WO2017009644A1 - Inhibiteurs de kinases pour une utilisation dans le traitement de la dystrophie facio-scapulo-humérale - Google Patents

Inhibiteurs de kinases pour une utilisation dans le traitement de la dystrophie facio-scapulo-humérale Download PDF

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WO2017009644A1
WO2017009644A1 PCT/GB2016/052119 GB2016052119W WO2017009644A1 WO 2017009644 A1 WO2017009644 A1 WO 2017009644A1 GB 2016052119 W GB2016052119 W GB 2016052119W WO 2017009644 A1 WO2017009644 A1 WO 2017009644A1
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group
alkyl
substituted
hydrogen
unsubstituted
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Robert David KNIGHT
Peter Steven ZAMMIT
Louise Anne MOYLE
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King's College London
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/517—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with carbocyclic ring systems, e.g. quinazoline, perimidine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system

Definitions

  • the present invention relates to compounds for use in the treatment of fascioscapulohumeral dystrophy (FSHD), to dosage forms for use in these methods and to methods of treating the disease in patients.
  • FSHD fascioscapulohumeral dystrophy
  • Muscle dystrophies are a group of disorders characterised by skeletal muscle weakness and wasting. Fascioscapulohumeral dystrophy (FSFID) is a degenerative muscle disease that leads to progressive muscle weakness, initially affecting muscles in the face, shoulders and hips, usually from teens to adulthood. It is associated with a genomic mutation on chromosome 4q35 that changes the number of repeat D4Z4 units in a macrosatellite. There is no clear consensus on how this contraction causes disease. However, these D4Z4 units contain an open reading frame encoding a transcription factor called DUX4. This gene is not normally expressed in muscle cells.
  • FSFID Fascioscapulohumeral dystrophy
  • SCs satellite cells
  • Muscle progenitor cells extracted from FSHD patients show a compromised ability to form muscle. This is thought to reflect sporadic expression of the DUX4 gene. Thus, it is thought that the sporadic expression of DUX4 in muscle causes muscle weakness and wasting, and results in a reduced capacity of muscle repair by SC, leading to compromised muscle function.
  • DUX4 often inhibits muscle formation by SCs and several reports have shown pro-apoptotic functions, but low levels of DUX4 does not promote rapid, overt apoptosis in mouse or in man (Vanderplanck et al, PLoS One. 2011; 6(10):e26820). There is therefore no clear route for devising pharmacological interventions to overcome DUX4-induced pathologies as there is no single mammalian model that encompasses the genetic and pathophysiological spectrum of FSFID (Lek et al. 2015; Trends in molecular medicine 21, 295-306).
  • DUX4 expression is considered to be sporadic in muscle. Any knockdown of DUX4 by this approach would only be transient and so not be able to compensate for prior DUX4- induced changes to muscle stem cells.
  • Another limitation is delivery, as the
  • oligonucleotides would have to penetrate the affected muscle in order to reach the muscle fibres and muscle stem cells and this would likely require high doses to be applied systematically and often.
  • the applicants have identified key DUX4 target genes that contribute to the pathogenic phenotype of FSHD and found that inhibitors of the receptor tyrosine kinases, in particular small molecule inhibitors, can be useful in the treatment of the FSHD phenotype. In particular, they may ameliorate FSHD-associated pathologies in muscle.
  • an inhibitor of a receptor tyrosine kinase for use in the treatment of fascioscapulohumeral dystrophy (FSHD).
  • Inhibitors of receptor tyrosine kinases have been found to rescue muscle differentiation in cells containing DUX4, in a dose dependent manner and therefore, may promote enhanced muscle function in FSHD patients.
  • inhibitors are suitably small molecule inhibitors and examples are known in the art. Some such inhibitors are approved for use in the treatment of proliferative diseases such as cancer.
  • the inhibitors may act on one or more typical single-pass type I receptor tyrosine kinases including, but not limited to, RET, PDGFRa/b, VEGFRl-3 or FLT3 and related proteins.
  • RET single-pass type I receptor tyrosine kinases
  • PDGFRa/b including, but not limited to, RET, PDGFRa/b, VEGFRl-3 or FLT3 and related proteins.
  • 'related proteins' may include proteins from similar receptor subfamilies.
  • the inhibitors may not specifically target structurally atypical receptor tyrosine kinases such as ALK-4, ALK-5, JAK1, JAK2, JAK3 and TYK2.
  • the inhibitor may inhibit a number of related RTKs including c-KIT and CSF-IR, but in those cases, it suitably also inhibits other receptor tyrosine kinases, in particular at least one of RET, PDGFRa/b, VEGFRl-3 or FLT3.
  • the receptor tyrosine kinase is RET, PDGFRa/b, VEGFRl-3 and FLT3.
  • the inhibitor is principally considered to be a RET inhibitor, where RET is the receptor tyrosine kinase encoded by the RET oncogene.
  • small molecule inhibitors include indolidinone derivatives such as pyrrole substituted indolinone derivatives as described for example in WO01/60814, pyridine derivatives such as bi-aryl meta pyridine derivatives for instance as described in WO2007/053452 or quinazoline derivatives such as aryl- amino quinazoline derivatives as described for example in WOO 1/32651.
  • indolidinone derivatives such as pyrrole substituted indolinone derivatives as described for example in WO01/60814
  • pyridine derivatives such as bi-aryl meta pyridine derivatives for instance as described in WO2007/053452
  • quinazoline derivatives such as aryl- amino quinazoline derivatives as described for example in WOO 1/32651.
  • the small molecule may be a pyrrole substituted indolinone of formula (I)
  • R 1 is selected from the group consisting of hydrogen, halo, alkyl, cyclkoalkyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy, -(CO)R 15 , - R 13 R 14 , -(CH 2 ) r R 16 and - C(0) R 8 R 9 ;
  • R 2 is selected from the group consisting of hydrogen, halo, alkyl, trihalomethyl, hydroxy, alkoxy, cyano,-NR3 ⁇ 4 14 ,- R 13 C(0)R 14 ,-C(0)R 15 , aryl, heteroaryl, and - S(0) 2 R 1 R 14 ;
  • R 3 is selected from the group consisting of hydrogen, halogen, alkyl, trihalomethyl, hydroxy, alkoxy, -(CO)R 15 , - R 13 R 14 , aryl, heteroaryl,-NR 13 S(0) 2 R 14 -S(0) 2 R 1 R 14 , - R 13 C(0)R 14 , - R 13 C(0)OR 14 and-SO 2 R 20 (wherein R 20 is alkyl, aryl, aralkyl, heteroaryl and heteroaralkyl);
  • R 4 is selected from the group consisting of hydrogen, halogen, alkyl, hydroxy, alkoxy and - R 13 R 14 ;
  • R 5 is selected from the group consisting of hydrogen, alkyl and -C(0)R K) ;
  • R 6 is selected from the group consisting of hydrogen, alkyl and -C(0)R 10 ;
  • R 7 is selected from the group consisting of hydrogen, alkyl, aryl, heteroaryl, -C(0)R 17 and -C(0)R 10 ; or
  • R 6 and R 7 may combine to form a group selected from the group consisting of-(CH 2 )4-, -(CH 2 ) 5 - and -(CH 2 ) 6 -; with the proviso that at least one of R 5 , R 6 or R 7 must be - C(0)R 10 ;
  • R 8 and R 9 are independently selected from the group consisting of hydrogen, alkyl and aryl;
  • R 10 is selected from the group consisting of hydroxy, alkoxy, aryloxy, -N(R u )(CH 2 ) n R 12 and- R 13 R 14 ;
  • R 11 is selected from the group consisting of hydrogen and alkyl
  • R 12 is selected from the group consisting of-NR 1 R 14 , hydroxy, -C(0)R 15 , aryl, heteroaryl, -N + (0 )R 13 R 14 ,- N(OH)R 13 , and -NHC(0)R a (wherein R a is unsubstituted alkyl, haloalkyl, or aralkyl);
  • R and R are independently selected from the group consisting of hydrogen, alkyl, Ci-4alkyl substituted with hydroxyalkylamino, cyanoalkyl, cycloalkyl, aryl and heteroaryl; or
  • R 13 and R 14 may combine to form a heterocyclo group
  • R 15 is selected from the group consisting of hydrogen, hydroxy, alkoxy and aryloxy
  • R 16 is selected from the group consisting of hydroxy, -C(0)R 15 , - R 13 R 14 and - C(0) R 13 R 14 ;
  • R 17 is selected from the group consisting of alkyl, cycloalkyl, aryl and heteroaryl; R 20 is alkyl, aryl, aralkyl or heteroaryl; and n and r are independently 1, 2, 3, or 4; or a pharmaceutically acceptable salt thereof.
  • alkyl refers to a saturated aliphatic hydrocarbon groups including straight chain or branched chains , which unless otherwise stated, may contain from 1 to 20 carbon atoms, in particular from 1 to 10 carbon atoms.
  • Cycloalkyl refers to alkyl groups as defined above which comprise one or more rings which may be fused.
  • Aryl refers to aromatic structures comprising carbon monocyclic or fused-ring polycyclic groups such as phenyl, naphthalenyl and anthracenyl.
  • Heteroaryl refers to a aromatic monocyclic or fused rings which contain at least one heteroatom such as N, O, or S.
  • Heteroalicyclic refers to a monocyclic or fused ring group having in the ring (s) of 5 to 9 ring atoms in which one or two ring atoms are heteroatoms selected from N, O, or S (O) n (where n is an integer from 0 to 2), the remaining ring atoms being C.
  • the rings may also have one or more double bonds.
  • Heterocycle or heterocyclo refers to saturated cyclic groups of from 3 to 8 ring atoms which contain at least one heteroatom selected from N, O, or S (O) n (where n is an integer from 0 to 2).
  • Alkoxy refers to both -O-alkyl or O-cycloalkyl groups and "aryloxy” refers to both an-0-aiyl and an-0-heteroaiyl group, as defined herein.
  • “Aralkyl” groups are alkyl groups substituted with an aryl group such as benzyl; and “heteroaralkyl” groups are alkyl groups substituted with a heteroaryl group as defined above.
  • the compound of formula (I) may be a compound in which:
  • R 1 is selected from hydrogen, Ci -4 alkyl, -(CH 2 ) r R 16 and -C(0) R 8 R 9;
  • R 2 is selected from hydrogen, halogen, aryl and -S(0) 2 R 13 R 14 ;
  • R 3 is selected from hydrogen, (C i- 4)alkyl, (C i- 4)alkoxy, aryl, heteroaryl, and - C(0)R 15 ;
  • R 4 is hydrogen
  • R 5 is selected from hydrogen and (C i-4)alkyl ;
  • R 6 is -C(0)R 10 ;
  • R 7 is selected from hydrogen, (C i-4)alkyl and aryl;
  • R 8 and R 9 are independently selected from hydrogen, alkyl and aryl
  • R 10 is -N(R u )(CH 2 )nR 12 , wherein n is 1, 2 or 3, R 11 is hydrogen and R 12 is selected from hydroxy, (C 1 - 4 )alkoxy, -C(0)R 15 , heteroaryl and - R 13 R 14 ;
  • R 13 and R 14 are independently selected from the group consisting of hydrogen. (C 1-C
  • R 13 and R 14 may combine to form a heterocyclo group
  • R 15 is selected from the group consisting of hydrogen, hydroxy, (C 1-C 4)alkoxy and aryloxy;
  • R 16 is selected from hydroxy and -C(0)R 15 ;
  • r 2 or 3;
  • alkoxy and aryloxy are as defined above;
  • heteroaryl refers to a monocyclic or fused ring group of 5 to 12 ring atoms containing one, two or three ring heteroatoms selected from N, O or S, the remaining ring atoms being C;
  • heterocyclo group refers to a saturated cyclic radical of 3 to 8 ring atoms in which one or two ring atoms are heteroatoms selected from N, O or S(0) n where n is an integer from 0 to 2, the remaining ring atoms being C, where one or two C atoms are optionally replaced by a carbonyl group;
  • alkyl, alkoxy and cycloalkyl groups are unsubstituted
  • the aryl and heteroaryl groups are optionally substituted with one or two substituents independently selected from halo, (Ci-4)alkyl, trihalo(Ci-4)alkyl, hydroxy, mercapto, cyano, N-amido, mono- or di(Ci-4)alkylamino, carboxy and N-sulfonamido;
  • the heterocyclo group is optionally substituted with one or two substituents independently selected from halo, -(Ci-4)alkyl, -(Ci-4)alkyl-carboxy, -(Ci-4)alkyl-ester, hydroxyl and mono- or di(Ci-4)alkylamino.
  • groups R and R are independently selected from hydrogen, (Ci 4 )alkyl, heteroaryl and, combined, -(CH 2 ) 4 -, -(CH 2 ) 5 -, -(CH 2 ) 2 -0-(CH 2 ) 2 - and -(CH 2 ) 2 N(CH 3 )(CH 2 ) 2 -, and in particular (Ci -4 )alkyl.
  • n is 2 or 3 and R 12 is - R 13 R 14 wherein R 13 and R 14 are independently (C i-4)alkyl, such as ethyl.
  • R 12 is - R 13 R 14 wherein R 13 and R 14 combine to form a group selected from -(CH 2 )4-, -(CH 2 ) 5-, - (CH 2 ) 2 -0-(CH 2 ) 2 - or - (CH 2 ) 2 N(CH 3 )(CH 2 ) 2 -.
  • R 2 is halo and in particular is fluoro.
  • R 1 , R 3 and R 4 are all hydrogen.
  • R 5 and R 7 are methyl and R 6 is a group -C(0)R 10 .
  • R 10 is a group -N(R u )(CH 2 ) n R 12 where R 11 is hydrogen, n is 2 and R 12 is a group R 13 R 14 .
  • R 13 and R 14 are both alkyl groups and in particular ethyl groups.
  • a particular example of a compound of formula (I) is sunitinib (N-(2- diethylaminoethyl)-5-[(Z)-(5-fluoro-2-oxo-lH-indol-3-ylidene)methyl]-2,4-dimethyl- lH-pyrrole-3-carboxamide) or a pharmaceutically acceptable salt thereof including an L-malate salt.
  • the small molecule ret inhibitor is a bi-aryl metapyridine derivative, for example as described in WO2007/053452.
  • the inhibitor may be a compound of formula (II)
  • Y b is selected from a group consisting of a bond or R 9b ; or
  • X b and Y b taken together is a bond;
  • each of R lb and R 2b is independently selected from a group consisting of H, C 1-6 substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycle, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl; or R lb and R 2b taken together is a bond; or R lb and R 2b taken together form a moiety selected from a group consisting of (CH 2 ) m ', (CH 2 )r ⁇ -S-(CH 2 )m ⁇ , (CH 2 ) r ⁇ -SO-(CH 2
  • each of p', q', r', n', m' is independently an integer having the value between O and 6,
  • R 9b is selected from a group consisting of H, Ci-C 6 alkyl, Ci-C 6 cycloalkyl, Ci-C 6 branched alkyl, Ci-C 6 substituted alkyl, Ci-C 6 aminoalkyl, and Ci-C 6 hydroxyalkyl;
  • Go is selected from a group consisting of N, O, H, and CH,
  • each of R 3b and R 4b is independently selected from a group consisting of H, Ci-C 6 alkyl, Ci-C 6 substituted or unsubstituted hydroxyalkyl or aminoalkyl, Ci-C 6 substituted or unsubstituted branched alkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl, or R 3b and R 4b taken together form a moiety selected from a group consisting of (CH 2 ) m >, (CH 2 ) r >-S-(CH 2 ) m >, (CH 2 ) r >-SO-(CH 2 ) m >, (CH 2 ) r -S0 2 - (CH 2 ) m ⁇ , (CH 2 ) - R 9b -(CH 2 ) m ⁇ , and (CH 2 )-0-(CH 2 ) m ⁇ ;
  • R 3b is selected from a group consisting of H, Ci-C6alkyl and Ci-C 6 substituted or unsubstituted hydroxyalkyl or aminoalkyl, substituted or unsubstituted branched alkyl, substituted or unsubstituted cycloalkyl, substituted heterocyclic connected through carbon or nitrogen, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl connected through carbon or nitrogen, with no group R ; R and R taken together form a moiety selected from a group consisting of (CH 2 ) m ', (CH 2 ) r '-S-(CH 2 ) m ', (CH 2 )r ⁇ -SO-(CH 2 )m ⁇ , (CH 2 ) r ⁇ -S0 2 -(CH 2 ) m ⁇ , (CH 2 ) - R 9b -(CH 2 ) m and (CH 2 ) -O-
  • each of R 3b and R 4b is independently selected from a group consisting of H, Ci-C 6 alkyl, Ci-C 6 substituted or unsubstituted hydroxyalkyl or aminoalkyl, Ci-C 6 substituted or unsubstituted branched alkyl, substituted or unsubstituted aryl, Ci-C 6 substituted or unsubstituted heterocycle connected through carbon or nitrogen, and substituted or unsubstituted heteroaryl connected through carbon or nitrogen, or R 3b and R 4b taken together form a moiety selected from a group consisting of (CHR 9b ) r ⁇ -(CHR 9b ) m ⁇ -(CHR 9b ) p ⁇ , (CHR 9b ) r ⁇ -S- (CHR 9 V, (CHR 9 VSO-(CHR 9 V, (CHR 9b ) r ⁇ -S0 2 (CHR 9
  • G is N or CR 6b , and each G is independent of each other G, with the further proviso that not more than two groups G can be N, with the further proviso that for each CR 6b , each R 6b is independent of each other group R 6b ;
  • each of R 6b , R 7b , R 8b is independently selected from a group consisting of H, Ci-C 6 substituted or unsubstituted alkyl, Ci-C 6 substituted or unsubstituted alkenyl, Ci- C 6 substituted or unsubstituted alkynyl, Ci-C 6 substituted or unsubstituted
  • Ci-C 6 substituted or unsubstituted branched alkyl Ci-C 6 substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl connected through carbon or a heteroatom, substituted or unsubstituted heteroaryl connected through carbon or a heteroatom, Ci-C 6 alkoxy, a halogen, CF 3 , -OCF 3 , CHR 3b R 4b , SR 3 , SOR 3b , S0 2 R 3b , S0 2 R 3b R 4b , S0 3 R 3b , POR 3b , P0 2 R 3b , P0 2 R 3b R 4b , P0 2 CR 3b R 4b , P0 3 CR 3b R 4b , P0 3 R 3b , R 3b R 4b , N0 2 , CN, OH, C0 R 3b R 4b , COR 3
  • R 3b COR 4b R 3b CO R 3b R 4b , OCO R 3b R 4b , CS R 3b R 4b , CSR 3b , R 3b CS R 3b R 4b , SCO R 3b R 4b , SCS R 3b R 4b , and SCS R 3b R 4b ; or any of R 6b and R 7b taken together, or R 7b and R 8b taken together, or R 6b and R 8b taken together form a moiety independently selected from a group consisting
  • A is selected from a group consisting of O, R 3b , CR 3b R 4b , S, SO, and S0 2 ;
  • Gi is selected from a group consisting of CH, N, H, S, and O;
  • G 2 is selected from a group consisting of CR 7b , N, NH, S, and O, with each group R 7b being independent of every other group R 7b ;
  • X or Go includes at least one heteroatom included with X and selected from O, S and N, or Go comprises at least four non-hydrogen atoms, inclusive of the heteroatom, and R 3b and R 4b , or R lb and R 9b , or R lb and R 4b , or R 9b and R 4b taken together form an aromatic, heteroaromatic, cyclic or heterocyclic ring system, or if a noncyclic system is present, then more than one heteroatom is present, and if A is NR 3b , then any of R 6b , R 7b or R 8b , or any combination thereof independently includes at least two non-hydrogen substituents, or if A is NR 3b , then Q forms a fused ring from R 6b to R 7b , or from R 7b to R 8b ,
  • Suitable examples of compounds of formula (II) are compounds of formula II wherein:
  • X b is selected from a group consisting of a bond, O, S0 2 , and CH 2 ; Y b is selected from a group consisting of a bond or NR 9b ; or X b and Y b taken together is a bond;
  • each of R lb and R 2b is independently selected from a group consisting of H, Ci-6 alkyl, cycloalkyl; or R lb and R 2b taken together is a bond; or R lb and R 2b taken together form a moiety selected from a group consisting of (CH 2 ) m ', (CH 2 ) r '-S-(CH 2 ) m ', (CH 2 ) r -SO- (CH 2 ) m % (CH 2 )r ⁇ -S02-(CH 2 )m ⁇ , (CH 2 ) r -NR 9 -(CH 2 ) m and (CH 2 ) r -0-(CH 2 ) m ';
  • R 9b is selected from a group consisting of H, Ci-6alkyl, Ci-6cycloalkyl, Ci-6 branched alkyl, Ci-6 aminoalkyl, and Ci-6 hydroxyalkyl;
  • Go is selected from a group consisting of N, O, H, and CH,
  • G is CH or C when bonded to X
  • R 5b is methyl
  • each of R 6b and R 7b is independently selected from a group consisting of Ci-6 alkenyl, Ci-6 alkynyl, Ci-6 hydroxyalkyl or aminoalkyl, Ci-6 cycloalkyl, Ci-6 alkoxy, a halogen, CF 3 , OCF 3 , S0 2 H, S0 2 (Ci-6 alkyl), S0 2 -heterocycle, S0 2 -cycloalkyl, S0 2 N(Ci -6 alkyl)H, S0 2 N(Ci -6 alkyl)(Ci- 6 alkyl), S0 2 H(Ci- 6 cycloalkyl), S0 2 H-heterocycle, (S0 2 N(Ci-6 branched alkyl)H, N0 2 , CN, OH, CO H 2 , CO-(Ci -6 alkyl), COOH, COO- (Ci-6 alkyl), and HCO-(Ci -6 alkyl), and
  • R 8b is independently selected from the group consisting of H, Ci-6 alkenyl, Ci-6 alkynyl, Ci-6 hydroxyalkyl or aminoalkyl, Ci-6 cycloalkyl, halogen, CF 3 , OCF 3 , S0 2 H ,S0 2 (ci-6 alkyl), S0 2 -heterocycle, S0 2 -cycloalkyl, S0 2 N(Ci -6 alkyl)H, S0 2 N(Ci -6 alkyl)(Ci- 6 alkyl), S0 2 H(Ci -6 cycloalkyl), S0 2 H-heterocycle, (S0 2 N(Ci -6 branched alkyl)HN0 2 , CN, OH, CONH 2 , CO-(Ci -6 alkyl), COOH, COO-(Ci -6 alkyl), and NHCO-(Ci-6 alkyl);
  • A is selected from a group consisting of NH, and N-(Ci-6 alkyl);
  • Gl is CH
  • G2 is CR 7b , with each group R 7b independent of every other group R 7b ;
  • all G groups are CH groups.
  • X b and Y b are a bond.
  • p', q' and n' are 0.
  • Go is N
  • R 3b and R 4b together form a group selected from (CH 2 ) m ', (CH 2 ) r -S-(CH 2 ) m ⁇ , (CH 2 ) r -SO-(CH 2 ) m ⁇ , (CH 2 ) r -S0 2 -(CH 2 ) m ⁇ , (CH 2 ) -NR 9b -(CH 2 ) m ⁇ , and (CH 2 ) -0-(CH 2 ) m '; and in particular (CH 2 ) r -NR 9b -(CH 2 ) m ⁇
  • r' and m' are both 2 and R 9b is suitably an alkyl group such as methyl.
  • A is a group NR 3b .
  • R 6b , R 7b and R 8b are all hydrogen.
  • Gi is CH.
  • G 2 is a group CR 7b and in this case, R 7b is a group of formula S0 2 NR 3b R 4b .
  • R and R are independently selected from H or Ci-6alkyl.
  • one of R 3b or R 4b is H and the other is tert-butyl.
  • the compound of formula (II) is TG101209 (N-(l, l-dimethylethyl)-3-[[5-methyl-2-[[4-(4-methyl- l-piperazinyl)phenyl]amino]-4- pyrimidinyl]amino]-benzenesulfonamide),( Ramakrishnan et al. Am J Hematol. 2010 Sep;85(9):675-86. doi: 10.1002/ajh.21785).
  • the small molecule may comprise a quinazoline derivative and in particular a compound of formula III)
  • m is an integer from 1 to 3 ;
  • R la represents halo or Ci-3alkyl
  • R 2a is selected from one of the following three groups :
  • Ci-5alkylR 3a (wherein R 3a is piperidin-4-yl which may bear one or two substituents selected from hydroxy, halogeno, Ci-4alkyl, Ci-4hydroxyalkyl and Ci-4alkoxy ;
  • m is 2 and each R la group is a halo group.
  • R 2a is a group (1) above, and in particular, is [(1- methyl)piperidin-4-yl]methyl.
  • a particular example of a compound of formula (III) is vandetanib (N-(4- bromo-2-fluorophenyl)-6-methoxy-7-[(l-methylpiperidin-4-yl)methoxy]quinazolin-4- amine), which may be available under the trade names Caprelsa or ZACTIMA
  • the small molecule inhibitor is one which will produce a dose related inhibitory response such as Sunitinib or TGI 01209 but in particular is Sunitinib.
  • the invention provides a method for treating
  • fascioscapulohumeral dystrophy FSHD
  • said method comprising administering to a patient in need thereof, an effective amount of an inhibitor of a receptor tyrosine kinase.
  • the receptor tyrosine kinase is selected from the group consisting of
  • RET RET
  • PDGFRa/b VEGFR1-3 and FLT3 and related proteins
  • Inhibitors are suitably small molecule inhibitors such as those listed above.
  • the inhibitor is suitably administered in the form of a pharmaceutical composition.
  • a pharmaceutical composition Such compositions form a further aspect of the invention.
  • Suitable pharmaceutical compositions will be in either solid or liquid form. They may be adapted for administration by any convenient route, such as parenteral, oral or topical administration or for administration by inhalation or insufflation.
  • the pharmaceutical acceptable carrier may include diluents or excipients which are physiologically tolerable and compatible with the active ingredient.
  • compositions are prepared for injection, for example either subcutaneously or intravenously. They may be liquid solutions or suspensions, or they may be in the form of a solid that is suitable for solution in, or suspension in, liquid prior to injection. Suitable diluents and excipients are, for example, water, saline, dextrose, glycerol, or the like, and combinations thereof. In addition, if desired the compositions may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, stabilizing or pH-buffering agents, and the like.
  • Oral formulations will be in the form of solids or liquids, and may be solutions, syrups, suspensions, tablets, pills, capsules, sustained-release formulations, or powders.
  • Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like.
  • Topical formulations will generally take the form of suppositories or intranasal aerosols.
  • traditional binders and excipients may include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient.
  • the amount of inhibitor administered will vary depending upon factors such as the precise nature of the inhibitor, the size and health of the patient, the nature of the condition being treated etc. in accordance with normal clinical practice. Typically, a dosage for an adult would be in the range of 5-100mg daily, for example a daily dosage of from lC ⁇ g-10mg/Kg such as from 5C ⁇ g-2mg/Kg would be expected to produce a suitable effect.
  • One or more such receptor tyrosine kinase inhibitors may be combined in a dosing regime for treatment of FSHD.
  • a screen to identify targets of DUX4 in mouse satellite cells (SCs) was carried out as described below and showed that some tyrosine kinase receptors are highly upregulated by DUX4, including the RET receptor. Ret was found to be detectable in proliferating mouse SCs, but then is down-regulated during differentiation.
  • SCs satellite cells
  • the applicants used the inhibition of muscle differentiation as a readout of DUX4 function and investigated whether this could be rescued by genetic inhibition of the Ret gene. They could show that knockdown of Ret in SCs expressing DUX4 lead to a recovery of the cells ability to differentiate. Subsequently, they showed that over-activation of the RET gene using a constitutively active version of RET could inhibit muscle differentiation in an opposite manner.
  • Sunitinb resulted in significantly diminished phospho-ERK relative to total ERK, but there was no apparent change to phospho-Akt relative to total Akt.
  • Sunitinib treatment resulted in reduced Akt activity, although there was no statistically significant change to ERK activity
  • Figure 1 A) Mechanism by which Ret signals
  • Figure 2 Knockdown of Ret rescues DUX4-mediated inhibition of myogenic differentiation.
  • A Immunolabelling of satellite cell-derived myoblasts infected with control or DUX4 retrovirus (eGFP+) and transfected with Control or Ret siRNA following culture for 24 hours in mitogen poor differentiation media.
  • A Infected cells were detected by eGFP and examined for MyHC labelling to identify terminally differentiated myoblasts.
  • the fusion index (the number of nuclei detected in cells with MyHC expression/ total nuclei) was calculated for cells expressing control or DUX4 retrovirus and transfected with control or Ret siRNA. Expression of DUX4 reduced the fusion index. However, blocking Ret expression in DUX4-expressing myoblasts increased the fusion index enough such that it was not significantly different to the control fusion index.
  • C Plot of probability that a cell has MyHC
  • FIG. 3 Small molecules inhibitors block the Ret-induced phenotype in murine myoblasts.
  • A Immunolabelling with antibodies to eGFP and MyHC of C2C12 myoblasts infected with control or CA RET51 expressing retroviruses and induced to differentiate for 60 hours. Cells were treated with either 1 ⁇ g/ml Sunitinib or DMSO. Lower panels show anti-GFP labelling only.
  • B-D Fusion index of C2C12 myoblasts expressing control (red) or CA RET51 expressing retrovirus (blue) and treated with Sunitinib, Zactima and TG101209 at varying doses is shown as the -log of odds.
  • the - log of odds (-log(ratio (1-ratio)) is calculated from the fusion index (ratio of MyHC+ nuclei/ total nuclei).
  • E-G Quantification of the number of C2C12 cells expressing control (red) or CA RET51 expressing retrovirus (blue) when treated with Sunitinib, Zactima and TG101209 at varying doses.
  • H Immunolabelling of C2C12 cells to detect MyHC following treatment with varying doses of Sunitinib. All quantification represents an average of three independent experiments. Statistical significance assessed using a mixed Binomial model (B-D) or a quasi-Poisson model (E-G).
  • FIG. 4 Sunitinib treatment enables differentiation of DUX4-expressing myoblasts.
  • A-D Quantification of SC-derived myoblasts infected with control or DUX4 retrovirus and treated with either 250ng/ml Sunitinib or DMSO vehicle. Cells were cultured at low density and incubated in (A, B) proliferation or (C, D) differentiation medium for 24 hours prior to immunolabelling to detect eGFP and a specific label: EdU (A), MyoD (B), Myogenin (C) and MyHC (D). All values are represented as the ratio of the labelled GFP+ cells to all GFP+ cells and are derived from 3 independent replicate platings from 3 mice.
  • E Control or DUX4-expressing myoblasts treated with varying concentrations Sunitinib or DMSO vehicle control and incubated in differentiation medium at high density for 24 hours before immunolabelling with antibodies against eGFP and MyHC.
  • RET is co-expressed with PAX7 in human myoblasts extracted from muscle.
  • A Relative expression of RET from QPCR using TATA Binding Protein (TBP) to normalise during proliferation (prolif) and differentiation at days 1, 2, 3 and 4 (DM1- 4).
  • B PAX7 expression from the same cells.
  • FIG. 6 Sunitinib improves the phenotype of FSHD cells.
  • A Immunolabelling of mosaic FSHD patient-derived myoblast cell lines 54.6 (control) and 54.12 (containing the FSHD D4Z4 contraction) exposed to DMSO or 500ng/ ml Sunitinib. Tubulin and EdU were labelled in cells after EdU incorporation (A); MyHC was detected in cells grown at high density in differentiation medium (B). EdU incorporation was measured in cells exposed to different doses of Sunitinib and represented as a proportion of total labelled cells (C). Cell shape (eccentricity) was assessed and plotted relative to
  • Figure 7 shows the results of an experiment in which murine iC2C12 myoblasts were untreated, or treated with SOOng/ ⁇ doxycycline (Dox) to induce DUX4 and/or
  • AKT AKT
  • phosphorylated (p) AKT total ERK1/2
  • phosphorylated (p) ERK1/2 total ERK1/2
  • Caveolin-1 used as a loading control shows the quantification of the ratio between pERKl/2 : total ERK1/2 and pAKT : total AKT compared to control.
  • Figure 8 shows the results of an experiment in which a human immortalised myoblast clone (54.12) derived from a patient mosaic for the D4Z4 contraction that is associated with FSFID were untreated (Ctrl), or treated with either 250ng ⁇ l or 2.5 ⁇ g/ ⁇ l Sunitinib for 20 hours, and proteins extracted; where (A) shows a membrane probed with antibodies against total (t) AKT, phosphorylated (p) AKT, tERKl/2 and pERKl/2, with Vinculin used as a loading control; and (B and C) are graphs showing quantification of the ratio between pERKl/2 : tERKl/2 and pAKT : tAKT in the Sunitinib -treated groups compared to control. Data is represented as mean ⁇ SEM from 3 biological replicates, where an asterisk denotes a significant difference (p ⁇ 0.05) from Control, using a two-tailed unpaired Student's t Test.
  • a microarray of murine satellite cell (SC)-derived myoblasts expressing DUX4, modified DUX4 versions, or its ortholog DUX4c was prepared as described in (Banerji,J R Soc Interface. 2015 Jan 6; 12(102):20140797).
  • a large range of possible DUX4 targets were indicated using a selection matrix.
  • One gene that showed a significant change in expression from the SC microarray was the receptor tyrosine kinase Ret. This was up-regulated by DUX4. Ret phosphorylation activates signalling pathways such as MAP-kinase and PI3K-Akt as illustrated in Figure 1 A.
  • Ret signalling is crucial for neural and kidney differentiation, and RET mutations are associated with Hirschsprung's disease, while gain-of-function mutations are associated with certain cancers (including medullary thyroid carcinoma, multiple endocrine neoplasias type 2A and 2B, phaeochromocytoma and parathyroid hyperplasia). Absence of RET ligands can trigger apoptosis. Ret has 2 main isoforms, Ret9 and Ret51, differing in the C-terminal region.
  • DUX4c a homologue of DUX4 containing the same homeodomain DNA binding sites
  • truncated DUX4 constructs was compared.
  • Muscle myofibres were isolated from the extensor digitorum longus (EDL) as previously described (Moyle et al. Methods Mol Biol, (2014). 1210: p. 63-78). Briefly, dissected EDLs were digested in DMEM
  • Non-adherent cultures were grown in DMEM (Glutamax) with 10% horse serum (HS) (Gibco), 0.5% chicken embryo extract (CEE) (ICN Flow), 1% penicllin/streptomycin (Sigma Aldrich) for up to 72 hours.
  • HS horse serum
  • CEE chicken embryo extract
  • ICN Flow 1% penicllin/streptomycin
  • myofibres were plated on lmg/ml Matrigel (Collaborative research) coated dishes in DMEM (Glutamax) with 20% foetal calf serum (FBS) Gold (PAA), 10% HS, 1%) CEE, 1/10,000 basic FGF (bFGF) for 72 hours. Subsequently, myofibres were removed by pipette agitation and satellite cells re-plated by trypsinisation in 0.25% Trypsin-EDTA and myoblasts re-plated to expand for experimentation.
  • HS horse serum
  • CEE chicken embryo
  • Ret was increased over 3 fold in DUX4 expressing SCs, relative to controls, while expression of RET co-receptors GFRa2, GFRa3 and GFRa4 were unaltered, although GFRal expression was reduced 0.5-fold.
  • Expression of DUX4c did not alter Ret or GFRa co-receptor expression, suggesting that Ret may not be a direct DUX4 homeodomain-binding target.
  • DUX4 or DUX4c can induce Ret in inducible iC2C12 myoblasts, which contain a doxycycline-regulated DUX4 cassette.
  • DUX4 or DUX4c were induced with 200ng doxycycline and cultured for 6, 12, 24 and 48 hours and analysed by qRT-PCR. Expression of Ret was significantly increased after 12 hours in the iC2C12-DUX4 line, compared to un-induced myoblasts, and maintained at 24 and 48 hours. Induction of DUX4c did not significantly alter Ret expression at any time-point.
  • Ret is a DUX4 target gene in SCs, we infected SCs with DUX4 and DUX4c.
  • DUX4 increased Ret transcription after 48 hours relative to control cells.
  • DUX4 activation of Ret leads to the production of membrane-located RET protein by immunostaining with an antibody to RET51 in DUX4-infected cells.
  • Ret and Ret co-receptors are expressed in satellite cells
  • myofibres with their associated satellite cells were isolated and either immediately fixed, or cultured.
  • floating myofibres or plated satellite cells were fixed in 4% paraformaldehyde/PBS, permeabilised with 0.5% Triton/PBS (Sigma Aldrich) and blocked in PBS containing 5% swine serum + 5% goat serum (DAKO) for 1 hour (except for samples using goat Ret51 antibody, which were blocked in 10% swine serum).
  • Ret51 was barely detectable in murine quiescent satellite cells, but expression increased after 24 hours of activation. This correlates with the expression profile of phospho-Ret Tyl062, which identifies active Ret signalling. After 72 hours of culture, a proportion of satellite cell-derived myoblasts (SCs) stop expressing Pax7 and commit to differentiation, expressing Myogenin. Ret51 was found in both Myogenin-negative uncommitted myoblasts and Myogenin-positive differentiating myoblasts.
  • SCs satellite cell-derived myoblasts
  • siRNA-mediated knock-down To test whether satellite cells require Ret for normal function, the applicants used siRNA-mediated knock-down.
  • Satellite cells were transfected with 20nM of a scrambled-sequence control siRNA or Ret Silencer® Select siRNA (Life Technologies) (directed at both Ret isoforms) in the presence of Lipofectamine RNAiMAX (Life Technologies) for 6 hours at 37°C, 5% CO2 in proliferation medium.
  • the siRNA sequence 5'- GCUUGUACAUCGGGACUUATT-3' (ID: s72895) (SEQ ID NO 3) was used to knockdown murine Ret expression, and control siRNA was supplied by Life
  • SiRNA-mediated knock-down of Ret was also associated with a decrease in the proportion of SCs expressing Pax7, from a mean+SEM of 90.7 ⁇ 2.2% in control myoblasts to 84.0 ⁇ 2.0% with Ret siRNA, which was associated with lower Pax7 mRNA levels.
  • the reduced number of proliferating Pax7+ SCs and lower expression of Pax7 when Ret is knocked down suggests that Ret contributes to maintaining SCs in an undifferentiated state. Ret knockdown did not alter My/5 orMyoD expression.
  • satellite cell-derived myoblasts were cultured in mitogen- poor medium, containing DMEM + 2% HS + penicillin/streptomycin.
  • SCs treated with Ret siRNA had reduced Pax7 levels and proliferation rate, implying that the cells may be differentiating prematurely.
  • cultures of SCs treated with control or Ret siRNA as described in Example 2B above were plated at high density and incubated in differentiation medium for 48 hours, before immunostaining with antibodies to Myogenin and Myosin Heavy Chain (MyHC).
  • MyHC Myogenin and Myosin Heavy Chain
  • SCs were transfected with siRNA against GFRal, GFRa2 or GFRa4 and the expressions of Pax7, phospho-Hi stone H1/H3, Myogenin and MyHC was quantified as described above.
  • Ret siRNA knock-down of GFRa4 (but not GFRal or GFRal) was associated with fewer Pax7+ SCs in proliferation medium.
  • phospho-Hi stone H1/H3 immunostaining revealed that knock-down of GFRal, GFRa2 and GFRa4 were all associated with fewer cells in the active stages of the cell cycle.
  • knockdown of GFRal and GFRa4 significantly increased the number of Myogenin-positive nuclei, and knockdown of GFRa4 significantly increased the fusion index.
  • knockdown of all co-receptors recapitulated aspects of Ret knockdown, indicating that RET may signal through multiple co-receptors.
  • Plasmids encoding human RET9 and RET51 and constitutively active (CA) forms containing the Cys634Lys mutation (causing ligand-independent dimerisation of the RET receptor) that occurs in multiple endocrine neoplasia type 2A (MEN2A) patients were obtained 1 .
  • These coding sequences were sub-cloned into a modified pMSCV-puro vector (Clontech), in which the puromycin resistance gene was replaced with an internal ribosomal entry site (IRES) and enhanced green fluorescent protein
  • Retroviruses were produced using HEK 293 T packaging cells, by co- transfecting the expression vectors in the presence of an ectopic helper plasmid.
  • Replication incompetent viral particles were harvested from the culture medium and functionality confirmed by western blot and immunofluorescence.
  • HEK 293T cells were transfected with RET expression vectors for 24 hours and total protein extracted in the presence of complete protease inhibitor cocktail (Roche).
  • Samples were quantified using the BioRad Protein Assay system (BioRad) and equal quantifies run on pre-cast 4-20% electrophoresis gels (Invitrogen) with 0.35 ⁇ 1 dithiothreitol + bromophenol blue dye at 120V. Gels were transferred to PVDF membranes using the iBlot dry-blotting system (Invitrogen), blocked in 5% milk powder/PBS and incubated overnight at 4°C in primary antibody dissolved in PBS+ 1% milk powder.
  • HRP horseradish peroxidase
  • ECL enhanced chemiluminescence
  • the log of odds is calculated by:
  • CA RET constructs significantly increased proliferation rate from 47.6 ⁇ 0.8% in control to 56.9 ⁇ 1.8% (CA RET9) and 59.2 ⁇ 1.7% (CA RET51) respectively.
  • CA RET9 constitutive expression of the RET constructs affected muscle differentiation
  • the applicants analysed expression of Myogenin and MyHC in SCs cultured in differentiation medium. After 24 hours, CA RET51 expression reduced the percentage of myoblasts that were Myogenin+ (49.5 ⁇ 1.2%) relative to controls (58.9 ⁇ 3.1%). At later stages of differentiation, there was no change to the number of Myogenin+ myoblasts or of the fusion index.
  • DUX4 also suppresses myogenic regulatory factor (MRF) gene expression and inhibits proliferation and myogenic differentiation.
  • MRF myogenic regulatory factor
  • DUX4-induced pathology could be rescued by inhibiting RET signalling
  • the applicants measured the effects of siRNA mediated inhibition of Ret in DUX4-expressing SCs. The applicants initially tested whether knockdown of Ret could rescue SC proliferation rate.
  • Murine SCs infected with control or DUX4 retrovirus were transfected with 20nM control or Ret siRNA for 48 hours in proliferation medium and treated with EdU for two hours. As expected, the proportion of myoblasts incorporating EdU was significantly reduced when DUX4 was expressed; transfection of Ret siRNA transfection also resulted in a reduced EdU uptake.
  • Table 1 (a) Maximum likelihood parameters for a logistic model containing an interaction term, and a random effect term (the mouse) describing the probability of a nuclei being present in a MyHC+ cells the presence or absence of DUX4 and RET. (b) Corresponding ratios computed from the model, for all 4 tested conditions with the ratio representing the probability of a GFP+ cell expressing MyHC. In all condition but the control (Intercept), the error contribution of the baseline (Intercept) has been omitted when computing the confidence intervals (C.I.).
  • y represents the log-of-odds of the fusion index
  • ⁇ represents the intercept parameter (representing the control: MIG control retrovirus, control siRNA)
  • ⁇ are the parameters representing the effects of each treatment, or the interaction as specified and ⁇ indicates whether the effect is present or absent.
  • RET signalling potentiates DUX4 activity
  • small molecular inhibitors of RET were evaluated for their ability to affect myogenesis in the presence or absence of DUX4.
  • Three kinase inhibitors that block RET phosphorylation were tested: Zactima
  • Zactima and Sunitinib have been clinically approved as therapeutics for treating cancers arising from over-activation of RET signalling.
  • these drugs also inhibit several receptor tyrosine kinases other than RET at a higher IC50, including VEGFR, EGF, MET, and c- Kit, the applicants first aimed to show whether they could overcome the effects of a specific over-activation of RET in myoblasts.
  • C2C12 myoblasts were maintained in DMEM supplemented with 10% FBS with 1% L-Glutamine (Sigma Aldrich) and 1% pen/strep.
  • C2C12s were plated at 70% confluency and infected with RET or DUX4-encod g retrovirus in the presence of 4 ⁇ g/ml Polybrene for 6 hours at 37°C, 5% CO2 in proliferation medium, To induce differentiation, C2C12 myoblasts were cultured in mitogen-poor medium, containing DMEM with 2% HS, pen/strep and L-Glutamine, C2C12.
  • Table 2 Maximum likelihood parameters for a logistic model describing the effect on myoblast fusion following expression of RET51-MEN2A (RET51CA) or MIG control retrovirus in murine C2C12 cells when treated with different concentrations of Sunitinib, TG101209 or ZACTIMA (indicated by ng/ml).
  • the model contains interaction terms for RET51-MEN2A and Sunitinib (RET51C A: Sunitinib), Ret51- ME 2A with TGI 01209 (RET51CA: TGI 01209) or Ret5-MEN2A with ZACTIMA (RET51CA:Zactima) that reveals the gradient of response of muscle fusion.
  • RET51C A Sunitinib 1.574679 0.032506 48.44 ⁇ 2e-16 *** TG101209 -2.873471 0.342331 -8.39 ⁇ 2e-16 *** RET51CA:TG101209 3.295843 0.386403 8.53 ⁇ 2e-16 *** Zactima -0.304531 0.040307 -7.56 4.18e-14 *** RET51CA:Zactima 2.923664 0.045197 64.69 ⁇ 2e-16 ***
  • Sunitinib appeared to be the most effective compound tested for inhibition of RET, as it was the compound that promoted an effective rescue of fusion in the presence of CA RET51 without causing large changes to cell number. Therefore subsequent efforts focused on the use of Sunitinib.
  • Sunitinib does not rescue proliferation in DUX4-expressing murine myoblasts but does rescue myogenic induction
  • DUX4 expression in SCs also leads to repression of MyoD, as previously described.
  • Sunitinib is unable to rescue proliferation of murine SCs in the presence of DUX4, but can improve myogenic commitment.
  • Table 3 (a) Maximum likelihood parameters for a logistic model containing an interaction term to describe the effect of DUX4 expression and Sunitinib during fusion of satellite-cells grown at high density, y represents the log-of-odds of the fusion index, ⁇ represents the intercept parameter (representing the control treatment), ⁇ are the parameters representing the effects of each treatment, or the interaction as specified and ⁇ indicates whether the effect is present or absent, y represents the log-of-odds of the fusion index, ⁇ represents the intercept parameter (representing the control treatment: no retrovirus, with no drug present), ⁇ are the parameters representing the effects of each treatment, or the interaction as specified and ⁇ indicates whether the effect is present or absent, (b) Corresponding log of odds ratios computed from the model for all 4 tested conditions. y— ⁇ + ⁇ 3 ⁇ 4UX4 1 /3 ⁇ 4>UX4 + a)
  • Table 4 (a) Maximum likelihood parameters for a logistic model containing an interaction term, and a random effect term (the mouse) that describes the fusion index of cells infected with DUX4 or MIG control retrovirus and grown at high density when exposed to Sunitinib at varying concentrations, y represents the log-of-odds of the fusion index, ⁇ represents the intercept parameter (representing the control treatment: MIG control retrovirus with no drug), ⁇ are the parameters representing the effects of each treatment, or the interaction as specified and ⁇ indicates whether the effect is present or absent, (b) Corresponding log of odds ratios computed from the model, for all 4 tested conditions.
  • Sunitinib enables DUX4-expressing SCs to differentiate independent of fusion to unaffected myoblasts
  • DUX4 infection rate approximately 40-50%.
  • this approach does not allow the applicants to determine whether the increased rate of fusion of DUX4 expressing cells in the presence of Sunitinib was due to Sunitinib rescuing the differentiation defect directly, or simply enabling DUX4-expressing myoblasts to fuse to uninfected myoblasts.
  • the applicants seeded SCs at low density before treating with 250ng/ml Sunitinib and inducing differentiation for 24 and 48 hours. Samples were then analysed on an individual cell basis using immunolabelling to detect Myogenenin and MyHC.
  • Sunitinib treatment was observed to cause only a small change in the number or myoblasts expressing Myogenin (27.4 ⁇ 1.1%) relative to vector control treated cells (27.6 ⁇ 0.9%) and this was not significant.
  • Table 5 (a) Maximum likelihood parameters for a logistic model containing an interaction term, and a random effect term (the mouse) to describe MyHC expression in SCs at 1 or 2 days of culture when grown at low density and exposed to Sunitinib or drug vector DMSO and infected with DUX4 or MIG control retrovirus, y represents the log-of-odds of the fusion index, ⁇ represents the intercept parameter (representing the control treatment: MIG control retrovirus with no drug), ⁇ are the parameters representing the effects of each treatment, or the interaction as specified and ⁇ indicates whether the effect is present or absent, (b) Corresponding ratios computed from the model, for all 4 tested conditions.
  • P values are approximate and are based on the t-value and represent the probability that there is a difference in cell shape at a specific concentration of Sunitnib.
  • y represents the eccentricity
  • ⁇ represents the intercept parameter (representing the control treatment: 54.6 cells with no drug)
  • ⁇ are the parameters representing the effects of each treatment, or the interaction as specified and ⁇ indicates whether the effect is present or absent.
  • Table 7 a) Maximum likelihood parameters for a logistic model containing an interaction term between the cell line and Sunitinib and incorporating a random effect term (the experiment). The model is a binomial model that tests the relationship between the proliferation of 54.6 (control) and 54.12 (FSHD) cells relative to different doses of Sunitinib. P values represent the probability of a difference in proliferation between the control cells with varying doses of Sunitinib and between control and FSHD cells at different doses of Sunitinib. y represents the proliferation index, ⁇ represents the intercept parameter (representing the control treatment: 54.6 cells with no drug), ⁇ are the parameters representing the effects of each treatment, or the interaction as specified and ⁇ indicates whether the effect is present or absent, (b)
  • Sunitinib 500 control 0. .365 0. .386 0. .407
  • Sunitinib750 control 0.358 0.377 0.396
  • Table 8 a) Maximum likelihood parameters for a logistic model containing an interaction term between the cell line and Sunitinib and incorporating a random effect term (the experiment).
  • the model is a binomial model that tests the relationship between the fusion of 54.6 (control) and 54.12 (FSFID) cells relative to different doses of Sunitinib.
  • Estimate represents the relative change in fusion between conditions.
  • P values represent the probability of a difference in fusion between the control cells with varying doses of Sunitinib and between control and FSFID cells at different doses of Sunitinib.
  • y represents the log-of-odds of the fusion index
  • ⁇ represents the intercept parameter (representing the control treatment: 54.6 cells with no drug)
  • ⁇ are the parameters representing the effects of each treatment, or the interaction as specified and ⁇ indicates whether the effect is present or absent
  • Sunitinib 500 control 0.730 0.763 0.794
  • Murine iC2C12 myoblasts were untreated, or treated with SOOng/ ⁇ doxycycline (Dox) to induce DUX4 and/or 250ng ⁇ l Sunitinib for 20 hours, and proteins then electrophoresed onto a PVDF membrane.
  • the membrane was probed with antibodies against DUX4, total AKT, phosphorylated (p) AKT, total ERKl/2 and phosphorylated (p) ERKl/2, with Caveolin-1 used as a loading control.
  • Protein bands were visualised by incubating with horseradish peroxidase-conjugated secondary antibodies and visualised using clarity western ECL substrate. Protein band intensity was quantified with the ChemiDocTM MP System and normalised to housekeeping protein Caveolin-1.
  • Results are shown in Figures 7 B and C and show that compared to control cells with no Dox or addition of Sunitinib, Sunitinib suppresses DUX4-mediated pERKl/2 signalling.
  • a human immortalised myoblast clone (54.12) derived from a patient mosaic for the D4Z4 contraction that is associated with FSHD were untreated (Ctrl), or treated with either 250ng ⁇ l or 2.5 ⁇ g/ ⁇ l Sunitinib for 20 hours, and proteins extracted onto a membrane.
  • the membrane was probed with antibodies against total (t) AKT, phosphorylated (p) AKT, tERKl/2 and pERKl/2, with Vinculin used as a loading control. Protein bands were visualised by incubating with horseradish peroxidase- conjugated secondary antibodies and visualised using clarity western ECL substrate. The results are shown in Figure 8A.
  • Protein band intensity was quantified with the ChemiDocTM MP System and normalised to housekeeping protein Vinculin and the results are shown in Figures 8B and 8C.

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Abstract

L'invention concerne un inhibiteur d'un récepteur aux tyrosine kinases, en particulier sélectionné entre les composés de formule (I), (II) ou (III) tels que définis dans la description, pour une utilisation dans le traitement de la dystrophie musculaire facio-scapulo-humérale (dystrophie FSH), la formule (I) étant développée en (N-(2- diéthylaminoéthyl)-5-[(Z)-(5-fluoro-2-oxo-1H-indol-3-ylidène)méthyl]-2,4-diméthyl-H-pyrrole-3-carboxamide (sunitinib), la formule (II) étant développée en (N-(1,1- diméthyléthyl)-3-[[5-méthyl-2-[[4-(4-méthyl-1-pipérazinyl)phényl]amino]-4- pyrimidinyl]amino]-benzènesulfonamide (TG101209) et la formule (III) étant développée en N-(4-bromo-2-fluorophényl)-6-méthoxy-7-[(1-méthylpipéridine-4- yl)methoxy]quinazoline-4-amine) (Vandétanib).
PCT/GB2016/052119 2015-07-15 2016-07-14 Inhibiteurs de kinases pour une utilisation dans le traitement de la dystrophie facio-scapulo-humérale WO2017009644A1 (fr)

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US10023570B2 (en) 2015-07-16 2018-07-17 Array Biopharma Inc. Substituted pyrazolo[1,5-A]pyridine compounds as RET kinase inhibitors
WO2018136663A1 (fr) 2017-01-18 2018-07-26 Array Biopharma, Inc. Inhibiteurs de ret
WO2018136661A1 (fr) 2017-01-18 2018-07-26 Andrews Steven W Composés de pyrazolo[1,5-a]pyrazine substitués utilisés en tant qu'inhibiteurs de la kinase ret
US10112942B2 (en) 2016-10-10 2018-10-30 Array Biopharma Inc. Substituted pyrazolo[1,5-A]pyridine compounds as RET kinase inhibitors
US10144734B2 (en) 2016-10-10 2018-12-04 Array Biopharma Inc. Substituted pyrazolo[1,5-A]pyridine compounds as RET kinase inhibitors
WO2019075114A1 (fr) 2017-10-10 2019-04-18 Mark Reynolds Formulations comprenant du 6-(2-hydroxy-2-méthylpropoxy)-4-(6-(6-((6-méthoxypyridin-3-yl)méthyl)-3,6-diazabicyclo[3.1.1]heptan-3-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile
WO2019075108A1 (fr) 2017-10-10 2019-04-18 Metcalf Andrew T Formes cristallines
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WO2019143977A1 (fr) 2018-01-18 2019-07-25 Array Biopharma Inc. Composés de pyrrolo[2,3-d]pyrimidines substitués utilisés en tant qu'inhibiteurs de la kinase ret
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US11851434B2 (en) 2017-01-18 2023-12-26 Array Biopharma Inc. Substituted pyrazolo[1,5-A]pyrazine compounds as ret kinase inhibitors
WO2018136663A1 (fr) 2017-01-18 2018-07-26 Array Biopharma, Inc. Inhibiteurs de ret
WO2018136661A1 (fr) 2017-01-18 2018-07-26 Andrews Steven W Composés de pyrazolo[1,5-a]pyrazine substitués utilisés en tant qu'inhibiteurs de la kinase ret
US10966985B2 (en) 2017-03-16 2021-04-06 Array Biopharma Inc. Macrocyclic compounds as ROS1 kinase inhibitors
WO2019075108A1 (fr) 2017-10-10 2019-04-18 Metcalf Andrew T Formes cristallines
WO2019075114A1 (fr) 2017-10-10 2019-04-18 Mark Reynolds Formulations comprenant du 6-(2-hydroxy-2-méthylpropoxy)-4-(6-(6-((6-méthoxypyridin-3-yl)méthyl)-3,6-diazabicyclo[3.1.1]heptan-3-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile
WO2019143977A1 (fr) 2018-01-18 2019-07-25 Array Biopharma Inc. Composés de pyrrolo[2,3-d]pyrimidines substitués utilisés en tant qu'inhibiteurs de la kinase ret
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