WO2017149306A1 - Polythérapie - Google Patents

Polythérapie Download PDF

Info

Publication number
WO2017149306A1
WO2017149306A1 PCT/GB2017/050553 GB2017050553W WO2017149306A1 WO 2017149306 A1 WO2017149306 A1 WO 2017149306A1 GB 2017050553 W GB2017050553 W GB 2017050553W WO 2017149306 A1 WO2017149306 A1 WO 2017149306A1
Authority
WO
WIPO (PCT)
Prior art keywords
tnfa
polypeptide
bmp9
seq
bmpr
Prior art date
Application number
PCT/GB2017/050553
Other languages
English (en)
Inventor
Nicholas W MORRELL
Paul D UPTON
Original Assignee
Cambridge Enterprise Limited
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Cambridge Enterprise Limited filed Critical Cambridge Enterprise Limited
Publication of WO2017149306A1 publication Critical patent/WO2017149306A1/fr

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1875—Bone morphogenic factor; Osteogenins; Osteogenic factor; Bone-inducing factor
    • 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
    • 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
    • A61P9/12—Antihypertensives

Definitions

  • the invention relates to a combination therapy for the treatment of vascular or respiratory diseases.
  • Vascular disease is a pathological state of large and medium sized muscular arteries and is triggered by endothelial cell dysfunction. Because of factors like pathogens, oxidized LDL particles and other inflammatory stimuli, endothelial cells become activated. This leads to changes in their characteristics: endothelial cells start to excrete cytokines and chemokines and express adhesion molecules on their surface. This in turn results in recruitment of white blood cells (monocytes and lymphocytes), which can infiltrate the blood vessel wall. Stimulation of the smooth muscle cell layer with cytokines produced by endothelial cells and recruited white blood cells causes smooth muscle cells to proliferate and migrate towards the blood vessel lumen.
  • endothelial cells start to excrete cytokines and chemokines and express adhesion molecules on their surface. This in turn results in recruitment of white blood cells (monocytes and lymphocytes), which can infiltrate the blood vessel wall. Stimulation of the smooth muscle cell layer with cytokines produced by endotheli
  • This process causes thickening of the vessel wall, forming a plaque consisting of proliferating smooth muscle cells, macrophages and various types of lymphocytes.
  • This plaque results in obstructed blood flow leading to diminished amounts of oxygen and nutrients that reach the target organ.
  • the plaque may also rupture causing the formation of clots, and as a result, strokes.
  • Respiratory disease encompasses pathological conditions affecting the organs and tissues that make gas exchange possible in higher organisms, and includes conditions of the upper respiratory tract, trachea, bronchi, bronchioles, alveoli, pleura and pleural cavity, and the nerves and muscles of breathing. Respiratory diseases range from mild and self-limiting, such as the common cold, to life-threatening entities like bacterial pneumonia, pulmonary embolism, and lung cancer. Pulmonary arterial hypertension (PAH) is a rare progressive disease defined by elevated pulmonary arterial pressure, often causing death from right heart failure. The pathology is characterized by increased muscularization and obliteration of small pulmonary arteries.
  • PAH Pulmonary arterial hypertension
  • BMPR-II bone morphogenetic protein type II receptor
  • HPAH heritable
  • IPH idiopathic
  • PAH patients exhibit heightened circulating levels of inflammatory cytokines, including for example tumor necrosis factor a (TNFa), IL-1 ⁇ , IL-6 and IL-8, that correlate with poor survival.
  • TNFa tumor necrosis factor a
  • IL-1 ⁇ IL-1 ⁇
  • IL-6 IL-6
  • IL-8 inflammatory cytokines
  • Henriques-Coelho et al. 2013, Rev. Port. Cardiol. 27: 341 ) reported that administration of etanercept, an anti- TNFa antibody, to rats with monocrotaline-induced pulmonary hypertension, did not lead to a significant improvement in pulmonary hypertension symptoms.
  • WO2016/005756 discloses a composition for treating a vascular or respiratory disease such as PAH, comprising a polypeptide selected from bone morphogenetic protein 10 (BMP10), or a bone morphogenetic protein 9 (BMP9) variant lacking osteogenic activity.
  • BMP10 bone morphogenetic protein 10
  • BMP9 bone morphogenetic protein 9
  • the present invention is directed to improved methods and compositions for treating a vascular or respiratory disease such as PAH.
  • a pharmaceutical composition comprising: (1 ) a polypeptide selected from bone morphogenetic protein 10 (BMP10), including the prodomain bound form of BMP10 (pro.BMPI O), and a bone morphogenetic protein 9 (BMP9) variant lacking osteogenic activity; and (2) a TNFa inhibitor.
  • BMP10 bone morphogenetic protein 10
  • pro.BMPI O prodomain bound form of BMP10
  • BMP9 bone morphogenetic protein 9
  • TNFa inhibitor a pharmaceutical composition as defined herein for use in the treatment of a vascular disease or a respiratory disease, wherein the polypeptide and the TNFa inhibitor are prepared to be administered to a patient in need thereof simultaneously, contemporaneously or concomitantly.
  • the TNFa inhibitor may be administered: (a) prior to administration of the polypeptide as defined herein; (b) in doses alternating with administration of the polypeptide as defined herein; and/or (c) in reducing doses together or alternating with administration of the polypeptide as defined herein.
  • the vascular disease may be selected from: pulmonary hypertension; pulmonary arterial hypertension (PAH); hereditary haemorrhagic telangiectasia (HHT, also known as Osler-Weber-Rendu syndrome); atherosclerosis; and hepatopulmonary syndrome.
  • PAH pulmonary arterial hypertension
  • HHT hereditary haemorrhagic telangiectasia
  • atherosclerosis and hepatopulmonary syndrome.
  • the respiratory disease may be selected from: obstructive lung diseases such as chronic obstructive pulmonary disease (COPD), chronic bronchitis and emphysema; pulmonary vascular diseases such as pulmonary edema and pulmonary hemorrhage; respiratory failure and respiratory distress syndrome, such as acute lung injury and acute respiratory distress syndrome; and interstitial lung diseases, such as idiopathic pulmonary fibrosis.
  • COPD chronic obstructive pulmonary disease
  • COPD chronic obstructive pulmonary disease
  • pulmonary vascular diseases such as pulmonary edema and pulmonary hemorrhage
  • respiratory failure and respiratory distress syndrome such as acute lung injury and acute respiratory distress syndrome
  • interstitial lung diseases such as idiopathic pulmonary fibrosis.
  • a package comprising:
  • FIG. 1 TNFa reduces full length BMPR-II protein expression and induces BMPR-II cleavage in human PASMCs.
  • Y-axis shows BMPR2 mRNA fold change (relative to 0.1 % in A and relative to 2% in B).
  • E Representative confocal images of TNFa staining in lung sections from control, idiopathic and heritable PAH subjects. Scale bars, 100 ⁇ .
  • TNFa reduces BMPR-II protein and mRNA expression via NF-KB//?£LA
  • Y-axis shows BMPR2 mRNA fold change (relative to 0.1 % in C and relative to 2% in D).
  • E BMPR2 mRNA expression in human control dPASMCs and PAECs transfected with DharmaFectl alone (DH1 ), s RELA or non-targeting siRNA control (siCP).
  • Y-axis shows BMPR2 mRNA fold change (relative to Control siCP).
  • TNFa specifically reduces BMPR-II protein expression and induces a lower molecular mass band in SMCs.
  • A Assessment of right ventricular hypertrophy (Fulton index). Y-axis shows Fulton index (RV/(LV+S)).
  • Y-axis shows % wall thickness for all vessels.
  • D Bmpr2 mRNA expression in mouse lungs and livers.
  • Y-axis shows Bmpr2 mRNA Relative Expression.
  • F Bmp6 and
  • G Bmp2 mRNA expression in mouse lungs and livers.
  • Y-axis in F shows Bmp6 mRNA Relative Expression.
  • G shows Bmpr2 mRNA Relative Expression.
  • FIG. 5 Release of sBMPR-ll can be inhibited by a pan-metalloprotease inhibitor.
  • ADAM10, ADAM12, ADAM15, ADAM17, MMP14, MMP15, MMP16, MMP17 and MMP24 mRNA expression in human control dPASMCs (A) and PAECs (B) treated with TNFa (1 ng/ml) for 24 h. (n 4; Student's f-test). Y-axis shows mRNA fold change (relative to 0.1 %).
  • D Schematic of pharmacological and siRNA inhibition of ADAM10 and 17.
  • FIG. 1 Schematic of BMPR-II (SP - signal peptide; ECD - ectodomain; TM - transmembrane; ICD - intracellular cytoplasmic domain).
  • Valines in bold in the 5'-myc-BMPR-ll WT transmembrane domain sequence (SEQ ID NO: 8) were converted to alanine (in bold) generating the V158A (SEQ ID NO: 9), V160A (SEQ ID NO: 10), V163A (SEQ ID NO: 1 1 ) and V166A (SEQ ID NO: 12) constructs (marked with " * ").
  • (B) Representative immunoblots (n 3 experiments) of BMPR-II, myc and immunoprecipitated sBMPR-ll from 24 h TNFa- treated (1 ng/ml) control human dPASMCs transfected with 5'-myc-BMPR-ll constructs.
  • C and D Luciferase assessment of ligand trap activity in C2C12-BRE cells.
  • Y-axis shows Luciferase activity (RLU/ g protein).
  • TNFa alters BMP2 and BMP6 signaling dynamics and influences PASMC proliferation.
  • B and C Signaling in dPASMCs with or without TNFa (1 ng/ml) for 23 h prior to 1 h BMP2 (10 ng/ml) or BMP6 (10 ng/ml) stimulation.
  • (C) ID1 mRNA expression (n 3). Y-axis shows ID1 mRNA fold change (relative to 0.1 %).
  • (D) Proliferation of human control and HPAH dPASMCs at 6 days after BMP2 (10 ng/ml), BMP4 (10 ng/ml) or BMP6 (10, 25 or 50 ng/ml) treatment (n 3).
  • (E) Proliferation of human control and HPAH dPASMCs at 6 days after TNFa (1 ng/ml) and/or BMP6 (10 or 50 ng/ml) treatment (n 3 control and HPAH cell lines). Y-axis in D and E shows Cell proliferation (%) (normalised to 5% FBS).
  • TNFa alters BMP2 and BMP6 signaling dynamics.
  • BMP2 and BMP6 are the most abundant expressed ligands in vascular cells.
  • Y-axis in A and B shows Relative Transcript Abundance.
  • Y-axis in C shows BMP2 mRNA (relative to 0.1 %).
  • Y-axis in D shows BMP6 mRNA (relative to 0.1 %).
  • E shows BMP mRNA (relative to 0.1 %).
  • Y-axis in F shows BMP2 mRNA fold change (relative to Control siCP).
  • Y-axis in G shows BMP6 mRNA fold change (relative to Control siCP).
  • Y-axis in H shows BMP2 mRNA fold change (relative to serum control).
  • Y-axis in I shows BMP6 mRNA fold change (relative to serum control).
  • LDN193189 inhibits enhanced BMP6 signaling by TNFa in PASMCs but does not affect the induction of interleukins.
  • Y-axis shows Luciferase activity (RLU/mg protein).
  • Y-axis in C shows ID1 mRNA fold change (relative to 0.1 %).
  • Y-axis in D shows IL8 mRNA fold change (relative to 0.1 %).
  • ALK2 and ACTR-IIA are required for BMP6 mediated HPAH PASMC proliferation and altered signaling.
  • Y-axis in A and B shows Cell proliferation (%) (normalised to control).
  • E and F Human HPAH dPASMCs following s ACVR2A transfection and treatment with TNFa (1 ng/ml) and/or BMP2 (10ng/ml) or BMP6 (10 ng/ml) for 24 h.
  • TNFa induces ACVR2A expression in HPAH PASMCs.
  • B ALK2, ALK3 and ALK6 mRNA expression in human dPASMCs from disease-free controls and HPAH patients stimulated with TNFa (1 ng/ml) for 24 h.
  • Y- axis shows mRNA fold change (relative to ALK2 0.1 %).
  • *** P ⁇ 0.001 Error bars represent mean + s.e.m.
  • TNFa alters NOTCH expression.
  • A Immunoblotting for NOTCH1 , NOTCH2 and NOTCH3 expression in control and HPAH dPASMCs treated with TNFa (1 ng/ml) and/or BMP2 or BMP6 (both 10 ng/ml) for 1 h. Representative experiment of three control and HPAH dPASMCs.
  • NOTCH and its targets are altered by TNFa in HPAH PASMCs.
  • A-F mRNA expression of NOTCH1 (A), NOTCH2 (B), NOTCH3 (C), HEY1 (D), HEY 2 (E) and HES1 (F) in control and HPAH dPASMCs stimulated with TNFa (1 ng/ml) and/or BMP2 or BMP6 (both 10 ng/ml) for 1 h.
  • Y-axis in A shows NOTCH1 mRNA fold change (relative to 0.1 %).
  • Y-axis in B shows NOTCH2 mRNA fold change (relative to 0.1 %).
  • Y- axis in C shows NOTCH3 mRNA fold change (relative to control 0.1 %).
  • Y-axis in D shows HEY1 mRNA fold change (relative to 0.1 %).
  • Y-axis in E shows HEY2 mRNA fold change (relative to control 0.1 %).
  • Y-axis in F shows HES1 mRNA fold change (relative to 0.1 %).
  • * P ⁇ 0.05, ** P ⁇ 0.01 , *** P ⁇ 0.001 . Error bars represent mean + s.e.m.
  • HEY1 and HEY2 are targets of NOTCH2; HES1 is a target of NOTCH3.
  • D - F mRNA expression of HEY1 (D), HEY2 (E) and HES1.
  • Y-axis in A and D shows HEY1 mRNA fold change (relative to 0.1 % siCP).
  • Y-axis in B and E shows HEY 2 mRNA fold change (relative to 0.1 % siCP).
  • TNFa alters NOTCH expression.
  • Y-axis in C shows Notch2 mRNA Relative Expression.
  • Y-axis in D shows Notch3 mRNA Relative Expression.
  • E Representative images of immunohistochemical staining for NOTCH2, NOTCH3 and aSMA in lung sections from control and HPAH subjects. Scale bars, 100 ⁇ .
  • FIG. 18 TNFa differentially alters NOTCH expression in BMPR2 deficiency compared to the normal state.
  • Y-axis in A shows NOTCH1 mRNA fold change (relative to 0.1 % siCP).
  • Y-axis in B shows NOTCH2 mRNA fold change (relative to siCP 0.1 %).
  • Y-axis in C shows NOTCH3 mRNA fold change (relative to siCP 0.1 %).
  • Y-axis in E shows Notch2 mRNA Relative Expression.
  • Y-axis in F shows Notch3 mRNA Relative Expression.
  • DAPT inhibits TNFa influence on PASMC proliferation.
  • Y-axis in A and B shows Cell proliferation (%) normalised to vehicle control.
  • FIG. 20 SRC kinases are activated by TNFa and BMP6, and can regulate Notch.
  • A Schematic depicting SRC phosphorylation.
  • Y-axis in E shows NOTCH2 mRNA Relative Expression.
  • Y-axis in F shows NOTCH3 mRNA Relative Expression.
  • Y- axis in G and H shows Cell proliferation (normalised to siCP).
  • Y- axis in A and D shows NOTCH1 mRNA fold change (relative to control 0.1 %).
  • Y-axis in B shows NOTCH2 mRNA fold change (relative to control 0.1 %).
  • Y-axis in C shows NOTCH3 mRNA fold change (relative to control 0.1 %).
  • Y-axis in E shows NOTCH1 mRNA fold change (relative to siCP 0.1 %).
  • Error bars represent mean + s.e.m.
  • a and B Assessment of RVSP (A) and right ventricular hypertrophy (Fulton index) (B). Y-axis in A shows RVSP (mmHg).
  • Y-axis in B shows Fulton Index (RV/(LV+S))
  • Y-axis in F shows Notch2 mRNA fold change (relative to control).
  • Y-axis in G shows Notch3 mRNA fold change (relative to control).
  • H Representative immunohistochemical images of Notch2, Notch3 and aSMA in control and S/H rat lung sections. Scale bars, 100 ⁇ .
  • FIG. 23 The anti-TNFa therapeutic, etanercept, reverses established pulmonary hypertension in the Sugen-hypoxia model.
  • B Assessment of pulmonary arterial wall thickness as a percentage of luminal diameter. Y-axis shows % wall thickness for all vessels.
  • Y-axis in C shows Bmp6 mRNA fold change (relative to control).
  • Y-axis in D shows Tnf mRNA fold change (relative to control).
  • Y-axis in E shows Notch l mRNA fold change (relative to control).
  • Y-axis in F shows mRNA fold change (relative to control).
  • Y-axis in G shows Hes1 mRNA fold change (relative to control).
  • Control human dPASMCs were transfected with BMPR2 siRNA (siBMPR2) or control siRNA (siCP) using DharmaFECTITM (DH1 ) followed by treatment with BMP6 (10 ng/ml) in 0.1 % FBS for 1 , 4 or 24 hours.
  • B Protein lysates were immunoblotted for phospho-Smad 1/5 and total Smad 1 followed by reprobing for a-tubulin to ensure equal loading. Data are representative of three separate experiments.
  • B Immunoblotting for BMPR-II to confirm the loss of protein in siBMPR2-transfected PASMCs. Blots were reprobed for ⁇ -tubulin to ensure equal loading. Data are representative of three separate experiments.
  • a pharmaceutical composition comprising: (1 ) a polypeptide selected from bone morphogenetic protein 10 (BMP10) and a bone morphogenetic protein 9 (BMP9) variant lacking osteogenic activity; and (2) a TNFa inhibitor.
  • BMP10 bone morphogenetic protein 10
  • BMP9 bone morphogenetic protein 9
  • the present invention involves a combination of a TNFa inhibitor in combination with a bone morphogenetic protein which maintains endothelial cell signalling activity (for example, as which may be evidenced by the induction of ID1 , ID2 and/or BMPR-II gene expression) but which lacks osteogenic activity (for example as which may be measured by alkaline phosphatase (ALP) activity in the mouse myoblast cell line C2C12).
  • ALP alkaline phosphatase
  • BMP10 and the BMP9 variants herein not only maintain endothelial cell signalling activity but are synergistically devoid of osteogenic activity.
  • native BMP10 and the BMP9 variants as described herein represent a more desirable agonist than native BMP9 for treating, in combination with a TNFa inhibitor, vascular disease or a respiratory disease, in particular PAH, by virtue of lacking the ability to promote bone formation.
  • a therapeutic polypeptide which is either BMP10 or a BMP9 variant lacking osteogenic activity for the treatment of a vascular or respiratory disease such as PAH.
  • the present application discloses for the first time that TNFa drives the development of PAH by repressing BMPRII transcription and activity.
  • anti-TNFa immunotherapy can reverse the disease progression and restore normal signaling in affected pathways.
  • the present invention relates to a composition which combines the therapeutic polypeptide disclosed in WO2016/005756 with a TNFa inhibitor, as well as medical uses of this composition for the treatment of PAH and other vascular and respiratory diseases.
  • composition and methods of the invention involving the use of a therapeutic polypeptide which is BMP10 or a BMP9 variant lacking osteogenic activity, as disclosed in WO2016/005756, and a TNFa inhibitor is more effective, and has wider applicability in different patient groups, than use of either active ingredient alone.
  • BMP10 and "bone morphogenetic protein 10" encompass a human polypeptide belonging to the TGF- ⁇ superfamily of proteins which is encoded by the BMP10 gene (having the sequence shown in SEQ ID NO: 1 ) and which has the 424 amino acid sequence shown in SEQ ID NO: 2, wherein amino acid residues 1 to 21 comprise the signal peptide, amino acid residues 22 to 316 comprise the propeptide, and amino acid residues 317 to 424 comprise mature BMP10. Specific amino acids within BMP10 are numbered herein with reference to the full length sequence.
  • references herein to "a BMP9 variant” and "bone morphogenetic protein 9 variant” encompass a human polypeptide belonging to the TGF- ⁇ superfamily of proteins which is encoded by the BMP9 gene (having the sequence shown in SEQ ID NO: 3) and which has a variant of the 429 amino acid sequence shown in SEQ ID NO: 4 wherein amino acid residues 1 to 22 comprise the signal peptide, amino acid residues 23 to 319 comprise the propeptide and amino acid residues 320 to 429 comprise mature BMP9.
  • BMP9 variants maintain endothelial cell signalling activity but lack osteogenic activity. Specific amino acids within BMP9 are numbered herein with reference to the full length sequence.
  • variant include a genetic variation in the native, non-mutant or wild type sequence of BMP9. Examples of such genetic variations include mutations selected from: substitutions, deletions, insertions and the like.
  • polypeptide refers to a polymer of amino acids. The term does not refer to a specific length of the polymer, so peptides, oligopeptides and proteins are included within the definition of polypeptide.
  • polypeptide may include polypeptides with post-expression modifications, for example, glycosylations, acetylations, phosphorylations and the like. Included within the definition of “polypeptide” are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids), polypeptides with substituted linkages, as well as other modifications known in the art both naturally occurring and non-naturally occurring.
  • references to "lacking osteogenic activity” or “lack osteogenic activity” as used herein may refer to a BMP9 variant comprising one or more, mutations of the sequence of SEQ ID NO: 4 which results in elimination, minimization and/or suppression of osteogenic activity (for example, which may be measured by alkaline phosphatase (ALP) activity in the mouse myoblast cell line C2C12).
  • BMP9 variants included are those which maintain endothelial specific signaling (i.e. those which have at least 0.75 fold ID1 induction compared to wild type BMP9, as measured by ID1 gene expression in HMEC-1 cells) and which have a lower value of osteogenic activity (i.e.
  • Suitable BMP9 variants are those which maintain endothelial specific signaling (i.e. those which have at least 0.75 fold ID1 induction compared to wild type BMP9, as measured by ID1 gene expression in HMEC-1 cells) and negligible osteogenic activity (i.e. less than 0.1 fold compared to wild type BMP9, as measured by ALP activity in the mouse myoblast cell line C2C12).
  • BMP9 variants which have increased endothelial specific signaling (i.e. those which have higher levels of ID1 induction compared to wild type BMP9, as measured by ID1 gene expression in HMEC-1 cells) and negligible osteogenic activity (i.e. less than 0.1 fold compared to wild type BMP9, as measured by ALP activity in the mouse myoblast cell line C2C12).
  • the polypeptide may be BMP10.
  • BMP10 is as potent as BMP9 in inducing ID1 , ID2 and BMPR-II gene expression (see Figures 3A to 3C of WO2016/005756).
  • BMP10 has been shown in WO2016/005756 to exhibit the same anti-apoptotic activity as BMP9 in protecting hPAECs against TNFa-CHX induced apoptosis (see Figure 3D of WO2016/005756).
  • BMP10 did not induce any ALP activity at the highest concentration tested (see Figure 3F of WO2016/005756) unlike BMP9.
  • the polypeptide may be BMP10 comprising the amino acid sequence of SEQ ID NO: 2.
  • the polypeptide may be BMP10 encoded by the nucleotide sequence of SEQ ID NO: 1 .
  • the polypeptide may be the prodomain bound form of BMP10 (pro.BMPI O).
  • pro.BMPI O pro.BMPI O
  • Data are provided in WO2016/005756 which demonstrate that the pro.BMPI O complex is very stable (see Figures 4B and 4C of WO2016/005756) and is likely to be a suitable form of BMP10 for the treatment of vascular and respiratory diseases, such as PAH.
  • the pro.BMPI O may comprise a propeptide sequence having the amino acid sequence of residues 22-316 of SEQ ID NO: 2 non-covalently bound to a mature BMP10 sequence having the amino acid sequence of residues 317-424 of SEQ ID NO: 2.
  • the pro.BMPI O may comprise a tetramer containing two of the above-mentioned propeptide sequences and two of the above-mentioned mature BMP10 sequences.
  • the polypeptide may be a BMP9 variant lacking osteogenic activity.
  • the polypeptide may be a variant of the prodomain bound form of BMP9 (pro.BMP9) lacking osteogenic activity.
  • the variant of pro.BMP9 may comprise a variant of: the propeptide sequence having the amino acid sequence of residues 23-319 of SEQ ID NO: 4 non-covalently bound to a mature BMP9 sequence having the amino acid sequence of residues 320-429 of SEQ ID NO: 4.
  • the variant of pro.BMP9 may comprise a tetramer containing two of the above- mentioned propeptide sequences and two of the above-mentioned mature BMP9 sequences.
  • the BMP9 variant lacking osteogenic activity may comprise a substitution, deletion or insertion mutant of the amino acid sequence of SEQ ID NO: 4.
  • the BMP9 variant lacking osteogenic activity may comprise a substitution mutant of the amino acid sequence of SEQ ID NO: 4.
  • the substitution mutant of the amino acid sequence of SEQ ID NO: 4 may comprise one or more (i.e. single, double, triple mutants etc.) of the following substitutions: H326A, D342A, S343A, W344A, I346A, K349A, F362A, D366A, K372A, I375A, L379A, H381 A, L382A, K383A, K390A, S402A, L404A, K406A, D408A, V41 1A, T413A, L414A, Y416A and Y418A.
  • the BMP9 variant lacking osteogenic activity may be selected from one of the following BMP9 variants of SEQ ID NO: 4: H326A, D342A, S343A, W344A, I346A, K349A, F362A, D366A, K372A, I375A, L379A, H381A, L382A, K383A, K390A, S402A, L404A, K406A, D408A, V41 1 A, T413A, L414A, Y416A and Y418A.
  • the substitution mutant of the amino acid sequence of SEQ ID NO: 4 may comprise one or more (i.e.
  • the BMP9 variant lacking osteogenic activity may be selected from one of the following BMP9 variants of SEQ ID NO: 4: H326A, S343A, K349A, F362A, D366A, I375A, L379A, L382A, K390A, S402A, D408A, Y416A and Y418A.
  • substitution mutant of the amino acid sequence of SEQ ID NO: 4 may comprise one or more (i.e. single, double, triple mutants etc.) of the following substitutions: F362A, D366A, I375A, L379A, S402A, D408A, Y416A and Y418A.
  • the BMP9 variant lacking osteogenic activity may be selected from one of the following BMP9 variants of SEQ ID NO: 4: F362A, D366A, I375A, L379A, S402A, D408A, Y416A and Y418A.
  • Data are provided in WO2016/005756 which demonstrate that these mutant sequences maintain the beneficial effect of endothelial specific signaling but lack osteogenic signaling (as evidenced by at least 0.75 fold ID1 induction and negligible (i.e. less than 0.1 fold) ALP activity when compared to wild type BMP9; see Figure 5 in WO2016/005756).
  • the substitution mutant of the amino acid sequence of SEQ ID NO: 4 may comprise one or both (i.e. a single or double mutant) of the following substitutions: D366A or D408A.
  • the BMP9 variant lacking osteogenic activity may be selected from one of the following BMP9 variants of SEQ ID NO: 4: D366A or D408A.
  • Data are provided in WO2016/005756 which demonstrate that these mutant sequences maintain the beneficial effect of BMP9 but are not able to initiate the osteogenic signaling and hence remove the potential risk of bone formation by administration of BMP9 in vivo (see the results shown in Figure 2 of WO2016/005756).
  • Data are also provided in WO2016/005756 which demonstrate that these mutant sequences have increased endothelial specific signaling but lack osteogenic signaling (as evidenced by a greater than 1 fold ID1 induction and negligible (i.e. less than 0.1 fold) ALP activity when compared to wild type BMP9; see Figure 5 of WO2016/005756).
  • the BMP9 variant lacking osteogenic activity may be selected from a D408A BMP9 variant of SEQ ID NO: 4.
  • Data are provided in WO2016/005756 which demonstrate that this mutant sequence has been shown to be able to rescue PAEC early apoptosis induced by tumor necrosis factor a (TNFa) and cycloheximide (CHX) (see the results shown in Figure 7 of WO2016/005756).
  • TNFa tumor necrosis factor a
  • CHX cycloheximide
  • the BMP9 variant lacking osteogenic activity may be selected from a D366A BMP9 variant comprising the amino acid sequence of SEQ ID NO: 5 or a D408A BMP9 variant comprising the amino acid sequence of SEQ ID NO: 6.
  • the BMP9 variant lacking osteogenic activity may be selected from a D366A/D408A double mutant BMP9 variant comprising the amino acid sequence of SEQ ID NO: 7.
  • Data concerning the biological activity of the D366A/D408A double mutant BMP9 variant comprising the amino acid sequence of SEQ ID NO: 7 can be found in US patent application no. 15/404,265 filed on 12 January 2017 as a continuation-in-part application from US patent application no. 15/324,864 (a US national phase application of WO2016/005756).
  • the double mutant BMP9 variant of SEQ ID NO: 7 has potent endothelial cell signalling activity, comparable with D366A and D408A single mutants and the wild type, but does not show show any osteogenic signalling activity in vitro.
  • the TNFa inhibitor of the composition may be selected from the group consisting of etanercept, infliximab (or "biosimilar inf!iximabs” such as "Inflectra” or “Remsima”), adalimumab, golimumab, certolizumab pegol, thalidomide, a thalidomide derivative (such as lenalidomide), a xanthine derivative (such as pentoxifylline), bupropion, a phosphodiesterase IV inhibitor, a pegylated soluble TNFa Receptor Type I (PEGs TNFa-R1 ), an agent containing a soluble TNFa receptor, and CDP571 (a humanized monoclon
  • the polypeptide and TNFa inhibitor may be in amounts effective in combination to treat a vascular disease or a respiratory disease.
  • the amount of polypeptide may be from about 1 ng to about 2 g.
  • the amount of TNFa inhibitor may be from about 1 ng to about 2 g, for example about 12.5 mg or about 25 mg or about 50 mg.
  • TNFa inhibitor for use in the invention is illustrated by the following specific examples:
  • - golimumab (Simponi®) - Initially: 50 mg once per month as a self-administered subcutaneous injection. Maintenance: same; - golimumab (Simponi Aria®) - Initially: Given as an IV at a dose of 2 mg/kg (according to body weight) at weeks 0 and 4. Maintenance: IV infusions every 8 weeks; and - certolizumab (Cimzia®) - Intially: Given as treatment starts with two (200mg) subcutaneous injections. Two further injections at the same dose, at 2 and 4 weeks later. Maintenance: 200 mg given as a single injection every fortnight.
  • the pharmaceutical composition of the invention may be sterile.
  • the invention further provides pharmaceutical compositions, as defined above, including one or more pharmaceutically acceptable excipients and optionally other therapeutic or prophylactic agents.
  • the pharmaceutically acceptable excipient(s) can be selected from, for example, carriers (e.g. a solid, liquid or semi-solid carrier), adjuvants, diluents, fillers or bulking agents, granulating agents, coating agents, release-controlling agents, binding agents, disintegrants, lubricating agents, preservatives, antioxidants, buffering agents, suspending agents, thickening agents, flavoring agents, sweeteners, taste masking agents, stabilizers or any other excipients conventionally used in pharmaceutical compositions. Examples of excipients for various types of pharmaceutical compositions are set out in more detail below.
  • compositions containing the polypeptides and TNFa inhibitor of the invention can be formulated in accordance with known techniques, see for example, Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA, USA.
  • compositions can be in any form suitable for oral, parenteral, topical, intranasal, intrabronchial, sublingual, ophthalmic, otic, rectal, intra-vaginal, or transdermal administration.
  • compositions are intended for parenteral administration, they can be formulated for intravenous, intramuscular, intraperitoneal, subcutaneous administration or for direct delivery into a target organ or tissue by injection, infusion or other means of delivery.
  • the delivery can be by bolus injection, short term infusion or longer term infusion and can be via passive delivery or through the utilization of a suitable infusion pump or syringe driver.
  • compositions adapted for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats, co-solvents, surface active agents, organic solvent mixtures, cyclodextrin complexation agents, emulsifying agents (for forming and stabilizing emulsion formulations), liposome components for forming liposomes, gellable polymers for forming polymeric gels, lyophilization protectants and combinations of agents for, inter alia, stabilizing the active ingredient in a soluble form and rendering the formulation isotonic with the blood of the intended recipient.
  • Pharmaceutical formulations for parenteral administration may also take the form of aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents (see for example Strickly 2004, Pharmaceutical Research, 21 :201 -230).
  • a method of making a pharmaceutical composition comprising admixing the composition ingredients as described herein is also encompassed by the invention.
  • Formulations of the pharmaceutical composition of the invention may be presented in unit-dose or multi-dose containers, for example sealed ampoules, vials and prefilled syringes, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use.
  • a formulation can be prepared by lyophilizing a polypeptide and/or TNFa inhibitor of the invention. Lyophilization refers to the procedure of freeze-drying a composition. Freeze-drying and lyophilization are used herein as synonyms.
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets.
  • compositions of the present invention for parenteral injection can also comprise pharmaceutically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use.
  • aqueous and nonaqueous carriers, diluents, solvents or vehicles examples include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (such as sunflower oil, safflower oil, corn oil or olive oil), and injectable organic esters such as ethyl oleate.
  • polyols such as glycerol, propylene glycol, polyethylene glycol, and the like
  • carboxymethylcellulose and suitable mixtures thereof examples include vegetable oils (such as sunflower oil, safflower oil, corn oil or olive oil), and injectable organic esters such as ethyl oleate.
  • vegetable oils such as sunflower oil, safflower oil, corn oil or olive oil
  • injectable organic esters such as ethyl oleate.
  • Proper fluidity can be maintained, for example, by the use of thickening or coating materials such as lecit
  • compositions of the present invention may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include agents to adjust tonicity such as sugars, sodium chloride, and the like. Prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.
  • the pharmaceutical composition may be in a form suitable for IV administration, for example by injection or infusion.
  • the solution can be dosed as is, or can be injected into an infusion bag (containing a pharmaceutically acceptable excipient, such as 0.9% saline or 5% dextrose), before administration.
  • a pharmaceutically acceptable excipient such as 0.9% saline or 5% dextrose
  • the pharmaceutical composition may be in a form suitable for sub-cutaneous (s.c.) administration.
  • Pharmaceutical dosage forms suitable for oral administration include tablets (coated or uncoated), capsules (hard or soft shell), caplets, pills, lozenges, syrups, solutions, powders, granules, elixirs and suspensions, sublingual tablets, wafers or patches such as buccal patches.
  • tablet compositions can contain a unit dosage of active polypeptide and/or TNFa inhibitor together with an inert diluent or carrier such as a sugar or sugar alcohol, e.g.; lactose, sucrose, sorbitol or mannitol; and/or a non-sugar derived diluent such as sodium carbonate, calcium phosphate, calcium carbonate, or a cellulose or derivative thereof such as microcrystalline cellulose (MCC), methyl cellulose, ethyl cellulose, hydroxypropyl methyl cellulose, and starches such as corn starch.
  • Tablets may also contain such standard ingredients as binding and granulating agents such as polyvinylpyrrolidone, disintegrants (e.g.
  • swellable crosslinked polymers such as crosslinked carboxymethylcellulose
  • lubricating agents e.g. stearates
  • preservatives e.g. parabens
  • antioxidants e.g. BHT
  • buffering agents for example phosphate or citrate buffers
  • effervescent agents such as citrate/bicarbonate mixtures.
  • excipients are well known and do not need to be discussed in detail here. Tablets may be designed to release the drug either upon contact with stomach fluids (immediate release tablets) or to release in a controlled manner (controlled release tablets) over a prolonged period of time or with a specific region of the Gl tract.
  • Capsule formulations may be of the hard gelatin or soft gelatin variety and can contain the active component in solid, semi-solid, or liquid form.
  • Gelatin capsules can be formed from animal gelatin or synthetic or plant derived equivalents thereof.
  • the solid dosage forms e.g.; tablets, capsules etc.
  • Coatings may act either as a protective film (e.g. a polymer, wax or varnish) or as a mechanism for controlling drug release or for aesthetic or identification purposes.
  • the coating e.g. a EudragitTM type polymer
  • the coating can be designed to release the active component at a desired location within the gastro-intestinal tract.
  • the coating can be selected so as to degrade under certain pH conditions within the gastrointestinal tract, thereby selectively release the polypeptide and/or TNFa inhibitor in the stomach or in the ileum, duodenum, jejunum or colon.
  • the pharmaceutical composition may be presented in a solid matrix comprising a release controlling agent, for example a release delaying agent which may be adapted to release the polypeptide in a controlled manner in the gastrointestinal tract.
  • the composition can be presented in a polymer coating e.g. a polymethacrylate polymer coating, which may be adapted to selectively release the polypeptide under conditions of varying acidity or alkalinity in the gastrointestinal tract.
  • the matrix material or release retarding coating can take the form of an erodible polymer (e.g. a maleic anhydride polymer) which is substantially continuously eroded as the dosage form passes through the gastrointestinal tract.
  • the coating can be designed to disintegrate under microbial action in the gut.
  • the active polypeptide can be formulated in a delivery system that provides osmotic control of the release of the polypeptide. Osmotic release and other delayed release or sustained release formulations (for example formulations based on ion exchange resins) may be prepared in accordance with methods well known to those skilled in the art.
  • the polypeptides and/or TNFa inhibitor of the invention may be formulated with a carrier and administered in the form of nanoparticles, the increased surface area of the nanoparticles assisting their absorption.
  • nanoparticles offer the possibility of direct penetration into the cell.
  • Nanoparticle drug delivery systems are described in "Nanoparticle Technology for Drug Delivery", edited by Ram B Gupta and Uday B. Kompella, Informa Healthcare, ISBN 9781574448573, published 13th March 2006. Nanoparticles for drug delivery are also described in J. Control. Release, 2003, 91 : 167-172, and in Sinha et al., 2006, Mol. Cancer Ther. 5: 1909.
  • compositions of the invention may typically comprise from approximately 1 % (w/w) to approximately 95% (w/w) active ingredients and from 99% (w/w) to 5% (w/w) of a pharmaceutically acceptable excipient or combination of excipients.
  • the compositions may comprise from approximately 20% (w/w) to approximately 90% (w/w) active ingredients and from 80% (w/w) to 10% of a pharmaceutically acceptable excipient or combination of excipients.
  • the pharmaceutical compositions may comprise from approximately 1 % to approximately 95%, particularly from approximately 20% to approximately 90%, active ingredients.
  • Pharmaceutical compositions according to the invention may be, for example, in unit dose form, such as in the form of ampoules, vials, suppositories, pre-filled syringes, dragees, tablets or capsules.
  • the pharmaceutically acceptable excipient(s) can be selected according to the desired physical form of the formulation and can, for example, be selected from diluents (e.g. solid diluents such as fillers or bulking agents; and liquid diluents such as solvents and co-solvents), disintegrants, buffering agents, lubricants, flow aids, release controlling (e.g. release retarding or delaying polymers or waxes) agents, binders, granulating agents, pigments, plasticizers, antioxidants, preservatives, flavoring agents, taste masking agents, tonicity adjusting agents and coating agents.
  • diluents e.g. solid diluents such as fillers or bulking agents; and liquid diluents such as solvents and co-solvents
  • disintegrants e.g. solid diluents such as fillers or bulking agents
  • lubricants such as solvents and co-solvents
  • flow aids
  • tablets and capsules typically contain 0-20% disintegrants, 0-5% lubricants, 0-5% flow aids and/or 0-99% (w/w) fillers/ or bulking agents (depending on drug dose). They may also contain 0-10% (w/w) polymer binders, 0-5% (w/w) antioxidants, 0-5% (w/w) pigments. Slow release tablets would in addition contain 0- 99% (w/w) release-controlling (e.g. delaying) polymers (depending on dose).
  • the film coats of the tablet or capsule typically contain 0-10% (w/w) polymers, 0-3% (w/w) pigments, and/or 0-2% (w/w) plasticizers.
  • Parenteral formulations typically contain 0-20% (w/w) buffers, 0-50% (w/w) cosolvents, and/or 0-99% (w/w) Water for Injection (WFI) (depending on dose and if freeze dried).
  • WFI Water for Injection
  • Formulations for intramuscular depots may also contain 0-99% (w/w) oils.
  • compositions for oral administration can be obtained by combining the active ingredients with solid carriers, if desired granulating a resulting mixture, and processing the mixture, if desired or necessary, after the addition of appropriate excipients, into tablets, dragee cores or capsules. It is also possible for them to be incorporated into a polymer or waxy matrix that allow the active ingredients to diffuse or be released in measured amounts.
  • the polypeptides and/or TNFa inhibitor of the invention can also be formulated as solid dispersions.
  • Solid dispersions are homogeneous extremely fine disperse phases of two or more solids.
  • Solid solutions molecularly disperse systems, one type of solid dispersion, are well known for use in pharmaceutical technology (see Chiou and Riegelman, 1971 , J. Pharm. Sci., 60, 1281 -1300) and are useful in increasing dissolution rates and increasing the bioavailability of poorly water-soluble drugs.
  • Solid dosage forms include tablets, capsules, chewable tablets and dispersible or effervescent tablets.
  • Known excipients can be blended with the solid solution to provide the desired dosage form.
  • a capsule can contain the solid solution blended with (a) a disintegrant and a lubricant, or (b) a disintegrant, a lubricant and a surfactant.
  • a capsule can contain a bulking agent, such as lactose or microcrystalline cellulose.
  • a tablet can contain the solid solution blended with at least one disintegrant, a lubricant, a surfactant, a bulking agent and a glidant.
  • a chewable tablet can contain the solid solution blended with a bulking agent, a lubricant, and if desired an additional sweetening agent (such as an artificial sweetener), and suitable flavors.
  • Solid solutions may also be formed by spraying solutions of drug and a suitable polymer onto the surface of inert carriers such as sugar beads ('non-pareils'). These beads can subsequently be filled into capsules or compressed into tablets.
  • the pharmaceutical composition may be presented to a patient in "patient packs" containing an entire course of treatment in a single package, usually a blister pack.
  • compositions for topical use and nasal delivery include ointments, creams, sprays, patches, gels, liquid drops and inserts (for example intraocular inserts). Such compositions can be formulated in accordance with known methods.
  • formulations for rectal or intra-vaginal administration include pessaries and suppositories which may be, for example, formed from a shaped moldable or waxy material containing the active polypeptide. Solutions of the active polypeptide and/or TNFa inhibitor may also be used for rectal administration.
  • compositions for administration by inhalation may take the form of inhalable powder compositions or liquid or powder sprays, and can be administrated in standard form using powder inhaler devices or aerosol dispensing devices. Such devices are well known.
  • the powdered formulations typically comprise the active polypeptide and/or TNFa inhibitor together with an inert solid powdered diluent such as lactose.
  • the polypeptide and/or TNFa inhibitor active ingredients of the invention may be presented in unit dosage form and, as such, will typically contain sufficient polypeptide and/or TNFa inhibitor to provide a desired level of biological activity.
  • a formulation may contain from 1 ng to 2 g of active ingredient, e.g.
  • polypeptide and/or TNFa inhibitor from 1 ng to 2 mg of active ingredient.
  • particular sub-ranges of polypeptide and/or TNFa inhibitor are 0.1 mg to 2 g of active ingredient (more usually from 10 mg to 1 g, e.g. 50 mg to 500 mg), or 1 ⁇ g to 20 mg (for example, 1 ⁇ g to 10 mg, e.g. 0.1 mg to 2 mg of active ingredient).
  • a unit dosage form may contain from 1 mg to 2 g, more typically 10 mg to 1 g, for example 50 mg to 1 g, e.g. 100 mg to 1 g, of active polypeptide and/or TNFa inhibitor.
  • the active polypeptide and/or TNFa inhibitor may be administered to a patient in need thereof (for example a human or animal patient) in an amount sufficient to achieve the desired therapeutic effect.
  • compositions as defined herein in the manufacture of a medicament for the treatment of a vascular disease or a respiratory disease, wherein the polypeptide and the TNFa inhibitor of the composition are prepared to be administered to a patient in need thereof simultaneously, contemporaneously or concomitantly.
  • vascular disease or a respiratory disease in a patient in need thereof, the method comprising administering to the patient an effective amount of a polypeptide as defined herein, in combination with an effective amount of a TNFa inhibitor as defined herein.
  • the polypeptide and TNFa inhibitor of the invention may be administered simultaneously, contemporaneously or concomitantly.
  • the TNFa inhibitor may be administered: (a) prior to administration of the polypeptide of the invention; (b) in doses alternating with administration of the polypeptide of the invention (such as on/off dosing); and/or (c) in reducing doses together or alternating with administration of the polypeptide of the invention.
  • the effects of the TNFa inhibitor may be assessed in patients by determining the levels of cytokines such as for example TNFa, IL-6, IL8 and/or MCP1 . Lower levels of one or more of these cytokines, for example IL-6, may be associated with improved patient survival.
  • the vascular disease treated by the composition for use or method may be selected from: pulmonary hypertension; pulmonary arterial hypertension (PAH); hereditary haemorrhagic telangiectasia; atherosclerosis; and hepatopulmonary syndrome.
  • the vascular disease may be PAH.
  • the respiratory disease treated by the composition for use or method may be selected from: obstructive lung diseases such as chronic obstructive pulmonary disease (COPD), chronic bronchitis and emphysema; pulmonary vascular diseases such as pulmonary edema and pulmonary hemorrhage; respiratory failure and respiratory distress syndrome, such as acute lung injury and acute respiratory distress syndrome; and interstitial lung diseases, such as idiopathic pulmonary fibrosis.
  • COPD chronic obstructive pulmonary disease
  • COPD chronic obstructive pulmonary disease
  • pulmonary vascular diseases such as pulmonary edema and pulmonary hemorrhage
  • respiratory failure and respiratory distress syndrome such as acute lung injury and acute respiratory distress syndrome
  • interstitial lung diseases such as idiopathic pulmonary fibrosis.
  • Gene therapy comprising the BMP10 or BMP9 variant of the invention together with administration of the TNFa inhibitor is also encompassed by the present invention.
  • a vector encoding the BMP10 or BMP9 variant nucleotide sequence may be administered to the host human subject resulting in endogenous expression (such as endogenous expression in the liver) of the BMP10 or BMP9 variant polypeptide for release into the circulation.
  • the TNFa inhibitor may then be administered to the subject with endogenous expression of the BMP10 or BMP9 variant polypeptide.
  • a vector comprising a nucleotide sequence encoding BMP10 or a BMP9 variant for use in the treatment together with a TNFa inhibitor of a vascular disease or a respiratory disease (such as PAH).
  • the vector may comprise the nucleotide sequence of SEQ ID NO: 1 .
  • the vector may be a viral vector, for example a retrovirus, adenovirus, lentivirus, herpes simplex, vaccinia or adeno-associated virus.
  • the vector may be a non-viral vector.
  • the use of non-viral vectors has a number of advantages over the use of viral vectors, such as ease of large scale production and low immunogenicity in the host.
  • Examples of non-viral gene therapy methods include: injection of naked DNA, electroporation, gene gun, sonoporation, magnetofection and the use of oligonucleotides, lipoplexes, dendrimers, and inorganic nanoparticles.
  • a package comprising:
  • a) a first pharmaceutical composition comprising a polypeptide as defined herein;
  • EXAMPLE 1 - TNFa drives pulmonary hypertension via BMPR-II suppression and NOTCH dysregulation Introduction
  • BMPR-II forms heteromeric cell surface receptor complexes with activin-like kinase (ALK) type I receptors, mediating BMP2, BMP4 and BMP6 signaling with ALK3 pulmonary artery smooth muscle cells (PASMCs), or mediating endothelial BMP9/10 responses with ALK1 .
  • ALK activin-like kinase
  • the activated receptors phosphorylate the canonical SMAD1/5/8 proteins that promote the transcription of genes including the Inhibitor of DNA binding (ID) gene family and NOTCH pathways.
  • BMPs can also signal independently of SMADs, through mitogen activated protein kinases (MAPKs), and proto-oncogene protein tyrosine kinase c-SRC (c-SRC) phosphorylation.
  • MAPKs mitogen activated protein kinases
  • c-SRC proto-oncogene protein tyrosine kinase c-SRC
  • TNFa reduces endothelial BMPR-II expression
  • TNFa might critically reduce BMPR-II expression in vascular cells harboring BMPR2 mutations and switch BMP signaling to recruit ACTR-IIA and ALK2, with potentially pathological consequences.
  • TNFa reduces BMPR-II in vascular cells and promotes ADAM 10/17-dependent BMPR-II cleavage in PASMCs, releasing the soluble ectodomain which acts a ligand trap.
  • the combined impact of genetic loss-of-function of BMPR-II with TNFa-mediated suppression of BMPR-II levels in PASMCs drives inappropriate proliferation through c-SRC family members and dysregulated NOTCH2/3 signaling. Similar signaling changes were observed in the lungs of rat and mouse PAH models.
  • therapeutic etanercept administration reversed the progression of PAH in the rat Sugen-hypoxia model and inhibited proliferative NOTCH signaling.
  • PAECs Human pulmonary artery endothelial cells
  • Lonza Basel, Switzerland
  • FBS 2% FBS
  • FBS foetal bovine serum
  • Distal human pulmonary arterial smooth muscle cells were derived from small vessels ( ⁇ 2mm diameter) lung resection specimens and proximal PASMCs isolated from vessel segments (5-8mm diameter) as described previously (Davies et al., 2012, Am.J. Physiol Lung Cell Mol. Physiol 302:L604-L615).
  • a summary of clinical information of the control and HPAH PASMCs used is provided in Table 1 .
  • DMEM Dulbecco's Modified Eagle Media
  • FBS FBS
  • A/A DMEM/10%.
  • Rat PASMCs were isolated from small pulmonary arteries, as described previously (Phillips et al., 2005, Am J Physiol Lung Cell Mol Physiol 288:L103-1 15).
  • Mouse PASMCs were isolated from small pulmonary arteries, as described previously (Long et al., 2006, Circ.Res. 98:818-827).
  • Human aortic smooth muscle cells isolated from patients under local ethics approval, were kindly provided by Dr Murray Clarke (University of Cambridge, UK). All smooth muscle cell lines were used between passages 4 and 8.
  • C2C12-BRE cells were cultured in DMEM/10% containing 2 mM L-glutamine and 700 Mg/ml G418) (Herrera & Inman, 2009, BMC.Cell Biol. 10:20). Smooth muscle cells and C2C12-BRE cells were quiesced in DMEM containing 0.1 % FBS and A/A (DMEM/0.1 %) overnight prior to treatments.
  • Recombinant human TNFa, BMP2, BMP4, BMP6, IL-1 ⁇ , IL-6 and IL-8 were purchased from R&D Systems (Oxfordshire, UK).
  • Recombinant mouse TNFa was purchased from PeproTech (NJ, USA).
  • TNFa (1 ng/mL) for 24 h, unless otherwise indicated.
  • Cells were stimulated with BMP2, BMP4 or BMP6 (10 ng/mL unless otherwise stated) or co-stimulated with TNFa and BMP ligand as indicated.
  • the metalloprotease inhibitor batimastat (BB94) (10 ng/mL) was a kind gift from Dr. Murray Clarke (University of Cambridge, UK).
  • ADAM 10 inhibitor GI254023X (10 ⁇ ) was a kind gift from Prof. Andreas Ludwig (RWTH AACHEN, Germany).
  • the anti-ADAM17 antibody, D1 (A12) (50 nM) was a kind gift from Prof. Gillian Murphy (Cancer Research UK Research Institute, Cambridge, UK).
  • ADAM10/17 inhibitor TAPI-1 (10 ⁇ ) was purchased from Enzo Life Sciences (Devon, UK).
  • the ⁇ -secretase inhibitor, DAPT was from Sigma. Unless otherwise stated, cells were pretreated with inhibitors for 30 minutes before TNFa stimulation and then added to cells for a total of 24 h. For immunoneutralization studies, treatments were preincubated with ⁇ g/ml monoclonal anti-BMP2 or anti-BMP6 (R&D Systems) for 1 h prior to adding to cells. CelS proliferation.
  • the pcDNA3 expression plasmid encoding 5'- myc-tagged BMPR-II wild type was prepared as previously described (Rudarakanchana et al., 2002, Hum. Mol. Genet. 1 1 :1517-1525). Mutant myc-tagged BMPR-II V158A, V160A, V163A and V166A plasmids were created using the QuikChangeTM Site-Directed Mutagenesis kit (Agilent Technologies, Cheshire, UK) according to the manufacturer's instructions (see Table 2). The presence of each mutation was verified by sequencing.
  • AdCMVBMPR2myc and kinase-dead AdCMVBMPR2(D485G)myc The synthesis of the AdCMVBMPR2myc and kinase-dead AdCMVBMPR2(D485G)myc, replication incompetent serotype 5 adenoviral vectors and production and titration of viral particles was described previously (Southwood et al., 2008, J.Pathol. 214:85-95). Cells were infected with 50 plaque-forming units (pfu) per cell for 4 h in serum-free DMEM and this was then replaced with DM EM/ 10% for 16 h.
  • pfu plaque-forming units
  • Plasmid Transfections Plasmids were prepared using the PureLinkTM maxiprep kit (Invitrogen, Renfrewshire, UK), according to the manufacturer's instructions. Prior to transfection, PASMCs were incubated with Opti-MEM-I for 2 h. Cells were transiently transfected with 4 ⁇ g of expression plasmid using 2 ⁇ LipofectamineTM 2000 reagent (Invitrogen) in Opti-MEM-I. Cells were incubated with transfection mixes for 4 h, followed by replacement with DMEM/10% for 48 h prior to quiescence and treatment as indicated. Transfection efficiency was confirmed via BMPR-II and Myc tag immunoblotting. siRNA Transfections.
  • PASMCs Prior to transfection, PASMCs were incubated with Opti-MEM-I serum-free medium (Life Technologies) for 3 h before adding 10 nM siRNA lipoplexed with DharmaFECTITM (Dharmacon, MA, USA) siRNA/Dharmafect complexes were allowed to form for 20 minutes at room temperature before being added to the cells. Cells were then incubated with the complexes for 4 h at 37 ° C before returning to DMEM/10% overnight. Knockdown efficiency was confirmed by immunoblotting.
  • siRNAs used were: ON-TARGETP/usTM siGENOMETM Smartpool oligos for (>x% values represent knockdown at RNA level): ACVR2A (>73%), ADAM10, ADAM17, ALK2 (>84%), BMPR2 (>75%), RELA (>82%, encoding NF- ⁇ p65) or a non-targeting control pool (siCP) (Thermo Fisher, Waltham, MA) or oligos targeting FYN (>64%), HEY1 (>63%), HEY 2 (>63%), NOTCH2 (>50%), NOTCH3 (>75%), SRC (>62%) or YES (>68%) from Sigma-Aldrich. For proliferation experiments, we confirmed that the level of knockdown was similar at Days 2,4 and 6 for each target.
  • Cell lysates (20-100 ⁇ g protein) were separated by SDS-PAGE and proteins transferred to polyvinylidene fluoride membranes by semidry blotting (GE Healthcare, Buckinghamshire, UK). Membranes were then blocked and probed with rabbit polyclonal antibodies toward total Smadl , phosphorylated SRC(Y527) (all Cell Signaling Technology, Danvers, MA), ADAM10, ADAM 17 (Abeam, Cambridgeshire, UK); rabbit monoclonal antibodies toward phosphorylated Smad1/5, caspase-3, cleaved caspase-3, NOTCH1 , NOTCH2, NOTCH3, phospho-SRC(Y416) and SRC (Cell Signaling Technology, Danvers, MA), ID1 (CalBioreagents, San Mateo, CA); or mouse monoclonal antibodies toward BMPR-II (BD Transduction Laboratories, Franklin Lakes, NJ) or c-Myc (Santa Cruz Biotechnology, TX, USA).
  • BMPR-II BD Transduction Labor
  • blots were incubated with secondary anti-mouse/rabbit horseradish peroxidase antibody (Dako, Glostrup, Denmark) for 1 h at room temperature. As a loading control, all blots were re-probed with a monoclonal antibody toward either a-tubulin (Sigma) or ⁇ -actin (Sigma). Densitometry was performed using imaged software. Membranes were developed using enhanced chemiluminescence (GE Healthcare).
  • Protein was deglycosylated using PNGase F according to the manufacturer's instructions (New England Biolabs). Approximately 60-80 ⁇ g of protein was deglycosylated and then fractionated by SDS-PAGE.
  • Immunoprecipitation Conditioned media were taken from transfected PASMCs prior to lysis. Media were centrifuged at 2,000 x g (4°C) to remove cellular debris and stored in 1 ml aliquots at -80°C. For immunoprecipitation, 1 ml culture media were incubated with a mouse monoclonal toward BMPR-II (R&D Systems, Oxford, UK) overnight on a rotary mixer at 4°C. Antibody:protein complexes were isolated by incubation with protein-G sepharose beads (Sigma-Aldrich) for 4 h on a rotary mixer at 4°C.
  • BMPR-II ECD was measured using an in-house ELISA.
  • ELISA was performed as previously described (Farahi et al., 2007, J.Immunol. 179:1264-1273) with the following modifications. Briefly, flat-bottom high binding 96-well ELISA plates (Greiner, South Lanarkshire, UK) were coated with 1 ⁇ g/ml mouse monoclonal anti-human BMPR-II antibody (R&D Systems) for 2 h at room temperature.
  • C2C12-BRE cells were treated with BMP2 or BMP4 (at 1 and 10 ng/ml) in the presence or absence of commercially available glycosylated His-tagged BMPR-II-ECD (Sino Biologicals, Beijing, P. R. China) or conditioned media from transfected PASMCs stimulated with TNFa. Luciferase activity in the cells was assessed using a luciferase reporter assay kit (Roche).
  • RNA Preparation and Quantitative RT-PCR Total RNA was extracted using the RNeasy Mini Kit with DNAse digestion (Qiagen, West Wales, UK). cDNA was prepared from ⁇ 1 ⁇ g of RNA using the High Capacity Reverse Transcriptase kit (Applied Biosystems, California, USA), according to the manufacturer's instructions. All qPCR reactions were prepared in MicroAmp® optical 96-well reaction plates (Applied Biosystems) using 50 ng/ ⁇ cDNA with SYBR®Green JumpstartTM Taq ReadymixTM (Sigma-Aldrich), ROX reference dye (Invitrogen) and custom sense and anti-sense primers (all 200 nM).
  • Primers for human ACTB (encoding ⁇ -actin), ADAM10, ADAM12, ADAM15, ADAM17, ALK3, BMPR2, HES1, HEY1, HEY2, ID1, MMP14, MMP15, MMP16, MMP17, MMP24, NOTCH3; mouse Acvr2a (encoding Actr-lla), Alk2, Bmpr2, Notch i, Notch2, Notch3 were all designed using Primer3 (http://primer3.sourceforge.net/) (Tables 3-5).
  • QuantiTect primers for: human ACVR2A (encoding ACTR-IIA), ALK2, ALK3, ALK6, BMP2, BMP4, BMP6, BMP7, BMP9, IL8, NOTCH1, NOTCH 2; mouse Bmp2, Bmp6; and rat Bmpr2, Notchi, Notch2, Notch3, Tnf Reactions were amplified on a StepOnePlusTM Real-Time PCR system (Applied Biosystems).
  • Target gene expression was normalized to ACTB/Actb and the difference in the amount of product produced was expressed as a fold change.
  • the relative abundance of BMP ligands was calculated, on the assumption of equal copy number, by calculating the expression of each BMP gene relative to ACTB after normalization to B2M.
  • mice were anesthetized with 0.5mg/kg fentanyl and 25mg/kg fluanisone (Hypnorm®, VetaPharma Ltd, Leeds, UK) and 12.5mg/kg midazolam (Hypnovel®), and right ventricular pressures and volumes were recorded using a Millar PVR-1045 catheter (Millar Instruments, Houston, TX). Mice were then sacrificed and the hearts, lungs and livers were harvested. Right ventricular hypertrophy (RVH) was assessed by removing the heart and dissecting the right ventricle (RV) free wall from the left ventricle plus septum (LV+S) and weighing separately. The degree of right ventricular hypertrophy was determined from the ratio RV/LV+S.
  • RVH right ventricular hypertrophy
  • the right lung was snap frozen in liquid nitrogen.
  • the left lung was inflated with a 1 :1 mixture of saline and O.C.T. compound (Sakura, Zoeterwoude, Netherlands) and fixed with 4% paraformaldehyde in PBS before dehydration and paraffin embedding.
  • mice Male Sprague Dawley rats (-150 to 200 g, Charles River) were given a single i.p. injection of Sugen 5416 (SU-5416; 20 mg/kg, Tocris, Bristol, UK) in vehicle (0.5% carboxyi methylce!luiose sodium, 0.4% poiysorbate 80, 0.9% benzyl alcohol, all Sigma), placed immediately into a 10% Oa chamber and maintained in hypoxia for 3 weeks, followed by 5 weeks in a normoxic environment to develop pulmonary hypertension. At the 8-week timepoint, rats were randomized into 2 groups. One group received i.p.
  • Sugen 5416 SU-5416; 20 mg/kg, Tocris, Bristol, UK
  • vehicle 0.5% carboxyi methylce!luiose sodium, 0.4% poiysorbate 80, 0.9% benzyl alcohol, all Sigma
  • the primary smooth muscle a-actin antibody was labeled with a modified biotinylated anti-mouse before application to the specimen.
  • the primary antibody and biotinylation reagent were mixed in solution, resulting in binding of biotinylated secondary antibody to the primary antibody.
  • the blocking reagent containing normal mouse serum, was then added to the mixture to bind residual biotinylation reagent not bound to the primary antibody, minimizing the potential interaction of the biotinylated anti-mouse secondary reagent with endogenous immunoglobulin present in the specimen.
  • T he biotin-iabeled primary antibody was then applied to the specimen followed by incubation with streptavidin- peroxidase and reaction with diaminobenzidine ⁇ DAB)-hydrogen peroxide as substrate-chromogen.
  • Assessment of pulmonary arteriolar muscuiarization involved the identification of alveolar ducts and the subsequent categorization of the accompanying intraacinar artery as non- , partially or fully muscularized, as judged by the degree of immunostaining for smooth muscle a-actin.
  • a minimum of 20 vessels with diameters ranging from 25 to 75 ⁇ were counted from nonseriai lung sections and categorized as either fully, partially or non-muscularized.
  • Statistical significance was assessed by comparing the percentage of fully muscularized vessels between groups.
  • Evaluation of wall thickness involved the identification of small arteries ( ⁇ 1 00 ⁇ ) proximal to the terminal epithelial bronchioles. Using image J, the diameter and thickness of the artery was measured after immunostaining for smooth muscle a-actin. Thickness measurements were taken in four different positions of the artery. A minimum of 10 arteries were assessed in each lung section.
  • TNFa reduces BMPR-II expression in vitro and in vivo
  • cytokines including TNFa, IL-1 ⁇ , IL-6 and IL-8 have been implicated in the pathogenesis of PAH.
  • TNFa selectively reduced BMPR-II mRNA (BMPR2) and protein in distal PASMCs (dPASMCs) and pulmonary arterial endothelial cells (PAECs) (Fig. 1A-D and Fig. 2A-D), via NF- ⁇ p65 (RELA) (Fig. 2E).
  • BMPR2 BMPR-II mRNA
  • PAECs pulmonary arterial endothelial cells
  • Fig. 1A-D and Fig. 2A-D pulmonary arterial endothelial cells
  • RELA NF- ⁇ p65
  • immunofluorescent staining demonstrated local vascular expression of TNFa in both human IPAH and HPAH that was absent in unaffected controls (Fig. 1 E).
  • TNFa promotes BMPR-II cleavage and extracellular domain shedding in SMCs via ADAM 10/17
  • BMPR-II-ICP an intracellular 60kDa product
  • s BMPR2 a BMPR-II fragment using siRNA
  • TNFa also promoted the production of this 60kDa band and reduction of full length BMPR-II in rat and mouse PASMCs, human proximal PASMCs (pPASMCs), and human aortic smooth muscle cells (Fig. 3B,C).
  • BMPR-II-ICP in PASMCs and SP-C/Tnf ⁇ ung suggested TNFa-dependent cleavage of BMPR-II.
  • Fig. 5A immunoprecipitation of a myc-tagged BMPR-II ectodomain from conditioned media from TNFa-treated dPASMCs.
  • ELISA of conditioned media from TNFa-treated PASMCs revealed enhanced endogenous soluble BMPR-II (sBMPR-ll) generation (Fig. 5B). Since BMPR-II cleavage has not been reported previously, we determined the proteolytic mechanism of TN Fa-mediated cleavage of BMPR-II in PASMCs.
  • MMP-14 matrix metalloproteinase-14
  • ADAM17 Disintegrin and Metalloprotease-17
  • BB94 pan- MMP/ADAM inhibitor
  • Fig. 5C,D Transcriptional analysis of candidate metalloproteinases revealed that TNFa induced ADAM 10 and ADAM17 in dPASMCs, but not PAECs
  • BMPR-II ECD recombinant BMPR-II ECD
  • Fig. 7D conditioned media from TNFa-treated dPASMCs overexpressing wild-type BMPR-II
  • Fig. 7E media from the cleavage-resistant V163A mutant
  • BMPR-II ECD neutralized the anti-proliferative effects of BMP2 and BMP4 (Fig. 7E), in a similar manner to treatment with TNFa (Fig. 7F).
  • TNFa alters BMP2 and BMP6 signaling dynamics and influences PASMC proliferation
  • TNFa suppresses BMPR-II expression in vitro and in vivo
  • TNFa inhibited BMP2-dependent SMAD1 /5 phosphorylation and ID1 transcription in control dPASMCs, but augmented BMP6 signaling, particularly in HPAH PASMCs (Fig. 8B,C and Fig. 9B,C).
  • BMP2, BMP4 and BMP6 inhibited control dPASMC proliferation whereas BMP6 promoted HPAH dPASMC proliferation and TNFa enhanced the BMP6 responses (Fig. 8D,E).
  • the pivotal role of BMPR-II levels in this TNFa/BMP6 response was demonstrated by the switching from anti-proliferative to pro-proliferative responses following s BMPR2 in control dPASMCs (Fig. 8F), and restoration of the anti-proliferative response to BMP6 following overexpression of wild- type BMPR-II in HPAH dPASMCs (Fig. 8G).
  • BMP2 and BMP6 are relatively highly expressed (Fig. 10A,B).
  • TNFa repressed BMP2, but consistently induced BMP6 expression in dPASMCs and PAECs (Fig. 10C,D), while other BMP ligands were unaltered (Fig. 10E).
  • Fig. 10F,G NF-KB p65
  • BMP6 induction by TNFa was greater in HPAH dPASMCs than control cells whereas BMP2 expression was repressed equally (Fig. 10H,I).
  • Lung TNFa overexpression also promoted pulmonary arteriolar muscularization (Fig. 4B,C) and repressed Bmp2 (Fig. 4G), albeit to similar extents in SP-C/Tnf/Bmpr2 +/+ and SP-C/Tnf/Bmpr2 +/ - mice.
  • ALK2 and ACTR-IIA are required for BMP6-mediated HPAH PASMC proliferation.
  • Both LDN-193189 (Fig. 12A,B) and ALK2 siRNA (Fig. 12C,D) abolished the antiproliferative response to BMP6 in control PASMCs and the pro-proliferative response in HPAH PASMCs.
  • ACTR-IIA siRNA (s ACVR2A) eliminated the enhanced TNFa/BMP6-dependent SMAD1/5 phosphorylation (Fig. 12E), ID1 induction (Fig. 12F) and proliferative responses (Fig. 12G) of HPAH PASMCs.
  • TNFa enhanced ACVR2A expression in HPAH PASMCs without altering ALK2, ALK3 or ALK6 expression (Fig. 13A,B). From these observations, we conclude that ALK2 mediates BMP6 signaling in PASMCs and TNFa-induced loss of BMPR-II permits preferential ACTR-IIA signaling, thus driving HPAH PASMC proliferation.
  • TNFa alters NOTCH signaling in HPAH PASMCs
  • BMPR2 silencing also promoted the TNFa-dependent reduction of NOTCH3-ICD generation and NOTCH3 transcription, again enhanced by BMP6 (Fig. 17A and Fig. 18C).
  • TNFa-dependent NOTCH2 induction following s BMPR2 was inhibited by co-silencing with s ⁇ ACVR2A (Fig. 18B).
  • s ⁇ ACVR2A reduced BMP6-stimulated NOTCH2 expression regardless of s BMPR2 in control dPASMCs (Fig. 18B).
  • s ACVR2A reduced NOTCH2-ICD generation and abrogated NOTCH3-ICD reduction (Fig. 17B).
  • NOTCH signaling mediates the proliferative response of HPAH PASMCs to TNFa/BMP6.
  • the ⁇ -secretase inhibitor DAPT previously reported to inhibit PASMC proliferation through NOTCH3 blockade, both prevented the proliferative responses of HPAH PASMCs to TNFa and BMP6 (Fig. 19A) and inhibited the anti-proliferative BMP6 response in control PASMCs (Fig. 19B).
  • NOTCH2 siRNA reduced this proliferation of HPAH PASMCs to TNFa and BMP6, whereas NOTCH3 siRNA did not (Fig. 17F,G).
  • NOTCH3 siRNA did not attenuated the antiproliferative responses whereas NOTCH2 siRNA had no effect (Fig.
  • SRC antibodies detect multiple family members, including FYN and YES, so we determined the contributions of individual members to the NOTCH responses in HPAH PASMCs, particularly as SRC and FYN can interact with BMPR-II and ACTR-IIA, respectively.
  • siRNA targeting FYN prevented the TNFa-dependent NOTCH1 and NOTCH2 induction and NOTCH3 repression (Fig. 20E,F and Fig 21 D) whereas either FYN or YES mediated the repression of NOTCH3 by BMP6 (Fig. 20F).
  • FYN or YES siRNAs also abolished the proliferative response to TNFa or BMP6 alone, or in combination (Fig. 20G).
  • TNFa antagonism ameliorates experimental PAH and reverses aberrant TNF/BMP signaling
  • Etanercept reversed the progression of PAH, reducing RVSP, right ventricular hypertrophy and muscularization of alveolar duct-associated arterioles (Fig 22A-C), without altering left ventricular function (Table 6).
  • the development of PAH in the S/H model was associated with BMP and NOTCH signaling changes consistent with our in vitro data.
  • Bmpr2 expression and Smad1 /5 signaling were reduced (Fig 22D,E) and Acvr2a, Alk2, Bmp6 and Tnf expression were all increased (Fig. 22D and Fig. 23C,D).
  • Notch2, Hey1 and Hey2 expression and medial Notch2 staining were increased in S/H rats (Fig. 22E,F,H and Fig.
  • Inflammatory cytokines are associated with the pathogenesis of PAH and our demonstration that TNFa suppressed BMPR-II in pulmonary vascular cells confirms reports in osteoblasts and aortic endothelial cells. Furthermore, TNFa exacerbates the genetic BMPR2 haploinsufficiency in HPAH PASMCs causing the substantial reduction of BMPR-II levels that allow BMP6 to switch signaling to the alternative type II receptor, ACTR-IIA. Also, TNFa increased BMP6 expression in PASMCs and hPAECs and induced ACTR-IIA expression in HPAH PASMCs.
  • NOTCH inhibition by soluble JAGGED1 attenuates PAH in hypoxic and MCT-PAH rat models.
  • Our data suggest that, on the background of BMPR-II haploinsufficiency, inappropriate NOTCH2 responses to TNFa stimulate PASMC proliferation.
  • NOTCH2 is abundantly expressed in vascular SMCs and NOTCH2 deletion reduces SMC number and causes embryonic lethality.
  • the NOTCH3 reduction we observed was surprising given previous reports of NOTCH3 promoting PAH. However, these previous studies used DAPT as the therapeutic intervention in PAH models, which blocks NOTCH2 and NOTCH3 cleavage.
  • NOTCH2 blockade may be relevant to these previous reports.
  • SRC family kinases in linking the TNFa, BMP and NOTCH pathways.
  • BMPR-II reduction increases the availability of SRC kinases to interact with ACTR-IIA or TNFa receptors.
  • FYN as a key regulator of the aberrant NOTCH2 signaling and proliferation to TNFa and a dual role for FYN and YES in the proliferative response to TNFa and BMP6.
  • TNFa induces BMP6 and exacerbates the reduced BMPR-II expression in HPAH PASMCs, enabling BMP6 to recruit the ALK2/ACTR-IIA receptor complex.
  • TNFa promotes excessive PASMC proliferation via activation of FYN and the NOTCH2-HEY1 /2 axis, while simultaneously suppressing the antiproliferative NOTCH3-HES1 axis.
  • BMPR-II bone morphogenetic protein type-ll receptor
  • HPAH heritable pulmonary arterial hypertension
  • TNFa selectively reduces BMPR-II transcription and mediates post-translational BMPR-II cleavage via the sheddases, ADAM10 and ADAM17 in pulmonary artery smooth muscle cells (PASMCs).
  • TNFa-mediated suppression of BMPR-II subverts BMP signaling leading to BMP6- mediated PASMC proliferation via preferential activation of an ALK2/ACTR-IIA signaling axis.
  • TNFa acting via SRC family kinases, increased pro- proliferative NOTCH2 signaling in HPAH PASMCs with reduced BMPR-II expression.
  • anti-TNFa immunotherapy reverses disease progression, restoring normal BMP/NOTCH signaling.
  • This example describes administration of etanercept and a BMP9 variant or BMP10 to assess effect on PAH in the rat Sugen-hypoxia model.
  • mice Male Sprague Dawley rats (-150 to 200 g, Charles River) are given a single i.p. injection of Sugen 5416 (SU-5416; 20 mg/kg, Tocris, Bristol, UK) in vehicle (0.5% carboxyi methylcellulose sodium, 0.4% poiysorbate 80, 0.9% benzyl alcohol, all Sigma), placed immediately into a 10% 0 2 chamber and maintained in hypoxia for 3 weeks, followed by 5 weeks in a normoxic environment to develop pulmonary hypertension. At the 8-week timepoint, rats are randomized into 4 groups. Group 1 receive twice weekly i.p. injections of 2.5 mg/kg Etanercept (Enbrel® Pfizer) diluted in saline.
  • Etanercept Enbrel® Pfizer
  • Group 2 receive 600ng/day i.p of a candidate from BMP9 (or a variant thereof or B P10) in saline.
  • Group 3 receive receive twice weekly i.p. injections of 2.5 mg/kg Etanercept (Enbrel ⁇ Pfizer) in saline and 600ng/day i.p of a candidate from BMP9 (or a variant thereof or BMP10) in saline.
  • Group 4 receive saline alone.
  • rats are anesthetized with isofiuorane, body weight recorded and right and left ventricular function assessed using a Miliar SPR-869 pressure-volume catheter. Rats are then sacrificed and the hearts, lungs and livers harvested.
  • RVH Right ventricular hypertrophy
  • RV right ventricle
  • LV+S left ventricle plus septum
  • the degree of right ventricular hypertrophy is determined from the ratio RV/LV+S.
  • the right lung is snap frozen in liquid nitrogen.
  • the left lung is inflated with a 1 :1 mixture of saline and O.C.T. compound (Sakura, Zoeterwoude, Netherlands) and fixed with 4% paraformaldehyde in PBS before dehydration and paraffin embedding.
  • sections of fixed rat lung tissue (5 ⁇ in thickness) are labeled with monoclonal mouse-anti-mouse/rat/human smooth muscle a-actin (clone 1 A4, Dako, Glostrup, Denmark), followed by polyclonal goat anti-mouse HRP.
  • the Dako ARKTM kit ⁇ Dako, Glostrup, Denmark is used to detect staining of the mouse primary antibody in mouse lung tissue in accordance with the manufacturer's instructions.
  • the primary smooth muscle a-actin antibody is labeled with a modified biotinyiated anti-mouse before application to the specimen.
  • the primary antibody and biotinylation reagent are mixed in solution, resulting in binding of biotinyiated secondary antibody to the primary antibody.
  • the blocking reagent containing normal mouse serum, is then added to the mixture to bind residual biotinylation reagent not bound to the primary antibody, minimizing the potential interaction of the biotinyiated anti-mouse secondary reagent with endogenous immunoglobulin present in the specimen.
  • the biotin -labeled primary antibody is then applied to the specimen followed by incubation with streptavidin-peroxidase and reaction with diaminobenzidine (DAB)-hydrogen peroxide as substrate-chromogen.
  • DAB diaminobenzidine
  • Assessment of pulmonary arteriolar muscularization involves the identification of alveolar ducts and the subsequent categorization of the accompanying intraacinar artery as non-, partially or fully muscuiarized, as judged by the degree of immunostaining for smooth muscle a-actin.
  • a minimum of 20 vessels with diameters ranging from 25 to 75 ⁇ are counted from nonserial lung sections and categorized as either fully, partially or non-muscuiarized.
  • Statistical significance is assessed by comparing the percentage of fully muscuiarized vessels between groups.
  • Evaluation of wall thickness involves the identification of small arteries ( ⁇ 100 ⁇ ) proximal to the terminal epithelial bronchioles. Using image J the diameter and thickness of the artery is measured after immunostaining for smooth muscle a-actin. Thickness measurements are taken in four different positions of the artery. A minimum of 10 arteries are assessed in each lung section.
  • Membranes are then blocked and probed with rabbit polyclonal antibodies toward total Smadl , phosphorylated SRC(Y527) (all Cell Signaling Technology, Danvers, MA), ADAM10, ADAM 17 (Abeam, Cambridgeshire, UK); rabbit monoclonal antibodies toward phosphorylated Smad1/5, caspase-3, cleaved caspase- 3, NOTCH1 , NOTCH2, NOTCH3, phospho-SRC(Y416) and SRC (Cell Signaling Technology, Danvers, MA), ID1 (CalBioreagents, San Mateo, CA); or mouse monoclonal antibodies toward BMPR-II (BD Transduction Laboratories, Franklin Lakes, NJ).
  • Smadl total Smadl
  • phosphorylated SRC(Y527) all Cell Signaling Technology, Danvers, MA
  • ADAM10 ADAM 17
  • ADAM 17 Abeam, Cambridgeshire, UK
  • blots are incubated with secondary anti-mouse/rabbit horseradish peroxidase antibody (Dako, Glostrup, Denmark) for 1 h at room temperature.
  • secondary anti-mouse/rabbit horseradish peroxidase antibody Dako, Glostrup, Denmark
  • all blots are re-probed with a monoclonal antibody toward either a- tubulin (Sigma) or ⁇ -actin (Sigma)
  • Densitometry is performed using !mageJ software.
  • Membranes are developed using enhanced chemiluminescence (GE Healthcare).
  • RNA Preparation and Quantitative RT-PCR Total RNA is extracted using the RNeasy Mini Kit with DNAse digestion (Qiagen, West Wales, UK). cDNA is prepared from ⁇ 1 ⁇ g of RNA using the High Capacity Reverse Transcriptase kit (Applied Biosystems, California, USA), according to the manufacturer's instructions. All qPCR reactions are prepared in MicroAmp® optical 96-well reaction plates (Applied Biosystems) using 50 ng/ ⁇ cDNA with SYBR®Green JumpstartTM Taq ReadymixTM (Sigma-Aldrich), ROX reference dye (Invitrogen) and custom sense and anti-sense primers (all 200 nM). QuantiTect primers are used for rat Bmpr2, Notch l, Notch2, Notch3 and Tnf. Reactions are amplified on a StepOnePlusTM Real-Time PCR system (Applied Biosystems).
  • Target gene expression is normalized to ACTB/Actb and the difference in the amount of product produced expressed as a fold change.
  • the relative abundance of BMP ligands is calculated, on the assumption of equal copy number, by calculating the expression of each BMP gene relative to ACTB after normalization to B2M.
  • Statistics. All data are analysed using GraphPad Prism. Data are presented as mean +/- S.E.M. Data are analysed by one-way ANOVA with post-hoc Tukey's HSD analysis or paired two-tailed Student's f-test where indicated. P ⁇ 0.05 is considered significant.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Organic Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Cardiology (AREA)
  • Orthopedic Medicine & Surgery (AREA)
  • Pulmonology (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Immunology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Epidemiology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

La présente invention concerne, entre autres, une composition pharmaceutique comprenant : (1) un polypeptide choisi parmi la protéine morphogénétique osseuse 10 (BMP10) et un variant de la protéine morphogénétique osseuse 9 (BMP9), dépourvu d'activité ostéogénique ; et (2) un inhibiteur du TNFα. L'invention concerne également des utilisations et des méthodes médicales visant à traiter une maladie vasculaire ou une maladie respiratoire chez un patient au moyen de cette composition pharmaceutique.
PCT/GB2017/050553 2016-03-02 2017-03-02 Polythérapie WO2017149306A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB1603653.5 2016-03-02
GBGB1603653.5A GB201603653D0 (en) 2016-03-02 2016-03-02 Combination Therapy

Publications (1)

Publication Number Publication Date
WO2017149306A1 true WO2017149306A1 (fr) 2017-09-08

Family

ID=55807199

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB2017/050553 WO2017149306A1 (fr) 2016-03-02 2017-03-02 Polythérapie

Country Status (2)

Country Link
GB (1) GB201603653D0 (fr)
WO (1) WO2017149306A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111939245A (zh) * 2019-05-16 2020-11-17 龚笑海 一种心脏治疗和保护的药物组合物
EP4081240A4 (fr) * 2019-12-03 2023-12-06 Acceleron Pharma Inc. Compositions et méthodes de traitement de l'hypertension pulmonaire

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004067006A1 (fr) * 2003-01-27 2004-08-12 Pharmacia Corporation Combinaison d'un inhibiteur de la pde iv et d'un antagoniste du tnf-alpha
US20100016232A1 (en) * 2008-07-18 2010-01-21 Novozymes A/S Treatment Of Inflammatory Diseases With Mammal Beta Defensins
WO2016005756A1 (fr) * 2014-07-10 2016-01-14 Cambridge Enterprise Limited Utilisation thérapeutique de protéines morphogénétiques osseuses

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004067006A1 (fr) * 2003-01-27 2004-08-12 Pharmacia Corporation Combinaison d'un inhibiteur de la pde iv et d'un antagoniste du tnf-alpha
US20100016232A1 (en) * 2008-07-18 2010-01-21 Novozymes A/S Treatment Of Inflammatory Diseases With Mammal Beta Defensins
WO2016005756A1 (fr) * 2014-07-10 2016-01-14 Cambridge Enterprise Limited Utilisation thérapeutique de protéines morphogénétiques osseuses

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
GOPINATH SUTENDRA ET AL: "Pyruvate dehydrogenase inhibition by the inflammatory cytokine TNFÎ contributes to the pathogenesis of pulmonary arterial hypertension", JOURNAL OF MOLECULAR MEDICINE, SPRINGER, BERLIN, DE, vol. 89, no. 8, 2 August 2011 (2011-08-02), pages 771 - 783, XP019936151, ISSN: 1432-1440, DOI: 10.1007/S00109-011-0762-2 *
HURST LIAM A ET AL: "TNF alpha drives pulmonary arterial hypertension by suppressing the BMP type-II receptor and altering NOTCH signalling", NATURE COMMUNICATIONS, vol. 8, 13 January 2017 (2017-01-13), XP055368592, ISSN: 2041-1723 *
ORMISTON MARK L ET AL: "The promise of recombinant BMP ligands and other approaches targeting BMPR-II in the treatment of pulmonary arterial hypertension.", GLOBAL CARDIOLOGY SCIENCE & PRACTICE 2015, vol. 2015, no. 4, 2015, pages 47, XP055368595 *
SAWADA HIROFUMI ET AL: "Reduced BMPR2 Increases GM-CSF mRNA Translation by Inhibiting eIF2 alpha Mediated Stress Granule Formation and Propensity to Pulmonary Vascular Disease", CIRCULATION, vol. 124, no. 21, Suppl. S, November 2011 (2011-11-01), & SCIENTIFIC SESSIONS OF THE AMERICAN-HEART-ASSOCIATION/RESUSCITATION SCIENCE SYMPOSIUM; ORLANDO, FL, USA; NOVEMBER 12 -16, 2011, pages A11784, XP009194261 *
VENGETHASAMY LEANDA ET AL: "BMPRII influences the response of pulmonary microvascular endothelial cells to inflammatory mediators", PFLUEGERS ARCHIV: EUROPEAN JOURNAL OF PHYSIOLOGY, SPRINGER VERLAG, BERLIN, DE, vol. 468, no. 11, 6 November 2016 (2016-11-06), pages 1969 - 1983, XP036113173, ISSN: 0031-6768, [retrieved on 20161106], DOI: 10.1007/S00424-016-1899-1 *
ZHANG LING-LING ET AL: "Preventive and remedial application of etanercept attenuate monocrotaline-induced pulmonary arterial hypertension.", INTERNATIONAL JOURNAL OF RHEUMATIC DISEASES FEB 2016, vol. 19, no. 2, February 2016 (2016-02-01), pages 192 - 198, XP055368631, ISSN: 1756-185X *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111939245A (zh) * 2019-05-16 2020-11-17 龚笑海 一种心脏治疗和保护的药物组合物
WO2020228705A1 (fr) * 2019-05-16 2020-11-19 龚笑海 Composition pharmaceutique pour le traitement et la protection cardiaque
CN111939245B (zh) * 2019-05-16 2024-03-01 龚笑海 一种心脏治疗和保护的药物组合物
EP4081240A4 (fr) * 2019-12-03 2023-12-06 Acceleron Pharma Inc. Compositions et méthodes de traitement de l'hypertension pulmonaire

Also Published As

Publication number Publication date
GB201603653D0 (en) 2016-04-13

Similar Documents

Publication Publication Date Title
US11572396B2 (en) Therapeutic use of bone morphogenetic proteins
US20220098594A1 (en) Compositions comprising sasp modulators and senescence attenuators and uses thereof for modulating cellular senescence
Okamoto et al. Atrial natriuretic peptide protects against bleomycin-induced pulmonary fibrosis via vascular endothelial cells in mice: ANP for pulmonary fibrosis
JP2022551987A (ja) 肝疾患を治療するための組成物および方法
DK2904009T3 (en) RELATIONSHIPS FOR TREATING THE REMYELINIZATION BLOCK IN DISEASES RELATED TO THE EXPRESSION OF HERV-W COAT PROTEIN
WO2017149306A1 (fr) Polythérapie
CN108159423B (zh) 治疗和预防心血管疾病的组合物和方法
WO2014107484A1 (fr) Procédés pour traiter l'inflammation
TW201200151A (en) Methods and compositions related to reduced MET phosphorylation by leukocyte cell-derived chemotaxin 2 in tumor cells
WO2018231025A9 (fr) Agent thérapeutique pour maladie provoquée par la migration des cellules immunitaires et son procédé d'identification par criblage
KR20180005149A (ko) Ｃｄ９ 항체를 유효성분으로 함유하는 세포노화 또는 노화 관련 질환 예방 또는 치료용 약학조성물
JP2004536827A (ja) Ａｂｉｎにより媒介される肝炎防護
KR102536092B1 (ko) Lrp5 발현 또는 활성 억제제를 포함하는 허혈성 심장질환의 예방 또는 치료용 약학적 조성물
US9718879B2 (en) Methods of treating pain by inhibition of VGF activity
Su et al. Overexpression of Corin Ameliorates Kidney Fibrosis through Inhibition of Wnt/β-Catenin Signaling in Mice
Abeyrathna Calpain-2 Activates Akt via the TGF~ 1-mTORC2 Pathway in Pulmonary Artery Smooth Muscle Cells
Leslie Exploring the Role of EBP50 in Vascular Inflammation
EP2874629A1 (fr) Inhibiteurs de nkx2.5 destinés au remodelage vasculaire

Legal Events

Date Code Title Description
NENP Non-entry into the national phase

Ref country code: DE

121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 17709756

Country of ref document: EP

Kind code of ref document: A1

122 Ep: pct application non-entry in european phase

Ref document number: 17709756

Country of ref document: EP

Kind code of ref document: A1