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WO2020161477A1 - Traitement de la fibrose par des inhibiteurs du raf - Google Patents

Traitement de la fibrose par des inhibiteurs du raf Download PDF

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WO2020161477A1
WO2020161477A1 PCT/GB2020/050243 GB2020050243W WO2020161477A1 WO 2020161477 A1 WO2020161477 A1 WO 2020161477A1 GB 2020050243 W GB2020050243 W GB 2020050243W WO 2020161477 A1 WO2020161477 A1 WO 2020161477A1
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Prior art keywords
braf
fibrosis
compound
mice
raf
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Daniel MEIJLES
Angela CLERK
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University of Reading
City St Georges University of London
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University of Reading
St Georges Hospital Medical School
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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
    • A61K31/495—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
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/437—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a five-membered ring having nitrogen as a ring hetero atom, e.g. indolizine, beta-carboline
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/04—Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives

Definitions

  • the present invention relates to the treatment or prevention of fibrosis associated with the activity of Raf kinases, e.g. BRaf.
  • Fibrosis can occur in many tissues of the body, for example the heart, liver, lungs and kidney. Activation of pathways such as the extracellular signal regulated kinase 1/2 (ERK 1/2) pathway ultimately lead to fibroblast proliferation, which results in the deposition of extracellular matrix into surrounding connective tissue, i.e. fibrosis. Whilst fibrosis is important in normal tissue repair, excessive fibroblast proliferation is harmful and can, depending on the location of the fibrosis within the body, lead to (for example) shortness of breath, heart failure or loss of kidney function.
  • ERK 1/2 extracellular signal regulated kinase 1/2
  • cardiac fibrosis and/or heart failure One of the most important diseases associated with fibrosis is cardiac fibrosis and/or heart failure, with hypertension as a major contributing factor.
  • the heart adapts to the increased work-load to maintain cardiac output. This is achieved in part by hypertrophy of terminally-differentiated contractile cardiomyocytes which increase in size, adapting and increasing the myofibrillar apparatus. In the longer term, this is not sustained, contractile function is compromised and heart failure develops.
  • Pathological changes include cardiomyocyte cell death, and capillary rarefaction. This is associated with inflammation and increased fibrosis. Strategies to reduce cardiomyocyte death, improve contractility, increase angiogenesis and reduce fibrosis are all necessary to treat heart failure.
  • ERK1/2 extracellular signal regulated kinase 1/2 cascade is a key growth-promoting pathway in all cells. It is best characterised in proliferating cells in which ERK1/2 activation promotes cell division. ERK1/2 are phosphorylated/activated by MKK1/2 that are phosphorylated/activated by upstream Raf kinases (ARaf, BRaf, cRaf) or, in the context of inflammation, Cot/Tp12. Raf kinases are regulated at multiple levels. Of particular importance, activation requires interaction with activated Ras which induces a
  • Raf proteins are structurally similar to Raf proteins. They also operate as homo- or heterodimers and are subject to activating and inhibitory phosphorylations.
  • the ERK1/2 cascade is strongly implicated in promoting cardiomyocyte hypertrophy and, independent of this, is potently cardioprotective. All Raf kinases are activated in cultured cardiomyocytes by hypertrophic stimuli such as endothelin-1.
  • hypertrophic stimuli such as endothelin-1.
  • cRaf is required for cardiac hypertrophy (Muslin et al ., Trends Cardiovasc. Med., (2005), 15:225-229).
  • BRaf may be pro- hypertrophic (Rramann et al., Cardiovasc.
  • BRaf inhibitors such as dabrafenib (N- ⁇ 3-[5-(2-amino-4- pyrimidinyl)-2-( 1 , 1 -dimethylethyl)- 1 ,3 -thiazol-4-yl]-2-fluorophenyl ⁇ -2,6- difluorobenzenesulfonamide) are in clinical use (Roring et al., Crit. Rev.
  • dabrafenib Although dabrafenib was developed to target oncogenic BRaf v600E/K , it also inhibits wild-type BRaf and cRaf. As a Type 1.5 inhibitor, dabrafenib binds to and inhibits the "DFG-in" conformation of the kinase, generally viewed as the active conformation. However, dabrafenib stabilises another part of the structure in an "out” (i.e., inactive) conformation. Type 1 and Type 1.5 inhibitors may, paradoxically, activate ERK1/2 at low concentrations. This is potentially because they bind to one partner in a Raf dimer, locking the other in an active conformation that can activate MKK1/2 in the presence of activated Ras (the Raf paradox). At high concentrations, both partners are inhibited.
  • dabrafenib can be used to treat fibrosis, including cardiac fibrosis. Without being limited to theory, it is considered that the efficacy is achieved via inhibition of Raf kinases, for instance BRaf, cRaf and/or ARaf (ARaf, BRaf and cRaf are hereafter referred to collectively as Raf). Efficacy is not limited to patients having mutated forms of the underlying genes and in particular is achieved in patients who do not carry, for instance, any BRaf mutation. It has further been found that the efficacy of dabrafenib may be especially high relative to other potential BRaf inhibitors (e.g.
  • SB590885 a Type 1 inhibitor with greater preference for BRaf.
  • dabrafenib is active in reducing cardiac fibrosis, particularly that associated with hypertension. More generally, it has been found that inhibitors targeting BRaf (a known class of active agents previously studied and applied primarily in oncology), and/or other Raf kinases, may be suitable for the treatment of fibrosis, including but not limited to cardiac fibrosis.
  • the present invention provides a compound for use in treating or preventing fibrosis, for example cardiac fibrosis, kidney fibrosis, liver fibrosis, pulmonary fibrosis, or muscular fibrosis, in a patient, which compound is N- ⁇ 3-[5-(2-amino-4-pyrimidinyl)-2-(1,1- dimethylethyl)-1 ,3-thiazol-4-yl]-2-fluorophenyl ⁇ -2,6-difluoiObenzenesulfonamide, or a pharmaceutically acceptable salt thereof.
  • fibrosis for example cardiac fibrosis, kidney fibrosis, liver fibrosis, pulmonary fibrosis, or muscular fibrosis
  • the compound is for use as described herein in treating or preventing fibrosis by inhibiting BRaf (possibly in conjunction with cRaf and/or ARaf) activity.
  • the compound is for use as described herein in treating or preventing cardiac fibrosis in a patient wherein the patient does not have a BRaf mutation, by inhibiting BRaf activity (or Raf activity), which compound is N- ⁇ 3-[5-(2-amino-4- pyrimidinyl)-2-(1,1-dimethylethyl)-1 ,3-thiazol-4-yl]-2-fluorophenyl ⁇ -2,6- difluorobenzenesulfonamide, or a pharmaceutically acceptable salt thereof.
  • Raf (including BRaf) kinase inhibitors related to dabrafenib such as vemurafenib, encorafenib, lifirafenib, LY3009120, PLX8394, LXH254, MLN2480, Raf709, TAK632 and PLX7904 may also potentially be therapeutically useful for reducing (e.g. cardiac) fibrosis.
  • the present invention also provides a compound for use in treating or preventing fibrosis, for example cardiac fibrosis, kidney fibrosis, liver fibrosis, pulmonary fibrosis, or muscular fibrosis, in a patient, which compound is a Raf inhibitor or a pharmaceutically acceptable salt thereof.
  • the Raf inhibitor may, for example, be vemurafenib, encorafenib, lifirafenib, LY3009120, PLX8394, LXH254, MLN2480, Raf709, TAK632 or PLX7904.
  • the compound is for use as described herein in treating or preventing cardiac fibrosis in a patient wherein the patient does not have a BRaf mutation, wherein the compound is a BRaf (and/or ARaf, and/or cRaf) inhibitor, as described herein, or a pharmaceutically acceptable salt thereof.
  • BRaf v600E knock-in Representation of breeding strategy using the Myh6-MER-Cre- MER (MCM) line for a tamoxifen (Tam) inducible system for cardiomyocyte-specific genetic modification. Mice were crossed with floxed BRaf v600E mice producing double heterozygote BRaf v600E/MCM mice for experiments.
  • B Genotyping for MCM (upper panel) and BRaf v600E (lower panel).
  • MCM Genotyping for MCM (upper panel) and BRaf v600E (lower panel).
  • MCM Genotyping for MCM
  • W 295 bp product
  • transgenic M; ⁇ 300 bp product mice.
  • floxed BRaf v600E transgene for knock-in a single primer pair was used producing different sized products for wild-type (185 bp) and transgenic (308 bp) mice.
  • C
  • FIG. 2 Activated BRaf in cardiomyocytes signals through MKK1/2 to promote changes in gene expression.
  • A Protocol for study of mice with cardiomyocyte-specific knock-in of BRaf v600E by tamoxifen (Tam; 40 mg/kg, i.p.) relative to vehicle (Veh). Mice were heterozygote for both floxed BRaf v600E and Myh6-directed tamoxifen-inducible Cre (BRaf v600E/MCM ).
  • BL baseline.
  • B Immunoblotting of phosphorylated and total MKK1/2 and ERK1/2 in mouse hearts.
  • C Echocardiographic data for mice treated with vehicle (Veh) with/without tamoxifen.
  • LV left ventricular
  • ID internal diameter
  • PW posterior wall
  • s systole
  • d diastole
  • boxplots are for Cre MCM/WT + Veh, BRaf v600E/MCM + Veh, Cre MCM/WT + T am in order from left to right. * p ⁇ 0.05 relative to vehicle alone (unpaired t test). Boxplots are 10-90 percentiles; boxes show the interquartile range with the median marked.
  • FIG. 4 BRaf signalling promotes cardiac hypertrophy.
  • BRaf v600E/MCM or Cre MCM/WT mice were treated with tamoxifen (Tam; 40 mg/kg, i.p.) or vehicle (Veh).
  • Tam 40 mg/kg, i.p.
  • Veh vehicle
  • LV left ventricle; ID, internal diameter; PW, posterior wall;
  • s systole;
  • C Sections of BRaf v600E/MCM mouse hearts (10 d) stained with H&E (left) or Masson’s Trichrome (right).
  • Figure 5 SB590885 induces Raf paradox signalling to ERK1/2 in rat hearts and cardiac cells.
  • A-C Immunoblot analysis of phosphorylated and total ERK1/2 in
  • A Langendorff-perfused adult rat hearts (A) or cultured cells (B and C).
  • A Rat hearts were perfused without or with 1 mM SB590885 (SB59; 1 mM, 15 min); in the graph, left hand data are Control, right hand data are SB590885.
  • B Neonatal rat cardiomyocytes or adult human fibroblasts were exposed to the indicated concentrations of SB590885 (20 min); fibroblast data points on the graph are the lower values at 0.01 and 0.1 mM but the higher values at 1 and 10 mM.
  • C Neonatal rat cardiomyocytes were exposed to 1 mM SB590885 for the times shown.
  • FIG. 6 The Raf paradox-inducer, SB590885, increases nuclear-localised activated ERK1/2 , induces ERK1/2 -dependent gene expression and promotes cardiomyocyte hypertrophy.
  • C Cardiomyocytes were exposed to 1 mM SB590885 (24 h) and immunostained for troponin T.
  • FIG. 7 Images are representative of 6 independent myocyte preparations with analysis of cell size shown in the right panels. **p ⁇ 0.005 vs control (unpaired t test).
  • Figure 7 SB590885 promotes cardiac hypertrophy in vivo.
  • LV left ventricle; ID, internal diameter; PW, posterior wall; (s), systole; (d), diastole; in each graph, left hand data are Vehicle, right hand data are SB590885.
  • Figure 8 Generation of mice with tamoxifen-inducible cardiomyocyte-specific BRaf knockout.
  • A Representation of breeding strategy using the Myh6-Mer-Cre-Mer (MCM) line for tamoxifen-inducible cardiomyocyte-specific BRaf knock-out. Mice homozygous for the floxed BRaf gene and heterozygous for MCM were generated. BRaf KO/KO /Cre MCM/WT mice were treated with 40 mg/kg (i.p.) Tam or vehicle 3 d before minipumps were implanted for delivery of 0.8 mg/kg/d Angll or vehicle.
  • MCM Myh6-Mer-Cre-Mer
  • FIG. 9 Cardiomyocyte-specific BRaf knock-out inhibits cardiac adaptation to angiotensin II in mouse hearts in vivo.
  • A Protocol for study of mice with inducible cardiomyocyte-specific knock-out of BRaf. Mice were homozygote for BRaf K and heterozygote for Myh6-directed tamoxifen- (Tam-) inducible Cre ( BRaf KO/KO /Cre MCM/WT ). Mice were treated with 40 mg/kg (i.p.) Tam or vehicle 3 d before minipumps were implanted for delivery of 0.8 mg/kg/d Angll or vehicle.
  • RNA (B) or protein (C) was extracted from mouse hearts after treatment with Tam or vehicle (Control, Con).
  • Raf isoform mRNA expression was measured by qPCR (B).
  • Samples were immunoblotted for ARaf, BRaf and cRaf (together) or Gapdh (C).
  • B and right panel of C for each of ARaf/BRaf/cRaf, left hand data are Control, right hand data are Tamoxifen.
  • left panels show representative blots (positions of relative molecular mass markers are on the right); the right panel provides quantification of Raf isoforms relative to Gapdh. * p ⁇ 0.05 vs vehicle (unpaired t test).
  • D - F Echocardiography of hearts showing representative images of individual mice before and after (7 d) treatment (D) with enlargement of the posterior wall (E), and quantification (F).
  • LV left ventricle; AW, anterior wall; PW, posterior wall; IVS, interventricular septum; ID, internal diameter; in each graph of F, left hand data are Control, central data are Angll, right hand data are Tam + Angll. Results are means ⁇ SEM with individual values shown.
  • FIG. 10 Cardiomyocyte-specific BRaf knock-out reduces cardiac hypertrophy induced by angiotensin II in mouse hearts in vivo : systolic measurements.
  • BRaf KO/KO /Cre MCM/WT mice were treated with 40 mg/kg (i.p.) Tam or vehicle 3 d before minipumps were implanted for delivery of 0.8 mg/kg/d Angll or vehicle.
  • Echocardiography of hearts showing systolic measurements.
  • LV left ventricle
  • ID internal diameter
  • PW posterior wall
  • AW anterior wall, IVS interventricular septum
  • left hand data are Control
  • central data are Angll
  • right hand data are Tamoxifen + Angll.
  • Results are means ⁇ SEM with individual values shown. *p ⁇ 0.05 , **p ⁇ 0.005, ****p ⁇ 0.0001 vs Control, # p ⁇ 0.05 vs Angll (one-way ANOVA with Holm-Sidak’s post-test).
  • BRaf KO/KO /Cre MCM/WT mice were treated with 40 mg/kg (i.p.) Tam or vehicle, 3 d before minipumps were implanted for delivery acidified PBS (to control for Angll delivery). Cardiac function/dimensions were measured by echocardiography. A, Representative images. B, Quantification of echocardiograms. LV, left ventricle; ID, internal diameter; PW, posterior wall, AW anterior wall, IVS, interventricular septum; for each graph, left hand data are Control, right hand data are Tamoxifen. Results are means ⁇ SEM with individual values shown. There were no significant differences between vehicle only and tamoxifen treated mice (t test).
  • FIG. 12 Tamoxifen-treatment of Cre MCM/WT mice (i.e. heterozygous for Myh6- MerCreMer only) had no significant effect on cardiac function/dimensions.
  • Mice wild- type for BRaf and heterozygous for Myh6-MerCreMer were treated with 40 mg/kg (i.p.) Tam or vehicle, 3 d before minipumps were implanted for delivery acidified PBS (to control for Angll delivery).
  • Cardiac function/dimensions were measured by echocardiography. Data show quantification of echocardiograms.
  • LV left ventricle
  • ID internal diameter
  • PW posterior wall
  • IVS interventricular septum
  • results are means ⁇ SEM with individual values shown. There were no significant differences between vehicle only and tamoxifen treated mice (t test).
  • FIG 13 Cardio myocyte-specific BRaf knock-out modulates changes in gene expression induced by angiotensin II in mouse hearts in vivo.
  • BRaf KO/KO /Cre MCM/WT mice were treated with 40 mg/kg (i.p.) Tam or vehicle 3 d before minipumps were implanted for delivery of 0.8 mg/kg/d Angll or vehicle.
  • RNA was extracted from mouse hearts following treatment without vehicle only (Control), with 0.8 mg/kg/d Angll, or with tamoxifen plus Angll (Tam/Angll).
  • mRNA was measured by qPCR for expression of hypertrophy- associated (A), early (B), cytokine (C), fibrosis-associated (D) or extracellular matrix (E) genes; at each time interval for each gene, left hand data are Control, central data are Angll, right hand data are Tam/Angll. Results are means ⁇ SEM with individual values shown. * p ⁇ 0.05, **p ⁇ 0.005, ***p ⁇ 0.0005, ****p ⁇ 0.0001 vs Control, # p ⁇ 0.05, ## p ⁇ 0.005, ####p ⁇ 0.0001 vs Angll (one-way ANOVA with Holm-Sidak's post-test).
  • BRaf KO/KO /Cre MCM/WT mice were treated with 40 mg/kg (i.p.) Tam or vehicle, 3 d before minipumps were implanted for delivery acidified PBS (to control for Angll delivery).
  • mRNA expression was determined by qPCR for hypertrophy-associated genes (A), early genes (B), cytokines (C), fibrosis-associated genes (D) and extracellular matrix genes (E); at each time interval for each gene, left hand data are Control, right hand data are Tamoxifen. Date were normalised to the housekeeping gene Gapdh and then to means of the vehicle-treated controls. Results are means ⁇ SEM with individual values shown. There were no significant differences between vehicle and tamoxifen-treated mice (one-way ANOVA).
  • FIG. 15 Cardiomyocyte-specific BRaf knock-out increases focal damage and cardiac fibrosis in mouse hearts in vivo.
  • BRaf KO/KO /Cre MCM/WT mice were treated with 40 mg/kg (i.p.) Tam or vehicle, 3 d before minipumps were implanted for delivery of 0.8 mg/kg/d Angll or vehicle.
  • Hearts were fixed in formaldehyde and sections stained with haemotoxylin and eosin (H&E, upper panels) or Masson's tri chrome (lower panels). Images for each condition were taken from different areas from the same heart section and are representative of the average response.
  • Figure 16 Dabrafenib inhibits ERK1/2 signalling in in perfused adult rat hearts.
  • C57B1/6J male mice were treated with vehicle or 3 mg/kg/d dabrafenib for 3 or 7 d. Cardiac function/dimensions were measured by echocardiography. Data show quantification of echocardiograms. LV, left ventricle; ID, internal diameter; PW, posterior wall, AW anterior wall, I VS, interventricular septum; in each graph, left hand data are Control, right hand data are Dabrafenib. Results are means ⁇ SEM with individual values shown. There were no significant differences between vehicle only and dabrafenib-treated mice (t test).
  • FIG. 18 Dabrafenib reduces cardiac hypertrophy induced by angiotensin II in mouse hearts in vivo. Echocardiography of hearts from C57BL/6J male mice treated without dabrafenib or Angll (Control), with 0.8 mg/kg/d Angll or with 3 mg/kg/d dabrafenib with Angll. Representative images from individual mice are in (A) with enlargement of the posterior wall in (B). C and D, Quantification of echocardiograms taken at 3 (C) or 7 (D) days.
  • LV left ventricle
  • ID internal diameter
  • PW posterior wall
  • IVS interventricular septum
  • left hand data are Control
  • central data are Angll
  • right hand data are Dabrafenib + Angll.
  • N.B. Diastolic dimensions are shown; systolic dimensions are provided in Figure 19).
  • Results are means ⁇ SEM with individual values shown. *p ⁇ 0.05, **p ⁇ 0.005, ***p ⁇ 0.0005, ****p ⁇ 0.0001 vs control, # p ⁇ 0.05, ## p ⁇ 0.005, ### p ⁇ 0.0005 vs Angll (one-way ANOVA with Holm-Sidak post-test).
  • FIG. 19 Dabrafenib reduces cardiac hypertrophy induced by angiotensin II in mouse hearts in vivo : systolic measurements.
  • C57B1/6J male mice were treated with vehicle or 3 mg/kg/d dabrafenib) for 3 or 7 d.
  • Cardiac function/dimensions were measured by echocardiography. Echocardiography of hearts showing systolic measurements.
  • LV left ventricle
  • ID internal diameter
  • PW posterior wall, IVS, interventricular septum
  • left hand data are Control
  • central data are Angll
  • right hand data are Dabrafenib + Angll.
  • Results are means ⁇ SEM with individual values shown. *p ⁇ 0.05 , **p ⁇ 0.005, **** p0.0001 vs Control, # p ⁇ 0.05 v Angll (one-way ANOVA with Holm-Sidak's post-test).
  • Dabrafenib modulates changes in gene expression induced by angiotensin II in mouse hearts in vivo.
  • C57BL/6J male mice were treated without dabrafenib or Angll (Control), with 0.8 mg/kg/d Angll or with 3 mg/kg/d dabrafenib with Angll.
  • Proteins or RNA were extracted from mouse hearts after 7 d.
  • A Proteins were immunoblotted for phospho- or total ERK1/2. Representative images are shown with densitometirc
  • quantification quantification; quantification data are Control, Dabrafenib, Angll, Dabrafenib + Angll in order from left to right.
  • B - D mRNA expression of hypertrophy-associated (B), Cytokines (C), fibrosis-promoting and extracellular matrix (D) genes was measured by qPCR; in each graph, left hand data are Control, central data are Angll, right hand data are Dabrafenib + Angll. Results are means ⁇ SEM with individual values shown. *p ⁇ 0.05, **p ⁇ 0.005, ***p ⁇ 0.0005, ****p ⁇ 0.0001 vs control, # p ⁇ 0.05, ## p ⁇ 0.005 vs Angll (one-way ANOVA with Holm-Sidak post-test).
  • Dabrafenib reduces cardiomyocyte diameter and inhibits cardiac fibrosis induced by angiotensin II in mouse hearts in vivo.
  • C57BL/6J male mice were treated without dabrafenib or Angll (Control), with 0.8 mg/kg/d Angll or with 3 mg/kg/d dabrafenib with Angll.
  • a - D Hearts were fixed in formaldehyde and sections stained with haemotoxylin and eosin (H&E) or Masson's trichrome.
  • Figure 22 Effects of SB590885 on Angll-induced hypertension. Echocardiography of hearts from C57BL/6J male mice treated without SB590885 or Angll (Control), with 0.8 mg/kg/d Angll or with 0.5 mg/kg/d SB590885 with Angll. Quantification of
  • Figure 23 Raf inhibitors inhibit ERK1/2 activation in cultured cardiac myocytes. A-C.
  • Neonatal rat cardiomyocytes were exposed (20 min) to the indicated concentrations of dabrafenib (A), vemurafenib (B) or PLX7904 (C).
  • D Cardiomyocytes were unstimulated or exposed to 10 mM dabrafenib, 30 mM vemurafenib or 10 mM PLX7904 in the absence or presence of 100 nM endothelin 1 (ET-1).
  • E Cardiomyocytes were unstimulated or exposed to 10 mM dabrafenib or 10 mM encorafenib in the absence or presence of 50 nM A61603. Protein samples were immunoblotted for phosphorylated (phospho-) or total ERK1/2.
  • Dabrafenib N- ⁇ 3-[5-(2-amino-4-pyrimidmyl)-2-(1,1-dimethylethyl)-1,3-thiazol-4-yl]-2-fluorophenyl ⁇ - 2,6-difluorobenzenesulfonamide may be referred to as dabrafenib.
  • Dabrafenib has the following structure:
  • the compound used in the treatment of the invention is dabrafenib or a pharmaceutically acceptable salt thereof.
  • a pharmaceutically acceptable salt is a salt with a pharmaceutically acceptable acid or base.
  • Pharmaceutically acceptable acids include both inorganic acids such as hydrochloric, sulphuric, phosphoric, diphosphoric, hydrobromic or nitric acid and organic acids such as citric, fumaric, maleic, malic, ascorbic, succinic, tartaric, benzoic, acetic, methanesulphonic, ethanesulphonic, benzenesulphonic or p-toluenesulphonic acid.
  • Pharmaceutically acceptable bases include alkali metal (e.g. sodium or potassium) and alkali earth metal (e.g. calcium or magnesium) hydroxides and organic bases such as alkyl amines, aralkyl amines and heterocyclic amines.
  • dabrafenib can, if desired, be used in the form of a solvate. Further, for the avoidance of doubt, dabrafenib may be used in any tautomeric form.
  • the compound used in the treatment of the invention may be a BRaf inhibitor, or a pharmaceutically acceptable salt thereof (including, but not limited to, dabrafenib and its pharmaceutically acceptable salts).
  • a BRaf inhibitor is a compound which inhibits BRaf activity. Inhibiting BRaf activity includes reducing BRaf or modulating BRaf.
  • Small molecule protein kinase inhibitors are classified are classified according to mode of binding to the kinase, assessed by structural analysis of the inhibitor in complex with the relevant kinase, usually using crystallography or related technique.
  • Most protein kinase inhibitors interact with the ATP-binding site containing a DFG motif, the position/orientation of which is crucial.
  • the DFG motif moves inwards ("DFG-in") for the kinase to adopt an active conformation.
  • Type 1 inhibitors bind to the "DFG-in” structure whilst Type 2 inhibitors bind to a "DFG-out” conformation.
  • An active kinase requires inward movement of the aC-helix.
  • Type 1.5 inhibitors bind to the "DFG-in” conformation, but stabilise the enzyme with the helix in an inactive“out” conformation.
  • the BRaf inhibitor for use in the present invention can be a Type 1 BRaf inhibitor.
  • the BRaf inhibitor for use in the present invention can be a Type 1.5 BRaf inhibitor.
  • the BRaf inhibitor for use in the present invention can be a Type 2 BRaf inhibitor.
  • the BRaf inhibitor used in the treatment of the invention is typically selected from dabrafenib, vemurafenib, encorafenib, lifirafenib, LY3009120, PLX8394, LXH254, or MLN2480.
  • the BRaf inhibitor used in the treatment of the invention is dabrafenib. Pharmaceutically acceptable salts of all of these compounds can be used.
  • the BRaf inhibitor can, if desired, be used in the form of a solvate. Further, for the avoidance of doubt, the BRaf inhibitor may be used in any tautomeric form.
  • the compound used in the treatment of the invention may be a Raf inhibitor, or a pharmaceutically acceptable salt thereof (including, but not limited to, dabrafenib and its pharmaceutically acceptable salts).
  • a Raf inhibitor is a compound which inhibits Raf activity, namely BRaf activity, and/or cRaf activity, and/or ARaf, activity. Inhibiting Raf activity includes reducing Raf or modulating Raf.
  • the Raf inhibitor may inhibit any or all of BRaf, cRaf and ARaf, and may derive its pharmacological efficacy in treating fibrosis via its capacity to inhibit any or all of BRaf, cRaf and ARaf.
  • Table 1 provides IC 50 values for a number of Raf inhibitors showing the level of inhibition of mutated BRaf V600E compared to wild-type BRaf or cRaf.
  • Table 1 IC 50 values for inhibition of mutated BRaf V600E vs wild-type (WT) BRaf or cRaf using cell-free assays.
  • the Raf inhibitor used in the treatment of the invention is typically selected from dabrafenib, vemurafenib, encorafenib, or PLX8394.
  • the Raf inhibitor used in the treatment of the invention is dabrafenib. Pharmaceutically acceptable salts of all of these compounds can be used.
  • the Raf inhibitor can, if desired, be used in the form of a solvate. Further, for the avoidance of doubt, the Raf inhibitor may be used in any tautomeric form.
  • Dabrafenib can inhibit BRaf activity, in addition to cRaf and ARaf activity. Dabrafenib can therefore treat conditions by inhibiting BRaf (alone or in conjunction with cRaf and/or ARaf) activity, in particular fibrosis.
  • fibrosis This includes cardiac fibrosis, kidney fibrosis, liver fibrosis, pulmonary fibrosis, or muscular fibrosis.
  • the fibrosis is cardiac fibrosis.
  • the invention provides dabrafenib for use in the treatment or prevention of fibrosis.
  • the invention provides dabrafenib for use in the treatment or prevention of fibrosis by inhibiting BRaf activity. More preferably, the invention provides dabrafenib for use in the treatment or prevention of cardiac fibrosis by inhibiting BRaf activity.
  • Raf inhibitors can also treat conditions by inhibiting BRaf (and/or Raf) activity, in particular fibrosis.
  • fibrosis This includes cardiac fibrosis, kidney fibrosis, liver fibrosis, pulmonary fibrosis, or muscular fibrosis.
  • the fibrosis is cardiac fibrosis.
  • the invention provides a Raf inhibitor for use in the treatment or prevention of fibrosis.
  • the invention provides a Raf inhibitor for use in the treatment or prevention of cardiac fibrosis.
  • Dabrafenib and related BRaf and/or Raf inhibitors can inhibit activity of wild-type BRaf or mutant BRaf.
  • the patient treated in the treatment of the invention does not have a BRaf mutation.
  • the patient who does not have a BRaf mutation has only wild- type BRaf.
  • the patient may be suffering from hypertension, heart failure, cardiac hypertrophy, non alcoholic steatohepatitis, or muscular dystrophy.
  • the patient may be suffering from hypertension, heart failure, or cardiac hypertrophy. More preferably, the patient may be suffering from hypertension.
  • the compounds for use of the invention may be present in a pharmaceutical composition.
  • the pharmaceutical composition may comprise dabrafenib or a BRaf or Raf inhibitor as described herein and one or more pharmaceutically acceptable carriers, diluents or excipients.
  • the pharmaceutical composition may be as described below.
  • compositions may be administered to the subject by any acceptable route of administration including, but not limited to, inhaled, oral, nasal, topical (including transdermal) and parenteral modes of administration. Oral administration is preferred.
  • compositions of the invention may be administered in multiple doses per day, in a single daily dose or a single weekly dose. It will be understood that any form of the active agents used in the composition of the invention that is suitable for the particular mode of administration can be used in the pharmaceutical compositions discussed herein.
  • compositions of this invention typically contain a therapeutically effective amount of an active agent.
  • a pharmaceutical composition may contain more than a therapeutically effective amount, i.e., bulk compositions, or less than a therapeutically effective amount, i.e., individual unit doses designed for multiple administration to achieve a therapeutically effective amount.
  • any conventional carrier or excipient may be used in the pharmaceutical compositions of the invention.
  • the choice of a particular carrier or excipient, or combinations of carriers or excipients, will depend on the mode of administration being used to treat a particular subject or type of medical condition or disease state. In this regard, the preparation of a suitable composition for a particular mode of administration is well within the scope of those skilled in the pharmaceutical arts. Additionally, carriers or excipients used in such compositions are commercially available.
  • conventional formulation techniques are described in Remington: The Science and Practice of Pharmacy, 20 th Edition, Lippincott Williams & White, Baltimore, Md. (2000); and H. C. Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7 th Edition, Lippincott Williams & White, Baltimore, Md. (1999).
  • materials which can serve as pharmaceutically acceptable carriers include, but are not limited to, the following: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, such as microcrystalline cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid;
  • pyrogen-free water isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; compressed propellant gases, such as chlorofluorocarbons and hydrofluorocarbons; and other non-toxic compatible substances employed in pharmaceutical compositions.
  • compositions are typically prepared by thoroughly and intimately mixing or blending the active agent / active ingredient with a pharmaceutically acceptable carrier and one or more optional ingredients.
  • the resulting uniformly blended mixture may then be shaped or loaded into tablets, capsules, pills, canisters, cartridges, dispensers and the like using conventional procedures and equipment.
  • compositions may be suitable for oral administration.
  • suitable compositions for oral administration may be in the form of capsules, tablets, pills, lozenges, cachets, dragees, powders, granules; solutions or suspensions in an aqueous or non-aqueous liquid; oil-in-water or water-in-oil liquid emulsions; elixirs or syrups; and the like; each containing a predetermined amount of the active agent.
  • the composition When intended for oral administration in a solid dosage form (i.e., as capsules, tablets, pills and the like), the composition will typically comprise the active agent and one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate.
  • Solid dosage forms may also comprise: fillers or extenders, such as starches, microcrystalline cellulose, lactose, sucrose, glucose, mannitol, and/or silicic acid; binders, such as
  • disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and/or
  • Release agents, wetting agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants may also be present in the pharmaceutical compositions.
  • Exemplary coating agents for tablets, capsules, pills and like include those used for enteric coatings, such as cellulose acetate phthalate, polyvinyl acetate phthalate, hydroxypropyl methylcellulose phthalate, methacrylic acid-methacrylic acid ester copolymers, cellulose acetate trimellitate, carboxymethyl ethyl cellulose, hydroxypropyl methyl cellulose acetate succinate, and the like.
  • antioxidants examples include: water- soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfate sodium sulfite and the like; oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, lecithin, propyl gallate, alpha- tocopherol, and the like; and metal-chelating agents, such as citric acid, ethylenediamine tetraacetic acid, sorbitol, tartaric acid, phosphoric acid, and the like.
  • water- soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfate sodium sulfite and the like
  • oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, lecithin, propyl gallate, alpha- tocopherol,
  • compositions may also be formulated to provide slow or controlled release of the active agent using, by way of example, hydroxypropyl methyl cellulose in varying proportions or other polymer matrices, liposomes and/or microspheres.
  • the pharmaceutical compositions of the invention may contain opacifying agents and may be formulated so that they release the active agent only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes.
  • the active agent can also be in micro-encapsulated form, if appropriate, with one or more of the above-described excipients.
  • Suitable liquid dosage forms for oral administration include, by way of illustration, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs.
  • Liquid dosage forms typically comprise the active agent and an inert diluent, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (e.g., cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
  • inert diluent such as, for example, water or other solvents, solubilizing agents and emulsifier
  • Suspensions may contain suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminium metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
  • suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminium metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
  • the pharmaceutical compositions of the invention may be packaged in a unit dosage form.
  • unit dosage form refers to a physically discrete unit suitable for dosing a subject, i.e., each unit containing a predetermined quantity of the active agents calculated to produce the desired therapeutic effect either alone or in combination with one or more additional units.
  • unit dosage forms may be capsules, tablets, pills, and the like.
  • compositions of the invention can also be administered parenterally (e.g., by subcutaneous, intravenous, intramuscular, or intraperitoneal injection).
  • the active agents are provided in a sterile solution, suspension, or emulsion.
  • Exemplary solvents for preparing such formulations include water, saline, low molecular weight alcohols such as propylene glycol, polyethylene glycol, oils, gelatin, fatty acid esters such as ethyl oleate, and the like.
  • a typical parenteral formulation is a sterile pH 4-7 aqueous solution of the active agents.
  • Parenteral formulations may also contain one or more solubilizers, stabilizers, preservatives, wetting agents, emulsifiers, and dispersing agents. These formulations may be rendered sterile by use of a sterile injectable medium, a sterilizing agent, filtration, irradiation, or heat.
  • compositions of the invention can also be administered transdermally using known transdermal delivery systems and excipients.
  • the active agents can be admixed with permeation enhancers, such as propylene glycol, polyethylene glycol monolaurate, azacycloalkan-2-ones and the like, and incorporated into a patch or similar delivery system.
  • permeation enhancers such as propylene glycol, polyethylene glycol monolaurate, azacycloalkan-2-ones and the like
  • Additional excipients including gelling agents, emulsifiers and buffers, may be used in such transdermal compositions if desired.
  • compositions of the invention may be suitable for inhaled administration, which will typically be in the form of an aerosol or a powder, for instance a dry powder composition.
  • Such compositions are generally administered using well-known delivery devices, such as a nebulizer inhaler, a dry powder inhaler, or a metered-dose inhaler.
  • a therapeutically effective amount of a compound of the invention is administered to a patient.
  • dabrafenib or a Raf inhibitor as described herein or the pharmaceutically acceptable salt thereof may be provided in any suitable dosage.
  • a typical dose is from about 0.001 to 50 mg per kg of body weight, for example 0.01 to 10 mg, according to the activity of the specific compound, the age, weight and conditions of the subject to be treated, the type and severity of the disease and the frequency and route of administration.
  • the amount of dabrafenib or Raf inhibitor present in a single dose may be from 1 mg to 50 mg, from 1 mg to 10 mg, from 1 mg to 1 mg, from 1 mg to 500 mg, from 1 mg to 100 mg or from 1 mg to 50 mg (i.e. 0.001 mg to 0.050 mg).
  • the amount of dabrafenib or BRaf inhibitor present in a single dose may be from 1 mg to 50 mg, from 10 mg to 50 mg, from 100 mg to 50 mg or from 250 mg to 50 mg.
  • the dose of dabrafenib or Raf inhibitor may be from 1 mg/kg to 500 mg/kg, or from 50 mg/kg to 250 mg/kg (kg as weight of patient).
  • a dose may be administered as often as required.
  • dabrafenib is administered orally at a dose of 150 mg, twice daily (i.e. ⁇ 3-5 mg/kg/d).
  • Example 1 - BRaf signalling promotes cardiomvocyte hypertrophy
  • Raf kinases lie upstream of the extracellular signal-regulated kinases 1/2 (ERK1/2).
  • ERK1/2 extracellular signal-regulated kinases 1/2
  • ERK1/2 promote cardiomyocyte hypertrophy and cytoprotection, but the role of BRaf in the adult heart has until now been unclear.
  • BRaf mutations cause cancer and Raf inhibitors are used clinically but some activate ERK1/2 via the Raf paradox, and the cardiac consequences have not been established.
  • the aim of this study was to determine if activation of BRaf®ERK1/2 signalling promotes cardiac hypertrophy.
  • a mouse model was established for cardiomyocyte-specific tamoxifen-inducible heterozygous knock-in of the activating BRaf v600E mutation into the endogenous gene. Tamoxifen-treatment increased ERK1/2 cascade signalling with increased hypertrophic gene expression from 24 h. Echocardiography detected increases in ejection fraction and fractional shortening from 7 d, with significantly increased left ventricular posterior wall (LVPW) thickness by 10 d. This was associated with increased cardiomyocyte cross-sectional area without detectable fibrosis.
  • the Type-1 Raf inhibitor, SB590885 activated ERK1/2 signalling in rat perfused hearts and isolated cardiomyocytes via the Raf paradox, promoting cardiomyocyte hypertrophy.
  • SB590885 (0.5 mg/kg/d, 3 d) increased LVPW thickness and, as with BRaf v600E knock-in, this was associated with increased cardiomyocyte cross-sectional area without increased fibrosis, probably because of a selective effect on cardiomyocytes rather than fibroblasts.
  • ERK1/2 are cardioprotective in the heart, promoting cardiomyocyte hypertrophy.
  • This study demonstrates a direct and significant role for BRaf in promoting cardiomyocyte hypertrophy.
  • the Type-1 Raf inhibitor SB590885 activates ERK1/2 in cardiomyocytes via a Raf paradox signalling mechanism to induce cardiomyocyte hypertrophy.
  • This new paradigm for ERK.1/2 signalling in the heart raises the possibility that Raf paradox-inducers may provide short-term benefit for heart failure patients.
  • ERK1/2 extracellular signal-regulated kinase 1/2 pathway increases cell proliferation and is a target for cancer, but is also cardioprotective and promotes hypertrophy (i.e. growth in the absence of proliferation) in contractile
  • BRaf is one of three Raf kinases (the others being ARaf and cRaf) that phosphorylate and activate downstream kinases (MKK1/2), which phosphorylate and activate ERK1/2.
  • MKK1/2 downstream kinases
  • Oncogenic BRaf mutations [particularly of BRaf(Val600) commonly mutated to BRaf v600E ] are associated with ⁇ 15% of all cancers with high rates in melanoma (40-60%).
  • Raf inhibitors are already used clinically. These were intended to have specificity for BRaf v600E , but IC 50 values for BRaf v600E vs wild-type BRaf or cRaf indicate modest selectivity at best.
  • Use of these“first generation” Raf inhibitors is limited because cancer cells become resistant to the drugs and because of paradoxical activation of ERK1/2 signalling in cells with activated Ras.
  • mammalian cardiomyocytes are terminally-differentiated from birth.
  • adult cardiomyocytes hypertrophy, increasing in size and myofibrillar content. This leads to compensated cardiac hypertrophy, but prolonged stress may cause decompensation and heart failure as
  • BRaf signalling promotes cardiomyocyte hypertrophy using a mouse model with cardiomyocyte-specific knock-in of an activating mutation (V600E) into the endogenous BRaf gene. It was further demonstrated that paradoxical activation of ERK1/2 in cardiomyocytes by the Type-1 Raf inhibitor, SB590885, promotes hypertrophy.
  • mice Genetically-modified mice were from Jackson Laboratories. Mice with a floxed cassette for Cre-induced knock-in of the BRaf v600E mutation (B6.129P2(Cg)- Braf tm1Mmcm /J, strain no. 017837) were maintained on a C57B1/6J background. Myh6- MERCreMER mice expressing tamoxifen-activated Cre under control of the mouse Myh6 promoter (Tg(Myh6-cre) 1 Jmk/J, strain no. 009074) were backcrossed against the C57B1/6J background for at least 4 generations. BRaf v600E/WT /Cre MCM/WT double heterozygous mice and single heterozygous Cre MCM/WT littermates were generated for experimentation.
  • tamoxifen 40 mg/kg i.p.; Sigma-Aldrich
  • vehicle only controls corn oil; Sigma-Aldrich
  • Alzet osmotic pumps were used to deliver SB590885 (0.5 mg/kg/d; Selleck Chemicals) dissolved in DMSO/PEG [50% (v/v) DMSO, 20% (v/v) polyethylene glycol 400, 5% (v/v) propylene glycol, 0.5% (v/v) Tween 80] or DMSO/PEG alone to male C57B1/6J mice (8 wks). Mice were culled by CO 2 inhalation with cervical dislocation.
  • Hearts were snap-frozen in liquid N 2 or fixed in 10% formalin for histology. Echocardiography was performed on anaesthetised mice (maintained with 1.5% isoflurane delivered via a nose cone) using a Vevo 2100 system and MS400 18-38 MHz transducer (Visualsonics). Left ventricular (LV) cardiac function and dimensions was assessed from short axis M-mode images with the axis at the mid-level of the LV at the level of the papillary muscles. Data analysis (Vevo LAB version 1.7.1) was performed by independent assessors blinded to intervention. Please see Further Methodology section for full details of methodology plus information on genotyping, confirmation of recombination, minipump preparation and implantation, sample preparation and histology.
  • rat heart perfusions and cell cultures Hearts from adult male Sprague-Dawley rats were perfused retrogradely (37°C, 70 mmHg) with 15 min equilibration. SB590885 in DMSO or DMSO alone was added at the end of the equilibration period and perfusions continued for a further 15 min.
  • Neonatal rat ventricular myocytes (NRVMs) were cultured from 2-4 d Sprague-Dawley rats.
  • cardiomyocytes were plated at 4x 10 6 cells per 60 mm dish. After 18 h myocytes were beating spontaneously.
  • cardiomyocytes were plated at 1 x 10 6 cells per 35 mm dish containing glass coverslips. The plating medium was withdrawn after 18 h and cells incubated in serum-free maintenance medium (24 h). Human cardiac fibroblasts were grown in fibroblast growth medium-3 (PromoCell). Cells were seeded the day before experimentation at a density to achieve 90% confluence after 24 h and synchronised overnight in Ml 99 medium containing 0.1% (v/v) FCS and 100 units/ml penicillin and streptomycin. See Further Methodology > section for full details of perfusions, cell cultures and sample preparation.
  • RNA preparation qPCR, Raf assays, immunostaining and immunoblotting: See
  • mice were bred with Myh6-MERCreMER mice expressing nuclear-localised tamoxifen-activated Cre recombinase under control of the mouse Myh6 promoter (T g(Myh6-cre) 1 Jmk/J, strain no. 009074) backcrossed against the C57B1/6J background for at least 4 generations.
  • Genotyping - DNA was extracted from ear notches using Purelink genomic DNA (gDNA) mini-kits (Invitrogen) according to the manufacturer's instructions. Briefly, tissue was digested in genomic digestion buffer containing proteinase K (2-4 h, 55°C). Following centrifugation (12,000 x g, 3 min, 18°C), supernatants were incubated with RNAse A (2 min) before addition of genomic lysis binding buffer mixed with an equal volume of ethanol. gDNA was purified using Purelink spin columns and PCR amplified (up to 33 cycles) with specific primers (see Table 2 for primer sequences and annealing temperatures) and using GoTaq Hot Start Polymerase (Promega).
  • PCR conditions were 95 °C for 3 min, followed by 33 cycles of 95°C denaturation for 30 s, 30 s annealing, elongation at 72°C for 30 s, followed by a 7 min 72°C final extension.
  • PCR products were run on 2% (w/v) agarose gels (45 min, 80V) and visualised under UV light.
  • BRaf v600E 50% of the resulting product was digested with Xbal (New England Biolabs) (3 h, 37°C). Digested and undigested products were separated by electrophoresis on a 2% (w/v) agarose gel (85 V, 45 min). BRaf v600E knock-in introduces a novel Xbal site in the PCR product in the cDNA producing an additional product.
  • Xbal New England Biolabs
  • Drug delivery - Drug delivery used Alzet osmotic pumps (models 1007D or 1002; supplied by Charles River), filled according to the manufacturer’s instructions in a laminar flow hood using sterile technique.
  • Mice were treated with SB590885 (0.5 mg/kg/d) dissolved in DMSO/PEG mix [50% (v/v) DMSO, 20% (v/v) polyethylene glycol 400, 5% (v/v) propylene glycol, 0.5% (v/v) Tween 80] or DMSO/PEG alone.
  • SB590885 was from SelleckChem and vehicle components were from Sigma-Aldrich. Minipumps were incubated overnight in sterile PBS (37°C) prior to implantation.
  • Implantation was performed under continuous inhalation anaesthesia using isoflurane (induction at 5%, maintenance at 2-2.5%) mixed with 2 l/min O 2 .
  • a 1 cm incision was made in the mid-scapular region and mice were given 0.05 mg/kg (s.c.) buprenorphine (Vetergesic, Ceva Animal Health Ltd.) to repress any post- surgical discomfort.
  • Minipumps were implanted portal first in a pocket created in the left flank region of the mouse. Wound closure used a simple interrupted suture with
  • Cardiac ultrasound - Echocardiography was performed on anaesthetised mice using a Vevo 2100 imaging system equipped with a MS400 18-38 MHz transducer (Visualsonics). Mice were anaesthetised in an induction chamber with isoflurane (5% flow rate) with 1 l/min O 2 then transferred to the heated Vevo Imaging Station. Anaesthesia was maintained with 1.5% isoflurane delivered via a nose cone. Left ventricular cardiac function and structure was assessed from short axis M-mode images with the axis placed at the mid-level of the left ventricle at the level of the papillary muscles. Baseline scans were taken prior to
  • Tissue harvesting and processing - Mice were culled by schedule 1 (CO 2 followed by cervical dislocation). Hearts were excised quickly, washed in PBS, dried and snap-frozen in liquid N 2 or fixed for histology.
  • Histology and assessment of myocyte size and fibrosis Histological analysis was performed on hearts subjected to in situ perfusion fixation with 10% formalin. Hearts were immersed in 70% (v/v) ethanol, embedded in paraffin and sectioned at 10 mm. Sections were de-waxed using xylene and re-hydrated through sequential washes in decreasing ethanol gradients (100%, 100%, 90%, 75%, 50%) to distilled water. Sections were stained using kits for hematoxylin and eosin (H&E, Sigma) or Masson's trichrome (Polysciences).
  • Rat heart perfusions Adult male (300-350 g) Sprague-Dawley rats (Charles River) were anaesthetised with a lethal intraperitoneal dose of Euthatal (pentobarbital sodium, 60 mg/kg). After complete anaesthesia was induced, a saphenous vein was exposed and heparin (1000 U/kg) administered intravenously.
  • Euthatal pentobarbital sodium, 60 mg/kg
  • the chest cavity was opened and the heart and lungs were removed into modified ice-cold KHBBS (25 mM NaHCO 3 , 119 mM NaCl, 35 mM KC1, 2.5 mM CaC1 2 , 1.2 mM MgSO 4 , 1.2mM KH2PO4 equilibrated with 95% 02/5% CO 2 ) whilst the heart was still beating.
  • KHBBS modified ice-cold KHBBS
  • Neonatal rat ventricular myocytes were prepared and cultured from 2-4 d Sprague-Dawley rats (Charles River) essentially as previously described. Ventricles were dissected and dissociated by serial digestion at 37°C with 0.44 mg/ml (6800 U) Worthington Type II collagenase (supplied by Lonza) and 0.6 mg/ml pancreatin (Sigma- Aldrich, cat. No. P3292) in sterile digestion buffer (1 16 mM NaCl, 20 mM HEPES, 0.8 mM Na 2 HPO 4 , 5.6 mM glucose, 5.4 mM KC1 and 0.8 mM MgSO 4 , pH 7.35).
  • the first digestion supernatant (5 min, 37°C, 160 cycles/min in a shaking waterbath) was discarded.
  • Cell suspensions from subsequent digestions (4x25 min, 37°C 136 cycles/min shaking) were recovered by centrifugation (5 min, 60 xg) and the cell pellet resuspended in plating medium (Dulbecco's modified Eagle's medium (DMEM)/medium 199 [4:1 (v/v)]) containing 15% (v/v) foetal calf serum (FCS; Life Technologies) and 100 units/ml penicillin and
  • DMEM Dulbecco's modified Eagle's medium
  • FCS foetal calf serum
  • cardiomyocytes were plated at 1 x10 6 cells/dish on 35 mm Primaria dishes containing glass coverslips pre-coated with sterile 1% (w/v) gelatin followed by laminin (20 mg/ml in PBS; Sigma-Aldrich). For all experiments, the plating medium was withdrawn after 18 h and cells were incubated in serum-free maintenance medium (DMEM/medium [4:1 (v/v)] containing 100 units/ml penicillin and streptomycin) for a further 24 h.
  • serum-free maintenance medium DMEM/medium [4:1 (v/v)] containing 100 units/ml penicillin and streptomycin
  • H uman cardiac fibroblasts (PromoCell) were grown in Fibroblast growth medium-3 (FGM3, PromoCell). Cells were seeded the day before experimentation (at a density to achieve 90% confluence after 24 h) and synchronised overnight in Ml 99 medium containing 0.1% (v/v) FCS and 100 units/ml penicillin and streptomycin.
  • RNA preparation and qPCR Total RNA was prepared using RNA Bee (AMS)
  • RNA was prepared according to the manufacturer's instructions, dissolved in nuclease-free water and purity assessed from the A 260 /A 280 measured using an Implen NanoPhotometer (values of 1.8-2.1 were considered acceptable). RNA concentrations were determined from the A260 values. Quantitative PCR (qPCR) analysis was performed as previously described. Total RNA (2 mg) was reverse transcribed to cDNA using High Capacity cDNA Reverse
  • qPCR was performed using an ABI Real-Time PCR 7500 system (Applied Biosystems). Optical 96-well reaction plates were used with iTaq Universal SYBR Green Supermix (Bio-Rad Laboratories Inc.) according to the manufacturer’s instructions.
  • Rat or mouse heart powders were extracted in 8 vol (relative to powder weight) Buffer A plus inhibitors. Samples were vortexed and extracted on ice (10 min). Extracts were centrifuged (10,000xg, 4°C) for 5 or 10 min for NRVMs or heart powders, respectively. The supernatants were removed, a sample was taken for protein assay and the rest boiled with 0.33 vol SDS-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer [0.33 M Tris-HCl pH 6.8, 10% (w/v) SDS, 13% (v/v) glycerol, 133 mM dithiothreitol, 0.2 mg/mL bromophenol blue]. Protein concentrations were determined by BioRad Bradford assay using bovine serum albumin (BSA) standards.
  • BSA bovine serum albumin
  • Cytosolic and nuclear extracts were prepared essentially by the method of Dignam et al (Dignam et al., Nucleic Acids Res., (1983), 11 :1475-1489). Cells were washed in ice-cold PBS and harvested into 150 ml of hypotonic buffer (10 mM Hepes, pH 7.9, 10 mM KC1, 1.5 mM MgCl 2 , 0.3 mM Na 3 VO 4 ) containing protease and phosphatase inhibitors. Samples were centrifuged (10,000xg, 4°C, 5 min) and the supernatant (cytosolic) fractions boiled with 50 ⁇ l of sample buffer.
  • the pellets were resuspended in 50 ⁇ l of nuclear extraction buffer (20 mM Hepes pH 7.9, 420 mM NaCl, 1.5 mM MgCl 2 , 0.2 mM EDTA, 25% (v/v) glycerol, 0.3 mM Na 3 VO 4 ) containing protease and phosphatase inhibitors and extracted on ice for 1 h with occasional vortex-mixing.
  • the samples were centrifuged (10,000xg, 4°C, 5 min) and the supernatant nuclear extracts boiled with 20 ⁇ l sample buffer.
  • heart powders were extracted in Buffer A plus inhibitors as described above and centrifuged (10,000xg, 4°C, 10 min). The pellets were resuspended in 8 Vol Buffer B (10 mM HEPES, pH 7.9, 400 mM KC1, 1.5 mM MgCl 2 , 0.3 mM Na3VO4 5 mM dithiothreitol) containing protease and phosphatase inhibitors to remove contractile proteins. Samples were vortexed, incubated on ice (10 min) and then centrifuged (10,000 x g, 10 min, 4°C).
  • 8 Vol Buffer B (10 mM HEPES, pH 7.9, 400 mM KC1, 1.5 mM MgCl 2 , 0.3 mM Na3VO4 5 mM dithiothreitol) containing protease and phosphatase inhibitors to remove contractile proteins. Samples were vortexed, incubated on ice (10 min) and
  • the pellets were further extracted in 8 vol SDS-PAGE sample buffer diluted 1 :1 with Buffer A without bromophenol blue. Samples were boiled for 5 min and centrifuged (12,000 x g, 10 min, 4°C). The supernatants were used to assess collagen content. Protein concentrations were determined using a BCA protein assay (Thermo Scientific). Remaining samples were boiled with 0.33 volumes of diluted (1 :1) SDS-PAGE sample buffer containing bromophenol blue.
  • Raf kinase assays Assays were conducted largely as described in Clerk et al ., Mol. Cell. Biol., (2001), 21 :1173-1 184. Cells (4x 10 6 ) were washed with ice-cold PBS, scraped into 150 ⁇ l buffer C [20 mM Tris-HCl pH 7.4, 2 mM EDTA, 100 mM KC1, 10% (v/v) glycerol, 1% (v/v) Triton X-100, 0.5 % 2-mercaptoethanol, 10 mM benzamidine, 5 mM NaF, 0.2 mM Na 3 VO 4 ] containing protease and phosphatase inhibitors, and centrifuged (10,000 x g, 5 min, 4°C).
  • sample (10 ⁇ l) was taken for immunoblotting of total extracts and boiled with an equal volume of sample buffer. The remaining sample was equally distributed for immunoprecipitation of BRaf or cRaf. Samples were incubated (18 h, 4°C with rotation) with antibodies (2 mg) pre-bound to protein G-Sepharose (30 ⁇ l of a 1 :1 slurry equilibrated in buffer C; Sigma-Aldrich). Samples were centrifuged (10,000 x g, 5 min, 4°C) and supernatants boiled with 0.33 vol sample buffer.
  • Pellets were washed 3 times in buffer C, then with 300 ⁇ l buffer D [30 mM Tris-HCl pH 7.5, 0.1 mM EGTA, 0.1 % (v/v) 2- mercaptoethano), 0.03% Brij 35, 10 mM magnesium acetate, 20 mM n-octyl ⁇ -D- glucopyranoside, 200 mM ATP] and then resuspended in 30 ⁇ l buffer D. Assays were initiated by addition of 0.2 mg GST-MKK1. Following incubation (10 min, 30°C) assays were terminated by addition of 5 ⁇ l 0.5 M EDTA pH 8.0 and placed on ice. Sample buffer (15 ⁇ l) was added and samples boiled prior to immunoblotting (20 ⁇ l).
  • Proteins were transferred to nitrocellulose using a BioRad semi-dry transfer cell (12 V, lh) using a modified transfer buffer for high molecular weight proteins [36.5 mM Tris-HCl, 115.5 mM Glycine, 0.65 mM SDS, 10% (v/v) methanol]. Proteins were detected as previously described using primary antibodies as indicated in Table 4. Bands were detected by enhanced chemiluminescence using ECL Prime Western Blotting detection reagents with visualisation using an ImageQuant LAS4000 system (GE Healthcare). ImageQuant TL 8.1 software (GE Healthcare) was used for densitometric analysis.
  • NRVMs were washed with ice-cold PBS and fixed in 3.7% (v/v) formaldehyde in PBS (10 min, room temperature).
  • Cells were permeabilised with 0.3% (v/v) Triton X-100 (10 min, room temperature) in PBS, and non-specific binding blocked with 1% (w/v) fatty acid free BSA (Sigma-Aldrich UK) in PBS containing 0.1% (v/v) Triton X-100 (10 min, room temperature). All incubations were at room temperature in a humidified chamber, and coverslips were washed three times in PBS after each stage of the immunostaining procedure.
  • Cardiomyocytes were stained with mouse monoclonal primary antibodies to troponin T (60 min) with detection using anti-mouse immunoglobulin secondary antibodies coupled to Alexa-Fluor 488 (60 min).
  • Coverslips were mounted using fluorescence mounting medium (Dako) and viewed with a Zeiss Axioskop fluorescence microscope using a 40x objective. Digital images were captured using a Canon PowerShot G3 camera using a 1.8x digital zoom and cardiomyocyte sizes measured using ImageJ. Images were cropped for presentation using Adobe Photoshop CC 2018.
  • BRaf is expressed in cardiomyocytes potentially signalling to ERK1/2 and promoting cardiomyocyte hypertrophy.
  • mice were generated for cardiomyocyte-specific knock-in of the activating BRaf v600E mutation into the endogenous gene. Since activating mutations in the ERK1/2 cascade cause developmental cardiac abnormalities, an inducible system was used for gene manipulation in adult cardiomyocytes in vivo. Mice with a floxed gene for BRaf v600E knock-in were crossed with mice expressing tamoxifen-inducible Cre regulated by an Myh6 promoter for cardiomyocyte-specific expression. Double heterozygote male mice (i.e.
  • Nppb B-type natriuretic peptide
  • Myh7 b-myosin heavy chain
  • mRNAs were significantly increased within 1 d
  • atrial natriuretic factor ( Nppa ) mRNA increased to a lesser degree at later times.
  • Expression of mRNAs encoding proteins associated with fibrosis (Collal, Col4a1, Lox, Timpl, Ddr2, IL-11 ) was also increased over 10 d ( Figure 2E).
  • ERK1/2 signalling in tamoxifen-treated hearts from Cre MCM/WT mice with minor increases only in Myh7 and ARaf mRNAs ( Figure 3, A and B).
  • BRaf ⁇ ERK1/2 signalling promotes changes in gene expression consistent with
  • Echocardiography was used to assess the physiological effects of BRaf v600E knock-in in cardiomyocytes. There were no significant differences in cardiac function/dimensions between Cre MCM/WT mice with/without tamoxifen and vehicle-treated BRaf v600E/MCM mice ( Figure 3C). Cardiomyocyte BRaf v600E knock-in significantly increased ejection fraction (EF) and fractional shortening (FS) at 7 d, with significant increases in left ventricular (LV) systolic and diastolic posterior wall (PW) thickness over 7-10 d ( Figure 4, A and B). There was no evidence of cardiac dilatation with no increase in LV internal diameter (ID). The data are consistent with development of compensated, concentric hypertrophy.
  • EF ejection fraction
  • FS fractional shortening
  • LV left ventricular
  • PW diastolic posterior wall
  • SB590885 activates ERK1/2 in cardiomyocytes
  • SB590885 activated ERK1/2 in Langendorff-perfused adult rat hearts (Figure 5A).
  • a high concentration (10 mM) of SB590885 inhibited ERK1/2 phosphorylation in neonatal rat ventricular myocytes ( Figure 5B) or human cardiac fibroblasts ( Figure 5C), although this concentration may not be entirely selective for Raf kinases.
  • Lower concentrations (0.01-0.1 mM) activated ERK1/2 in both cell types, but relative activation in cardiomyocytes was substantially greater than in fibroblasts.
  • SB590885 activates ERK1/2 -dependent gene expression and promotes cardiomyocyte hypertrophy
  • Hypertrophic agonists e.g. endothelin-1
  • endothelin-1 increase activated ERK1/2 in the nucleus without net accumulation of total ERK1/2 and this regulates gene expression.
  • SB590885 behaved similarly although, in contrast to endothelin-1, the degree of nuclear ERK1/2 activity was significantly greater than that in the cytoplasm ( Figure 6A).
  • SB590885 (0.1 mM, 1 h) increased expression of immediate early genes ( Figure 6B ). This was inhibited by the MKK1/2 inhibitor PD184352, indicating the response was directly caused by increased ERK1/2 signalling.
  • SB590885 promoted cardiomyocyte hypertrophy as shown by increased cell area ( Figure 6C ).
  • mice were treated with SB590885 (0.5 mg/kg/d; 3d) or vehicle, and cardiac function/dimensions assessed by echocardiography.
  • SB590885 increased LVPW thickness in the absence of any significant change in LVID, resulting in an increase in calculated LV mass ( Figure 7, A and B). This was associated with a significant increase in cardiomyocyte cross-sectional area with no evidence of increased fibrosis ( Figure 7C).
  • downstream signalling from ERK1/2 may regulate contractility directly (e.g. activation of p90 ribosomal S6 kinases that phosphorylate NHE1 or myofilament proteins). Further studies to identify the full range of ERK1/2 cascade targets, whether nuclear transcription factors or cytosolic proteins, may provide clarification.
  • BRaf v600E/MCM mice provided a clear conclusion with respect to the role of BRaf in cardiomyocytes, but BRaf ⁇ ERK1/2 signalling is also important in proliferating cardiac cells including fibroblasts, and inhibitors of ERK1/2 signalling will affect all cell types.
  • Type-1 inhibitors such as SB590885 bind to Raf kinases in an active conformation and increase Raf dimerization potential. Inhibitor binding to one protomer may prevent inhibitor binding to the other and, in the context of activated Ras, the active protomer activates MKK1/2 and ERK1/2.
  • the responses of terminally-differentiated cells such as cardiomyocytes have been unexplored. SB590885 activated ERK1/2 in perfused hearts at low concentrations ( ⁇ 0.1 mM) but, surprisingly, had a more pronounced effect in
  • SB590885 promoted cardiomyocyte hypertrophy in vivo with little evidence of fibrosis ( Figure 7, A-C), an effect that may be attributed to the greater enhancement of ERK1/2 activity by the drug in cardiomyocytes than fibroblasts ( Figure 5B).
  • BRaf exists in preformed complexes with cRaf in cardiomyocytes and the Type- 1 inhibitor SB590885 activates ERK1/2 in cardiomyocytes to induce cardiomyocyte hypertrophy.
  • a schematic representation of this paradigm is in Figure 7D.
  • Example 2 Cardiac adaptation to angiotensin II in mice requires cardiomyocyte BRaf, and the Raf inhibitor, dabrafenib, inhibits cardiomyocyte hypertrophy and cardiac fibrosis
  • Raf kinases activate extracellular signal-regulated kinases 1/2 (ERK1/2) that are cardioprotective and promote cardiac hypertrophy. Activation of BRaf in cardiomyocytes promotes hypertrophy, but its role in a pathophysiological setting has not been established. The aim of this study was to determine if cardiac adaption to hypertension requires cardiomyocyte BRaf and whether it is inhibited by the Raf inhibitor, dabrafenib.
  • a mouse model was established for cardiomyocyte-specific tamoxifen-inducible homozygous knock-out of BRaf.
  • Cardiomyocyte-specific BRaf knock- out alone did not affect cardiac dimensions/function (assessed by echocardiography) or mRNA expression.
  • Mice were treated with angiotensin II (Angll; 0.8 mg/kg/d, 7 d) to induce hypertension.
  • Angll-induced increased left ventricular wall thickness and decreased internal diameter were moderated by cardiomyocyte BRaf knock-out. Most Angll-induced changes in cardiac mRNA expression were also inhibited.
  • cardiomyocyte BRaf knock-out did not reduce interstitial/perivascular fibrosis induced by Angll, and caused areas of focal damage with loss of cardiomyocytes and enhanced fibrosis/inflammation.
  • Dabrafenib is a Raf inhibitor used for cancer. In mice, 3 mg/kg/d dabrafenib alone did not affect cardiac function/dimensions, but inhibited Angll-induced hypertrophy and changes in cardiac mRNA expression. At a cellular level, dabrafenib inhibited the increase in cardiomyocyte size induced by Angll and, in contrast to cardiomyocyte BRaf knock-out, almost eliminated cardiac fibrosis without any evidence of focal damage.
  • Cardiomyocyte BRaf is required for cardiac adaptation to Angll-induced hypertension.
  • targeting Raf kinases in all cardiac cells with inhibitors such as dabrafenib may be a viable therapeutic option for reducing cardiac hypertrophy and fibrosis in hypertension.
  • BRaf activates extracellular signal-regulated kinases 1/2 that are cardioprotective and promote cardiac hypertrophy. Activation of BRaf in cardiomyocytes promotes hypertrophy, but its role in a pathophysiological setting was until now unknown. Here, it was established that BRaf is required for cardiac adaptation to hypertension induced by angiotensin II in mice since loss of cardiomyocyte BRaf compromised the adaptive response enhancing focal damage with loss of cardiomyocytes and increased fibrosis.
  • the Raf inhibitor dabrafenib
  • the Raf inhibitor inhibited hypertension-induced cardiomyocyte hypertrophy, attenuated interstitial and perivascular fibrosis and did not damage the myocardium.
  • the data suggest that targeting cardiac Raf kinases, irrespective of cell type, with inhibitors such as dabrafenib may be a viable therapeutic option for reducing cardiac hypertrophy and fibrosis in hypertension.
  • Heart failure is an increasing cause of morbidity and mortality worldwide, with hypertension as a major contributing factor. Initially, the heart adapts to the increased work-load to maintain cardiac output. This is achieved in part by hypertrophy of terminally-differentiated contractile cardiomyocytes which increase in size, adapting and increasing the myofibrillar apparatus. In the longer term, this is not sustained, contractile function is compromised and heart failure develops. Pathological changes include cardiomyocyte cell death, and capillary rarefaction. This is associated with inflammation and increased fibrosis. Consequently, strategies to reduce cardiomyocyte death, improve contractility, increase angiogenesis and reduce fibrosis are all necessary to treat heart failure.
  • ERK1/2 extracellular signal regulated kinase 1/2 cascade is a key growth-promoting pathway in all cells. It is best characterised in proliferating cells in which ERK1/2 activation promotes cell division. ERK1/2 are phosphorylated/activated by MKK1/2 that are phosphorylated/activated by upstream Raf kinases (ARaf, BRaf, cRaf) or, in the context of inflammation, Cot/Tpl2. Raf kinases are regulated at multiple levels. Of particular importance, activation requires interaction with activated Ras which induces a
  • Raf proteins conformational change in Raf proteins. They also operate as homo- or heterodimers and are subject to activating and inhibitory phosphorylations. Mutations that activate ERK1/2 cause cancer. Oncogenic mutations are highly prevalent in BRaf, and BRaf inhibitors (e.g.
  • dabrafenib are in clinical use. Dabrafenib was developed to target oncogenic BRaf v600E/K (IC 50 0.7 nM), but also inhibits wild-type BRaf and cRaf (IC 50 of 5.2 and 6.3 nM, respectively). As a Type 1.5 inhibitor, dabrafenib binds to the "DFG-in" active
  • the ERK1/2 cascade promotes cardiomyocyte hypertrophy and, independent of this, is potently cardioprotective.
  • All Raf kinases are activated in cultured cardiomyocytes by hypertrophic stimuli such as endothelin-1 (Example 1).
  • hypertrophic stimuli such as endothelin-1 (Example 1).
  • cardiomyocyte-specific expression of dominant-negative cRaf in mice increases cardiomyocyte apoptosis, and leads to cardiomyopathy in response to pressure-overload, indicating that cRaf is cardioprotective.
  • the studies described elsewhere herein demonstrated that activation of endogenous BRaf in cardiomyocytes (via genetic manipulation or a Raf paradox-inducer) promotes hypertrophy (Example 1).
  • BRaf cardiac adaptation to hypertension induced by angiotensin II (Angll) was investigated.
  • Cardiomyocyte-specific BRaf knock-out compromised cardiac hypertrophy induced by Angll with reduced left ventricular (LV) wall thickness, but this was associated with increased focal cardiac damage and fibrosis indicating that, on balance, cardiomyocyte BRaf signalling is necessary for cardiomyocyte adaptation.
  • the Raf inhibitor, dabrafenib, reduced cardiomyocyte hypertrophy and cardiac fibrosis induced by Angll with no indication of focal damage or evidence of cardiotoxicity.
  • dabrafenib and other related Raf kinase inhibitors may be therapeutically useful for reducing cardiac fibrosis.
  • mice were used for Cre- induced BRaf gene deletion (129-Braf tm1Sva /J, strain no. 006373) that were cryoresuscitated. These mice were on a 129T2/SvEmsJ background prior to cryopreservation and were then backcrossed against the C57B1/6J background at UoR for at least 4 generations prior to experimentation. Control experiments were conducted with wild-type littermates.
  • mice were bred with Myh6-MerCreMer mice expressing nuclear-localised tamoxifen- activated Cre recombinase under control of the mouse Myh6 promoter (B6.FVB(129)- A1cf Tg(Myh6-cre/Esrl *)1Jmk /J; strain 005657). These were maintained on a C57B1/6J background at Jackson Laboratories and were backcrossed against the C57B1/6J background at UoR for at least 4 further generations. Breeding protocols were used to produce BRaf KO/KO /Cre MCM/WT mice (i.e.
  • Genotyping DNA was extracted from ear notches using Purelink genomic DNA (gDNA) mini-kits (Invitrogen) according to the manufacturer's instructions. Briefly, tissue was digested in genomic digestion buffer containing proteinase K (overnight, 55°C).
  • RNAse A (2 min) before addition of genomic lysis binding buffer mixed with an equal volume of ethanol.
  • gDNA was purified using Purelink spin columns and PCR amplified with specific primers (see Table 5 for primer sequences and annealing temperatures) using GoTaq Hot Start Polymerase (Promega). PCR conditions were 95°C for 3 min, followed by 33 cycles of 95°C denaturation for 30 s, 30 s annealing, elongation at 72°C for 30 s, followed by a 7 min 72°C final extension. PCR products were run on 2% (w/v) agarose gels (80V) and visualised under UV light.
  • mice were treated with a single dose of tamoxifen (40 mg/kg i.p.; Sigma- Aldrich) or vehicle. Tamoxifen was dissolved in ethanol and then mixed with corn oil.
  • RNA was extracted from tissue powders and cDNA prepared as described below. cDNA (4 ⁇ l) was subjected to PCR analysis using GoTaq Hot Start Polymerase with specific primers and conditions ( Table 5).
  • Drug delivery used Alzet osmotic pumps (models 1007D or 1002; supplied by Charles River), filled according to the manufacturer’s instructions in a laminar flow hood using sterile technique. Mice were treated with angiotensin II (Angll, 0.8 mg/kd/d) or vehicle (acidified PBS) without or with dabrafenib (3.0 mg/kg/d) dissolved in DMSO/PEG mix [50% (v/v) DMSO, 20% (v/v) polyethylene glycol 400, 5% (v/v) propylene glycol, 0.5% (v/v) Tween 80] or DMSO/PEG alone. Angll was from Merck, dabrafenib was from
  • Cardiac ultrasound Echocardiography was performed on anaesthetised mice using a Vevo 2100 imaging system equipped with a MS400 18-38 MHz transducer (Visualsonics). Mice were anaesthetised in an induction chamber with isoflurane (5% flow rate) with 1 l/min O 2 then transferred to the heated Vevo Imaging Station. Anaesthesia was maintained with 1.5% isoflurane delivered via a nose cone. Left ventricular cardiac function and structure was assessed from short axis M-mode images with the axis placed at the mid-level of the left ventricle at the level of the papillary muscles. Baseline scans were taken prior to
  • Tissue harvesting and processing Mice were culled by schedule 1 (CO 2 followed by cervical dislocation). Hearts were excised quickly, washed in PBS, dried and snap-frozen in liquid N 2 or fixed for histology.
  • Histology and assessment of myocyte size and fibrosis Histological analysis was performed on hearts subjected to in situ perfusion fixation with 10% formalin. Following immersion in 70% (v/v) ethanol, hearts were embedded in paraffin and sectioned at 10 mm. Sections were de-waxed using xylene and re-hydrated through sequential washes in decreasing ethanol gradients (100%, 100%, 90%, 75%, 50%) to distilled water. Sections were stained using kits for hematoxylin and eosin (H&E, Sigma) or Masson's trichrome (Polysciences).
  • Rat heart perfusions Adult male (300-350 g) Sprague-Dawley rats (Charles River) were anaesthetised with a lethal intraperitoneal dose of Euthatal (pentobarbital sodium, 60 mg/kg). After complete anaesthesia was induced, a saphenous vein was exposed and heparin (1000 U/kg) administered intravenously.
  • Euthatal pentobarbital sodium, 60 mg/kg
  • bFGF human basic fibroblast growth factor
  • RNA preparation and qPCR Total RNA was prepared using RNA Bee (AMS)
  • RNA was prepared according to the manufacturer's instructions, dissolved in nuclease-free water and purity assessed from the A 260 /A 280 measured using an Implen NanoPhotometer (values of 1.8-2.1 were considered acceptable). RNA concentrations were determined from the A260 values. Quantitative PCR (qPCR) analysis was performed as previously described (Marshall et al., PLoS One, (2010), 5:e10027). Total RNA (2 mg) was reverse transcribed to cDNA using High Capacity cDNA Reverse Transcription Kits with random primers (Applied Biosystems) according to the manufacturer's instructions.
  • qPCR Quantitative PCR
  • qPCR was performed using an ABI Real-Time PCR 7500 system (Applied Biosystems). Optical 96-well reaction plates were used with iTaq Universal SYBR Green Supermix (Bio-Rad Laboratories Inc.) according to the manufacturer’s instructions. Primers were from Invitrogen by Thermo Fisher
  • Gapdh was used as the reference gene for the study using proprietory primers for rat and mouse from PrimerDesign. Results were normalized to Gapdh, and relative quantification was obtained using the ACt (threshold cycle) method; relative expression was calculated as 2 -DDCt , and normalised to vehicle or time 0.
  • Rat or mouse heart powders were extracted in 8 vol (relative to powder weight) Buffer A plus inhibitors. Samples were vortexed and extracted on ice (10 min). Extracts were centrifuged (10,000xg, 4°C) for 5 or 10 min for NRVMs or heart powders, respectively. The supernatants were removed, a sample was taken for protein assay and the rest boiled with 0.33 vol SDS- polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer [0.33 M Tris-HCl pH 6.8, 10% (w/v) SDS, 13% (v/v) glycerol, 133 mM dithiothreitol, 0.2 mg/mL bromophenol blue]. Protein concentrations were determined by BioRad Bradford assay using bovine serum albumin (BSA) standards.
  • BSA bovine serum albumin
  • Table 7 Antibodies used for immunoblotting and immunostaining.
  • CST Cell Signaling Technologies
  • SCBT Santa Cruz Biotechnology Inc.
  • BD BD Transduction Labs.
  • Cardiomyocyte BRaf is required for cardiac adaptation to hypertension induced by angiotensin II in mice
  • cardiomyocyte BRaf Activation of cardiomyocyte BRaf promotes hypertrophy (Example 1).
  • mice with a floxed gene for BRaf knock-out were crossed with mice expressing tamoxifen-inducible Cre regulated by an Myh6 promoter for cardiomyocyte-specific expression, producing mice for inducible cardiomyocyte-specific BRaf knock-out.
  • Male mice homozygous for the floxed BRaf gene and heterozygous for Cre i.e. BRaf KO/KO /Cre MCM/WT . Recombination was induced by a single tamoxifen injection ( Figure 8).
  • BRaf KO /KO /Cre MCM/WT mice induced a significant decrease in BRaf mRNA and protein ( Figure 9, A-C. cRaf (not ARaf) protein was also significantly downregulated (discussed below), so the consequences of BRaf knock-out potentially reflect additional loss of cRaf.
  • mRNAs for the pro-inflammatory cytokines, 11.1 b and TNFa were not inhibited at 7 d, indicative of ongoing inflammation.
  • mRNAs for extracellular matrix proteins and enzymes required for remodelling were upregulated by Angll, though in the absence of any increase in Ddr2, a fibroblast marker ( Figure 13, D and E).
  • upregulation of mRNAs for the enzymes ⁇ Lox, Timpl ), but not the matrix proteins themselves, was inhibited by BRaf knock-out. Tamoxifen alone did not affect cardiac mRNA expression of the genes studied in BRaf KO7KO /Cre MCM/WT mice ( Figure 14).
  • cardiomyocyte BRaf plays a significant role in the cardiac gene expression response to Angll.
  • cardiomyocyte BRaf knock-out mice were fixed and sections stained with H&E or Masson's trichrome. As expected, Angll alone increased interstitial and perivascular fibrosis throughout the heart with some areas of focal damage (Figure 15). This was also detected in hearts from mice with cardiomyocyte-specific BRaf knock-out, but these hearts had more extensive focal damage with increased fibrosis and loss of cardiomyocytes. Overall, the data indicate that cardiomyocyte BRaf is required for sustainable adaptation of the heart to hypertension.
  • Dabrafenib acutely inhibits ERK1/2 signalling in perfused adult rat hearts and inhibits Angll-induced cardiac hypertrophy in mice in vivo.
  • Dabrafenib is a Type- 1.5 Raf inhibitor that can activate (rather than inhibit) ERK1/2 signalling via the "Raf paradox" in some cancer cells.
  • Data described elsewhere herein (Example 1) showed that ERK1/2 are activated in cardiomyocytes by the Type-1 Raf inhibitor, SB590885, and this promotes hypertrophy. It was determined if dabrafenib activates or inhibits ERK1/2 signalling in Langendorff perfused adult male rat hearts by immunoblotting samples for phosphorylated (activated) ERK1/2.
  • Dabrafenib (5 mM) inhibited basal ERK1/2 phosphorylation and the increase in ERK1/2 phosphorylation induced by FGF2 ( Figure 16).
  • dabrafenib is administered orally at up to 150 mg, twice daily (i.e. ⁇ 3-5 mg/kg/d).
  • mice were implanted with osmotic minipumps for delivery of 3 mg/kg/d dabrafenib or vehicle in the
  • Cardiomyocyte BRaf knock-out inhibited cardiac adaptation to Angll (Figure 9; Figure 10), resulting in enhanced focal damage with loss of cardiomyocytes and increased connective tissue (Figure 15). Cardiomyocyte BRaf knock-out was associated with loss of cRaf protein ( Figure 9C ), probably because BRaf heterodimerises with cRaf in cardiomyocytes (Example 1), and loss of BRaf presumably destabilized cRaf protein. Of the Raf kinases, BRaf has the greatest MKK1 /2-activating activity and this is most likely its main function.
  • cardiomyocyte BRaf is an essential element required for cardiac adaptation to hypertension.
  • Dabrafenib a Raf inhibitor used clinically for cancer, also inhibited Angll-induced cardiac hypertrophy (Figure 18; Figure 19).
  • dabrafenib inhibited the increased expression of IL1 ⁇ and TNFa mRNAs suggesting it had an additive effect to reduce inflammation.
  • dabrafenib inhibited cardiomyocyte hypertrophy and suppressed cardiac fibrosis, while there was no indication of focal damage to the heart (Figure 21).
  • ERK1/2 signalling in cardiac fibroblasts contributes to cardiac fibrosis.
  • miR-21 downregulation of Spryl increases ERK1/2 activity in fibroblasts that enhances cardiac fibrosis.
  • the pathway should promote proliferation of cardiac fibroblasts and, thus, fibrosis.
  • Ddr2 mRNA a fibroblast marker
  • AngII a fibroblast marker
  • dabrafenib a fibroblast marker
  • IL11 also promotes extracellular matrix production from cardiac fibroblasts via ERK1/2, acting in a post-transcriptional manner.
  • dabrafenib may be due to inhibition of non-ERK1/2 signalling downstream of a BRaf/cRaf heterodimer (discussed above) together with an alternative input into MKK1/2 ERK1/2 (discussed below).
  • it could reflect an off-target effect such as the pro-apoptotic kinase, RIPK3, that is inhibited by dabrafenib at high concentrations.
  • Raf inhibitors Further studies with different Raf inhibitors are warranted.
  • Type 2 inhibitors e.g. PLX8394
  • a 1 -adrenergic agonists e.g. phenylephrine
  • oxidative stress appear to use an alternative input to MKK1/2 in cardiomyocytes rather than Ras-associated Raf kinases, that seems to require phosphoinositide 3’ kinase.
  • this could be why no significant decrease was detected in ERK1/2 phosphorylation in hearts from mice treated with dabrafenib.
  • dabrafenib alone is cardiotoxic, there are side effects which are generally managed by dose reduction. The most severe effect is probably an increase in cutaneous squamous cell carcinoma ( ⁇ 12% of patients).
  • dabrafenib were to be used as a therapy for cardiac fibrosis, it may be important to consider dosage monitoring and whether patients have a predisposition for other diseases.
  • BRaf plays a significant role in cardiac adaptation to Angll-induced hypertension.
  • targeting Raf kinases in general with inhibitors such as dabrafenib may be a viable therapeutic option for reducing cardiac hypertrophy and fibrosis in hypertension.
  • SB590885 did not significantly affect the changes in systolic or diastolic left ventricle (LV) internal diameter (ID) or posterior wall (PW) thickness or the LVPW/LVID ratio.
  • Example 4 effects of vemurafenib and encorafenib on the heart
  • vemurafenib and encorafenib (Type 1.5 BRaf inhibitors) on the heart, in addition to the Type 2 Raf inhibitor PLX7904 (related to PLX8394) on the cardiomyocytes were studied and compared with dabrafenib.
  • cardiomyocytes showed significant inhibition at 10 mM but not with lower concentrations (Figure 23).
  • Vemurafenib inhibited basal ERK1/2 phosphorylation in cardiomyocytes at 10- 30 mM ( Figure 23B).
  • PLX7904 was most effective at inhibiting basal ERK1/2

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Abstract

La présente invention concerne le traitement ou la prévention de la fibrose associée à l'activité de kinases Raf, par exemple la fibrose cardiaque.
PCT/GB2020/050243 2019-02-04 2020-02-03 Traitement de la fibrose par des inhibiteurs du raf Ceased WO2020161477A1 (fr)

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WO2014111584A1 (fr) * 2013-01-18 2014-07-24 Genfit Procédés de traitement de fibroses et de cancers
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