unsubstituted linear, branched, or cyclic alkoxy group having 1 to 6 carbon atoms;
  • 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;
  • X represents a hydrogen atom, a halogen atom, an amino group, an R 1 - and R 2 -substituted amino group, an azide group, a cyano group, a nitro group, a hydroxy group, a linear, branched, or cyclic alkyloxy group having 1 to 6 carbon atoms, 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 linear, branched, or cyclic alkyl group having 1 to 6 carbon atoms, a benzyl or heteroarylmethyl group, a substituted or unsubstituted aryl group, or a substituted or
  • the present disclosure relates to a pharmaceutical composition for preventing, ameliorating, suppressing progression of, and/or treating Fabry disease, the pharmaceutical composition containing, as an active ingredient, a compound represented by Formula (X) or (X′) below, a prodrug thereof, or a pharmaceutically acceptable salt thereof,
  • R 5 represents a hydrogen atom, a methoxy group, or a 2,2,2-trifluoroethoxy group
  • R 6 represents a 2-furyl group, a 2-thiazolyl group, or a 4-pyridyl group
  • X 1 represents N or CH
  • X 2 represents NH, NCH 3 , S, or O
  • X′ represents a hydrogen atom, a chlorine atom, an iodine atom, a bromine atom, or a fluorine atom.
  • the present disclosure relates to a method for preventing, ameliorating, suppressing progression of, and/or treating Fabry disease, and the method includes administering a pharmaceutical composition according to the present disclosure to a subject that requires the pharmaceutical compound.
  • 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 COL4A5gene in Alport syndrome and the TSC2 gene in tuberous sclerosis.
  • 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 ).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • any pseudo exon mutations are considered as targets for splicing therapeutic agents.
  • 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.
  • a PTC exon an exon
  • PTC premature termination codon
  • 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 NE peripheral NF, occipital horn syndrome, retinoblastoma, schizophrenia, and tuberous sclerosis.
  • Pompe disease mu
  • GLA Fabry disease
  • CFTR cystic fibrosis
  • MTRR homocystinuria
  • ATM hereditary breast/ovarian cancer syndrome
  • MSH2 ataxia-telangiectasia/Louis-Bar syndrome
  • NF1 neurofibromatosis type 1
  • TSC2 tuberous sclerosis
  • ADH7A1 atypical pyridoxine-dependent epilepsy
  • CEP290 Leber congenital amaurosis
  • COL4A3 chronic granulomatous disease (CYBB), 17 ⁇ -hydroxylase deficiency (CYP17A1), Marfan syndrome (FBN1), X-linked hypophosphatemia (PHEX), and polycystic kidney disease (PKHD1) (responsible genes with pseudo exon are indicated in parentheses).
  • CYBB chronic granulomatous disease
  • CYP17A1 17 ⁇ -hydroxylase deficiency
  • FBN1 Marfan syndrome
  • PHEX X-linked hypophosphatemia
  • PKHD1 polycystic kidney disease
  • 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
  • the present disclosure relates to a pharmaceutical composition for preventing, ameliorating, suppressing progression of, and/or treating genetic diseases caused by an aberrant splicing regulation, 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.
  • the pharmaceutical composition of the present disclosure contains, as an active ingredient, a compound capable of enhancing exon recognition in splicing in which exon recognition is incomplete due to a splicing mutation, and a compound capable of suppressing recognition of an exon created by a splicing mutation.
  • examples of the compound capable of enhancing exon recognition in splicing in which exon recognition is incomplete due to a splicing mutation include compounds represented by Formulae (II), (II′), and (III).
  • examples of the compound capable of suppressing exon recognition in the splicing include compounds represented by Formulae (IV), (V), (VI), (VII), and (VIII).
  • 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.
  • Z forms, together with atoms marked with a and b, a ring selected from the group consisting of one benzene ring, one heteroaromatic ring, an aromatic ring fused with one or more benzene rings, a heteroaromatic ring fused with one or more heteroaromatic rings, a mixed fused polycyclic ring in which one or more benzene rings and one or more heteroaromatic rings are fused, and cycloaliphatic compounds, and the ring may include one or more substituents, the substituents being a hydrogen atom, a halogen atom, or a C 1 -C 6 alkyl group, and an atomic bonding to which a wavy line is attached indicates a portion that binds to Formula (IV); and
  • Z forms, together with atoms marked with a and b, a ring selected from the group consisting of one benzene ring, one heteroaromatic ring, an aromatic ring fused with one or more benzene rings, a heteroaromatic ring fused with one or more heteroaromatic rings, a mixed fused polycyclic ring in which one or more benzene rings and one or more heteroaromatic rings are fused, and cycloaliphatic compounds, and the ring may include one or more substituents, the substituents being a hydrogen atom, a halogen atom, or a C 1 -C 6 alkyl group, and an atomic bonding to which a wavy line is attached indicates a portion that binds to Formula (V); and
  • R 26 , R 27 , and R 28 each independently represent a hydrogen atom, a halogen atom, a carboxyl group, an amino group, a hydroxy group, a C 1 -C 4 alkyl group, or a halogen-substituted C 1 -C 4 alkyl group, and an atomic bonding to which a wavy line is attached indicates a portion that binds to Formula (VIII);
  • R 29 , R 30 , R 31 , and R 32 each independently represent a hydrogen atom, a halogen atom, a carboxyl group, an amino group, a hydroxy group, a C 1 -C 4 alkyl group, or a halogen-substituted C 1 -C 4 alkyl group, and an atomic bonding to which a wavy line is attached indicates a portion that binds to Formula (VIII); and
  • 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
  • examples of the compound represented by Formula (II) or (II′) include the compounds below.
  • R 7 and R 8 each independently represent a hydrogen atom, a methyl group, a halogen-substituted methyl group, a trifluoromethyl group, an ethyl group, a halogen-substituted ethyl group, and a trifluoroethyl group
  • R 9 represents a hydrogen atom, a halogen atom, a methyl group, a trifluoromethyl group, an ethyl group, a trifluoroethyl group, —O—R 10 , —NHR 10 , or —N(R 10 ) 2
  • R 10 represents a hydrogen atom, a methyl group, or an ethyl group.
  • examples of the compound represented by Formula (III) include the compounds below,
  • R 9 represents a hydrogen atom, a halogen atom, or a halogen-substituted or unsubstituted C 1 -C 10 alkyl group, and preferably a hydrogen atom, a fluorine atom, a chlorine atom, a methyl group, or an ethyl group
  • R 10 represents a hydrogen atom or a C 1 -C 10 alkyl group, and preferably a hydrogen atom, a methyl group, or an ethyl group.
  • examples of the compound represented by Formula (III) include the compounds below.
  • examples of the compound represented by Formula (IV) include the compounds below,
  • R 14 and R 41 each independently represent a hydrogen atom or a C 1 -C 6 alkyl group, and preferably a hydrogen atom, a methyl group, or an ethyl group, and more preferably a methyl group
  • R 14 represents a hydrogen atom, a halogen atom, or a C 1 -C 6 alkyl group, and preferably a hydrogen atom, a fluorine atom, a chlorine atom, or a methyl group
  • R 33 and R 34 each independently represent a hydrogen atom, a halogen atom, or a C 1 -C 6 alkyl group, and preferably a hydrogen atom, a fluorine atom, a chlorine atom, or a methyl group, and more preferably, one of R 33 and R 34 represents a hydrogen atom and the other represents a chlorine atom or a methyl group, or both represent a methyl group.
  • examples of the compound represented by Formula (IV) include the compounds below,
  • R 33 and R 34 each independently represent a hydrogen atom, a halogen atom, or a C 1 -C 6 alkyl group, and preferably a hydrogen atom, a fluorine atom, a chlorine atom, or a methyl group, and more preferably, one of R 33 and R 34 represents a hydrogen atom and the other represents a chlorine atom or a methyl group, or both represent a methyl group.
  • Formula (IV) include the compounds below.
  • examples of the compound represented by Formula (V) include the compounds below,
  • R 16 represents a hydrogen atom, a halogen atom, or a C 1 -C 6 alkyl group, and preferably a hydrogen atom
  • X 4 represents S or NH
  • examples of the compound represented by Formula (VI) or (VII) include the compounds below.
  • R 18 represents a hydrogen atom, a hydroxy group, or a C 1 -C 6 alkyl group, and preferably a hydrogen atom, a hydroxy group, or a methyl group.
  • examples of the compound represented by Formula (VI) or (VII) include the compounds below.
  • examples of the compound represented by Formula (VIII) include the compounds below,
  • R 25 , R 26 , R 27 , R 28 , R 29 , R 30 , R 31 , and R 32 each independently represent a hydrogen atom, a halogen atom, a carboxyl group, an amino group, a hydroxy group, a C 1 -C 4 alkyl group, or a halogen-substituted C 1 -C 4 alkyl group, and preferably a hydrogen atom, a fluorine atom, a chlorine atom, or a methyl group.
  • examples of the compound represented by Formula (VIII) include the compounds below.
  • the pharmaceutical composition according to the present disclosure contains, as an active ingredient, a compound represented by Formula (II), (II′), (III), (IV), (V), (VI), (VII), or (VIII), or a combination thereof, and may further contain a medicinally acceptable carrier, an antiseptic, a diluent, an excipient, or other medicinally acceptable component.
  • the present disclosure relates to a method for preventing, ameliorating, suppressing progression of, and/or treating genetic diseases caused by aberrant splicing events, and the method includes administering a compound capable of suppressing a splicing abnormality that contributes to the development or progression of the genetic diseases to a subject that requires the compound.
  • examples of the compound include compounds capable of enhancing exon recognition in splicing in which exon recognition is incomplete due to a splicing abnormality or compounds capable of suppressing exon recognition, and specific examples thereof, which are not particularly limited, include compounds represented by Formula (II), (II′), (III), (IV), (V), (VI), (VII), or (VIII).
  • 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.
  • Gb3 glycolipids
  • GLA enzyme which is a lysosomal hydrolase
  • Fabry disease is classified into three types according to symptoms: classic, atypical, and heterozygous.
  • classic Fabry disease normally, the GLA enzyme activity is low or barely detectable.
  • 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.
  • 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.
  • IVS4+919G>A mutation 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.
  • the present disclosure relates to a pharmaceutical composition for 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).
  • the pharmaceutical composition according to the present disclosure may be used to suppress an aberrant splicing regulation that contributes to Fabry disease.
  • “An aberrant splicing regulation that contributes to Fabry disease” in the present disclosure results from a mutation in a gene to be spliced.
  • 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).
  • the pharmaceutical composition according to the present disclosure may be used to prevent, ameliorate, suppress progression of, and/or treat cardiac Fabry disease out of the Fabry diseases.
  • the pharmaceutical composition according to the present disclosure may be used to alter abnormal splicing that contributes to Fabry disease in mammalian cells or mammalian individuals.
  • the abnormal splicing that contributes to Fabry disease may result from a mutation within a gene to be spliced.
  • the abnormal splicing that contributes to Fabry disease may be splicing of a pre-mRNA of the mutant GLA gene with the IVS4+919G>A mutation.
  • the pharmaceutical composition according to the present disclosure may be used to increase the ratio of normal splicing to abnormal splicing that contributes to Fabry disease in mammalian cells or mammalian individuals.
  • the abnormal splicing that contributes to Fabry disease may result from a mutation in a gene to be spliced.
  • 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.
  • the pharmaceutical composition according to the present disclosure may be used to alter splicing of a pre -mRNA of the mutant GLA gene having the IVS4+919G>A mutation in human cells or human individuals. Also, in one or more embodiments, the pharmaceutical composition according to the present disclosure 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.
  • 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.
  • human cells and human individuals of the above-described embodiment may have the IVS4+919G>A mutation in the endogenous GLA gene.
  • the IVS4+919G>A mutation of the present disclosure is a single base substitution (G ⁇ A) in intron 4 of the GLA gene.
  • whether human cells and human individuals have an IVS4+919G>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.
  • the present disclosure relates to a pharmaceutical composition for preventing, ameliorating, suppressing progression of, and/or treating Fabry disease, the pharmaceutical composition containing, as an active ingredient, a compound represented by Formula (I) or (I′), a prodrug thereof, or a pharmaceutically acceptable salt thereof.
  • R 1 and R 2 each independently represent a hydrogen atom, a linear, branched, or cyclic alkyl group having 1 to 6 carbon atoms, a benzyl or heteroarylmethyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted heteroaryl group.
  • examples of the linear or branched alkyl group having 1 to 6 carbon atoms represented by R 1 and R 2 include a methyl group, an ethyl group, a 1-propyl group, a 2-propyl group, a 2-methyl-1-propyl group, a 2-methyl-2-propyl group, a 1-butyl group, a 2-butyl group, a 1-pentyl group, a 2-pentyl group, a 3-pentyl group, a 2-methyl-1-butyl group, a 3-methyl-1-butyl group, a 2-methyl-2-butyl group, a 3-methyl-2-butyl group, a 2,2-dimethyl-1-propyl group, a 1-hexyl group, a 2-hexyl group, 3-hexyl group, a 2-methyl-1-pentyl group, a 3-methyl-1-pentyl group, a 4-methyl-1-pentyl group, a 2-methyl-2-
  • examples of the cyclic alkyl group having 1 to 6 carbon atoms represented by R 1 and R 2 include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.
  • examples of the heteroaryl (including heteroaryl of the heteroarylmethyl group) represented by R 1 and R 2 include a 5- to 6-membered monocyclic group having 1 to 2 nitrogen atoms, a 5- to 6-membered monocyclic group having 1 to 2 nitrogen atoms and either 1 oxygen atom or 1 sulfur atom, a 5-membered monocyclic group having 1 oxygen atom or 1 sulfur atom, and a bicyclic group that has 1 to 4 nitrogen atoms and is formed through fusion of a 6-membered ring and a 5- or 6-membered ring.
  • examples of the heteroaryl include 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-thienyl, 3-thienyl, 3-oxadiazolyl, 2-imidazolyl, 2-thiazolyl, 3-isothiazolyl, 2-oxazolyl, 3-isoxazolyl, 2-furyl, 3-furyl, 3-pyrrolyl, 2-quinolyl, 8-quinolyl, 2-quinazolinyl, and 8-purinyl.
  • Examples of the aryl group represented by R 1 and R 2 include an aryl group having 10 or less carbon atoms, such as a phenyl group or a naphthyl group.
  • the number of substituents of the aryl group and the heteroaryl group represented by R 1 and R 2 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
  • R 3 represents a hydrogen atom, a linear, branched, or cyclic alkyl group having 1 to 6 carbon atoms, 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 linear, branched, or cyclic alkyl group having 1 to 6 carbon atoms, a benzyl or heteroaryl methyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted heteroaryl group.
  • examples of the linear or branched alkyl group having 1 to 6 carbon atoms represented by R 3 and R 4 include a methyl group, an ethyl group, a 1-propyl group, a 2-propyl group, a 2-methyl-1-propyl group, a 2-methyl-2-propyl group, a 1-butyl group, a 2-butyl group, a 1-pentyl group, a 2-pentyl group, a 3-pentyl group, a 2-methyl-1butyl group, a 3-ethyl-1-butyl group, a 2-methyl-2-butyl group, a 3-methyl-2-butyl group, a 2,2-dimethyl-1-propyl group, a 1-hexyl group, a 2-hexyl group, 3-hexyl group, a 2-methyl-1-pentyl group, a 3-methyl-1-pentyl group, a 4-methyl-1-pentyl group, a 2-methyl
  • examples of the cyclic alkyl group having 1 to 6 carbon atoms represented by R 3 and R 4 include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.
  • examples of the heteroaryl (including heteroaryl of the heteroarylmethyl group) represented by R 3 and R 4 include a 5- to 6-membered monocyclic group having 1 to 2 nitrogen atoms, a 5- to 6-membered monocyclic group having 1 to 2 nitrogen atoms and either 1 oxygen atom or 1 sulfur atom, a 5-membered monocyclic group having 1 oxygen atom or 1 sulfur atom, and a bicyclic group that has 1 to 4 nitrogen atoms and is formed through fusion of a 6-membered ring and a 5- or 6-membered ring.
  • examples of the heteroaryl include 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-thienyl, 3-thienyl, 3-oxadiazolyl, 2-imidazolyl, 2-thiazolyl, 3-isothiazolyl, 2-oxazolyl, 3-isoxazolyl, 2-furyl, 3-furyl, 3-pyrrolyl, 2-quinolyl, 8-quinolyl, 2-quinazolinyl, and 8-purinyl.
  • Examples of the aryl group represented by R 1 and R 2 include an aryl group having 10 or less carbon atoms, such as a phenyl group or a naphthyl group.
  • the number of substituents of the aryl group and the heteroaryl group represented by R 3 and R 4 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 alky
  • X represents a hydrogen atom, a halogen atom, an amino group, the above-described R 1 - and R 2 -substituted amino group, an azide group, a cyano group, a nitro group, a hydroxy group, a linear, branched, or cyclic alkyloxy group having 1 to 6 carbon atoms, 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 linear, branched, or cyclic alkyl group having 1 to 6 carbon atoms, a benzyl or heteroarylmethyl group, a substituted or unsubsti
  • examples of the linear or branched alkyl group (including alkyl groups of the alkyloxy group and the alkylthio group) having 1 to 6 carbon atoms represented by X include a methyl group, an ethyl group, a 1-propyl group, a 2-propyl group, a 2-methyl-1-propyl group, a 2-methyl-2-propyl group, a 1-butyl group, a 2-butyl group, a 1-pentyl group, a 2-pentyl group, a 3-pentyl group, a 2-methyl-1-butyl group, a 3-methyl-1-butyl group, a 2-methyl-2-butyl group, a 3-methyl-2-butyl group, a 2,2-dimethyl-1-propyl group, 1-hexyl group, a 2-hexyl group, a 3-hexyl group, a 2-methyl-1-pentyl group, a 3-methyl-1-pentyl group, a 2-methyl-1
  • examples of the heteroaryl (including heteroaryl of the heteroaryloxy group, the heteroarylthio group, and the heteroarylmethyl group) represented by X include a 5- to 6-membered monocyclic group having 1 to 2 nitrogen atoms, a 5- to 6-membered monocyclic group having 1 to 2 nitrogen atoms and either 1 oxygen atom or 1 sulfur atom, a 5-membered monocyclic group having 1 oxygen atom or 1 sulfur atom, and a bicyclic group that has 1 to 4 nitrogen atoms and is formed through fusion of a 6-membered ring and a 5- or 6-membered ring.
  • examples thereof include 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-thienyl, 3-thienyl, 3-oxadiazolyl, 2-imidazolyl, 2-thiazolyl, 3-isothiazolyl, 2-oxazolyl, 3-isoxazolyl, 2-furyl, 3-furyl, 3-pyrrolyl, 2-quinolyl, 8-quinolyl, 2-quinazolinyl, and 8-purinyl.
  • Examples of the aryl group (including heteroaryl of the aryloxy group and the arylthio group) represented by X include an aryl group having 10 or less carbon atoms, such as a phenyl group or a naphthyl group.
  • the number of substituents of the aryl group and the heteroaryl group represented by X 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 alkylsul
  • examples of the halogen atom represented by X include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom.
  • the present disclosure relates to a pharmaceutical composition for preventing, ameliorating, suppressing progression of, and/or treating Fabry disease, the pharmaceutical composition containing, as an active ingredient, a compound represented by Formula (IX) or (IX′), a prodrug thereof, or a pharmaceutically acceptable salt thereof.
  • R 1 and R 2 each independently represent a hydrogen atom, a linear, branched, or cyclic alkyl group having 1 to 6 carbon atoms, a benzyl or heteroarylmethyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted heteroaryl group.
  • R 5 represents a hydrogen atom, a halogen atom, a substituted or unsubstituted linear, branched, or cyclic alkoxy group having 1 to 6 carbon atoms.
  • X 1 represents N or CH.
  • X 2 represents —N(R 3 )—, S, or O.
  • R 3 represents a hydrogen atom, a linear, branched, or cyclic alkyl group having 1 to 6 carbon atoms, 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.
  • X represents a hydrogen atom, a halogen atom, an amino group, an R 1 - and R 2 -substituted amino group, an azide group, a cyano group, a nitro group, a hydroxy group, a linear, branched, or cyclic alkyloxy group having 1 to 6 carbon atoms, 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 linear, branched, or cyclic alkyl group having 1 to 6 carbon atoms, a benzyl or heteroarylmethyl group, a substituted or unsubstituted aryl group, or a substituted or
  • R 1 , R 2 , R 3 , R 4 , and X in Formulae (IX) and (IX′) are the same as those in Formulae (I) and (I′).
  • examples of the alkoxy group having 1 to 6 carbon atoms represented by R 5 include a methoxy group, an ethoxy group, a propoxy group, a butoxy group, a pentyloxy group, a hexyloxy group, a phenyloxy group, a cyclopropyloxy group, a cyclobutyloxyl group, a cyclopentyloxy group, and a cyclohexyloxy group.
  • the number of substituents of the alkoxy group represented by R 5 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,
  • the present disclosure relates to a pharmaceutical composition for preventing, ameliorating, suppressing progression of, and/or treating Fabry disease, the pharmaceutical composition containing, as an active ingredient, a compound represented by Formula (X) or (X′), a prodrug thereof, or a pharmaceutically acceptable salt thereof.
  • R 5 represents a hydrogen atom, a methoxy group, or a 2,2,2-trifluoroethoxy group.
  • R 6 represents a 2-furyl group, a 2-thiazolyl group, or a 4-pyridyl group.
  • X 1 represents N or CH.
  • X 2 represents NH, NCH 3 , S, or O.
  • X′ represents a hydrogen atom, a chlorine atom, an iodine atom, a bromine atom, or a fluorine atom.
  • examples of the compound according to the present disclosure include the compounds below,
  • Y represents a halogen atom in the formula.
  • examples of the halogen atom represented by Y include a chlorine atom, a fluorine atom, and an iodine atom.
  • examples of the compound according to the present disclosure include the compounds below.
  • the compound represented by Formula (I), (I′), (IX), (IX′), (X), or (X′) includes an asymmetric carbon atom, and/or if stereoisomers thereof are present, the compound is a mixture of isomers or an isolated compound, in one or more embodiments.
  • the compound represented by Formula (I), (I′), (IX), (IX′), (X), or (X′) in the present disclosure can be synthesized by referring to the method disclosed in WO2010/118367 or the method disclosed in WO2016/115434.
  • a “prodrug” in the present disclosure refers to compounds that are to be converted in a living body into compounds represented by General Formula (I), (I′), (II), (II′), (III), (IV), (V), (VI), (VII), (VIII), (IX), (IX′), (X), or (X′).
  • examples of the prodrug include a compound whose carboxyl group has changed to an alkoxycarbonyl group, a compound whose carboxyl group has changed to an alkylthio carbonyl group, and a compound whose carboxyl group has changed to an alkylaminocarbonyl group.
  • examples of the prodrug include a compound whose amino group is substituted with an alkanoyl group to form an alkanoylamino group, a compound whose amino group is substituted with an alkoxycarbonyl group to form an alkoxycarbonylamino group, a compound whose amino group has changed to an acyloxymethylamino group, and a compound whose amino group has changed to a hydroxylamine.
  • examples of the prodrug include a compound whose hydroxy group is substituted with the acyl group to form an acyloxy group, a compound whose hydroxy group has changed to a phosphoric acid ester, and a compound whose hydroxy group has changed to an acyloxymethyloxy group.
  • An example of an alkyl portion of a group used to form these prodrugs is the alkyl group, and the alkyl group may be substituted (by an alkoxy group having 1 to 6 carbon atoms, for example).
  • examples of a compound whose carboxyl group has changed to an alkoxycarbonyl group include a lower (e.g., 1 to 6 carbon atoms) alkoxycarbonyl such as methoxycarbonyl and ethoxycarbonyl, and a lower (e.g., 1 to 6 carbon atoms) alkoxycarbonyl obtained through substitution with an alkoxy group, such as methoxymethoxycarbonyl, ethoxymethoxycarbonyl, 2-methoxyethoxycarbonyl, 2-methoxyethoxymethoxycarbonyl, and pivaloyloxymethoxycarbonyl.
  • a lower alkoxycarbonyl such as methoxycarbonyl and ethoxycarbonyl
  • a lower (e.g., 1 to 6 carbon atoms) alkoxycarbonyl obtained through substitution with an alkoxy group such as methoxymethoxycarbonyl, ethoxymethoxycarbonyl, 2-methoxyethoxycarbonyl,
  • 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.
  • Preferred examples of the inorganic acid salts include hydrochloride, hydrobromide, sulfate, nitrate, and phosphate
  • preferred examples of the organic acid salts include acetate, succinate, fumarate, maleate, tartrate, citrate, lactate, stearate, benzoate, methanesulfonate, and p-toluenesulfonate.
  • 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.
  • 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.
  • 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.
  • a known drug preparation technique may be applied to the pharmaceutical composition according to the present disclosure 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.
  • 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.
  • 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.
  • the pharmaceutical composition according to the present disclosure does not contain other active ingredients having a therapeutic effect, or contains another one or more active ingredients.
  • excipient examples include, but not limited to, starches such as starch, potato starch, and corn starch, lactose, crystalline cellulose, and calcium hydrogen phosphate.
  • coating agent examples include, but not limited to, ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, shellac, talc, carnauba wax, and paraffin.
  • binder examples include, but not limited to, polyvinylpyrrolidone, macrogol, and compounds that are similar to the above-described excipients.
  • disintegrant examples 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.
  • the stabilizing agent examples 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.
  • 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 thimerosal
  • dehydroacetic acid and sorbic acid.
  • corrigent examples include, but not limited to, sweeteners, acidulants, and flavors that are usually used.
  • a solvent ethanol, phenol, chlorocresol, purified water, distilled water, and the like
  • a surfactant, an emulsifying agent, or the like can also be used as needed.
  • the surfactant or emulsifying agent include, but not limited to, polysorbate 80, polyoxyl 40 stearate, and lauromacrogol.
  • a method for using a pharmaceutical composition according to the present disclosure may change depending on the symptoms, age, administration method, and the like.
  • 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 represented by Formula (I) or (I′) is in a range of 100 nM to 1 mM.
  • the pharmaceutical composition in the case of oral administration, 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 represented by Formula (I) or (I′), once or over several times in a day according to a symptom.
  • the pharmaceutical composition in the case of intravenous administration, 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.
  • the present disclosure may relate to 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+919G>A mutation in human cells or human individuals.
  • These methods may be performed by bringing the compound represented by Formula (I), (I′), (IX), (IX′), (X), or (X′) or the pharmaceutical composition according to the present disclosure into contact with the human cells or the human individuals.
  • the compound represented by Formula (I), (I′), (IX), (IX′), (X) or (X′) or the pharmaceutical composition according to the present disclosure may be brought into contact with in vitro or ex vivo human cells through addition of the compound represented by Formula (I), (I′), (IX), (IX′), (X) or (X′), a salt thereof, or the pharmaceutical composition according to the present disclosure to a cell culture medium.
  • the addition is performed so that the concentration of the compound represented by Formula (I), (I′), (IX), (IX′), (X) or (X′) is in a range of 100 nM to 1 mM.
  • the compound represented by Formula (I), (I′), (IX), (IX′), (X) or (X′) or the pharmaceutical composition according to the present disclosure 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.
  • a pharmaceutical composition for 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 development or progression of the Fabry disease.
  • 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.
  • [A3] The pharmaceutical composition according to [A1] or [A2], in which the splicing abnormality is a splicing abnormality caused by the IVS4+919G>A mutation in the GLA gene.
  • a pharmaceutical composition for preventing, ameliorating, suppressing progression of, and/or treating Fabry disease the pharmaceutical composition containing, as an active ingredient, a compound represented by Formula (I) or (I′), a prodrug thereof, or a pharmaceutically acceptable salt thereof,
  • R 1 and R 2 each independently represent a hydrogen atom, a linear, branched, or cyclic alkyl group having 1 to 6 carbon atoms, a benzyl or heteroarylmethyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted heteroaryl group;
  • R 3 represents a hydrogen atom, a linear, branched, or cyclic alkyl group having 1 to 6 carbon atoms, 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 linear, branched, or cyclic alkyl group having 1 to 6 carbon atoms, a benzyl or heteroarylmethyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted heteroaryl group;
  • X represents a hydrogen atom, a halogen atom, an amino group, an R 1 - and R 2 -substituted amino group, an azide group, a cyano group, a nitro group, a hydroxy group, a linear, branched, or cyclic alkyloxy group having 1 to 6 carbon atoms, 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 linear, branched, or cyclic alkyl group having 1 to 6 carbon atoms, a benzyl or heteroarylmethyl group, a substituted or unsubstituted aryl group, or a substituted or
  • a pharmaceutical composition for preventing, ameliorating, suppressing progression of, and/or treating Fabry disease the pharmaceutical composition containing, as an active ingredient, a compound represented by Formula (IX) or (IX′), a prodrug thereof, or a pharmaceutically acceptable salt thereof,
  • a pharmaceutical composition for preventing, ameliorating, suppressing progression of, and/or treating Fabry disease the pharmaceutical composition containing, as an active ingredient, a compound represented by Formula (X) or (X′) below, a prodrug thereof, or a pharmaceutically acceptable salt thereof,
  • a method for preventing, ameliorating, suppressing progression of, and/or treating Fabry disease including
  • composition Capable of Preventing or Ameliorating GLA Deficiency or Decrease in GLA Activity
  • the present disclosure relates to a pharmaceutical composition for diseases of which GLA deficiency contributes to the development or progression thereof, the pharmaceutical composition containing an active ingredient capable of suppressing the splicing abnormality.
  • the present disclosure relates to a pharmaceutical composition for diseases of which GLA deficiency contributes to the development or progression thereof, the pharmaceutical composition containing, as an active ingredient, the compound represented by Formula (I), (I′), (IX), (IX′), (X), or (X′), a prodrug thereof, or a pharmaceutically acceptable salt thereof.
  • the inventors of the present invention made a new discovery that the compounds represented by Formulae (III), (VII), and (VIII) suppress pseudo exon resulting from the 3849+10kbC>T mutation in the CFTR gene in cystic fibrosis, and as a result of which normal CFTR splicing products are restored.
  • the present disclosure relates to a pharmaceutical composition for preventing, ameliorating, suppressing progression of, and/or treating cystic fibrosis, the pharmaceutical composition containing, as an active ingredient, a compound represented by Formula (III), (VI), (VII), or (VIII), a prodrug thereof, or a pharmaceutically acceptable salt thereof, and a method therefor. Also, in another aspect, the present disclosure relates to a method for preventing, ameliorating, suppressing progression of, and/or treating cystic fibrosis, in which a compound represented by Formula (III), (VI), (VII), or (VIII), a prodrug thereof, or a pharmaceutically acceptable salt thereof is included as an active ingredient.
  • Compound 1 was synthesized in the following manner with reference to the method disclosed in WO2010/118367.
  • 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).
  • 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.
  • the GLA gene pseudo exon skipping evaluation vector is shown in FIG. 2 .
  • pAM1 is constituted by a normal IVS4 sequence
  • 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.
  • HeLa cells of human epithelial origin were cultured on 6 cm-plates (0.5 ⁇ 10 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 ⁇ M, 5 ⁇ M, or 10 ⁇ M, 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 .
  • 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 (GAPDI-M 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.
  • Compound 1 suppressed aberrant splicing (incorporation of the pseudo exon) of GLA gene at a concentration as low as 5 ⁇ M.
  • splice correcting activity of Compound 1 was observed in a concentration-dependent manner.
  • 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.
  • 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.
  • RFP red fluorescent protein
  • GFP green fluorescent protein
  • 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)).
  • pseudo exon skipping products (4/5/GFP) were dominantly expressed in the cells transfected with the SPREADD vector with wild-type IVS4 (pAM17 (WT)
  • pseudo exon inclusion products 4/ps/5/RFP
  • 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.
  • Compound 1 suppressed the pseudo exon of GLA gene, caused by the IVS4+919 G>A splicing mutation in a concentration-dependent manner.
  • the SPREADD reporter system as shown in FIG. 5 for a splicing mutation in the IKBKAP gene in familial dysautonomia was produced.
  • 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).
  • HeLa cells transfected with the SPREADD reporter construct were brought into contact with the compounds shown in Table 3 and cultured (concentration: 10 ⁇ M or 50 ⁇ M), and cellular fluorescence was measured after 24 hours.
  • 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.
  • the compounds represented by Formula (II), such as Compound 1 was capable of suppressing abnormal splicing resulting from the IVS20+6T>C mutation.
  • 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.
  • 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.
  • HEK293 cells transfected with the SPREADD reporter construct were brought into contact with Compound III-1 and cultured (concentrations: 10 ⁇ M and 30 ⁇ M). Fluorescence observation was performed after 6 hours. Results thereof are shown in FIG. 6B .
  • 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.
  • GFP/(GFP+RFP) of Compound III-1 increased in a concentration-dependent manner.
  • the concentration was 30 ⁇ M
  • the fluorescence intensity ratio was at about the same level as that of the normal type.
  • 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 CPTR splicing products was restored.
  • 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 CPTRc. 3849+10 kb mutant vector was set to 0% and GFP/(GFP+RFP) of the wild-type ChM vector was 100%), and, in particular, Compounds H and Q exhibited higher activity values, compared to Compound III-1.
  • 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.
  • Compound III-1 had a concentration-dependent splicing induction effect on the COL4A5 gene and the TSC2 gene.

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US11970494B2 (en) 2021-11-09 2024-04-30 Ajax Therapeutics, Inc. 6-heteroaryloxy benzimidazoles and azabenzimidazoles as JAK2 inhibitors

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US11970494B2 (en) 2021-11-09 2024-04-30 Ajax Therapeutics, Inc. 6-heteroaryloxy benzimidazoles and azabenzimidazoles as JAK2 inhibitors

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