WO2017141032A1 - Treatment of fibrotic disorders - Google Patents

Treatment of fibrotic disorders Download PDF

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Publication number
WO2017141032A1
WO2017141032A1 PCT/GB2017/050397 GB2017050397W WO2017141032A1 WO 2017141032 A1 WO2017141032 A1 WO 2017141032A1 GB 2017050397 W GB2017050397 W GB 2017050397W WO 2017141032 A1 WO2017141032 A1 WO 2017141032A1
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Prior art keywords
inhibitor
proprotein convertase
unsubstituted
substituted
group
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PCT/GB2017/050397
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French (fr)
Inventor
Shoumo Bhattacharya
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Oxford University Innovation Limited
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Priority claimed from GBGB1602641.1A external-priority patent/GB201602641D0/en
Priority claimed from GBGB1602640.3A external-priority patent/GB201602640D0/en
Application filed by Oxford University Innovation Limited filed Critical Oxford University Innovation Limited
Publication of WO2017141032A1 publication Critical patent/WO2017141032A1/en

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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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/005—Enzyme inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/07—Tetrapeptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/542—Carboxylic acids, e.g. a fatty acid or an amino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0815—Tripeptides with the first amino acid being basic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0815—Tripeptides with the first amino acid being basic
    • C07K5/0817—Tripeptides with the first amino acid being basic the first amino acid being Arg
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/81—Protease inhibitors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/08—Hepato-biliairy disorders other than hepatitis
    • G01N2800/085—Liver diseases, e.g. portal hypertension, fibrosis, cirrhosis, bilirubin

Definitions

  • PCSKs Proprotein convertases
  • the inventors unexpectedly identified a substantial intersect between targets that are cleaved by PCSKs and candidate mediators of fibrosis, leading them to them to
  • PI is an arginine mimetic selected from the following structures:
  • Ri is selected from H, OH, O-CH
  • n is an integer from 1 to 5;
  • P5 is an N-terminal modification of the amino or imino acid P4, wherein P5 is selected from the following groups:
  • Ci-io alkyl which is unsubstituted or substituted with a halogen, NH 2 , guanidino, C(0)OH or C(0)OR 5 group,
  • z is an integer from 2 to 20;
  • R 7 is:
  • the invention also provides the use of a proprotein convertase inhibitor in the manufacture of a medicament for use in the treatment of a fibrotic disorder, which proprotein convertase inhibitor is a compound of formula (I) as defined above or a prodrug thereof or a pharmaceutically acceptable salt thereof, or comprises an oligonucleotide or variant sequence as defined above or any isoform thereof.
  • the invention also provides a method of treating a fibrotic disorder, which method comprises administering to a subject in need of such treatment a therapeutically effective amount of a proprotein convertase inhibitor, which proprotein convertase inhibitor is a compound of formula (I) as defined above or a prodrug thereof or a pharmaceutically acceptable salt thereof, or comprises an oligonucleotide or variant sequence as defined above or any isoform thereof.
  • a proprotein convertase inhibitor which proprotein convertase inhibitor is a compound of formula (I) as defined above or a prodrug thereof or a pharmaceutically acceptable salt thereof, or comprises an oligonucleotide or variant sequence as defined above or any isoform thereof.
  • the invention also provides a combination comprising (a) a proprotein convertase inhibitor and (b) a further antifibrotic agent, for use in the treatment of a fibrotic disorder.
  • the proprotein convertase inhibitor is typically a compound of formula (I) as defined above or a prodrug thereof or a pharmaceutically acceptable salt thereof, or comprises an oligonucleotide or variant sequence as defined above or any isoform thereof.
  • the proprotein convertase inhibitor and the further antifibrotic agent may be for separate, simultaneous, concomitant or sequential administration in the treatment of the fibrotic disorder.
  • PCSK inhibitors can robustly reduce collagen formation and procollagen propeptide production without significantly affecting fibronectin secretion. This is advantageous, as fibronectin plays a major role in cell adhesion, growth, migration, and differentiation and is important for processes such as wound healing and embryonic development. Also, altered fibronectin expression, degradation, and organization has been associated with a number of pathologies, including cancer. It can therefore be desirable not to alter fibronectin secretion.
  • the present invention further provides a proprotein convertase inhibitor for use in the treatment of a fibrotic disorder by inhibiting the formation of collagen matrix without inhibiting fibronectin secretion.
  • a proprotein convertase inhibitor capable of inhibiting collagen matrix formation without significantly inhibiting fibronectin secretion may be employed in this aspect of the invention.
  • the inhibitor employed may for instance be a compound of formula (I), as defined above, or a prodrug or pharmaceutically acceptable salt thereof.
  • proprotein convertase inhibitors could also be employed.
  • the invention also provides a composition for use in the treatment of a fibrotic disorder by inhibiting collagen matrix formation without inhibiting fibronectin secretion, which composition comprises a proprotein convertase inhibitor and a pharmaceutically acceptable carrier or diluent.
  • the invention also provides the use of a proprotein convertase inhibitor in the manufacture of a medicament for use in the treatment of a fibrotic disorder by inhibiting collagen matrix formation without inhibiting fibronectin secretion.
  • the invention also provides a method of treating a fibrotic disorder in a subject by inhibiting collagen matrix formation without inhibiting fibronectin secretion, which method comprises administering to the subject a therapeutically effective amount of a proprotein convertase inhibitor, which amount is effective to inhibit collagen matrix formation in the subject without inhibiting fibronectin secretion.
  • the invention also provides an in vitro method of inhibiting the formation of collagen matrix without inhibiting fibronectin secretion, the method comprising contacting an in vitro sample comprising collagen-secreting cells with an amount of a proprotein convertase inhibitor effective to inhibit the formation of collagen matrix by the cells without significantly inhibiting secretion by the cells of fibronectin.
  • the collagen- secreting cells typically comprise fibroblasts.
  • the invention also provides a novel assay method for identifying molecules that affect collagen secretion in vitro, and thus for identifying antifibrotic agents.
  • the assay was used in the present case to identify proprotein convertase inhibitors as inhibitors of collagen matrix formation.
  • the assay is miniaturized so that it is compatible with high-throughput screening with various established compound libraries. The miniaturization also allows comprehensive and systematic quantification of collagen signals for high-throughput imaging.
  • the assay can also provide for improved morphological contrast for quantitative imaging as compared to known collagen matrix assays.
  • the invention provides a method of identifying an agent as being capable of inhibiting collagen matrix formation, which method comprises (a) contacting an agent with an in vitro sample comprising collagen- secreting cells in the absence of exogenous transforming growth factor (TGF) and (b) measuring the amount of collagen matrix formed by the cells and thereby determining whether or not the agent is capable of inhibiting collagen matrix formation.
  • TGF transforming growth factor
  • Fig. 1 shows that Calbiochem 537076 specifically inhibits extracellular collagen levels.
  • the modified "scar-in-a-jar” assay was carried out as per Figure 6 on three different passages of juvenile HDF (j- HDF) treated with 0, 1.25, 2.5, 5, 10, or 20 ⁇
  • Fig. 2 is a graph containing dose response curves for Calbiochem 537076'
  • Fig. 3 shows the results of validation of siRNA against individual PCSKs; gene expression was determined by qPCR using Taqman gene expression assays for FURIN (Hs00965485_gl) (a), PCSK5 (Hs00196400_ml) (b), and PCSK6 (Hs00159844_ml) (c), compared against non-targeted control (NTC) RNA (D-001810).
  • FURIN Hs00965485_gl
  • PCSK5 Hs00196400_ml
  • PCSK6 Hs00159844_ml
  • Fig. 4 shows that knockdown of FURIN (PCSK3) inhibits extracellular collagen.
  • PCSK3 FURIN
  • PCSK5 PCSK5
  • PCSK6 were knocked down in j-NHDF cells as per Figure 3, and scar-in-a-jar assay was carried out.
  • Graphs show quantification from image analysis of (a) total collagen divided by cell number total, (b) fibronectin divided by cell number, and (c) total collagen divided by total fibronectin.
  • Fig. 5 shows that knockdown of FURIN suppresses COL1 Al mRNA expression.
  • Gene expression was determined by qPCR using Taqman gene expression assays for FURIN (Hs00965485_gl), COL1A1 (Hs00164004_ml), COL1A2 (Hs00164099_ml), and COL3A1 (Hs00943809_ml). Data were normalised to HPRT (Hs99999909_ml), and presented using the 2 A (-AACT) method, with the NTC control normalised to 100 and expressed as mean ⁇ SEM..
  • Fig. 6 is a schematic illustration of the modified "Scar-in-a-Jar" assay of the invention. DETAILED DESCRIPTION OF THE INVENTION
  • Ci-24 alkyl group is an unsubstituted or substituted, straight or branched chain saturated hydrocarbon radical having from 1 to 24 carbon atoms (unless otherwise specified). Often, it is C 1-20 alkyl, i.e. an unsubstituted or substituted, straight or branched chain saturated hydrocarbon radical having from 1 to 20 carbon atoms.
  • Ci-io alkyl for example methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl or decyl
  • Ci-6 alkyl for example methyl, ethyl, propyl, butyl, pentyl or hexyl
  • Ci-4 alkyl for example methyl, ethyl, i-propyl, n-propyl, t-butyl, s-butyl or n-butyl, or for instance C 1-3 alkyl.
  • a substituted alkenyl group carries 1, 2 or 3 substituents, for instance 1 or
  • a C3-25 cycloalkyl group is an unsubstituted or substituted alkyl group which is also a cyclyl group; that is, a monovalent moiety obtained by removing a hydrogen atom from an alicyclic ring atom of a carbocyclic ring of a carbocyclic compound, which moiety has from 3 to 25 carbon atoms (unless otherwise specified), including from 3 to 25 ring atoms.
  • cycloalkyl includes the sub-classes cycloalkyenyl and cycloalkynyl.
  • Examples of groups of C3-25 cycloalkyl groups include C3-20 cycloalkyl, C3-15 cycloalkyl, C3-10 cycloalkyl, C3-7 cycloalkyl.
  • a C3-25 cycloalkyl group When a C3-25 cycloalkyl group is substituted it typically bears one or more substituents selected from Ci-6 alkyl which is unsubstituted, aryl (as defined herein), cyano, amino, Ci-10 alkylamino, di(Ci-io)alkylamino, arylamino, diarylamino, arylalkylamino, amido, acylamido, hydroxy, oxo, halo, carboxy, ester, acyl, acyloxy, Ci-20 alkoxy, aryloxy, haloalkyl, sulfonic acid, sulfhydryl (i.e.
  • thiol -SH
  • a substituted C3-25 cycloalkyl group carries 1, 2 or 3 substituents, for instance 1 or 2.
  • C3-25 cycloalkyl groups include, but are not limited to, those derived from saturated monocyclic hydrocarbon compounds, which C3-25 cycloalkyl groups are unsubstituted or substituted as defined above:
  • camphene Cio
  • limonene Cio
  • pinene Cio
  • indene C9
  • indane e.g., 2,3-dihydro-lH-indene
  • tetraline e.g., 1,3-dihydro-lH-indene
  • a C3-20 heterocyclyl group is an unsubstituted or substituted monovalent moiety obtained by removing a hydrogen atom from a ring atom of a heterocyclic compound, which moiety has from 3 to 20 ring atoms (unless otherwise specified), of which from 1 to 10 are ring heteroatoms.
  • each ring has from 3 to 7 ring atoms, of which from 1 to 4 are ring heteroatoms.
  • a C3-7 heterocyclyl has from 3 to 7 ring atoms, of which from 1 to 4 are ring heteroatoms.
  • a C3-20 heterocyclyl group When a C3-20 heterocyclyl group (or a C3-7 heterocyclyl group) is substituted it typically bears one or more substituents selected from Ci-6 alkyl which is unsubstituted, aryl (as defined herein), cyano, amino, C 1-10 alkylamino, di(Ci- io)alkylamino, arylamino, diarylamino, arylalkylamino, amido, acylamido, hydroxy, oxo, halo, carboxy, ester, acyl, acyloxy, C 1-20 alkoxy, aryloxy, haloalkyl, sulfonic acid, sulfhydryl (i.e.
  • thiol -SH
  • Ci-io alkylthio arylthio
  • phosphoric acid phosphate ester
  • phosphonic acid phosphonate ester
  • sulfonyl Typically a substituted C3-20 heterocyclyl group carries 1, 2 or 3 substituents, for instance 1 or 2.
  • groups of heterocyclyl groups include C3-2oheterocyclyl,
  • Examples of (non-aromatic) monocyclic C3-20 heterocyclyl groups include, but are not limited to, those derived from:
  • Oi oxirane (C 3 ), oxetane (C 4 ), oxolane (tetrahydrofuran) (C5), oxole
  • O2 dioxolane (C5), dioxane (C 6 ), and dioxepane (C 7 );
  • N2 imidazolidine (C5), pyrazolidine (diazolidine) (C5), imidazoline (C5), pyrazoline (dihydropyrazole) (C5), piperazine (C 6 );
  • N1O1 tetrahydrooxazole (C5), dihydrooxazole (C5), tetrahydroisoxazole (C5), dihydroisoxazole (C5), morpholine (C 6 ), tetrahydrooxazine (C 6 ), dihydrooxazine (C 6 ), oxazine (C 6 );
  • N1S1 thiazoline (C5), thiazolidine (C5), thiomorpholine (C 6 );
  • O1S1 oxathiole (C5) and oxathiane (thioxane) (C 6 ); and,
  • N1O1S1 oxathiazine (C 6 ).
  • An aryl group is a substituted or unsubstituted, monocyclic or bicyclic aromatic group which typically contains from 5 to 20 carbon atoms, more typically from 5 to 14 carbon atoms, preferably from 6 to 14, or for instance from 6 to 10, or from 5 to 10, carbon atoms in the ring portion. Examples include phenyl, naphthyl, indenyl and indanyl groups. An aryl group is unsubstituted or substituted.
  • heteroaralkyl refers to a heteroaryl group (as defined herein) in which at least one hydrogen atom (e.g., 1, 2, 3) has been substituted with an unsubstituted or substituted Ci-20 alkyl group, which is typically an unsubstituted Ci-6 alkyl group.
  • Ci-20 alkylene and C 1-20 alkyl groups as defined herein are either uninterrupted or interrupted by one or more heteroatoms or heterogroups, such as S, O or N(R") wherein R" is H, Ci-6 alkyl or aryl (typically phenyl), or by one or more arylene (typically phenylene) groups.
  • the phrase "optionally interrupted” as used herein thus refers to a C 1-20 alkyl group or an alkylene group, as defined above, which is uninterrupted or which is interrupted between adjacent carbon atoms by a heteroatom such as oxygen or sulfur, by a heterogroup such as N(R") wherein R" is H, aryl or Ci-C 6 alkyl, or by an arylene group.
  • guanidino group is a group of the following structure:
  • a reference to a particular compound includes all such isomeric forms, including (wholly or partially) racemic and other mixtures thereof.
  • a reference to a particular compound also includes ionic, salt (e.g. acid addition salt), solvate, protected forms and prodrugs thereof.
  • Examples of pharmaceutically acceptable salts of the compounds for use in accordance with the present invention include salts with inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulphuric acid, nitric acid and phosphoric acid; and organic acids such as methanesulfonic acid, benzenesulphonic acid, formic acid, acetic acid, trifluoroacetic acid, propionic acid, butyric acid, isobutyric acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, ethanesulfonic acid, aspartic acid, benzoic acid and glutamic acid.
  • inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulphuric acid, nitric acid and phosphoric acid
  • organic acids such as methanesulfonic acid, benzenesulphonic acid,
  • the salt is a hydrochloride, an acetate, a propionate, a benzoate, a butyrate or an isobutyrate.
  • pharmaceutically acceptable salts are discussed in Berge et al., 1977, "Pharmaceutically Acceptable Salts," J. Pharm. Sci., Vol. 66, pp. 1-19.
  • a proprotein convertase inhibitor for use in the treatment of a fibrotic disorder, which proprotein convertase inhibitor is a compound of formula (I)
  • Ri is selected from H, OH, 0-CH 3 , H 2 , 0-C(0)-CH 3 and -C(0)-0-(CH 2 )m-CH 3 wherein m is an integer from 1 to 5;
  • Ci-10 alkyl which is unsubstituted or substituted with a halogen, NH 2 , guanidino, C(0)OH or C(0)OR 5 group,
  • a sphingosine acylated with a bifunctional group having the following structure:
  • sphingosyl-phosphorylcholine acylated with a bifunctional group having the followin structure:
  • z is an integer from 2 to 20;
  • R.5 is H or unsubstituted or substituted C 1-3 alkyl
  • R 7 is:
  • heteroaryl ring in said heteroaryl and in said heteroaralkyl contains from 1 to 3 heteroatoms selected from N, S and O,
  • substituent or substituents which may be present at R 7 are independently selected from - H2, -CH2- H2, -amidino, -hydroxyamidino, -guanidino, - CH2-guanidino, methylguanidino, -halogen, -CI, -Br, -I, -CN, -CF 3 , aryl, C1-3 alkyl, C3-20 cycloalkyl, C1-3 alkoxy, and -C(0)0-R 5 wherein R5 is H or unsubstituted or substituted C1-3 alkyl, or
  • P2 in the compound of formula (I) is an a-amino or a-imino acid residue, selected from the following structures
  • p is an integer from 0 to 5;
  • R 3 is an amino, guanidino or methylguanidino group, or an -O-guanidino group (such as, for instance, in a canavanine residue), or an unsubstituted or substituted (e.g.
  • P2 is often an arginine, N-methylarginine (( Me)Arg), lysine or canavanine residue in D- or L-configuration.
  • P2 may for instance be an arginine, lysine or canavanine residue in D- or L-configuration.
  • the P2 residue is in the L-configuration.
  • P2 has the following structure:
  • R 3 and p are as defined above.
  • R 3 is a guanidine group and p is 3.
  • P2 has the following structure:
  • P3 is any natural or unnatural a-amino or a-imino acid residue of L or D configuration.
  • P3 may for instance be any natural a-amino acid residue of L or D configuration, for instance any natural a-amino acid residue of L configuration.
  • P3 is selected from groups having the following structures:
  • P3 is often selected from the following amino acid residues in L or D configuration: Val, Lys, Phe, He, Leu, His, Asn, Asp, Arg, Pro, Ser, Thr, Ala, Gly and Dap.
  • Other possible groups for P3 are the groups defined herein for P4.
  • P3 typically has the following structure:
  • P4 in the compound of formula (I) is an a-amino or a-imino acid residue, selected from the followin structures
  • p is an integer from 0 to 5;
  • P 3 is an amino, guanidine, methylguanidino or C 1-3 alkyl group, or an -O-guanidino group (such as, for instance, in a canavanine residue), or an -N(H)C(0)OCH2Ph group, or an unsubstituted or substituted (e.g.
  • aryl or heteroaryl group having from 5 to 10 ring atoms, wherein the heteroaryl ring contains from 1 to 3 heteroatoms selected from N, S and O, wherein the or each substituent on the aryl or heteroaryl ring is independently selected from - H 2 , -CH2- H2, -amidino, - hydroxyamidino, -guanidino, -CH2-guanidino, -halogen, -CI, -Br, -I, -CN, -CF 3 , C1-3 alkyl, Ci-3 alkoxy and -COOR5, wherein R5 is a hydrogen or C1-3 alkyl; and
  • R4 is -H, -OH or -0-(CH2) q -R6 wherein q is an integer from 2 to 4 and R5 is an amino or guanidino group.
  • P4 is typically an arginine, ( Me)Arg (i.e. N-methylarginine), valine, lysine, N6-Cbz-lysine (N6-[(phenylmethoxy)carbonyl]lysine) or canavanine residue in L- or D- configuration.
  • P4 may for instance be L-arginine, N-methyl-L-arginine, D-Arginine, L- Valine, L-Lysine, N6-Cbz-L-lysine (N6-[(phenylmethoxy)carbonyl]-L-lysine, Lys(Cbz)) or L-canavanine.
  • R3 is preferably an amino, guanidino, or -O-guanidino group, or a substituted aryl group having 6 ring atoms or an unsubstituted pyridyl group (wherein the nitrogen may be present at any position of the pyridyl ring and, if appropriate, may be present as N-oxide), wherein the substituent on the aryl ring is selected from - H2, -CH2- H2, -amidino, - hydroxyamidino, -guanidino, and -CH2-guanidino.
  • P4 is often an arginine, lysine, or canavanine residue in L- or D- configuration.
  • P4 may for instance be L-arginine, D-Arginine, L-Lysine or L-canavanine.
  • P4 has the following structure:
  • R3 and p are as defined above.
  • R3 is a guanidine group and p is 3.
  • P4 has the following structure:
  • P4 is an arginine (Arg) residue.
  • PI is selected from the following structures:
  • Ri is usually H.
  • the hydrophobicity of the inhibitors is capable of being increased (Ettmayer et al, J. Med. Chem. 2004, 47, 2393-2404) by employing compounds wherein Ri is any of OH, 0-CH 3 , H 2 , 0-C(0)-CH 3 and
  • the P2 residue is in the L-configuration.
  • the P2 residue is an amino acid residue in the L-configuration. It is often a basic amino acid residue in the L- configuration. Particularly preferred are the amino acid residues arginine, lysine and their homo or nor-derivatives which have either one methylene group more or one methylene group less in the side chain.
  • canavanine, 3- and 4-amidinophenylalanine, as well as 3- and 4-aminomethylphenylalanine or 3- and 4-guanidinophenylalanine are preferred P2 residues. All phenylalanine derivatives mentioned, such as the homo and nor-derivatives, are also particularly suitable.
  • pyridylalanine and homopyridylalanine are preferable as P2 residues, wherein the nitrogen may be present in any possible position of the pyridyl ring.
  • P2 is present in the L configuration.
  • P2 is preferably selected from the following structures:
  • p is an integer from 0 to 5;
  • R 3 is an amino, guanidino or methylguanidino group, or an -O-guanidino group (such as, for instance, in a canavanine residue), or an unsubstituted or substituted (e.g.
  • aryl or heteroaryl group having from 5 to 10 ring atoms, wherein the heteroaryl ring contains from 1 to 3 heteroatoms selected from N, S and O, wherein the or each substituent on the aryl or heteroaryl ring is independently selected from - H 2 , -CH 2 - H 2 , -amidino, -hydroxyamidino, -guanidino, -CH 2 -guanidino, -halogen, -CI, -Br, -I, -CN, -CF 3 , Ci -3 alkyl, Ci -3 alkoxy and -COOR5, wherein R5 is a hydrogen or Ci- 3 alkyl; and
  • R4 is -H, -OH or -0-(CH 2 ) q -R6 wherein q is an integer from 2 to 4 and R5 is an amino or guanidino group.
  • R 3 is preferably an amino, guanidino, or -O-guanidino group, or a substituted aryl group having 6 ring atoms or an unsubstituted pyridyl group (wherein the nitrogen may be present at any position of the pyridyl ring and, if appropriate, may be present as N-oxide), wherein the substituent on the aryl ring is selected from - H 2 , -CH2- H2, -amidino, - hydroxyamidino, -guanidino, and -CH 2 -guanidino.
  • R3 is an amino, guanidino, or -O-guanidino group.
  • P2 has the following structure:
  • R3 and p are as defined anywhere above.
  • R3 is a guanidine group.
  • p is 3.
  • P2 is often an arginine, N-methylarginine ((NMe)Arg), lysine or canavanine residue in L-configuration.
  • P2 may for instance be an arginine, lysine or canavanine residue in L- configuration.
  • the P3 residue is typically an arbitrary natural or unnatural a-amino or a-imino acid residue in the L- or D- configuration.
  • P3 may be a basic a-amino acid residue in the D- configuration.
  • P3 may be D-arginine, D-homoarginine, D-norarginine, D-lysine, D-homolysine, D-norlysine, D-3-amidinophenylalanine, D-4-amidinophenylalanine, a D- pyridylalanine, or a D-homopyridylalanine, wherein the nitrogen may be in any possible position of the pyridyl ring.
  • P3 may for example be a group of the following structure.
  • P3 may be d-Arg.
  • P3 is a group of the following structure:
  • P3 is an L-valine residue.
  • the P4 residue is in the L-configuration.
  • the P4 residue is an amino acid residue in the L-configuration. It is usually a basic amino acid residue in the L- configuration. Particularly suitable are the amino acid residues arginine, lysine, and their homo or nor-derivatives which have either one methylene group more or one methylene group less in the side chain.
  • canavanine, 3- and 4-amidinophenylalanine, 3- and 4- aminomethylphenylalanine, as well as, 3 and 4-guanidinophenylalanine are preferred P4 residues, wherein also all phenylalanine derivatives mentioned, such as the homo and nor- derivatives, are particularly suitable.
  • pyridylalanine and homopyridylalanine are preferable as P4 residues, wherein the nitrogen is capable of standing in every possible position of the pyridyl ring.
  • P4 is present in the L configuration.
  • P4 is preferably selected from the following structures:
  • p is an integer from 0 to 5;
  • R.3 is an amino, guanidine, methylguanidino or C 1-3 alkyl group, or an -O-guanidino group (such as, for instance, in a canavanine residue), or an -N(H)C(0)OCH2Ph group, or an unsubstituted or substituted (e.g.
  • aryl or heteroaryl group having from 5 to 10 ring atoms, wherein the heteroaryl ring contains from 1 to 3 heteroatoms selected from N, S and O, wherein the or each substituent on the aryl or heteroaryl ring is independently selected from - H 2 , -CH2- H2, -amidino, - hydroxyamidino, -guanidino, -CH2-guanidino, -halogen, -CI, -Br, -I, -CN, -CF 3 , C1-3 alkyl, Ci-3 alkoxy and -COOR5, wherein R5 is a hydrogen or C1-3 alkyl; and
  • R4 is -H, -OH or -0-(CH2) q -R6 wherein q is an integer from 2 to 4 and R5 is an amino or guanidine group.
  • P4 is often an arginine, N-methylarginine ((NMe)Arg), valine, lysine, N6-Cbz- lysine (N6-[(phenylmethoxy)carbonyl]lysine, Lys(Cbz)) or canavanine residue in L- configuration.
  • R3 is preferably an amino, guanidino, or -O-guanidino group, or a substituted aryl group having 6 ring atoms or an unsubstituted pyridyl group (wherein the nitrogen may be present at any position of the pyridyl ring and, if appropriate, may be present as N-oxide), wherein the substituent on the aryl ring is selected from - H2, -CH2- H2, -amidino, - hydroxyamidino, -guanidino, and -CH2-guanidino.
  • P4 is preferably an arginine residue in L-configuration (L-arginine).
  • PI is a 4-amidinobenzylamide group
  • P3 is selected from groups having the following structures:
  • P5 is as defined anywhere herein. Often, however, in this embodiment, P5 is selected from -CO-R7, -CO-X-R7 and -SO2-R7, wherein R7 and X are as defined above. In particular, P5 is often:
  • R7 is either unsubstituted or substituted C 1-24 alkyl or
  • R7 is either unsubstituted or substituted phenyl or
  • P5 may for instance in this embodiment be:
  • Preferred compounds of formula (I) are the following: 97
  • Arg is an L-arginine amino acid residue
  • d-Arg is a D-arginine amino acid residue
  • Lys(Cbz) is an N6-[(phenylmethoxy)carbonyl]-L-lysine amino acid residue; Val is an L-valine amino acid residue; Phe is an L-phenylalanine amino acid residue;
  • Leu is an L-leucine amino acid residue
  • His is an L-histidine amino acid residue
  • Asn is an L-asparagine amino acid residue
  • Asp is an L-aspartic acid amino acid residue
  • Pro is an L-proline amino acid residue
  • Ser is an L-serine amino acid residue
  • Thr is an L-threonine amino acid residue
  • Ala is an L-alanine amino acid residue
  • the proprotein convertase inhibitor may be any one of the compounds of formula (I) shown above, or a prodrug thereof, or a pharmaceutically acceptable salt thereof.
  • the proprotein convertase inhibitor may be a compound of formula (la) as shown above, or a prodrug thereof, or a pharmaceutically acceptable salt thereof.
  • proprotein convertase inhibitors which inhibit collagen matrix formation do not significantly affect fibronectin secretion.
  • fibronectin plays a major role in cell adhesion, growth, migration, and differentiation and is important for processes such as wound healing and embryonic development.
  • altered fibronectin expression, degradation, and organization has been associated with a number of pathologies, including cancer. It can therefore be desirable not to alter fibronectin secretion.
  • the invention provides a proprotein convertase inhibitor for use in the treatment of a fibrotic disorder by inhibiting the formation of collagen matrix without inhibiting fibronectin secretion.
  • the invention also provides a composition for use in the treatment of a fibrotic disorder by inhibiting collagen matrix formation without inhibiting fibronectin secretion, which composition comprises a proprotein convertase inhibitor and a pharmaceutically acceptable carrier or diluent.
  • the invention also provides the use of a proprotein convertase inhibitor in the manufacture of a medicament for use in the treatment of a fibrotic disorder by inhibiting collagen matrix formation without inhibiting fibronectin secretion.
  • the invention also provides a method of treating a fibrotic disorder in a subject by inhibiting collagen matrix formation without inhibiting fibronectin secretion, which method comprises administering to the subject a therapeutically effective amount of a proprotein convertase inhibitor, which amount is effective to inhibit collagen matrix formation in the subject without inhibiting fibronectin secretion.
  • a proprotein convertase inhibitor capable of inhibiting collagen matrix formation without significantly inhibiting fibronectin secretion may be employed in these aspects of the invention.
  • proprotein convertase inhibitor that is capable of inhibiting collagen matrix formation without significantly inhibiting fibronectin secretion is the class of compounds of formula (I) and formula (la) as defined herein.
  • the proprotein convertase inhibitor employed is a compound of formula (I), or formula (la), or a prodrug thereof or a pharmaceutically acceptable salt thereof.
  • proprotein convertase inhibitors may also be employed in the present invention, to treat fibrotic disorders by inhibiting collagen matrix formation without inhibiting fibronectin secretion.
  • the skilled person is readily able to identify other suitable proprotein convertase inhibitors, as many proprotein convertase inhibitors are known in the art, as are procedures for screening compounds for proprotein convertase inhibitory activity.
  • proprotein convertase inhibitors may be identified using the screening methods described in the following references: Becker, G. L., et al. (2012). "Highly potent inhibitors of proprotein convertase furin as potential drugs for treatment of infectious diseases.” Journal of Biological Chemistry 287(26): 21992-22003; Becker, G. L., et al. (2010). "Potent Inhibitors of Furin and Furin-like Proprotein Convertases
  • proprotein convertase inhibitors that may be employed in the present invention include compounds that prevent the transcription, translation or expression of a proprotein convertase.
  • proprotein convertase inhibitors include, for instance, ribozymes and antisense and RNA interference molecules, such as an antisense RNA, small interfering RNA (siRNA) or small hairpin RNA (shRNA).
  • RNAi Antisense and RNA interference (RNAi) technology for knocking down protein expression are well known in the art and standard methods can be employed to knock down expression of PCSKs.
  • oligonucleotides interfere with mRNA by binding to (hybridising with) a section of the mRNA.
  • the antisense oligonucleotide is therefore designed to be complementary to the mRNA (although the oligonucleotide does not have to be 100% complementary as discussed below).
  • the antisense oligonucleotide may be a section of the cDNA.
  • the oligonucleotide sequence may not be 100% identical to the cDNA sequence. This is also discussed below.
  • RNAi involves the use of double-stranded RNA, such small interfering RNA (siRNA) or small hairpin RNA (shRNA), which can bind to the mRNA and inhibit protein expression.
  • siRNA small interfering RNA
  • shRNA small hairpin RNA
  • the inhibitor comprises an oligonucleotide which
  • the inhibitor comprises an oligonucleotide which specifically hybridises to a part of SEQ ID NO: 5 (human PCSK 3 mRNA) or any isoform thereof.
  • Oligonucleotides are short nucleotide polymers which typically have 50 or fewer nucleotides, such 40 or fewer, 30 or fewer, 22 or fewer, 21 or fewer, 20 or fewer, 10 or fewer or 5 or fewer nucleotides.
  • the oligonucleotide used in the invention is preferably 20 to 25 nucleotides in length, more preferably 21 or 22 nucleotides in length.
  • the nucleotides can be naturally occurring or artificial.
  • a nucleotide typically contains a nucleobase, a sugar and at least one linking group, such as a phosphate, 2'O-methyl, 2' methoxy-ethyl, phosphoramidate, methylphosphonate or phosphorothioate group.
  • the nucleobase is typically heterocyclic.
  • Nucleobases include, but are not limited to, purines and pyrimidines and more specifically adenine (A), guanine (G), thymine (T), uracil (U) and cytosine (C).
  • the sugar is typically a pentose sugar.
  • Nucleotide sugars include, but are not limited to, ribose and deoxyribose.
  • nucleotides may contain additional modifications.
  • suitable modified nucleotides include, but are not limited to, 2'amino pyrimidines (such as 2'- amino cytidine and 2 '-amino uridine), 2'-hyrdroxyl purines (such as , 2'-fluoro
  • oligonucleotide may comprise a pyrimidine dimer.
  • dimers are typically associated with damage by ultraviolet light.
  • the nucleotides in the oligonucleotide may be attached to each other in any manner.
  • the nucleotides may be linked by phosphate, 2'0-methyl, 2' methoxy-ethyl, phosphoramidate, methylphosphonate or phosphorothioate linkages.
  • the nucleotides are typically attached by their sugar and phosphate groups as in nucleic acids.
  • the nucleotides may be connected via their nucleobases as in pyrimidine dimers.
  • An oligonucleotide “specifically hybridises” to a target sequence when it hybridises with preferential or high affinity to the target sequence but does not substantially hybridise, does not hybridise or hybridises with only low affinity to other sequences.
  • An oligonucleotide “specifically hybridises” if it hybridises to the target sequence with a melting temperature (T m ) that is at least 2 °C, such as at least 3 °C, at least 4 °C, at least 5 °C, at least 6 °C, at least 7 °C, at least 8 °C, at least 9 °C or at least 10 °C, greater than its T m for other sequences.
  • T m melting temperature
  • the inhibitor preferably comprises an oligonucleotide which comprises 50 or fewer consecutive nucleotides from (a) SEQ ID NO: 2, 3, 6, 8, 10, 12, 14, 16 or 18 (human PCSK 1 to 9 cDNA respectively) or any isoform thereof or (b) a variant sequence which has at least 95%, such as at least 97%, at least 98% or at least 99%, homology to SEQ ID NO: 2, 3, 6, 8, 10, 12, 14, 16 or 18 or any isoform thereof based on nucleotide identity over the entire sequence.
  • the oligonucleotide may be any of the lengths discussed above. It is preferably from 16 to 22 nucleotides in length, for instance from 18 to 20 nucleotides in length. It may for instance be 19 nucleotides in length.
  • the oligonucleotide may comprise any of the nucleotides discussed above, including the modified nucleotides.
  • the inhibitor comprises an oligonucleotide which comprises 50 or fewer consecutive nucleotides from (a) SEQ ID NO: 6 (human PCSK 3 cDNA) or any isoform thereof or (b) a variant sequence which has at least 95%, such as at least 97%, at least 98% or at least 99%, homology to SEQ ID NO: 6 or any isoform thereof based on nucleotide identity over the entire sequence.
  • the oligonucleotide may be single stranded.
  • the oligonucleotide may be double stranded.
  • the oligonucleotide may compirse a hairpin.
  • SEQ ID NOs: 19 to 30 are oligonucleotides which specifically hybridise to a part of SEQ ID NO: 5, 9 or 11 (human PCSK 3, 5 and 6 mRNA respectively).
  • the inhibitor preferably comprises an oligonucleotide which is (a) any one of SEQ ID NOs: 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30, or (b) a variant sequence which has at least 95%, such as at least 97%, at least 98% or at least 99%, homology to any one of SEQ ID NOs: 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30 or any isoform thereof based on nucleotide identity over the entire sequence.
  • an oligonucleotide which is (a) any one of SEQ ID NOs: 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30, or (b) a variant sequence which has at least 95%, such as at least 97%, at least 98% or at least 99%, homology to any one of SEQ ID NOs: 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30 or any isoform thereof based on nucleotide identity over the entire sequence.
  • the inhibitor may for instance comprise any one of SEQ ID NOs: 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30.
  • the inhibitor could for instance comprise two or more different oligonucleotides, for instance two, three or four different oligonucleotides, each of which is (a) any one of SEQ ID NOs: 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30, or (b) a variant sequence which has at least 95%, such as at least 97%, at least 98% or at least 99%, homology to any one of SEQ ID NOs: 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30 or any isoform thereof based on nucleotide identity over the entire sequence.
  • the inhibitor may for instance comprise two or more different oligonucleotides, for instance two, three or four different oligonucleotides, each of which is any one of SEQ ID NOs: 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30.
  • the inhibitor may comprise two or more of, for instance two, three or four of the following oligonucleotides: SEQ ID NOs: 23, 24, 25 and 26.
  • the inhibitor may for instance comprise two or more of, for instance two, three or four of the following oligonucleotides: SEQ ID NOs: 27, 28, 29 and 30.
  • the inhibitor comprises an oligonucleotide which specifically hybridises to a part of SEQ ID NO: 5 (human PCSK 3, FURIN) or any isoform thereof.
  • the inhibitor may for instance comprise any one of SEQ ID NOs: 19, 20, 21 and 22.
  • the inhibitor could for instance comprise two or more different oligonucleotides, for instance two, three or four different oligonucleotides, each of which is (a) any one of SEQ ID NOs: 19, 20, 21 and 22, or (b) a variant sequence which has at least 95%, such as at least 97%, at least 98% or at least 99%, homology to any one of SEQ ID NOs: 19, 20, 21 and 22 or any isoform thereof based on nucleotide identity over the entire sequence.
  • the inhibitor may for instance comprise two or more different oligonucleotides, for instance two, three or four different oligonucleotides, selected from the oligonucleotides having SEQ ID NOs: 19, 20, 21 and 22.
  • the inhibitor may for example comprise the oligonucleotide of SEQ ID NO: 19, the oligonucleotide of SEQ ID NO: 20, the oligonucleotide of SEQ ID NO: 21 and the oligonucleotide of SEQ ID NO: 22.
  • Oligonucleotides may be synthesised using standard techniques known in the art.
  • the proprotein convertase inhibitor employed in the present invention is usually other than the two compounds whose structures are shown above. It is also typically also other than salts, solvates, enantiomers, diastereomers and racemates of those compounds.
  • Fibrotic disorders can be treated with a proprotein convertase inhibitor in accordance with the present invention.
  • fibrotic disorder refers to a disorder in which excessive fibrosis leads to pathological derangement and malfunctioning of tissue.
  • Atrial fibrillation cardiac diastolic dysfunction, heart failure, cardiomyopathies, dermal fibrosis, a fibrotic skin disease, cirrhosis, Crohn's disease, cirrhosis of the liver, liver fibrosis, progressive kidney disease, glomerulonephritis, renal fibrosis, pulmonary fibrosis, idiopathic pulmonary fibrosis, cardiovascular fibrosis, myocardial fibrosis, myocardial hibernation, fibrosis following myocardial infarction, systemic sclerosis, central nervous system fibrosis following a stroke or following a neuro-degenerative disorder (e.g.
  • Fibrotic disorders which can be treated in accordance with the present invention therefore include, but are not limited to, the following conditions:
  • CFIF chronic heart failure
  • a fibrotic skin disease for example: scleroderma, nephrogenic fibrosing dermopathy, mixed connective tissue disease, scleromyxedema, scleredema, eosinophilic fasciitis, or a fibrotic skin disease caused by exposure to a chemical or physical agent;
  • cirrhosis for instance cirrhosis of the liver
  • CD Crohn's Disease
  • treating and “treatment” as used herein in the context of treating a fibrotic disorder refers generally to treatment and therapy, whether of a human subject or an animal subject (e.g. in veterinary applications), in which some desired therapeutic effect is achieved, such as, for example, the inhibition of the progress of the condition.
  • the term includes a reduction in the rate of progress, a halt in the rate of progress, regression of the condition, amelioration of the condition, and cure of the condition.
  • Palliative treatment or treatment as a prophylactic measure i.e. prophylaxis, prevention
  • prophylactic measure i.e. prophylaxis, prevention
  • Fibrotic disorders can be treated with a proprotein convertase inhibitor in accordance with the present invention either alone or in combination with a further active ingredient.
  • the further active ingredient may for instance be a further antifibrotic agent.
  • Further classes of antifibrotic agents that may be used in combination with a proprotein convertase inhibitor include PDE6D inhibitors, the drug pirfenidone, Endothelin antagonists, Endothelin receptor inhibitors (e.g.
  • the invention further provides the use of (b) a further antifibrotic agent in the manufacture of a medicament for use in the treatment of a fibrotic disorder by
  • the invention further provides the use of (a) proprotein convertase inhibitor and (b) a further antifibrotic agent in the manufacture of a medicament for use in the treatment of a fibrotic disorder.
  • the invention also provides a method of treating a fibrotic disorder which method comprises administering to a subject in need of such treatment therapeutically effective amounts of (a) a proprotein convertase inhibitor and (b) a further antifibrotic agent.
  • the invention also provides a kit of parts comprising a proprotein convertase inhibitor together with instructions for simultaneous, concurrent, separate or sequential use in combination with a further antifibrotic agent, for the treatment of a fibrotic disorder.
  • the invention also provides a kit of parts comprising a further antifibrotic agent together with instructions for simultaneous, concurrent, separate or sequential use in combination with a proprotein convertase inhibitor, for the treatment of a fibrotic disorder.
  • the invention also provides a kit of parts comprising a proprotein convertase inhibitor, a further antifibrotic agent, and instructions for their simultaneous, concurrent, separate or sequential use for the treatment of a fibrotic disorder.
  • the proprotein convertase inhibitor employed in these "combination" aspects of the invention may be any of the proprotein convertase inhibitors described herein.
  • Endothelin antagonists include Endothelin antagonists, Endothelin receptor inhibitors (e.g. Bosentan), Transforming growth factor beta (TGFbeta) antagonists, TGFbeta receptor inhibitors, Platelet-derived growth factor (PDGF) antagonists, PDGF receptor inhibitors, chemokine antagonists, chemokine receptor inhibitors, cytokine antagonists, cytokine receptor inhibitors, N-acetylcysteine, prednisone, azathioprine, and tyrosine kinase inhibitors.
  • Endothelin antagonists e.g. Bosentan
  • TGFbeta Transforming growth factor beta
  • PDGF Platelet-derived growth factor
  • chemokine antagonists chemokine receptor inhibitors
  • cytokine antagonists cytokine antagonists
  • cytokine receptor inhibitors N-acetylcysteine
  • prednisone azathioprine
  • azathioprine
  • the further antifibrotic agent is an Endothelin antagonist, an Endothelin receptor inhibitor (e.g. Bosentan), a Transforming growth factor beta (TGFbeta) antagonist, a TGFbeta receptor inhibitor, a Platelet-derived growth factor (PDGF) antagonist, a PDGF receptor inhibitor, a chemokine antagonist, a chemokine receptor inhibitor, a cytokine antagonist, a cytokine receptor inhibitor, N- acetylcysteine, prednisone, azathioprine, or a tyrosine kinase inhibitor.
  • Another class of antifibrotic agent that can be employed in combination with the proprotein convertase inhibitor in the treatment of a fibrotic disorder is a PDE6D inhibitor.
  • the further antifibrotic agent is a PDE6D inhibitor.
  • 1602641.1 are incorporated herein by reference in their entirety.
  • the further antifibrotic agent is a PDE6D inhibitor which is a compound of formula (X)
  • R 5 , R 5 ' and R 5 " are independently selected from -H, -OCH 3 , -OC2H5,
  • R 8 and R 8 ' are independently selected fr m -H, -C0 2 C(CH 3 ) 3 ,
  • R 9 and R 10 are independently selected from -H, -F, -Br, -CI, -I, -CN, -OH, -OCH 3 , -OC 2 H 5 , -CH 3 and -C 2 H 5 ;
  • R 12 is -Ph or -OH
  • the compound of formula (X) may be selected from 2-(4-(Allyloxy)phenyl)-l- benzyl- lH-benzo[d]imidazole, 2-(4-(Allyloxy)phenyl)-l-(pyridin-2-ylmethyl)-lH- benzo[d]imidazole, 2-(4-(Allyloxy)phenyl)- 1 -(2-methylbenzyl)- lH-benzo[d]imidazole, 2- (4-(Allyloxy)phenyl)-l-(3-methylbenzyl)-lH-benzo[d]imidazole, 2-(4-(Allyloxy)phenyl)-
  • the further antifibrotic agent may for instance be a PDE6D inhibitor of the following formula (Xa) which is deltarasin:
  • R 21 and R 22 may together form an unsubstituted or substituted Ci-20 alkylene group, wherein said Ci-20 alkylene is optionally interrupted by N(R'), O or S wherein R' is H, Ci-6 alkyl or aryl;
  • R is OH, NHR 20 or R 21 R 22 ;
  • R 20 is -CH 2 -
  • R 1 is H, unsubstituted or substituted aryl, unsubstituted or substituted C3-10 cycloalkyl, unsubstituted or substituted C3-7 heterocyclyl, unsubstituted or substituted C3-10 heteroaryl, and unsubstituted or substituted Ci-10 alkyl; and
  • R 1 is H, unsubstituted or substituted phenyl, or unsubstituted or substituted C3-10 cycloalkyl; and R 2 is H or unsubstituted or substituted Ci-6 alkyl. More typically, R 2 is zso-propyl and R 1 is phenyl, cyclopropyl or cyclopentyl.
  • oligonucleotide which specifically hybridises to a part of SEQ ID NO: 5 (human PCSK 3 mRNA) or any isoform thereof;
  • the further antifibrotic compound is a PDE6D inhibitor of the following formula:
  • the proprotein convertase inhibitor comprises an oligonucleotide which is (a) any one of SEQ ID NOs: 19, 20, 21 and 22, or (b) a variant sequence which has at least 95% homology to any one of SEQ ID NOs: 19, 20, 21 and 22 or any isoform thereof based on nucleotide identity over the entire sequence; and
  • the further antifibrotic compound is a PDE6D inhibitor of the following formula:
  • the further antifibrotic compound is a PDE6D inhibitor of the following formula:
  • the dosage depends on a variety of factors including the age, weight and condition of the patient and the route of administration. Daily dosages can vary within wide limits and will be adjusted to the individual requirements in each particular case. Typically, however, the dosage adopted for each route of administration when a compound is administered alone to adult humans is 0.0001 to 50 mg/kg, most commonly in the range of 0.001 to 10 mg/kg, body weight, for instance 0.01 to 1 mg/kg. Such a dosage may be given, for example, from 1 to 5 times daily. For intravenous injection a suitable daily dose is from 0.0001 to 1 mg/kg body weight, preferably from 0.0001 to 0.1 mg/kg body weight. A daily dosage can be administered as a single dosage or according to a divided dose schedule.
  • the tablets may be uncoated or they may be coated by known techniques to delay disintegration and adsorption in the gastrointestinal tract and thereby provide a sustained action over a longer period.
  • a time delay material such as glyceryl monostearate or glyceryl distearate may be employed.
  • Such preparations may be manufactured in a known manner, for example by means of mixing, granulating, tableting, sugar coating or film coating processes.
  • the emulsion may also contain sweetening and flavouring agents.
  • Syrups and elixirs may be formulated with sweetening agents, for example glycerol, sorbitol or sucrose.
  • a syrup for diabetic patients can contain as carriers only products, for example sorbitol, which do not metabolise to glucose or which only metabolise a very small amount to glucose.
  • the invention provides an in vitro method of inhibiting the formation of collagen matrix without inhibiting fibronectin secretion, the method comprising contacting an in vitro sample comprising collagen-secreting cells with an amount of a proprotein convertase inhibitor effective to inhibit the formation of collagen matrix by the cells without inhibiting fibronectin secretion.
  • the in vitro method comprises contacting said sample with an amount of a proprotein convertase inhibitor effective to inhibit the formation of collagen matrix by the cells without inhibiting the secretion by the cells of fibronectin.
  • it comprises contacting said sample with an amount of a proprotein convertase inhibitor effective to inhibit the formation of collagen matrix by the cells without altering the secretion by the cells of fibronectin.
  • dermal fibroblasts are employed.
  • An amount of a proprotein convertase inhibitor which is effective to inhibit the formation of collagen matrix by the cells may be easily determined by the skilled person.
  • the amount of proprotein convertase inhibitor which is effective to inhibit the formation of collagen matrix by the cells is in the micromolar range, for instance from 0.01 ⁇ to 500 ⁇ , or for example from 0.1 to 50 ⁇ , for instance from 0.5 to 25 ⁇ .
  • the invention also provides a novel assay method for identifying molecules that affect collagen secretion in vitro, and thus for identifying antifibrotic agents.
  • the invention provides a method of identifying an agent as being capable of inhibiting collagen matrix formation, which method comprises (a) contacting an agent with an in vitro sample comprising collagen- secreting cells in the absence of exogenous transforming growth factor (TGF) and (b) measuring the amount of collagen matrix formed by the cells and thereby determining whether or not the agent is capable of inhibiting collagen matrix formation.
  • TGF transforming growth factor
  • the agent used in the method of the invention may be any of the types discussed above.
  • the agent may be a compound or an oligonucleotide as defined above.
  • the agent is contacted with an in vitro sample comprising collagen-secreting cells. Such samples and cells are defined above.
  • the cells are preferably human cells.
  • the cells are preferably fibroblasts.
  • the fibroblasts may be any of those discussed above.
  • the cells are preferably normal human dermal fibroblast ( HDF).
  • the cells are preferably not WI-38 human lung fibroblasts.
  • NHDF cells provide better morphological contrast for quantitative imaging as compared to WI-38 lung fibroblasts.
  • the agent is contacted with the in vitro sample comprising collagen-secreting cells in the absence of exogenous TGF.
  • Exogenous TGF is TGF that is added to the in vitro sample, such as to the culture medium, comprising the cells. In other words, exogenous TGF is TGF that is not produced by the cells themselves.
  • the culture medium added to the cells does not contain exogenous TGF.
  • the TGF is preferably human TGF.
  • the TGF is preferably TGFp, such as TGFpi, TGFP2 or TGFP3.
  • the agent is preferably contacted with the in vitro sample comprising collagen-secreting cells in the absence of human TGFpi .
  • the contacting may be carried out in any suitable volume. Typical volumes range from about ⁇ to about 1ml, preferably from about 50 ⁇ 1 to about 500 ⁇ 1, more preferably from about ⁇ to about 200 ⁇ 1. Typically, the length of time for which the cells are contacted with the agent is from about 1 day to about 5 days, such as about 2 days, about 3 days or about 4 days.
  • the cells may be contacted with the agent at any suitable temperature.
  • the suitable temperature is typically in the same range as the normal body temperature of the human or animal from which the cells are derived.
  • the incubation is carried out at a fixed temperature between about 4°C and about 38°C, preferably at about 37°C.
  • the cells are typically cultured under standard conditions of 37°C, 5% CO 2 in medium supplemented with serum.
  • the cells such as fibroblasts, are preferably conditioned before they are contacted with the agent.
  • Fibroblasts are preferably conditioned with an optimal conditioned medium (Fibroblast Growth Medium, Lonza CC-3131) supplemented with insulin and fibroblast growth factor (FGF) before they are contacted with the agent.
  • the optimal conditioned medium preferably comprises 2% fetal bovine serum (FBS).
  • the cells, such as fibroblasts may be conditioned for at least about one day.
  • the cells may be contacted with the agent in any culture medium.
  • the culture medium preferably comprises Ficoll PM-400.
  • the culture medium preferably comprises Ficoll PM-400 and ascorbic acid.
  • the cells, such as fibroblasts are preferably contacted with the agent in Fibroblast Growth Medium (Lonza CC-3131) supplemented with insulin, FGF, 0.5% FBS, ascorbic acid and Ficoll PM400.
  • the method may be carried out using any number of agents. For instance, the method may be carried out using 1, 2, 5, 10, 15, 20, 30, 40, 50 , 100, 150, 200, 300, 500 or more agents. The method is preferably carried out using 6, 12, 24, 48, 96 or 384 or 1526 agents. This allows high-throughput screening of agents.
  • the method may be carried out using any number of in vitro samples. For instance, the method may be carried out using 1, 2, 5, 10, 15, 20, 30, 40, 50 , 100, 150, 200, 300, 500 or more samples. The method is preferably carried out using 6, 12, 24, 48, 96 or 384 or 1526 samples. This allows high-throughput screening of agents.
  • Collagen matrix formation may be measured using standard techniques, such as those disclosed in the Examples. Collagen matrix formation is preferably measured by immunofluorescence. It may for instance measured using dyes, e.g. Sirius dye picrosirius red or Sirius Red F3BA.
  • the Sircol collagen assay may be employed, or for instance, enzyme linked immunoassays, gas chromatography, mass spectrometry, polyacrylamide gel electrophoresis following stable metabolic labelling with radiolabelled glycine and proline, or followed by silver staining, or by western blotting with an antibody, among other methods; see, for instance: Chen, C. Z. and M. Raghunath (2009); Fibrogenesis Tissue Repair 2(1): 7.
  • the agent is capable of inhibiting collagen matrix formation if the cells produce less collagen matrix ⁇ i.e. a decreased amount of collagen matrix) in the presence of the agent than in the absence of the agent.
  • the method preferably comprises comparing the amount of collagen matrix produced by the cells in the presence of the agent with the amount of collagen produced by the cells in the absence of the agent.
  • the amount of collagen matrix produced in the absence of the agent i.e. the control value
  • the control value may be obtained beforehand and recorded, for instance on a computer.
  • the control value is typically used for comparison purposes in method of the invention.
  • the control value may be used for multiple repetitions of the method of the invention.
  • the control value is preferably obtained under the same conditions, such as cell number, cell type and culture conditions, under which the method of the invention is carried out.
  • the amount of collagen matrix produced in the absence of the agent is obtained at the same time as carrying out the method of the invention (i.e. using one or more control samples of cells which are not contacted with the agent).
  • the test samples and control sample(s) are present in the wells of a standard 96 or 384 well plate. The test and control samples are then assayed using the same conditions.
  • PCSKs Proprotein convertases
  • PCSKs are responsible for the cleavage and activation of proteins that biochemically play a role in collagen secretion.
  • Example 1 Modified scar-in-a-iar assay and evaluation of Calbiochem 537076 (and decanoyl-RVKR-CMK and hexa-D-arginine)
  • Figure 6 A miniaturized "Scar-in-a-Jar" assay.
  • the "Scar-in-a-Jar” assay is a well-validated in vitro assay that uses WI-38 human embryonic lung fibroblasts, macromolecular crowding, and exogenous TGFB 1 to achieve rapid deposition of cross-linked collagen in vitro (Chen, C. et al. The Scar-in-a-Jar:
  • Calbiochem 537076 markedly inhibits reticular collagen formation ( Figure 1, 2) while two other proprotein convertase inhibitors - dec-RVKR-cmk and hexa-D- arginine amide - had a smaller effect. This is possibly due to the -1000 fold excess inhibitory activity of Calbiochem 537076 in comparison to the other two compounds.
  • Calbiochem 537076 has an IC50 of 2.17 ⁇ ( Figure 2). This is substantially greater than its published Ki on FURIN ( ⁇ 17 pM (Becker, G.L. et al. Highly potent inhibitors of proprotein convertase furin as potential drugs for treatment of infectious diseases. Journal of Biological Chemistry 287, 21992-22003 (2012))).
  • the proprotein convertase inhibitor for use in accordance with the invention, is dissolved in most of the water (35° to 40° C) and the pH adjusted to between 4.0 and 7.0 with the hydrochloric acid or the sodium hydroxide as appropriate.
  • the batch is then made up to volume with water and filtered through a sterile micropore filter into a sterile 10 ml amber glass vial (type 1) and sealed with sterile closures and overseals.
  • SEQ ID NOs: 1, 3, 5, 7, 9, 1 1, 13, 15 and 17 set forth human PCSKl, 2, 3, 4, 5, 6, 7, 8 and 9 mRNA respectively.
  • SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16 and 18 set forth human PCSKl, 2, 3, 4, 5, 6, 7, 8 and 9 cDNA respectively.
  • SEQ ID NO: 19 sets forth anti- human PCSK 3 (FURIN) siRNA no. 1.
  • SEQ ID NO: 21 sets forth anti- human PCSK 3 (FURIN) siRNA no. 3.
  • SEQ ID NO: 22 sets forth anti- human PCSK 3 (FURIN) siRNA no. 4.
  • SEQ ID NO: 24 sets forth anti- human PCSK 5 siRNA no. 2.
  • SEQ ID NO: 25 sets forth anti- human PCSK 5 siRNA no. 3.
  • SEQ ID NO: 26 sets forth anti- human PCSK 5 siRNA no. 4.
  • SEQ ID NO: 27 sets forth anti- human PCSK 6 siRNA no. 1.
  • SEQ ID NO: 28 sets forth anti- human PCSK 6 siRNA no. 2.
  • SEQ ID NO: 29 sets forth anti- human PCSK 6 siRNA no. 3.
  • SEQ ID NO: 30 sets forth anti- human PCSK 6 siRNA no. 4.
  • SEQ ID NO: 31 sets forth non-targeting control siRNA no. 1.
  • SEQ ID NO: 32 sets forth non-targeting control siRNA no. 2.
  • SEQ ID NO: 34 sets forth non-targeting control siRNA no. 4.
  • SEQ ID NO: 35 sets forth the commercially-available proprotein convertase inhibitor decanoyl-Arg-Val-Lys-Arg-CMK.

Abstract

The present invention relates to proprotein convertase inhibitors for use in the treatment of fibrotic disorders. It also provides a novel assay method for identifying antifibrotic agents.

Description

TREATMENT OF FIBROTIC DISORDERS
FIELD OF THE INVENTION
The present invention relates to the treatment of fibrotic disorders.
BACKGROUND TO THE INVENTION
Fibrosis is characterised by the overgrowth, hardening, and/or scarring of various tissues and is attributed to the deposition of extracellular matrix components including collagen. Disorders in which excessive fibrosis leads to pathological derangement and malfunctioning of tissue are known as fibrotic disorders. Fibrotic disorders are characterised by the accumulation of fibrous tissue (predominately collagens) in an abnormal fashion within the tissue. Accumulation of such fibrous tissues may result from a variety of disease processes. These diseases do not necessarily have to be caused by surgery, traumatic injury or wounding. Fibrotic disorders are usually chronic. Examples of fibrotic disorders include cirrhosis of the liver, liver fibrosis, progressive kidney disease, glomerulonephritis, pulmonary fibrosis, idiopathic pulmonary fibrosis, dermal fibrosis, fibrotic skin disease, cardiovascular fibrosis, myocardial fibrosis, fibrosis following myocardial infarction, systemic sclerosis, central nervous system fibrosis following a stroke or neuro-degenerative disorders (e.g. Alzheimer's Disease), proliferative vitreoretinopathy (PVR), restenosis (for example following
angioplasty), arthritis and Crohn's disease.
The excessive fibrosis in such disorders can result from a chronic inflammatory reaction, which may itself be induced by a variety of stimuli, including for example a persistent infection, an autoimmune reaction, an allergic response, a chemical insult, radiation, and tissue injury. Current strategies for treating fibrotic disorders typically focus on targeting the inflammatory response.
Current treatments for Crohn's Disease (CD), for instance, are mainly focussed on the blockade of inflammation, although advanced fibrosis in CD is typically treated by either surgery, which affects patients' quality of life, or by endoscopic dilation (H. Szabo et al., Aliment Pharmacol Ther 31, 189-201).
Although the pathological accumulation of extracellular matrix is also a key
contributor to chronic heart failure (CHF), current therapeutic strategies for CFIF do not target the profibrotic inflammatory processes occurring in the heart that contribute to accumulation of extracellular matrix. Strategies designed to target proinflammatory cytokine (TGF-β) activation and thereby prevent pathological fibrosis in the heart are discussed in A. J. Edgley et al. Cardiovascular Therapeutics 30 (2012) e30-e40, but an effective therapy along these lines has yet to be identified.
Boor, P. et al. Nat. Rev. Nephrol. 6, 643-656 (2010) discusses current insights into mechanisms and therapeutic targets for renal fibrosis, which is the common end point of virtually all progressive kidney diseases. Possible mechanisms and potential treatment targets based on different cell types are discussed, as are the ongoing difficulties in the field, which include specific therapeutic targeting of the kidney, the development of improved diagnostic methods to assess renal fibrosis and the shortcomings of available animal models. However, there remains an urgent need for antifibrotic therapies for treating such progressive kidney disorders.
Evolving therapies for liver fibrosis are discussed D. Schuppan et al, Journal of Clinical Investigation; vol. 123, no. 5, May 2013. In the liver, fibrosis is thought mainly to be due to chronic viral hepatitis B or C, autoimmune and biliary diseases, alcoholic
steatohepatitis (ASH) and, increasingly, nonalcoholic steatohepatitis (NASH). The progression of fibrosis in the liver toward cirrhosis is the major cause of liver-related morbidity and mortality. Current preventive measures include antiviral regimens for hepatitis B or C.
However other causes, such as NASH (which is linked to obesity and type 2 diabetes) remain unaddressed. Moreover, numerous patients present initially in the clinic with advanced fibrosis or cirrhosis, which are largely irreversible. Therefore, antifibrotics, especially ones that prevent progression toward cirrhosis, or induce regression of advanced fibrosis and cirrhosis, are urgently needed.
Systemic sclerosis (SSc) is a rare autoimmune disorder characterized by immune activation, vascular damage and an excessive accumulation of extracellular matrix proteins in the skin and internal organs. Treatment concepts for SSc are discussed in M. Antic et al., Current Opinion in Pharmacology 2013, 13 :455-462, which presents the most promising targets for treatment of fibrotic disorders as including inhibitors of B-cells, tyrosine kinases, 5- hydroxytryptamin receptors, interleukin-6 and Wnt signalling. However, Antic et al. indicate that the currently available treatment options for fibrotic manifestations of SSc remain limited and their clinical antifibrotic effects are borderline.
There is clearly therefore an important need for identifying molecules that inhibit collagen matrix formation in the search for effective treatments for fibrotic disorders.
Pirfenidone is the only antifibrotic that is currently available, but that is toxic and is currently only licensed for idiopathic pulmonary fibrosis. T. A. Wynn, J Pathol 2008; 214: 199-210 discusses the current understanding of the cellular and molecular mechanisms of fibrogenesis, including that the key cellular mediator of fibrosis is the myofibroblast, which when activated serves as the primary collagen-producing cell. Wynn indicates that myofibroblasts are activated by a variety of mechanisms, and that cytokines (IL-13, IL-21, TGF-βΙ), chemokines (MCP-1, MIP-Ιβ), angiogenic factors (VEGF), growth factors (PDGF), peroxisome proliferator-activated receptors (PPARs), acute phase proteins (SAP), caspases, and components of the renin-angiotensin-aldosterone system (ANG II) have been identified as being regulators of fibrosis and are being investigated as potential targets of antifibrotic drugs.
Proprotein convertases (PCSKs) cleave target peptides in the secretory pathway, at the cell surface and in the extra cellular matrix (Seidah, N. G. & Prat, A., The biology and therapeutic targeting of the proprotein convertases, 1-17 (2012). doi: 10.1038/nrd3699).
US2011/0059896 indicates that the commercially-available proprotein convertase inhibitors decanoyl-Arg-Val-Lys-Arg-CMK (Cayman Chemical Item Number 14965; CAS 150113-99- 8) and hexa-D-arginine (Calbiochem® catalogue number 344931 ; CAS 673202-67-0) would block TGF-β activation, and it was suggested on this basis that these two compounds would be effective in reducing scarring and fibrosis. However, no direct evidence of this was provided.
Accordingly, the fact remains that there are few or no effective therapies available for fibrotic disorders, and there therefore remains an urgent need to develop improved treatments for such disorders by regulating (i.e. preventing, inhibiting or reversing) fibrosis in these conditions. Identification of molecules that inhibit collagen matrix formation is thus of great importance in the search for effective treatments for fibrotic disorders.
SUMMARY OF THE INVENTION
The inventors unexpectedly identified a substantial intersect between targets that are cleaved by PCSKs and candidate mediators of fibrosis, leading them to them to
hypothesise that PCSK inhibition is effective in inhibiting collagen matrix formation.
Upon then testing various commercially available PCSK inhibitors, it was surprisingly found that a class of N-terminally modified tetrapeptide mimetics having a C-terminal arginine mimetic robustly reduce collagen formation and procollagen propeptide
production, and that such inhibitors are therefore useful as antifibrotic agents.
Accordingly, the present invention provides a proprotein convertase inhibitor for use in the treatment of a fibrotic disorder, which proprotein convertase inhibitor is a compound of formula (I) P5-P4-P3-P2-P1 (I)
wherein:
PI is an arginine mimetic selected from the following structures:
Figure imgf000005_0001
wherein
Ri is selected from H, OH, O-CH
wherein m is an integer from 1 to 5;
R2 is H or an amidino group;
n is equal to 3, 4 or 5; and
s is equal to 2 or 3; P2, P3 and P4 are independently selected from amino acid residues and imino acid residues; and
P5 is an N-terminal modification of the amino or imino acid P4, wherein P5 is selected from the following groups:
-H, -C(0)-R7, -C(0)-X-R7,
a pyroglutamyl residue or another amino or imino acid residue,
-S(0)2-R7,
Ci-io alkyl which is unsubstituted or substituted with a halogen, NH2, guanidino, C(0)OH or C(0)OR5 group,
a sphingosine ac lated with a bifunctional roup, having the following structure:
Figure imgf000006_0001
a sphingosyl-phosphorylcholine acylated with a bifunctional group, having the followin structure:
Figure imgf000006_0002
wherein
z is an integer from 2 to 20;
X is O, CH2 or NH; R.5 is H or unsubstituted or substituted C1-3 alkyl; and
R7 is:
(i) unsubstituted or substituted C1-24 alkyl, or unsubstituted or substituted C2-24 alkenyl,
(ii) unsubstituted or substituted aryl, unsubstituted or substituted heteroaryl, unsubstituted or substituted aralkyl, unsubstituted or substituted heteroaralkyl or unsubstituted or substituted C3-20 cycloalkyl,
wherein the heteroaryl ring in said heteroaryl and in said heteroaralkyl contains from 1 to 3 heteroatoms selected from N, S and O,
and wherein the substituent or substituents which may be present at R7 are independently selected from -NH2, -CH2-NH2, -amidino, -hydroxyamidino, -guanidino, - CH2-guanidino, methylguanidino, -halogen, -CI, -Br, -I, -CN, -CF3, aryl, C1-3 alkyl, C3-20 cycloalkyl, C1-3 alkoxy, and -C(0)0-R5 wherein R5 is H or unsubstituted or substituted C1-3 alkyl, or
(iii) a cholesterol group;
or a prodrug thereof, or a pharmaceutically acceptable salt thereof.
The invention also provides a proprotein convertase inhibitor for use in the treatment of a fibrotic disorder, which proprotein convertase inhibitor comprises an oligonucleotide which specifically hybridises to a part of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15 or 17 (human PCSK 1 to 9 mRNA respectively) or any isoform thereof.
The invention further provides a proprotein convertase inhibitor for use in the treatment of a fibrotic disorder, which proprotein convertase inhibitor comprises an oligonucleotide which comprises 50 or fewer consecutive nucleotides from (a) SEQ ID NO: 2, 3, 6, 8, 10, 12, 14, 16 or 18 (human PCSK 1 to 9 cDNA respectively) or any isoform thereof or (b) a variant sequence which has at least 95% homology to SEQ ID NO: 2, 3, 6, 8, 10, 12, 14, 16 or 18 or any isoform thereof based on nucleotide identity over the entire sequence.
The invention also provides a composition for use in the treatment of a fibrotic disorder, which composition comprises a pharmaceutically acceptable carrier or diluent and a proprotein convertase inhibitor which is a compound of formula (I) as defined above or a prodrug thereof or a pharmaceutically acceptable salt thereof.
The invention also provides the use of a proprotein convertase inhibitor in the manufacture of a medicament for use in the treatment of a fibrotic disorder, which proprotein convertase inhibitor is a compound of formula (I) as defined above or a prodrug thereof or a pharmaceutically acceptable salt thereof, or comprises an oligonucleotide or variant sequence as defined above or any isoform thereof.
The invention also provides a method of treating a fibrotic disorder, which method comprises administering to a subject in need of such treatment a therapeutically effective amount of a proprotein convertase inhibitor, which proprotein convertase inhibitor is a compound of formula (I) as defined above or a prodrug thereof or a pharmaceutically acceptable salt thereof, or comprises an oligonucleotide or variant sequence as defined above or any isoform thereof.
The invention also provides an in vitro method of inhibiting the formation of collagen matrix, the method comprising contacting an in vitro sample comprising collagen- secreting cells with an amount of a proprotein convertase inhibitor effective to inhibit the formation of collagen matrix by the cells, wherein the proprotein convertase inhibitor is a compound of formula (I) as defined above or a prodrug thereof or a pharmaceutically acceptable salt thereof, or comprises an oligonucleotide or variant sequence as defined above or any isoform thereof. The collagen-secreting cells typically comprise fibroblasts.
The invention also provides a combination comprising (a) a proprotein convertase inhibitor and (b) a further antifibrotic agent, for use in the treatment of a fibrotic disorder. The proprotein convertase inhibitor is typically a compound of formula (I) as defined above or a prodrug thereof or a pharmaceutically acceptable salt thereof, or comprises an oligonucleotide or variant sequence as defined above or any isoform thereof. The proprotein convertase inhibitor and the further antifibrotic agent may be for separate, simultaneous, concomitant or sequential administration in the treatment of the fibrotic disorder.
It is a further surprising finding of the invention that PCSK inhibitors can robustly reduce collagen formation and procollagen propeptide production without significantly affecting fibronectin secretion. This is advantageous, as fibronectin plays a major role in cell adhesion, growth, migration, and differentiation and is important for processes such as wound healing and embryonic development. Also, altered fibronectin expression, degradation, and organization has been associated with a number of pathologies, including cancer. It can therefore be desirable not to alter fibronectin secretion.
Accordingly, the present invention further provides a proprotein convertase inhibitor for use in the treatment of a fibrotic disorder by inhibiting the formation of collagen matrix without inhibiting fibronectin secretion. Any proprotein convertase inhibitor capable of inhibiting collagen matrix formation without significantly inhibiting fibronectin secretion may be employed in this aspect of the invention. The inhibitor employed may for instance be a compound of formula (I), as defined above, or a prodrug or pharmaceutically acceptable salt thereof. However, other proprotein convertase inhibitors could also be employed.
The invention also provides a composition for use in the treatment of a fibrotic disorder by inhibiting collagen matrix formation without inhibiting fibronectin secretion, which composition comprises a proprotein convertase inhibitor and a pharmaceutically acceptable carrier or diluent.
The invention also provides the use of a proprotein convertase inhibitor in the manufacture of a medicament for use in the treatment of a fibrotic disorder by inhibiting collagen matrix formation without inhibiting fibronectin secretion.
The invention also provides a method of treating a fibrotic disorder in a subject by inhibiting collagen matrix formation without inhibiting fibronectin secretion, which method comprises administering to the subject a therapeutically effective amount of a proprotein convertase inhibitor, which amount is effective to inhibit collagen matrix formation in the subject without inhibiting fibronectin secretion.
The invention also provides an in vitro method of inhibiting the formation of collagen matrix without inhibiting fibronectin secretion, the method comprising contacting an in vitro sample comprising collagen-secreting cells with an amount of a proprotein convertase inhibitor effective to inhibit the formation of collagen matrix by the cells without significantly inhibiting secretion by the cells of fibronectin. The collagen- secreting cells typically comprise fibroblasts.
The invention also provides a combination comprising (a) a proprotein convertase inhibitor and (b) a further antifibrotic agent, for use in the treatment of a fibrotic disorder by inhibiting collagen matrix formation without inhibiting fibronectin secretion.
The invention also provides a novel assay method for identifying molecules that affect collagen secretion in vitro, and thus for identifying antifibrotic agents. The assay was used in the present case to identify proprotein convertase inhibitors as inhibitors of collagen matrix formation. Advantageously, the assay is miniaturized so that it is compatible with high-throughput screening with various established compound libraries. The miniaturization also allows comprehensive and systematic quantification of collagen signals for high-throughput imaging. The assay can also provide for improved morphological contrast for quantitative imaging as compared to known collagen matrix assays.
Accordingly, the invention provides a method of identifying an agent as being capable of inhibiting collagen matrix formation, which method comprises (a) contacting an agent with an in vitro sample comprising collagen- secreting cells in the absence of exogenous transforming growth factor (TGF) and (b) measuring the amount of collagen matrix formed by the cells and thereby determining whether or not the agent is capable of inhibiting collagen matrix formation. BRIEF DESCRIPTION OF THE FIGURES
Fig. 1 shows that Calbiochem 537076 specifically inhibits extracellular collagen levels. The modified "scar-in-a-jar" assay was carried out as per Figure 6 on three different passages of juvenile HDF (j- HDF) treated with 0, 1.25, 2.5, 5, 10, or 20 μΜ
Calbiochem 537076. [a] Immunocytochemistry of collagen type 1 (COL-1) and
fibronectin, and nuclear staining with Hoechst as imaged on the Operetta, [b-d] Graphs show quantification from image analysis of total COL-1 divided by total fibronectin (b), total COL-1 divided by cell number (c), and total fibronectin divided by cell number (d). Data were expressed as mean ±SEM, with n = 2..
Fig. 2 is a graph containing dose response curves for Calbiochem 537076'
(Calbiochem 537076, 'PCi'), dec-RVKR-cmk (Cayman 14965, 'Fil'), and hexa-D-arginine amide (Calbiochem 344931, 'Fill'), showing that Calbiochem 537076 inhibits extracellular collagen in a dosage dependent manner. The graph shows quantification from image analysis of total collagen divided by total fibronectin. The dose response curves were fitted by a nonlinear sigmoidal dose response function to a variable slope without constraints, based on which an IC50 for each drug was determined..
Fig. 3 shows the results of validation of siRNA against individual PCSKs; gene expression was determined by qPCR using Taqman gene expression assays for FURIN (Hs00965485_gl) (a), PCSK5 (Hs00196400_ml) (b), and PCSK6 (Hs00159844_ml) (c), compared against non-targeted control (NTC) RNA (D-001810).
Fig. 4 shows that knockdown of FURIN (PCSK3) inhibits extracellular collagen.
PCSK3 (FURIN), PCSK5, and PCSK6 were knocked down in j-NHDF cells as per Figure 3, and scar-in-a-jar assay was carried out. Graphs show quantification from image analysis of (a) total collagen divided by cell number total, (b) fibronectin divided by cell number, and (c) total collagen divided by total fibronectin. Fig. 5 shows that knockdown of FURIN suppresses COL1 Al mRNA expression. Gene expression was determined by qPCR using Taqman gene expression assays for FURIN (Hs00965485_gl), COL1A1 (Hs00164004_ml), COL1A2 (Hs00164099_ml), and COL3A1 (Hs00943809_ml). Data were normalised to HPRT (Hs99999909_ml), and presented using the 2A(-AACT) method, with the NTC control normalised to 100 and expressed as mean ±SEM..
Fig. 6 is a schematic illustration of the modified "Scar-in-a-Jar" assay of the invention. DETAILED DESCRIPTION OF THE INVENTION
The following definitions apply to the compounds defined herein.
A Ci-24 alkyl group is an unsubstituted or substituted, straight or branched chain saturated hydrocarbon radical having from 1 to 24 carbon atoms (unless otherwise specified). Often, it is C1-20 alkyl, i.e. an unsubstituted or substituted, straight or branched chain saturated hydrocarbon radical having from 1 to 20 carbon atoms. Typically it is Ci-io alkyl, for example methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl or decyl, or Ci-6 alkyl, for example methyl, ethyl, propyl, butyl, pentyl or hexyl, or Ci-4 alkyl, for example methyl, ethyl, i-propyl, n-propyl, t-butyl, s-butyl or n-butyl, or for instance C1-3 alkyl. When an alkyl group is substituted it typically bears one or more substituents selected from unsubstituted or substituted (but typically unsubstituted) C1-24 alkyl, unsubstituted or substituted C2-24 alkenyl (as defined herein), substituted or unsubstituted aryl (as defined herein), cyano, amino (-NH2), C1-10 alkylamino, di(Ci-io)alkylamino, arylamino, diarylamino, arylalkylamino, amido, acylamido, hydroxy, oxo (i.e. keto), halo (i.e. halogen, -X', wherein X' is usually F, CI, Br or I), carboxy, ester, acyl, acyloxy, Ci-20 alkoxy, aryloxy, haloalkyl, sulfonic acid, sulfhydryl (i.e. thiol, -SH), Ci-10 alkylthio, arylthio, sulfonyl, phosphoric acid, phosphate ester, phosphonic acid and phosphonate ester. Examples of substituted alkyl groups include haloalkyl, hydroxyalkyl, aminoalkyl, alkoxyalkyl and alkaryl groups. The term alkaryl, as used herein, pertains to a Ci-20 alkyl group in which at least one hydrogen atom has been replaced with an aryl group.
Examples of such groups include, but are not limited to, benzyl (phenylmethyl, PhCH2-), benzhydryl (Ph2CH-), trityl (triphenylmethyl, Ph3C-), phenethyl (phenylethyl, Ph-CH2CH2- ), styryl (Ph-CH=CH-), cinnamyl (Ph-CH=CH-CH2-).
Typically a substituted alkyl group carries 1, 2 or 3 substituents, for instance 1 or 2. A C2-24 alkenyl group is an unsubstituted or substituted, straight or branched chain unsaturated hydrocarbon radical, which contains from 2 to 24 carbon atoms. One or more carbon-carbon double bonds are present in the alkenyl group, typically one, two or three carbon-carbon double bonds, and more typically one carbon-carbon double bond. Often, it is a C2-20 alkenyl group i.e. it contains from 2 to 20 carbon atoms. A C2-20 alkenyl group is typically ethenyl or a C3-10 alkenyl group, i.e. a C2-10 alkenyl group, more typically a C2-6 alkenyl group. A C3-10 alkenyl group is typically a C3-6 alkenyl group, for example allyl, propenyl, butenyl, pentenyl or hexenyl. A C2-4 alkenyl group is ethenyl, propenyl or butenyl. An alkenyl group may be unsubstituted or substituted by one to four (e.g. one, two, three or four) substituents, the substituents, unless otherwise specified, being selected from those listed above for Ci-20 alkyl groups. Where two or more substituents are present, these may be the same or different.
Typically a substituted alkenyl group carries 1, 2 or 3 substituents, for instance 1 or
2.
A C3-25 cycloalkyl group is an unsubstituted or substituted alkyl group which is also a cyclyl group; that is, a monovalent moiety obtained by removing a hydrogen atom from an alicyclic ring atom of a carbocyclic ring of a carbocyclic compound, which moiety has from 3 to 25 carbon atoms (unless otherwise specified), including from 3 to 25 ring atoms. Thus, the term "cycloalkyl" includes the sub-classes cycloalkyenyl and cycloalkynyl. Examples of groups of C3-25 cycloalkyl groups include C3-20 cycloalkyl, C3-15 cycloalkyl, C3-10 cycloalkyl, C3-7 cycloalkyl. When a C3-25 cycloalkyl group is substituted it typically bears one or more substituents selected from Ci-6 alkyl which is unsubstituted, aryl (as defined herein), cyano, amino, Ci-10 alkylamino, di(Ci-io)alkylamino, arylamino, diarylamino, arylalkylamino, amido, acylamido, hydroxy, oxo, halo, carboxy, ester, acyl, acyloxy, Ci-20 alkoxy, aryloxy, haloalkyl, sulfonic acid, sulfhydryl (i.e. thiol, -SH), Ci-10 alkylthio, arylthio, phosphoric acid, phosphate ester, phosphonic acid and phosphonate ester and sulfonyl. Typically a substituted C3-25 cycloalkyl group carries 1, 2 or 3 substituents, for instance 1 or 2.
Examples of C3-25 cycloalkyl groups include, but are not limited to, those derived from saturated monocyclic hydrocarbon compounds, which C3-25 cycloalkyl groups are unsubstituted or substituted as defined above:
cyclopropane (C3), cyclobutane (C4), cyclopentane (C5), cyclohexane (C6), cycloheptane (C7), methylcyclopropane (C4), dimethylcyclopropane (C5),
methylcyclobutane (C5), dimethylcyclobutane (C6), methylcyclopentane (C6), dimethylcyclopentane (C7), methylcyclohexane (C7), dimethylcyclohexane (C8), menthane unsaturated monocyclic hydrocarbon compounds:
cyclopropene (C3), cyclobutene (C4), cyclopentene (C5), cyclohexene (C6), methylcyclopropene (C4), dimethylcyclopropene (C5), methylcyclobutene (C5), dimethylcyclobutene (C6), methylcyclopentene (C6), dimethylcyclopentene (C7), methylcyclohexene (C7), dimethylcyclohexene (C8);
saturated polycyclic hydrocarbon compounds:
thujane (Cio), carane (Cio), pinane (Cio), bornane (Cio), norcarane (C7), norpinane (C7), norbornane C7), adamantane (Cio decalin (decahydronaphthalene) (Cio);
Figure imgf000013_0001
unsaturated polycyclic hydrocarbon compounds: camphene (Cio), limonene (Cio), pinene (Cio
Figure imgf000013_0002
polycyclic hydrocarbon compounds having an aromatic ring:
indene (C9), indane (e.g., 2,3-dihydro-lH-indene) (C9), tetraline
(1,2,3,4-tetrahydronaphthalene) (Cio), acenaphthene (C12), fluorene (C13), phenalene (C13), acephenanthrene (C15), aceanthrene (C16), cholanthrene (C20).
A C3-20 heterocyclyl group is an unsubstituted or substituted monovalent moiety obtained by removing a hydrogen atom from a ring atom of a heterocyclic compound, which moiety has from 3 to 20 ring atoms (unless otherwise specified), of which from 1 to 10 are ring heteroatoms. Preferably, each ring has from 3 to 7 ring atoms, of which from 1 to 4 are ring heteroatoms. A C3-7 heterocyclyl has from 3 to 7 ring atoms, of which from 1 to 4 are ring heteroatoms. When a C3-20 heterocyclyl group (or a C3-7 heterocyclyl group) is substituted it typically bears one or more substituents selected from Ci-6 alkyl which is unsubstituted, aryl (as defined herein), cyano, amino, C1-10 alkylamino, di(Ci- io)alkylamino, arylamino, diarylamino, arylalkylamino, amido, acylamido, hydroxy, oxo, halo, carboxy, ester, acyl, acyloxy, C1-20 alkoxy, aryloxy, haloalkyl, sulfonic acid, sulfhydryl (i.e. thiol, -SH), Ci-io alkylthio, arylthio, phosphoric acid, phosphate ester, phosphonic acid and phosphonate ester and sulfonyl. Typically a substituted C3-20 heterocyclyl group carries 1, 2 or 3 substituents, for instance 1 or 2.
Examples of groups of heterocyclyl groups include C3-2oheterocyclyl,
C5-2oheterocyclyl, Cs-isheterocyclyl, Cs-isheterocyclyl, C3-i2heterocyclyl, Cs-nheterocyclyl, C3-ioheterocyclyl, Cs-ioheterocyclyl, C3-7heterocyclyl, C5-7heterocyclyl, and
C5-6heterocyclyl.
Examples of (non-aromatic) monocyclic C3-20 heterocyclyl groups include, but are not limited to, those derived from:
Ni: aziridine (C3), azetidine (C4), pyrrolidine (tetrahydropyrrole) (C5), pyrroline (e.g., 3-pyrroline, 2,5-dihydropyrrole) (C5), 2H-pyrrole or 3H-pyrrole (isopyrrole, isoazole) (C5), piperidine (C6), dihydropyridine (C6), tetrahydropyridine (C6), azepine (C7);
Oi: oxirane (C3), oxetane (C4), oxolane (tetrahydrofuran) (C5), oxole
(dihydrofuran) (C5), oxane (tetrahydropyran) (C6), dihydropyran (C6), pyran (C6), oxepin (C7);
Si: thiirane (C3), thietane (C4), thiolane (tetrahydrothiophene) (C5), thiane
(tetrahydrothiopyran) (C6), thiepane (C7);
O2: dioxolane (C5), dioxane (C6), and dioxepane (C7);
O3 : trioxane (C6);
N2: imidazolidine (C5), pyrazolidine (diazolidine) (C5), imidazoline (C5), pyrazoline (dihydropyrazole) (C5), piperazine (C6);
N1O1 : tetrahydrooxazole (C5), dihydrooxazole (C5), tetrahydroisoxazole (C5), dihydroisoxazole (C5), morpholine (C6), tetrahydrooxazine (C6), dihydrooxazine (C6), oxazine (C6);
N1S1 : thiazoline (C5), thiazolidine (C5), thiomorpholine (C6);
N2O1 : oxadiazine (C6);
O1S1 : oxathiole (C5) and oxathiane (thioxane) (C6); and,
N1O1S1 : oxathiazine (C6).
An aryl group is a substituted or unsubstituted, monocyclic or bicyclic aromatic group which typically contains from 5 to 20 carbon atoms, more typically from 5 to 14 carbon atoms, preferably from 6 to 14, or for instance from 6 to 10, or from 5 to 10, carbon atoms in the ring portion. Examples include phenyl, naphthyl, indenyl and indanyl groups. An aryl group is unsubstituted or substituted. When an aryl group as defined above is substituted it typically bears one or more substituents selected from unsubstituted or substituted Ci-20 alkyl (to form an aralkyl group), aryl, cyano, amino, Ci-10 alkylamino, di(Ci-io)alkylamino, arylamino, diarylamino, arylalkylamino, amido, acylamido, hydroxy, halo, carboxy, ester, acyl, acyloxy, Ci-20 alkoxy, aryloxy, haloalkyl, sulfhydryl (i.e. thiol, - SH), Ci-10 alkylthio, arylthio, sulfonic acid, phosphoric acid, phosphate ester, phosphonic acid and phosphonate ester and sulfonyl. Typically it carries 0, 1, 2 or 3 substituents. A substituted aryl group may be substituted in two positions with a single Ci-6 alkylene group, or with a bidentate group represented by the formula -X-Ci-6 alkylene, or -X-Ci-6 alkylene-X-, wherein X is selected from O, S and R, and wherein R is H, aryl or Ci-6 alkyl. Thus a substituted aryl group may be an aryl group fused with a cycloalkyl group or with a heterocyclyl group. The term aralkyl as used herein, pertains to an aryl group (as defined herein) in which at least one hydrogen atom (e.g., 1, 2, 3) has been substituted with an unsubstituted or substituted Ci-20 alkyl group, which is typically an unsubstituted Ci-6 alkyl group. Examples of such groups include, but are not limited to, tolyl (from toluene), xylyl (from xylene), mesityl (from mesitylene), and cumenyl (or cumyl, from cumene), and duryl (from durene). Similarly, the term heteroaralkyl, as used herein, pertains to a heteroaryl group (as defined herein) in which at least one hydrogen atom (e.g., 1, 2, 3) has been substituted with an unsubstituted or substituted Ci-20 alkyl group, which is typically an unsubstituted Ci-6 alkyl group.
The ring atoms of an aryl group may include one or more heteroatoms (as in a heteroaryl group). Such an aryl group (a "heteroaryl group") is a substituted or
unsubstituted mono- or bicyclic heteroaromatic group which typically contains from 5 to 10 atoms in the ring portion (i.e. it is a 5- to 10-membered ring) including one or more heteroatoms. A heteroaryl group which contains from 5 to 10 atoms in the ring portion including one or more heteroatoms may be termed a C5-10 heteroaryl group. It is generally a 5- or 6-membered ring, containing at least one heteroatom selected from O, S, N, P, Se and Si. It may contain, for example, 1, 2 or 3 heteroatoms. Usually, each heteroatom is either N, O or S. Examples of heteroaryl groups include pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, furanyl, thienyl, pyrazolidinyl, pyrrolyl, oxazolyl, oxadiazolyl, isoxazolyl, thiadiazolyl, thiazolyl, isothiazolyl, imidazolyl, pyrazolyl, quinolyl and isoquinolyl. A heteroaryl group may be unsubstituted or substituted, for instance, as specified above for aryl. Typically it carries 0, 1, 2 or 3 substituents. A Ci-20 alkylene group is an unsubstituted or substituted bidentate moiety obtained by removing two hydrogen atoms, either both from the same carbon atom, or one from each of two different carbon atoms, of a hydrocarbon compound having from 1 to 20 carbon atoms (unless otherwise specified), which may be aliphatic or alicyclic, and which may be saturated, partially unsaturated, or fully unsaturated. Thus, the term "alkylene" includes the sub-classes alkenylene, alkynylene, cycloalkylene, etc., discussed below. Typically it is Ci-io alkylene, for instance Ci-6 alkylene. Typically it is Ci-4 alkylene, for example methylene, ethylene, i-propylene, n-propylene, t-butylene, s-butylene or n- butylene. It may also be pentylene, hexylene, heptylene, octylene and the various branched chain isomers thereof. An alkylene group may be unsubstituted or substituted, for instance, as specified above for alkyl. Typically a substituted alkylene group carries 1, 2 or 3 substituents, for instance 1 or 2.
In this context, the prefixes (e.g., Ci-4, C1-7, C1-20, C2-7, C3-7, etc.) denote the number of carbon atoms, or range of number of carbon atoms. For example, the term
"Ci-4alkylene," as used herein, pertains to an alkylene group having from 1 to 4 carbon atoms. Examples of groups of alkylene groups include Ci-4 alkylene ("lower alkylene"), Ci-7 alkylene, Ci-io alkylene and C1 -20 alkylene.
Examples of linear saturated C1-7 alkylene groups include, but are not limited to, -(0¾)η- where n is an integer from 1 to 7, for example, -CH2- (methylene), -CH2CH2- (ethylene), -CH2CH2CH2- (propylene), and -CH2CH2CH2CH2- (butylene).
Examples of branched saturated C1-7 alkylene groups include, but are not limited to, -CH(CH3)-, -CH(CH3)CH2-, -CH(CH3)CH2CH2-, -CH(CH3)CH2CH2CH2-, -CH2CH(CH 3)CH2-, -CH2CH(CH3)CH2CH2-, -CH(CH2CH3)-, -CH(CH2CH3)CH2-,
and -CH2CH(CH2CH3)CH2-.
Examples of linear partially unsaturated C1-7 alkylene groups include, but is not limited to, -CH=CH- (vinylene), -CH=CH-CH2-, -CH2-
CH=CH2-, -CH=CH-CH2-CH2-, -CH=CH-CH2-CH2-CH2-, -CH=CH-CH=CH- , -CH=CH-CH=CH-CH2-, -CH=CH-CH=CH-CH2-CH2-, -CH=CH-CH2-CH=CH-, and -CH=CH-CH2-CH2-CH=CH-.
Examples of branched partially unsaturated C1-7 alkylene groups include, but is not limited to, -C(CH3)=CH-, -C(CH3)=CH-CH2-, and -CH=CH-CH(CH3)-.
Examples of alicyclic saturated C1-7 alkylene groups include, but are not limited to, cyclopentylene (e.g., cyclopent-l,3-ylene), and cyclohexylene (e.g., cyclohex-l,4-ylene). Examples of alicyclic partially unsaturated C1-7 alkylene groups include, but are not limited to, cyclopentenylene (e.g., 4-cyclopenten-l,3-ylene), cyclohexenylene (e.g., 2-cyclohexen- 1 ,4-ylene; 3 -cy clohexen- 1 ,2-ylene; 2,5-cyclohexadien- 1 ,4-ylene).
Ci-20 alkylene and C1-20 alkyl groups as defined herein are either uninterrupted or interrupted by one or more heteroatoms or heterogroups, such as S, O or N(R") wherein R" is H, Ci-6 alkyl or aryl (typically phenyl), or by one or more arylene (typically phenylene) groups. The phrase "optionally interrupted" as used herein thus refers to a C1-20 alkyl group or an alkylene group, as defined above, which is uninterrupted or which is interrupted between adjacent carbon atoms by a heteroatom such as oxygen or sulfur, by a heterogroup such as N(R") wherein R" is H, aryl or Ci-C6 alkyl, or by an arylene group.
For instance, a C1-20 alkyl group such as n-butyl may be interrupted by the heterogroup N(R") as follows: -CH2N(R")CH2CH2CH3, -CH2CH2N(R")CH2CH3,
or -CH2CH2CH2N(R")CH3. Similarly, an alkylene group such as n-butylene may be interrupted by the heterogroup N(R") as follows: -CH2N(R")CH2CH2CH2- , -CH2CH2N(R")CH2CH2-, or -CH2CH2CH2N(R")CH2-. Typically an interrupted group, for instance an interrupted C1-20 alkylene or C1-20 alkyl group, is interrupted by 1, 2, 3 or 4 heteroatoms or heterogroups or by 1, 2, 3 or 4 arylene (typically phenylene) groups. More typically, an interrupted group, for instance an interrupted C1-20 alkylene or C1-20 alkyl group, is interrupted by 1 or 2 heteroatoms or heterogroups or by 1 or 2 arylene (typically phenylene) groups. For instance, a C1-20 alkyl group such as n-butyl may be interrupted by 2 heterogroups N(R") as follows: -CH2N(R")CH2N(R")CH2CH3.
An arylene group is an unsubstituted or substituted bidentate moiety obtained by removing two hydrogen atoms, one from each of two different aromatic ring atoms of an aromatic compound, which moiety has from 5 to 14 ring atoms (unless otherwise specified). Typically, each ring has from 5 to 7 or from 5 to 6 ring atoms. An arylene group may be unsubstituted or substituted, for instance, as specified above for aryl.
In this context, the prefixes (e.g., C5-2o, C6-2o, C5-14, C5-7, C5-6, etc.) denote the number of ring atoms, or range of number of ring atoms, whether carbon atoms or heteroatoms. For example, the term "C5-6 arylene," as used herein, pertains to an arylene group having 5 or 6 ring atoms. Examples of groups of arylene groups include C5-2o arylene, C6-2o arylene, C5-14 arylene, C6-14 arylene, C6-io arylene, C5-12 arylene, C5-10 arylene, C5-7 arylene, C5-6 arylene, C5 arylene, and C6 arylene.
The ring atoms may be all carbon atoms, as in "carboarylene groups" (e.g., C6-2o carboarylene, C6-14 carboarylene or C6-io carboarylene). Examples of C6-2o arylene groups which do not have ring heteroatoms (i.e., C6-2o carboarylene groups) include, but are not limited to, those derived from the compounds discussed above in regard to aryl groups, e.g. phenylene, and also include those derived from aryl groups which are bonded together, e.g. phenylene-phenylene (diphenylene) and phenylene-phenylene-phenylene (triphenylene).
Alternatively, the ring atoms may include one or more heteroatoms, as in
"heteroarylene groups" (e.g., C5-10 heteroarylene).
Examples of C5-10 heteroarylene groups include, but are not limited to, those derived from the compounds discussed above in regard to heteroaryl groups.
As used herein the term oxo represents a group of formula: =0
As used herein the term acyl represents a group of formula: -C(=0)R, wherein R is an acyl substituent, for example, a substituted or unsubstituted Ci-20 alkyl group, a substituted or unsubstituted C3-20 heterocyclyl group, or a substituted or unsubstituted aryl group. Examples of acyl groups include, but are not limited to, -C(=0)CH3
(acetyl), -C(=0)CH2CH3 (propionyl), -C(=0)C(CH3)3 (t-butyryl), and -C(=0)Ph (benzoyl, phenone).
As used herein the term acyloxy (or reverse ester) represents a group of formula: -OC(=0)R, wherein R is an acyloxy substituent, for example, substituted or unsubstituted Ci-20 alkyl group, a substituted or unsubstituted C3-2oheterocyclyl group, or a substituted or unsubstituted aryl group, typically a Ci-6 alkyl group. Examples of acyloxy groups include, but are not limited to, -OC(=0)CH3
(acetoxy), -OC(=0)CH2CH3, -OC(=0)C(CH3)3, -OC(=0)Ph, and -OC(=0)CH2Ph.
As used herein the term ester (or carboxylate, carboxylic acid ester or oxycarbonyl) represents a group of formula: -C(=0)OR, wherein R is an ester substituent, for example, a substituted or unsubstituted Ci-20 alkyl group, a substituted or unsubstituted C3-20 heterocyclyl group, or a substituted or unsubstituted aryl group (typically a phenyl group).
Examples of ester groups include, but are not limited
to, -C(=0)OCH3, -C(=0)OCH2CH3, -C(=0)OC(CH3)3, and -C(=0)OPh.
As used herein the term phosphonic acid represents a group of the formula:
-P(=0)(OH)2. As would be understood by the skilled person, a phosphonic acid group can exist in protonated and deprotonated forms (i.e. -P(=0)(OH)2, -P(=0)(0")2 and
-P(=0)(OH)(0")) all of which are within the scope of the term "phosphonic acid".
As used herein the term phosphonic acid salt represents a group which is a salt of a phosphonic acid group. For example a phosphonic acid salt may be a group of the formula -P(=0)(OH)(0"X+) wherein X is a monovalent cation. X+ may be an alkali metal cation. X+ may be Na+ or K+, for example.
As used herein the term phosphonate ester represents a group of one of the formulae:
-P(=0)(OR)2 and -P(=0)(OR)0" wherein each R is independently a phosphonate ester substituent, for example, -H, substituted or unsubstituted C1-20 alkyl, substituted or unsubstituted C3-2o heterocyclyl, C3-20 heterocyclyl substituted with a further C3-20 heterocyclyl, substituted or unsubstituted Ci-2o alkylene-C3-2o heterocyclyl, substituted or unsubstituted C3-25 cycloalkyl, substituted or unsubstituted Ci-20 alkylene-C3-25 cycloalkyl, aryl, substituted or unsubstituted Ci-20 alkylene-aryl. Examples of phosphonate ester groups include, but are not limited to, -P(=0)(OCH3)2, -P(=0)(OCH2CH3)2, -P(=0)(0- t-Bu)2, and -P(=0)(OPh)2,
As used herein the term phosphoric acid represents a group of the formula:
-OP(=0)(OH)2.
As used herein the term phosphate ester represents a group of one of the formulae:
-OP(=0)(OR)2 and -OP(=0)(OR)0" wherein each R is independently a phosphate ester substituent, for example, -H, substituted or unsubstituted Ci-20 alkyl, substituted or unsubstituted C3 -20 heterocyclyl, C3-20 heterocyclyl substituted with a further C3-20 heterocyclyl, substituted or unsubstituted Ci-2o alkylene-C3-2o heterocyclyl, substituted or unsubstituted C3-25 cycloalkyl, substituted or unsubstituted Ci-20 alkylene-C3-25 cycloalkyl, aryl, substituted or unsubstituted Ci-20 alkylene-aryl. Examples of phosphate ester groups include, but are not limited
to, -OP(=0)(OCH3)2, -OP(=0)(OCH2CH3)2, -OP(=0)(0-t-Bu)2, and -OP(=0)(OPh)2.
As used herein the term amino represents a group of formula -NH2. The term Ci- Cio alkylamino represents a group of formula -NHR' wherein R' is a Ci-10 alkyl group, preferably a Ci-6 alkyl group, as defined previously. The term di(Ci-io)alkylamino represents a group of formula -NR'R' ' wherein R' and R' ' are the same or different and represent Ci-10 alkyl groups, preferably Ci-6 alkyl groups, as defined previously. The term arylamino represents a group of formula -NHR' wherein R' is an aryl group, preferably a phenyl group, as defined previously. The term diarylamino represents a group of formula -NR'R' ' wherein R' and R' ' are the same or different and represent aryl groups, preferably phenyl groups, as defined previously. The term arylalkylamino represents a group of formula -NR'R" wherein R' is a Ci-10 alkyl group, preferably a Ci-6 alkyl group, and R" is an aryl group, preferably a phenyl group. As used herein the term amido represents a group of formula: -C(=0) R'R", wherein R and R are independently amino substituents, as defined for di(Ci-io)alkylamino groups. Examples of amido groups include, but are not limited
to, -C(=0) H2, -C(=0) HCH3, -C(=0)N(CH3)2, -C(=0) HCH2CH3,
and -C(=0)N(CH2CH3)2, as well as amido groups in which R and R , together with the nitrogen atom to which they are attached, form a heterocyclic structure as in, for example, piperidinocarbonyl, morpholinocarbonyl, thiomorpholinocarbonyl, and
piperazinocarbonyl .
As used herein the term acylamido represents a group of formula: - R1C(=0)R2, wherein R1 is an amide substituent, for example, hydrogen, a Ci-2oalkyl group, a C3-2o heterocyclyl group, an aryl group, preferably hydrogen or a C1-20 alkyl group, and R2 is an acyl substituent, for example, a C1-20 alkyl group, a C3-2o heterocyclyl group, or an aryl group, preferably hydrogen or a C1-20 alkyl group. Examples of acylamide groups include, but are not limited to, - HC(=0)CH3
,
Figure imgf000020_0001
Thus, a substituted C1-20 alkyl group may comprise an acylamido substituent defined by the formula
Figure imgf000020_0002
R1 and R2 may together form a cyclic structure, as in, for example, succinimidyl, maleimidyl, and phthalimid l:
Figure imgf000020_0003
succinimidyl maleimidyl phthalimidyl
A Ci-io alkylthio group is a said Ci-io alkyl group, preferably a Ci-6 alkyl group, attached to a thio group. An arylthio group is an aryl group, preferably a phenyl group, attached to a thio group.
A Ci-20 alkoxy group is a said substituted or unsubstituted C1-20 alkyl group attached to an oxygen atom. A Ci-6 alkoxy group is a said substituted or unsubstituted Ci-6 alkyl group attached to an oxygen atom. A Ci-4 alkoxy group is a substituted or unsubstituted Ci-4 alkyl group attached to an oxygen atom. Said C1-20, Ci-6 and Ci-4 alkyl groups are optionally interrupted as defined herein. A C1-3 alkoxy group is a substituted or unsubstituted C1-3 alkyl group attached to an oxygen atom. Examples of Ci-4 alkoxy groups include, -OMe (methoxy), -OEt (ethoxy), -O(nPr) (n-propoxy), -O(iPr)
(isopropoxy), -O(nBu) (n-butoxy), -O(sBu) (sec-butoxy), -O(iBu) (isobutoxy), and -O(tBu) (tert-butoxy). Further examples of Ci-20 alkoxy groups are -O(Adamantyl), -O-CH2- Adamantyl and -O-CH2-CH2- Adamantyl. An aryloxy group is a substituted or
unsubstituted aryl group, as defined herein, attached to an oxygen atom. An example of an aryloxy group is -OPh (phenoxy).
An amidino group is a group of the following structure:
Figure imgf000021_0001
A hydroxyamidino group is a group of the following structure:
Figure imgf000021_0002
guanidino group is a group of the following structure:
Figure imgf000021_0003
A methylguanidino group is a group of the following structure:
Figure imgf000021_0004
The term "a cholesterol group" as used herein refers to a group of the following structure:
Figure imgf000022_0001
The term "-PEG-", as used herein, refers to a linker group comprising polyethylene glycol, i.e. comprising a unit of formula [CH2CH20]n wherein n is an integer greater than 2. The integer n is typically greater than or equal to 3, and may for instance be from 3 to 50, for instance from 3 to 20, or from 3 to 10, or from 3 to 6.
The term "Biotin", as used herein, refers to a Biotin group, i.e. a group of the following formula:
Figure imgf000022_0002
Unless otherwise specified, included in the above are the well known ionic, salt, solvate, and protected forms of these substituents. For example, a reference to carboxylic acid or carboxyl group (-COOH) also includes the anionic (carboxylate) form (-COO"), a salt or solvate thereof, as well as conventional protected forms. Similarly, a reference to an amino group includes the protonated form (-ISTFIR1] 2), a salt or solvate of the amino group, for example, a hydrochloride salt, as well as conventional protected forms of an amino group. Similarly, a reference to a hydroxyl group also includes the anionic form (- O"), a salt or solvate thereof, as well as conventional protected forms.
Certain compounds may exist in one or more particular geometric, optical, enantiomeric, diasteriomeric, epimeric, atropic, stereoisomeric, tautomeric,
conformational, or anomeric forms, including but not limited to, cis- and trans-forms; E- and Z-forms; c-, t-, and r- forms; endo- and exo-forms; R-, S-, and meso-forms; D- and Informs; d- and 1-forms; (+) and (-) forms; keto-, enol-, and enolate-forms; syn- and anti- forms; synclinal- and anticlinal-forms; a- and β-forms; axial and equatorial forms; boat-, chair-, twist-, envelope-, and halfchair- forms; and combinations thereof, hereinafter collectively referred to as "isomers" (or "isomeric forms").
Note that, except as discussed below for tautomeric forms, specifically excluded from the term "isomers," as used herein, are structural (or constitutional) isomers
(i.e., isomers which differ in the connections between atoms rather than merely by the position of atoms in space). For example, a reference to a methoxy group, -OCH3, is not to be construed as a reference to its structural isomer, a hydroxymethyl group, -CH2OH.
Similarly, a reference to ortho-chlorophenyl is not to be construed as a reference to its structural isomer, meta-chlorophenyl. However, a reference to a class of structures may well include structurally isomeric forms falling within that class (e.g., Ci-7alkyl includes n-propyl and iso-propyl; butyl includes n-, iso-, sec-, and tert-butyl; methoxyphenyl includes ortho-, meta-, and para-methoxyphenyl).
The above exclusion does not pertain to tautomeric forms, for example, keto, enol, and enolate forms, as in, for example, the following tautomeric pairs: keto/enol (illustrated below), imine/enamine, amide/imino alcohol, amidine/amidine, nitroso/oxime,
thioketone/enethiol, N-nitroso/hyroxyazo, and nitro/aci-nitro.
Figure imgf000023_0001
keto enol enolate
Note that specifically included in the term "isomer" are compounds with one or more isotopic substitutions. For example, H may be in any isotopic form, including ¾, 2H (D), and 3H (T); C may be in any isotopic form, including 12C, 13C, and 14C; O may be in any isotopic form, including 160 and 180; and the like.
Unless otherwise specified, a reference to a particular compound includes all such isomeric forms, including (wholly or partially) racemic and other mixtures thereof.
Methods for the preparation (e.g., asymmetric synthesis) and separation (e.g., fractional crystallisation and chromatographic means) of such isomeric forms are either known in the art or are readily obtained by adapting known methods, in a known manner.
Unless otherwise specified, a reference to a particular compound also includes ionic, salt (e.g. acid addition salt), solvate, protected forms and prodrugs thereof.
Examples of pharmaceutically acceptable salts of the compounds for use in accordance with the present invention include salts with inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulphuric acid, nitric acid and phosphoric acid; and organic acids such as methanesulfonic acid, benzenesulphonic acid, formic acid, acetic acid, trifluoroacetic acid, propionic acid, butyric acid, isobutyric acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, ethanesulfonic acid, aspartic acid, benzoic acid and glutamic acid. Typically the salt is a hydrochloride, an acetate, a propionate, a benzoate, a butyrate or an isobutyrate. Examples of pharmaceutically acceptable salts are discussed in Berge et al., 1977, "Pharmaceutically Acceptable Salts," J. Pharm. Sci., Vol. 66, pp. 1-19.
A prodrug of a proprotein convertase inhibitor, is a compound which, when metabolised (e.g., in vivo), yields the desired active compound. Typically, the prodrug is inactive, or less active than the active compound, but may provide advantageous handling, administration, or metabolic properties.
In one aspect of the present invention, a proprotein convertase inhibitor is provided, for use in the treatment of a fibrotic disorder, which proprotein convertase inhibitor is a compound of formula (I)
P5-P4-P3-P2-P1 (I)
wherein:
PI is an arginine mimetic selected from the following structures:
Figure imgf000024_0001
Figure imgf000024_0002
Figure imgf000025_0001
wherein
Ri is selected from H, OH, 0-CH3, H2, 0-C(0)-CH3 and -C(0)-0-(CH2)m-CH3 wherein m is an integer from 1 to 5;
R2 is H or an amidino group;
n is equal to 3, 4 or 5; and
s is equal to 2 or 3;
P2, P3 and P4 are independently selected from amino acid residues and imino acid residues; and
P5 is an N-terminal modification of the amino or imino acid P4, wherein P5 is selected from the following groups:
-H, -C(0)-R7, -C(0)-X-R7,
a pyroglutamyl residue or another amino or imino acid residue,
-S(0)2-R7,
Ci-10 alkyl which is unsubstituted or substituted with a halogen, NH2, guanidino, C(0)OH or C(0)OR5 group,
a sphingosine acylated with a bifunctional group, having the following structure:
Figure imgf000025_0002
a sphingosyl-phosphorylcholine acylated with a bifunctional group, having the followin structure:
Figure imgf000026_0001
wherein
z is an integer from 2 to 20;
X is O, CH2 or H;
R.5 is H or unsubstituted or substituted C1-3 alkyl; and
R7 is:
(i) unsubstituted or substituted C1-24 alkyl, or unsubstituted or substituted C2-24 alkenyl,
(ii) unsubstituted or substituted aryl, unsubstituted or substituted heteroaryl, unsubstituted or substituted aralkyl, unsubstituted or substituted heteroaralkyl or unsubstituted or substituted C3-20 cycloalkyl,
wherein the heteroaryl ring in said heteroaryl and in said heteroaralkyl contains from 1 to 3 heteroatoms selected from N, S and O,
and wherein the substituent or substituents which may be present at R7 are independently selected from - H2, -CH2- H2, -amidino, -hydroxyamidino, -guanidino, - CH2-guanidino, methylguanidino, -halogen, -CI, -Br, -I, -CN, -CF3, aryl, C1-3 alkyl, C3-20 cycloalkyl, C1-3 alkoxy, and -C(0)0-R5 wherein R5 is H or unsubstituted or substituted C1-3 alkyl, or
(iii) a cholesterol group;
or a prodrug thereof, or a pharmaceutically acceptable salt thereof.
Typically, P2 in the compound of formula (I) is an a-amino or a-imino acid residue, selected from the following structures
Figure imgf000027_0001
wherein:
p is an integer from 0 to 5;
R3 is an amino, guanidino or methylguanidino group, or an -O-guanidino group (such as, for instance, in a canavanine residue), or an unsubstituted or substituted (e.g.
mono- or poly-substituted) aryl or heteroaryl group preferably having from 5 to 10 ring atoms, wherein the heteroaryl ring contains from 1 to 3 heteroatoms selected from N, S and O, wherein the or each substituent on the substituted aryl or heteroaryl ring is
independently selected from - H2, -CH2- H2, -amidino, -hydroxyamidino, -guanidino, - CH2-guanidino, -halogen, -CI, -Br, -I, -CN, -CF3, C1-3 alkyl, Ci-3 alkoxy and -COOR5, wherein R5 is a hydrogen or Ci-3 alkyl; and
R4 is -H, -OH or -0-(CH2)q-R6 wherein q is an integer from 2 to 4 and R5 is an amino or guanidino group.
P2 is often an arginine, N-methylarginine (( Me)Arg), lysine or canavanine residue in D- or L-configuration. P2 may for instance be an arginine, lysine or canavanine residue in D- or L-configuration. Preferably, the P2 residue is in the L-configuration.
R3 is preferably an amino, guanidino, or -O-guanidino group, or a substituted aryl group having 6 ring atoms or an unsubstituted pyridyl group (wherein the nitrogen may be present at any position of the pyridyl ring and, if appropriate, may be present as N-oxide), wherein the substituent on the aryl ring is selected from - H2, -CH2- H2, -amidino, - hydroxyamidino, -guanidino, and -CH2-guanidino.
In a preferred embodiment, P2 has the following structure:
Figure imgf000027_0002
wherein R3 and p are as defined above. Preferably, R3 is a guanidine group and p is 3. Thus, preferably, P2 has the following structure:
Figure imgf000028_0001
In a preferred embodiment, P2 is an arginine (Arg) residue.
Usually, P3 is any natural or unnatural a-amino or a-imino acid residue of L or D configuration. P3 may for instance be any natural a-amino acid residue of L or D configuration, for instance any natural a-amino acid residue of L configuration.
Typically, P3 is selected from groups having the following structures:
Figure imgf000028_0002
Figure imgf000029_0001
Thus, P3 is often selected from the following amino acid residues in L or D configuration: Val, Lys, Phe, He, Leu, His, Asn, Asp, Arg, Pro, Ser, Thr, Ala, Gly and Dap. Other possible groups for P3 are the groups defined herein for P4.
P3 typically has the following structure:
Figure imgf000030_0001
Typically, P4 in the compound of formula (I) is an a-amino or a-imino acid residue, selected from the followin structures
Figure imgf000030_0002
wherein:
p is an integer from 0 to 5;
P 3 is an amino, guanidine, methylguanidino or C1-3 alkyl group, or an -O-guanidino group (such as, for instance, in a canavanine residue), or an -N(H)C(0)OCH2Ph group, or an unsubstituted or substituted (e.g. mono- or poly-substituted) aryl or heteroaryl group having from 5 to 10 ring atoms, wherein the heteroaryl ring contains from 1 to 3 heteroatoms selected from N, S and O, wherein the or each substituent on the aryl or heteroaryl ring is independently selected from - H2, -CH2- H2, -amidino, - hydroxyamidino, -guanidino, -CH2-guanidino, -halogen, -CI, -Br, -I, -CN, -CF3, C1-3 alkyl, Ci-3 alkoxy and -COOR5, wherein R5 is a hydrogen or C1-3 alkyl; and
R4 is -H, -OH or -0-(CH2)q-R6 wherein q is an integer from 2 to 4 and R5 is an amino or guanidino group.
Thus, P4 is typically an arginine, ( Me)Arg (i.e. N-methylarginine), valine, lysine, N6-Cbz-lysine (N6-[(phenylmethoxy)carbonyl]lysine) or canavanine residue in L- or D- configuration. P4 may for instance be L-arginine, N-methyl-L-arginine, D-Arginine, L- Valine, L-Lysine, N6-Cbz-L-lysine (N6-[(phenylmethoxy)carbonyl]-L-lysine, Lys(Cbz)) or L-canavanine.
R3 is preferably an amino, guanidino, or -O-guanidino group, or a substituted aryl group having 6 ring atoms or an unsubstituted pyridyl group (wherein the nitrogen may be present at any position of the pyridyl ring and, if appropriate, may be present as N-oxide), wherein the substituent on the aryl ring is selected from - H2, -CH2- H2, -amidino, - hydroxyamidino, -guanidino, and -CH2-guanidino.
Thus, P4 is often an arginine, lysine, or canavanine residue in L- or D- configuration. P4 may for instance be L-arginine, D-Arginine, L-Lysine or L-canavanine.
In a preferred embodiment, P4 has the following structure:
Figure imgf000031_0001
wherein R3 and p are as defined above. Preferably, R3 is a guanidine group and p is 3. Thus, preferably, P4 has the following structure:
Figure imgf000031_0002
Thus, in a preferred embodiment, P4 is an arginine (Arg) residue.
Typically, in the compound of formula (I), PI is selected from the following structures:
Figure imgf000031_0003
wherein
Ri is selected from H, OH, O-CH3, H2, 0-C(0)-CH3 and -C(0)-0-(CH2)m-CH3 wherein m is an integer from 1 to 5; R2 is H or an amidino group; and
n is equal to 3, 4 or 5.
Typically, Ri is H or OH. More typically, Ri is H.
Often, n is 4.
Typically, R2 is an amidino group.
Thus, often, in the compound of formula (I), PI is selected from the following structures:
Figure imgf000032_0001
wherein Ri is selected from H, OH, 0-CH3, H2, 0-C(0)-CH3 and -C(0)-0-(CH2) wherein m is an integer from 1 to 5. Typically, Ri is H.
In a preferred embodiment of the compound of formula (I), PI is a 4- amidinobenzyl amide group of the followin structure
Figure imgf000032_0002
wherein Ri is selected from H, OH, O-CH3, H2, 0-C(0)-C¾ and -C(0)-0-(CH2)m-CH3 wherein m is an integer from 1 to 5. Typically, Ri is H.
As mentioned above, Ri is usually H. However, in order to achieve improved membrane permeability of the inhibitors, the hydrophobicity of the inhibitors is capable of being increased (Ettmayer et al, J. Med. Chem. 2004, 47, 2393-2404) by employing compounds wherein Ri is any of OH, 0-CH3, H2, 0-C(0)-CH3 and
-C(0)-0-(CH2)m-CH3, wherein m is an integer from 1 to 5. Such 'prodrugs' are converted in vivo into inhibitorially effective compounds with a free amidino group. Accordingly, Ri may alternatively be any of OH, O-CH3, H2, 0-C(0)-CH3 and -C(0)-0-(CH2)m-CH3, wherein m is an integer from 1 to 5.
Typically, the P2 residue is in the L-configuration. Usually, the P2 residue is an amino acid residue in the L-configuration. It is often a basic amino acid residue in the L- configuration. Particularly preferred are the amino acid residues arginine, lysine and their homo or nor-derivatives which have either one methylene group more or one methylene group less in the side chain. Likewise, canavanine, 3- and 4-amidinophenylalanine, as well as 3- and 4-aminomethylphenylalanine or 3- and 4-guanidinophenylalanine are preferred P2 residues. All phenylalanine derivatives mentioned, such as the homo and nor-derivatives, are also particularly suitable. Furthermore, pyridylalanine and homopyridylalanine are preferable as P2 residues, wherein the nitrogen may be present in any possible position of the pyridyl ring.
Preferably, P2 is present in the L configuration.
Thus P2 is preferably selected from the following structures:
Figure imgf000033_0001
wherein
p is an integer from 0 to 5;
R3 is an amino, guanidino or methylguanidino group, or an -O-guanidino group (such as, for instance, in a canavanine residue), or an unsubstituted or substituted (e.g.
mono- or poly-substituted) aryl or heteroaryl group having from 5 to 10 ring atoms, wherein the heteroaryl ring contains from 1 to 3 heteroatoms selected from N, S and O, wherein the or each substituent on the aryl or heteroaryl ring is independently selected from - H2, -CH2- H2, -amidino, -hydroxyamidino, -guanidino, -CH2-guanidino, -halogen, -CI, -Br, -I, -CN, -CF3, Ci-3 alkyl, Ci-3 alkoxy and -COOR5, wherein R5 is a hydrogen or Ci-3 alkyl; and
R4 is -H, -OH or -0-(CH2)q-R6 wherein q is an integer from 2 to 4 and R5 is an amino or guanidino group.
R3 is preferably an amino, guanidino, or -O-guanidino group, or a substituted aryl group having 6 ring atoms or an unsubstituted pyridyl group (wherein the nitrogen may be present at any position of the pyridyl ring and, if appropriate, may be present as N-oxide), wherein the substituent on the aryl ring is selected from - H2, -CH2- H2, -amidino, - hydroxyamidino, -guanidino, and -CH2-guanidino.
Often, for instance, R3 is an amino, guanidino, or -O-guanidino group.
In particularly preferred embodiments, P2 has the following structure:
Figure imgf000034_0001
wherein R3 and p are as defined anywhere above. Preferably, R3 is a guanidine group. Typically, p is 3.
P2 is often an arginine, N-methylarginine ((NMe)Arg), lysine or canavanine residue in L-configuration. P2 may for instance be an arginine, lysine or canavanine residue in L- configuration.
Preferabl P2 has the following structure:
Figure imgf000034_0002
Thus, P2 is preferably an arginine residue in L-configuration (L-arginine).
The P3 residue is typically an arbitrary natural or unnatural a-amino or a-imino acid residue in the L- or D- configuration.
The P3 residue may for instance be a non-basic a-amino or a-imino acid residue in the L-configuration. It may for instance be the residue of a proteinogenic a-amino or a-imino acid.
P3 may be selected from groups having the following structures:
Figure imgf000035_0001
Figure imgf000036_0001
Thus, P3 is often selected from the following amino acid residues in L
configuration: Val, Lys, Phe, He, Leu, His, Asn, Asp, Arg, Pro, Ser, Thr, Ala, Gly and
Dap.
Alternatively, for instance, P3 may be a basic a-amino acid residue in the D- configuration. For instance, P3 may be D-arginine, D-homoarginine, D-norarginine, D-lysine, D-homolysine, D-norlysine, D-3-amidinophenylalanine, D-4-amidinophenylalanine, a D- pyridylalanine, or a D-homopyridylalanine, wherein the nitrogen may be in any possible position of the pyridyl ring.
P3 may for example be a group of the following structure.
Figure imgf000037_0001
Thus, P3 may be d-Arg.
Preferably, however, P3 is a group of the following structure:
Figure imgf000037_0002
Thus, preferably, P3 is an L-valine residue.
Typically, the P4 residue is in the L-configuration. Usually, the P4 residue is an amino acid residue in the L-configuration. It is usually a basic amino acid residue in the L- configuration. Particularly suitable are the amino acid residues arginine, lysine, and their homo or nor-derivatives which have either one methylene group more or one methylene group less in the side chain. Likewise, canavanine, 3- and 4-amidinophenylalanine, 3- and 4- aminomethylphenylalanine, as well as, 3 and 4-guanidinophenylalanine, are preferred P4 residues, wherein also all phenylalanine derivatives mentioned, such as the homo and nor- derivatives, are particularly suitable. Furthermore, pyridylalanine and homopyridylalanine are preferable as P4 residues, wherein the nitrogen is capable of standing in every possible position of the pyridyl ring.
Preferably, P4 is present in the L configuration.
Thus P4 is preferably selected from the following structures:
Figure imgf000038_0001
wherein
p is an integer from 0 to 5;
R.3 is an amino, guanidine, methylguanidino or C1-3 alkyl group, or an -O-guanidino group (such as, for instance, in a canavanine residue), or an -N(H)C(0)OCH2Ph group, or an unsubstituted or substituted (e.g. mono- or poly-substituted) aryl or heteroaryl group having from 5 to 10 ring atoms, wherein the heteroaryl ring contains from 1 to 3 heteroatoms selected from N, S and O, wherein the or each substituent on the aryl or heteroaryl ring is independently selected from - H2, -CH2- H2, -amidino, - hydroxyamidino, -guanidino, -CH2-guanidino, -halogen, -CI, -Br, -I, -CN, -CF3, C1-3 alkyl, Ci-3 alkoxy and -COOR5, wherein R5 is a hydrogen or C1-3 alkyl; and
R4 is -H, -OH or -0-(CH2)q-R6 wherein q is an integer from 2 to 4 and R5 is an amino or guanidine group.
P4 is often an arginine, N-methylarginine ((NMe)Arg), valine, lysine, N6-Cbz- lysine (N6-[(phenylmethoxy)carbonyl]lysine, Lys(Cbz)) or canavanine residue in L- configuration.
R3 is preferably an amino, guanidino, or -O-guanidino group, or a substituted aryl group having 6 ring atoms or an unsubstituted pyridyl group (wherein the nitrogen may be present at any position of the pyridyl ring and, if appropriate, may be present as N-oxide), wherein the substituent on the aryl ring is selected from - H2, -CH2- H2, -amidino, - hydroxyamidino, -guanidino, and -CH2-guanidino.
Often, for instance, R3 is an amino, guanidino, or -O-guanidino group.
In particularly preferred embodiments, P4 has the following structure:
Figure imgf000038_0002
wherein R3 and p are as defined anywhere above. Preferably, R3 is a guanidino Typically, p is 3.
Preferably, P4 is an arginine, lysine or canavanine residue in L-configuration.
More preferably, P4 has the following structure:
Figure imgf000039_0001
Thus, P4 is preferably an arginine residue in L-configuration (L-arginine).
The P5 group is highly variable and is either a hydrogen atom in the simplest case or, preferably, it is a hydrophobic, saturated or (where applicable) a mono- or polyunsaturated acyl group derived from naturally occurring fatty acids. However, aralkyl-CO groups, heteroaralkyl-CO groups or sulfonyl groups are also preferred, wherein all these groups are unsubstituted (where applicable) or substituted with up to three substituents. The following are particularly suitable as substituents: halogens, in particular chlorine atoms, amino
groups, carboxyl groups that are also capable of being available as esters, particularly as ethyl esters, amidino, hydroxyamidino, guanidino, CH2-guanidino, CF3 and C1-3 alkyl groups or C1-3 alkoxy groups.
P5 may in some embodiments be a pyroglutamyl residue. Thus, P5 may be a group having the following structure
Figure imgf000039_0002
P5 is typically however selected from H, -CO-R7, -CO-X-R7 and -SO2-R7, wherein R7 and X are as defined above.
P5 is often for instance selected from -CO-R7, -CO-X-R7 and -SO2-R7, wherein R7 and X are as defined above.
P5 may for instance be a group having any of the following structures:
Figure imgf000040_0001
wherein z is an integer from 1 to 18. Often, z is equal to 8, 14 or 16.
Alternatively, P5 may be a sphingosine acylated with a bifunctional group, having the following structure:
Figure imgf000041_0001
a sphingosyl-phosphorylcholine acylated with a bifunctional group, having the followin structure:
Figure imgf000041_0002
wherein z is an integer from 2 to 20.
P5 is typically however a phenylacetyl or another acetyl group.
In a preferred embodiment P5 is:
H;
-C(0)-CH2-R7 wherein R7 is unsubstituted or substituted aryl (usually unsubstituted or sub stituted phenyl) ;
-C(0)-R7 wherein R7 is either unsubstituted or substituted C1-24 alkyl or unsubstituted or substituted C2-24 alkenyl;
-C(0)-0-R7 wherein R7 is either unsubstituted or substituted aryl (usually unsubstituted or substituted phenyl) or unsubstituted or substituted C1-24 alkyl; or
S(0)2-R7 wherein R7 is unsubstituted or substituted aryl (usually unsubstituted or substituted phenyl), wherein the substituent or substituents which may be present at R7 are
independently selected from - H2, -CH2- H2, -amidino, -hydroxyamidino, -guanidino, - CH2-guanidino, methylguanidino, -halogen, -CI, -Br, -I, -CN, -CF3, aryl, C1-3 alkyl, C3-20 cycloalkyl, C1-3 alkoxy, and -C(0)0-R5 wherein R5 is H or unsubstituted or substituted C1-3 alkyl.
In a further preferred embodiment P5 is -C(0)-CH2-R7 wherein R7 is unsubstituted or substituted aryl, or P5 is -CO-R7 wherein R7 is either unsubstituted or substituted C1-24 alkyl or unsubstituted or substituted C2-24 alkenyl. Preferably the substituents, where present, in R7 are independently selected from - H2, -CH2- H2, -amidino, -guanidino, and -CH2-guanidino.
Preferably, P5 is either of the following structures
Figure imgf000042_0001
P5 may for instance be a group of the following structure:
Figure imgf000042_0002
Thus, in one preferred embodiment:
PI is a 4-amidinobenzylamide group;
P2 is an arginine, N-methylarginine ((NMe)Arg), lysine or canavanine residue in L- configuration;
P3 is selected from groups having the following structures:
Figure imgf000042_0003
Figure imgf000043_0001
P4 is L-arginine, N-methyl-L-arginine, D-Arginine, L- Valine, L-Lysine, N6-Cbz-L- lysine (N6-[(phenylmethoxy)carbonyl]-L-lysine) or L-canavanine; and
P5 is as defined anywhere herein. Often, however, in this embodiment, P5 is selected from -CO-R7, -CO-X-R7 and -SO2-R7, wherein R7 and X are as defined above. In particular, P5 is often:
H;
-C(0)-CH2-R7 wherein R7 is unsubstituted or substituted phenyl;
-C(0)-R7 wherein R7 is either unsubstituted or substituted C1-24 alkyl or
unsubstituted or substituted C2-24 alkenyl;
-C(0)-0-R7 wherein R7 is either unsubstituted or substituted phenyl or
unsubstituted or substituted C1-24 alkyl; or
S(0)2-R7 wherein R7 is unsubstituted or substituted phenyl,
wherein the substituent or substituents which may be present at R7 are
independently selected from -NH2, -CH2-NH2, -amidino, -hydroxyamidino, -guanidino, - CH2-guanidino, methylguanidino, -halogen, -CI, -Br, -I, -CN, -CF3, aryl, C1-3 alkyl, C3-20 cycloalkyl, C1-3 alkoxy, and -C(0)0-R5 wherein R5 is H or unsubstituted or substituted C1-3 alkyl.
P5 may for instance be selected from:
Figure imgf000044_0001
P5 may for instance in this embodiment be:
Figure imgf000044_0002
Preferred compounds of formula (I) are the following:
Figure imgf000045_0001
97
Figure imgf000046_0001
Figure imgf000047_0001
Figure imgf000048_0001
Figure imgf000049_0001
Figure imgf000050_0001
In the compounds shown above:
Arg is an L-arginine amino acid residue;
d-Arg is a D-arginine amino acid residue;
( Me)Arg is an N-methyl-L-arginine amino acid residue, having the following structure:
Figure imgf000050_0002
Lys is an L-lysine amino acid residue;
Lys(Cbz) is an N6-[(phenylmethoxy)carbonyl]-L-lysine amino acid residue; Val is an L-valine amino acid residue; Phe is an L-phenylalanine amino acid residue;
He is an L-isoleucine amino acid residue;
Leu is an L-leucine amino acid residue;
His is an L-histidine amino acid residue;
Asn is an L-asparagine amino acid residue;
Asp is an L-aspartic acid amino acid residue;
Pro is an L-proline amino acid residue;
Ser is an L-serine amino acid residue;
Thr is an L-threonine amino acid residue;
Ala is an L-alanine amino acid residue;
Gly is a glycine amino acid residue; and
Dap is a diaminopimelic acid amino acid residue, having the following structure:
Figure imgf000051_0001
Thus, the proprotein convertase inhibitor may be any one of the compounds of formula (I) shown above, or a prodrug thereof, or a pharmaceutically acceptable salt thereof.
Preferably, the compound of formula (I) is Calbiochem 537076, which has the following structure:
Figure imgf000051_0002
The full structural chemical formula of Calbiochem 537076 is shown below, as formula (la):
Figure imgf000052_0001
Thus, the proprotein convertase inhibitor may be a compound of formula (la) as shown above, or a prodrug thereof, or a pharmaceutically acceptable salt thereof.
The synthesis of the compounds of formula (I) and formula (la), and their activity as proprotein convertase inhibitors, is described in US 2011/0312873 Al .
It is also a finding of the present invention that proprotein convertase inhibitors which inhibit collagen matrix formation do not significantly affect fibronectin secretion. This is advantageous, as fibronectin plays a major role in cell adhesion, growth, migration, and differentiation and is important for processes such as wound healing and embryonic development. Also, altered fibronectin expression, degradation, and organization has been associated with a number of pathologies, including cancer. It can therefore be desirable not to alter fibronectin secretion.
Thus, in another aspect, the invention provides a proprotein convertase inhibitor for use in the treatment of a fibrotic disorder by inhibiting the formation of collagen matrix without inhibiting fibronectin secretion.
The invention also provides a composition for use in the treatment of a fibrotic disorder by inhibiting collagen matrix formation without inhibiting fibronectin secretion, which composition comprises a proprotein convertase inhibitor and a pharmaceutically acceptable carrier or diluent.
The invention also provides the use of a proprotein convertase inhibitor in the manufacture of a medicament for use in the treatment of a fibrotic disorder by inhibiting collagen matrix formation without inhibiting fibronectin secretion.
The invention also provides a method of treating a fibrotic disorder in a subject by inhibiting collagen matrix formation without inhibiting fibronectin secretion, which method comprises administering to the subject a therapeutically effective amount of a proprotein convertase inhibitor, which amount is effective to inhibit collagen matrix formation in the subject without inhibiting fibronectin secretion. Any proprotein convertase inhibitor capable of inhibiting collagen matrix formation without significantly inhibiting fibronectin secretion may be employed in these aspects of the invention.
One class of proprotein convertase inhibitor that is capable of inhibiting collagen matrix formation without significantly inhibiting fibronectin secretion is the class of compounds of formula (I) and formula (la) as defined herein. Thus, in one embodiment, the proprotein convertase inhibitor employed is a compound of formula (I), or formula (la), or a prodrug thereof or a pharmaceutically acceptable salt thereof.
Other, different classes of proprotein convertase inhibitor may also be employed in the present invention, to treat fibrotic disorders by inhibiting collagen matrix formation without inhibiting fibronectin secretion. The skilled person is readily able to identify other suitable proprotein convertase inhibitors, as many proprotein convertase inhibitors are known in the art, as are procedures for screening compounds for proprotein convertase inhibitory activity. For instance, proprotein convertase inhibitors may be identified using the screening methods described in the following references: Becker, G. L., et al. (2012). "Highly potent inhibitors of proprotein convertase furin as potential drugs for treatment of infectious diseases." Journal of Biological Chemistry 287(26): 21992-22003; Becker, G. L., et al. (2010). "Potent Inhibitors of Furin and Furin-like Proprotein Convertases
Containing Decarboxylated PI Arginine Mimetics." J. Med. Chem. 53(3): 1067-1075; and Hardes, K., et al. (2015). "Novel Furin Inhibitors with Potent Anti-infectious Activity. " ChemMedChem 10(7): 1218-1231. The skilled person can therefore apply such methods without undue experimentation to identify other classes of proprotein convertase inhibitor that can be employed in the invention.
Classes of proprotein convertase inhibitor that can be employed in the present invention include, but are not limited to, compounds that bind to a proprotein convertase and thereby inhibit its activity. Such compounds include competitive inhibitors of a proprotein convertase and allosteric inhibitors of a proprotein convertase, and these can be identified by the skilled person without undue burden using known assay methods as described above.
Other classes of proprotein convertase inhibitors that may be employed in the present invention include compounds that prevent the transcription, translation or expression of a proprotein convertase. Such proprotein convertase inhibitors include, for instance, ribozymes and antisense and RNA interference molecules, such as an antisense RNA, small interfering RNA (siRNA) or small hairpin RNA (shRNA). Antisense and RNA interference (RNAi) technology for knocking down protein expression are well known in the art and standard methods can be employed to knock down expression of PCSKs.
Both antisense and siRNA technology interfere with mRNA. Antisense
oligonucleotides interfere with mRNA by binding to (hybridising with) a section of the mRNA. The antisense oligonucleotide is therefore designed to be complementary to the mRNA (although the oligonucleotide does not have to be 100% complementary as discussed below). In other words, the antisense oligonucleotide may be a section of the cDNA. Again, the oligonucleotide sequence may not be 100% identical to the cDNA sequence. This is also discussed below.
RNAi involves the use of double-stranded RNA, such small interfering RNA (siRNA) or small hairpin RNA (shRNA), which can bind to the mRNA and inhibit protein expression.
Accordingly, the inhibitor preferably comprises an oligonucleotide which specifically hybridises to a part of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15 or 17 (human PCSK 1 to 9 mRNA repectively) or any isoform thereof.
Preferably, for instance, the inhibitor comprises an oligonucleotide which specifically hybridises to a part of SEQ ID NO: 5, 9 or 11 (human PCSK 3, 5 and 6 mRNA respectively) or any isoform thereof.
It is also preferred that the inhibitor comprises an oligonucleotide which
specifically hybridises to a part of SEQ ID NO: 1, 3, 5, 7, 13, 15 or 17 (human PCSK 1, 2, 3, 4, 7, 8 or 9 mRNA respectively) or any isoform thereof.
More preferably, however, the inhibitor comprises an oligonucleotide which specifically hybridises to a part of SEQ ID NO: 5 (human PCSK 3 mRNA) or any isoform thereof.
Oligonucleotides are short nucleotide polymers which typically have 50 or fewer nucleotides, such 40 or fewer, 30 or fewer, 22 or fewer, 21 or fewer, 20 or fewer, 10 or fewer or 5 or fewer nucleotides. The oligonucleotide used in the invention is preferably 20 to 25 nucleotides in length, more preferably 21 or 22 nucleotides in length. The nucleotides can be naturally occurring or artificial.
A nucleotide typically contains a nucleobase, a sugar and at least one linking group, such as a phosphate, 2'O-methyl, 2' methoxy-ethyl, phosphoramidate, methylphosphonate or phosphorothioate group. The nucleobase is typically heterocyclic. Nucleobases include, but are not limited to, purines and pyrimidines and more specifically adenine (A), guanine (G), thymine (T), uracil (U) and cytosine (C). The sugar is typically a pentose sugar. Nucleotide sugars include, but are not limited to, ribose and deoxyribose. The nucleotide is typically a ribonucleotide or deoxyribonucleotide. The nucleotide typically contains a monophosphate, diphosphate or triphosphate. Phosphates may be attached on the 5' or 3' side of a nucleotide.
Nucleotides include, but are not limited to, adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), guanosine monophosphate (GMP), guanosine diphosphate (GDP), guanosine triphosphate (GTP), thymidine monophosphate (TMP), thymidine diphosphate (TDP), thymidine triphosphate (TTP), uridine monophosphate (UMP), uridine diphosphate (HDP), uridine triphosphate (UTP), cytidine monophosphate (CMP), cytidine diphosphate (CDP), cytidine triphosphate (CTP), 5-methylcytidine monophosphate, 5-methylcytidine diphosphate, 5 -methyl cytidine triphosphate, 5-hydroxymethylcytidine monophosphate, 5-hydroxymethylcytidine diphosphate, 5-hydroxymethylcytidine triphosphate, cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), deoxyadenosine monophosphate (dAMP), deoxyadenosine diphosphate (dADP), deoxyadenosine triphosphate (dATP), deoxyguanosine monophosphate (dGMP), deoxyguanosine diphosphate (dGDP), deoxyguanosine triphosphate (dGTP), deoxythymidine monophosphate (dTMP), deoxythymidine diphosphate (dTDP), deoxythymidine triphosphate (dTTP), deoxyuridine monophosphate (dUMP), deoxyuridine diphosphate (dUDP), deoxyuridine triphosphate (dUTP), deoxycytidine monophosphate (dCMP), deoxycytidine diphosphate (dCDP) and deoxycytidine triphosphate (dCTP), 5 -methyl-2' -deoxycytidine monophosphate, 5-methyl- 2 '-deoxycytidine diphosphate, 5-methyl-2'-deoxycytidine triphosphate, 5-hydroxymethyl- 2 '-deoxycytidine monophosphate, 5 -hydroxymethyl-2' -deoxycytidine diphosphate and 5- hydroxymethyl-2' -deoxycytidine triphosphate. The nucleotides are preferably selected from AMP, TMP, GMP, UMP, dAMP, dTMP, dGMP or dCMP.
The nucleotides may contain additional modifications. In particular, suitable modified nucleotides include, but are not limited to, 2'amino pyrimidines (such as 2'- amino cytidine and 2 '-amino uridine), 2'-hyrdroxyl purines (such as , 2'-fluoro
pyrimidines (such as 2'-fluorocytidine and 2'fluoro uridine), hydroxyl pyrimidines (such as 5'-a-P-borano uridine), 2'-0-methyl nucleotides (such as 2'-0-methyl adenosine, 2'-0- methyl guanosine, 2'-0-methyl cytidine and 2'-0-methyl uridine), 4'-thio pyrimidines (such as 4'-thio uridine and 4'-thio cytidine) and nucleotides have modifications of the nucleobase (such as 5-pentynyl-2'-deoxy uridine, 5-(3-aminopropyl)-uridine and 1,6- diaminohexyl-N-5-carbamoylmethyl uridine).
One or more nucleotides in the oligonucleotide can be oxidized or methylated. One or more nucleotides in the oligonucleotide may be damaged. For instance, the
oligonucleotide may comprise a pyrimidine dimer. Such dimers are typically associated with damage by ultraviolet light.
The nucleotides in the oligonucleotide may be attached to each other in any manner. The nucleotides may be linked by phosphate, 2'0-methyl, 2' methoxy-ethyl, phosphoramidate, methylphosphonate or phosphorothioate linkages. The nucleotides are typically attached by their sugar and phosphate groups as in nucleic acids. The nucleotides may be connected via their nucleobases as in pyrimidine dimers.
The oligonucleotide can be a nucleic acid, such as deoxyribonucleic acid (DNA) or a ribonucleic acid (RNA). The oligonucleotide may be any synthetic nucleic acid known in the art, such as peptide nucleic acid (PNA), glycerol nucleic acid (GNA), threose nucleic acid (TNA), locked nucleic acid (LNA), morpholino nucleic acid or other synthetic polymers with nucleotide side chains.
An oligonucleotide preferably specifically hybridises to a part of any one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15 or 17 or any isoform thereof, hereafter called the target sequence. The length of the target sequence typically corresponds to the length of the oligonucleotide. For instance, a 21 or 22 nucleotide oligonucleotide typically specifically hybridises to a 21 or 22 nucleotide target sequence. The target sequence may therefore be any of the lengths discussed above with reference to the length of the oligonucleotide. The target sequence is typically consecutive nucleotides within the target polynucleotide.
An oligonucleotide "specifically hybridises" to a target sequence when it hybridises with preferential or high affinity to the target sequence but does not substantially hybridise, does not hybridise or hybridises with only low affinity to other sequences.
An oligonucleotide "specifically hybridises" if it hybridises to the target sequence with a melting temperature (Tm) that is at least 2 °C, such as at least 3 °C, at least 4 °C, at least 5 °C, at least 6 °C, at least 7 °C, at least 8 °C, at least 9 °C or at least 10 °C, greater than its Tm for other sequences. More preferably, the oligonucleotide hybridises to the target sequence with a Tm that is at least 2 °C, such as at least 3 °C, at least 4 °C, at least 5 °C, at least 6 °C, at least 7 °C, at least 8 °C, at least 9 °C, at least 10 °C, at least 20 °C, at least 30 °C or at least 40 °C, greater than its Tm for other nucleic acids. Preferably, the portion hybridises to the target sequence with a Tm that is at least 2 °C, such as at least 3 °C, at least 4 °C, at least 5 °C, at least 6 °C, at least 7 °C, at least 8 °C, at least 9 °C, at least 10 °C, at least 20 °C, at least 30 °C or at least 40 °C, greater than its Tm for a sequence which differs from the target sequence by one or more nucleotides, such as by 1, 2, 3, 4 or 5 or more nucleotides. The portion typically hybridises to the target sequence with a Tm of at least 90 °C, such as at least 92 °C or at least 95 °C. Tm can be measured experimentally using known techniques, including the use of DNA microarrays, or can be calculated using publicly available Tm calculators, such as those available over the internet.
Conditions that permit the hybridisation are well-known in the art (for example, Sambrook et al., 2001, Molecular Cloning: a laboratory manual, 3rd edition, Cold Spring Harbour Laboratory Press; and Current Protocols in Molecular Biology, Chapter 2, Ausubel et al, Eds., Greene Publishing and Wiley-lnterscience, New York (1995)).
Hybridisation can be carried out under low stringency conditions, for example in the presence of a buffered solution of 30 to 35% formamide, 1 M NaCl and 1 % SDS (sodium dodecyl sulfate) at 37 °C followed by a 20 wash in from IX (0.1650 M Na+) to 2X (0.33 M Na+) SSC (standard sodium citrate) at 50 °C. Hybridisation can be carried out under moderate stringency conditions, for example in the presence of a buffer solution of 40 to 45% formamide, 1 M NaCl, and 1 % SDS at 37 °C, followed by a wash in from 0.5X (0.0825 M Na+) to IX (0.1650 M Na+) SSC at 55 °C. Hybridisation can be carried out under high stringency conditions, for example in the presence of a buffered solution of 50% formamide, 1 M NaCl, 1% SDS at 37 °C, followed by a wash in 0. IX (0.0165 M Na+) SSC at 60 °C.
The oligonucleotide may comprise a sequence which is substantially
complementary to the target sequence. Typically, the oligonucleotides are 100% complementary. However, lower levels of complementarity may also be acceptable, such as 95%), 90%, 85% and even 80%. Complementarity below 100% is acceptable as long as the oligonucleotides specifically hybridise to the target sequence. An oligonucleotide may therefore have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mismatches across a region of 5, 10, 15, 20, 21, 22, 30, 40 or 50 nucleotides.
Alternatively, the inhibitor preferably comprises an oligonucleotide which comprises 50 or fewer consecutive nucleotides from (a) SEQ ID NO: 2, 3, 6, 8, 10, 12, 14, 16 or 18 (human PCSK 1 to 9 cDNA respectively) or any isoform thereof or (b) a variant sequence which has at least 95%, such as at least 97%, at least 98% or at least 99%, homology to SEQ ID NO: 2, 3, 6, 8, 10, 12, 14, 16 or 18 or any isoform thereof based on nucleotide identity over the entire sequence. The oligonucleotide may be any of the lengths discussed above. It is preferably from 16 to 22 nucleotides in length, for instance from 18 to 20 nucleotides in length. It may for instance be 19 nucleotides in length. The oligonucleotide may comprise any of the nucleotides discussed above, including the modified nucleotides.
Preferably, for instance, the inhibitor comprises an oligonucleotide which comprises 50 or fewer consecutive nucleotides from (a) SEQ ID NO: 6, 10 or 12 (human PCSK 3, 5 or 6 cDNA respectively) or any isoform thereof or (b) a variant sequence which has at least 95%, such as at least 97%, at least 98% or at least 99%, homology to SEQ ID NO: 6, 10 or 12 or any isoform thereof based on nucleotide identity over the entire sequence.
It is also preferred that the inhibitor comprises an oligonucleotide which comprises 50 or fewer consecutive nucleotides from (a) SEQ ID NO: 2, 3, 6, 8, 14, 16 or 18 (human PCSK 1, 2, 3, 4, 7, 8 or 9 cDNA respectively) or any isoform thereof or (b) a variant sequence which has at least 95%, such as at least 97%, at least 98% or at least 99%, homology to SEQ ID NO: 2, 3, 6, 8, 14, 16 or 18 or any isoform thereof based on nucleotide identity over the entire sequence.
More preferably, however, the inhibitor comprises an oligonucleotide which comprises 50 or fewer consecutive nucleotides from (a) SEQ ID NO: 6 (human PCSK 3 cDNA) or any isoform thereof or (b) a variant sequence which has at least 95%, such as at least 97%, at least 98% or at least 99%, homology to SEQ ID NO: 6 or any isoform thereof based on nucleotide identity over the entire sequence.
The oligonucleotide can be a nucleic acid, such as any of those discussed above. The oligonucleotide is preferably RNA.
The oligonucleotide may be single stranded. The oligonucleotide may be double stranded. The oligonucleotide may compirse a hairpin.
SEQ ID NOs: 19 to 30 are oligonucleotides which specifically hybridise to a part of SEQ ID NO: 5, 9 or 11 (human PCSK 3, 5 and 6 mRNA respectively).
Accordingly, the inhibitor preferably comprises an oligonucleotide which is (a) any one of SEQ ID NOs: 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30, or (b) a variant sequence which has at least 95%, such as at least 97%, at least 98% or at least 99%, homology to any one of SEQ ID NOs: 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30 or any isoform thereof based on nucleotide identity over the entire sequence.
The inhibitor may for instance comprise any one of SEQ ID NOs: 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30. The inhibitor could for instance comprise two or more different oligonucleotides, for instance two, three or four different oligonucleotides, each of which is (a) any one of SEQ ID NOs: 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30, or (b) a variant sequence which has at least 95%, such as at least 97%, at least 98% or at least 99%, homology to any one of SEQ ID NOs: 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30 or any isoform thereof based on nucleotide identity over the entire sequence.
The inhibitor may for instance comprise two or more different oligonucleotides, for instance two, three or four different oligonucleotides, each of which is any one of SEQ ID NOs: 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30.
The inhibitor may for instance comprise two or more of, for instance two, three or four of the following oligonucleotides: SEQ ID NOs: 19, 20, 21 and 22.
Alternatively, the inhibitor may comprise two or more of, for instance two, three or four of the following oligonucleotides: SEQ ID NOs: 23, 24, 25 and 26.
The inhibitor may for instance comprise two or more of, for instance two, three or four of the following oligonucleotides: SEQ ID NOs: 27, 28, 29 and 30.
In a preferred embodiment the inhibitor comprises an oligonucleotide which specifically hybridises to a part of SEQ ID NO: 5 (human PCSK 3, FURIN) or any isoform thereof.
Accordingly, the inhibitor preferably comprises an oligonucleotide which is (a) any one of SEQ ID NOs: 19, 20, 21 and 22, or (b) a variant sequence which has at least 95%, such as at least 97%, at least 98% or at least 99%, homology to any one of SEQ ID NOs: 19, 20, 21 and 22 or any isoform thereof based on nucleotide identity over the entire sequence.
The inhibitor may for instance comprise any one of SEQ ID NOs: 19, 20, 21 and 22.
The inhibitor could for instance comprise two or more different oligonucleotides, for instance two, three or four different oligonucleotides, each of which is (a) any one of SEQ ID NOs: 19, 20, 21 and 22, or (b) a variant sequence which has at least 95%, such as at least 97%, at least 98% or at least 99%, homology to any one of SEQ ID NOs: 19, 20, 21 and 22 or any isoform thereof based on nucleotide identity over the entire sequence.
The inhibitor may for instance comprise two or more different oligonucleotides, for instance two, three or four different oligonucleotides, selected from the oligonucleotides having SEQ ID NOs: 19, 20, 21 and 22. The inhibitor may for example comprise the oligonucleotide of SEQ ID NO: 19, the oligonucleotide of SEQ ID NO: 20, the oligonucleotide of SEQ ID NO: 21 and the oligonucleotide of SEQ ID NO: 22.
Oligonucleotides may be synthesised using standard techniques known in the art.
In experiments described in the Examples below, it was found that two particular known proprotein convertase inhibitors showed little activity against collagen matrix formation under the conditions tested. These were hexa-D-arginine amide (CAS no.
-67-0) which has the following chemical structure:
Figure imgf000060_0001
and decanoyl-Arg-Val-Lys-Arg-CMK (CAS 150113-99-8), which has the following chemical structure:
Figure imgf000060_0002
Accordingly, the proprotein convertase inhibitor employed in the present invention is usually other than the two compounds whose structures are shown above. It is also typically also other than salts, solvates, enantiomers, diastereomers and racemates of those compounds.
Fibrotic disorders
Fibrotic disorders can be treated with a proprotein convertase inhibitor in accordance with the present invention.
The term "fibrotic disorder", as used herein, refers to a disorder in which excessive fibrosis leads to pathological derangement and malfunctioning of tissue.
Fibrotic disorders are typically characterised by the accumulation of fibrous tissue (predominately collagen) in an abnormal fashion. Thus, fibrotic disorders are usually characterised by an excess of collagen matrix formation (Rosenbloom, J., Mendoza, F. A. & Jimenez, S. A. Strategies for anti-fibrotic therapies. Biochim. Biophys. Acta 1832, 1088-1103; 2013). This occurs, for example, in atrial fibrillation, cardiac diastolic dysfunction, heart failure, cardiomyopathies, dermal fibrosis, a fibrotic skin disease, cirrhosis, Crohn's disease, cirrhosis of the liver, liver fibrosis, progressive kidney disease, glomerulonephritis, renal fibrosis, pulmonary fibrosis, idiopathic pulmonary fibrosis, cardiovascular fibrosis, myocardial fibrosis, myocardial hibernation, fibrosis following myocardial infarction, systemic sclerosis, central nervous system fibrosis following a stroke or following a neuro-degenerative disorder (e.g. Alzheimer's Disease), proliferative vitreoretinopathy (PVR), restenosis (for example following angioplasty), and arthritis. Fibrotic skin diseases are characterised by excessive scarring of the skin, and is a result of a pathologic wound healing response. There is a wide spectrum of fibrotic skin diseases, including but not limited to: scleroderma, nephrogenic fibrosing dermopathy, mixed connective tissue disease, scleromyxedema, scleredema, and eosinophilic fasciitis. Exposures to chemicals or physical agents are also potential causes of fibrotic skin disease.
Fibrotic disorders which can be treated in accordance with the present invention therefore include, but are not limited to, the following conditions:
atrial fibrillation;
cardiac diastolic dysfunction;
heart failure, for instance chronic heart failure (CFIF);
cardiomyopathy;
cardiovascular fibrosis;
myocardial fibrosis;
myocardial hibernation; fibrosis following myocardial infarction;
dermal fibrosis;
a fibrotic skin disease, for example: scleroderma, nephrogenic fibrosing dermopathy, mixed connective tissue disease, scleromyxedema, scleredema, eosinophilic fasciitis, or a fibrotic skin disease caused by exposure to a chemical or physical agent;
liver fibrosis;
cirrhosis, for instance cirrhosis of the liver;
Crohn's Disease (CD);
progressive kidney disease;
glomerulonephritis;
renal fibrosis;
pulmonary fibrosis, for instance idiopathic pulmonary fibrosis;
systemic sclerosis;
central nervous system fibrosis following a stroke or following a neuro-degenerative disorder (e.g. Alzheimer's Disease);
proliferative vitreoretinopathy (PVR);
restenosis (for example following angioplasty); and
arthritis.
It is a further finding of the present invention that proprotein convertase inhibitors, including for instance the inhibitors of formula (I), can reduce the secretion of the precursor of collagen: procollagen. Also, as explained elsewhere herein, another finding of the invention is that proprotein convertase inhibitors which inhibit collagen matrix formation do not significantly affect fibronectin secretion.
Accordingly, the proprotein convertase inhibitor which is for use in the treatment of a fibrotic disorder in accordance with the invention, is preferably for use in the treatment of said fibrotic disorder by inhibiting the secretion of procollagen.
More preferably, the proprotein convertase inhibitor which is for use in the treatment of a fibrotic disorder in accordance with the invention, is for use in said treatment of the fibrotic disorder by inhibiting the secretion of procollagen without inhibiting fibronectin secretion. The proprotein convertase inhibitor may for instance be for use in said treatment of the fibrotic disorder by inhibiting the secretion of procollagen without altering (for instance, without inhibiting or increasing) the secretion of fibronectin.
The invention also provides the use of a proprotein convertase inhibitor in the manufacture of a medicament for use in the treatment of a fibrotic disorder. In the use of the invention, the proprotein convertase inhibitor, the fibrotic disorder, and/or the treatment of the fibrotic disorder may be as further defined herein.
The invention also provides a method of treating a fibrotic disorder, which method comprises administering to a subject in need of such treatment a therapeutically effective amount of a proprotein convertase inhibitor. In the method of the invention, the PDE6D inhibitor, the fibrotic disorder, and/or the treatment of the fibrotic disorder may be as further defined herein.
The terms "treating" and "treatment" as used herein in the context of treating a fibrotic disorder refers generally to treatment and therapy, whether of a human subject or an animal subject (e.g. in veterinary applications), in which some desired therapeutic effect is achieved, such as, for example, the inhibition of the progress of the condition. The term includes a reduction in the rate of progress, a halt in the rate of progress, regression of the condition, amelioration of the condition, and cure of the condition. Palliative treatment or treatment as a prophylactic measure (i.e. prophylaxis, prevention) are also included.
Generally, the invention relates to the treatment of mammals, particularly humans.
The term "therapeutically effective amount" as used herein, refers to the amount of the proprotein convertase inhibitor, whether as part of a pharmaceutical composition, product, combination or otherwise, which is effective for producing the desired therapeutic effect when administered in accordance with a desired treatment regimen. As will be discussed further below, administration can be effected in one dose, continuously or intermittently (e.g. in divided doses at appropriate intervals) throughout the course of treatment. Methods of determining the most effective means and dosage of administration are well known to those of skill in the art and will vary with the formulation used for therapy, the purpose of the therapy, the target tissue or cells being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician, veterinarian, or clinician.
It is a further finding of the present invention that proprotein convertase inhibitors potently inhibit collagen matrix formation in dermal fibroblasts, and reduce the secretion of procollagen by dermal fibroblasts. Accordingly, in some embodiment the fibrotic disorder is dermal fibrosis or a fibrotic skin disease. The fibrotic skin disease may for instance be any one of the following fibrotic skin diseases: scleroderma, nephrogenic fibrosing dermopathy, mixed connective tissue disease, scleromyxedema, scleredema, eosinophilic fasciitis, and a fibrotic skin disease caused by exposure to a chemical or physical agent. Fibrotic disorders can be treated with a proprotein convertase inhibitor in accordance with the present invention either alone or in combination with a further active ingredient. The further active ingredient may for instance be a further antifibrotic agent. Further classes of antifibrotic agents that may be used in combination with a proprotein convertase inhibitor include PDE6D inhibitors, the drug pirfenidone, Endothelin antagonists, Endothelin receptor inhibitors (e.g. Bosentan), Transforming growth factor beta (TGFbeta) antagonists, TGFbeta receptor inhibitors, Platelet-derived growth factor (PDGF) antagonists, PDGF receptor inhibitors, chemokine antagonists, chemokine receptor inhibitors, cytokine antagonists, cytokine receptor inhibitors, N-acetylcysteine, prednisone, azathioprine, and tyrosine kinase inhibitors.
Accordingly, the invention further provides a combination comprising (a) a proprotein convertase inhibitor and (b) a further antifibrotic agent, which combination is for use in the treatment of a fibrotic disorder.
The combination of the invention may comprise: a first pharmaceutical
composition comprising the proprotein convertase inhibitor, and a second pharmaceutical composition comprising the further antifibrotic agent, wherein the first and second pharmaceutical compositions are for separate, simultaneous, concomitant or sequential administration in the treatment of said fibrotic disorder.
The first and second pharmaceutical compositions may each further comprise a pharmaceutically acceptable carrier or diluent.
The invention also provides a composition for use in treating a fibrotic disorder, which composition comprises (a) a proprotein convertase inhibitor, (b) a further
antifibrotic agent, and (c) a pharmaceutically acceptable carrier or diluent.
The invention also provides (a) a proprotein convertase inhibitor for use in the treatment of a fibrotic disorder by coadministration with (b) a further antifibrotic agent.
The invention also provides (b) a further antifibrotic agent for use in the treatment of a fibrotic disorder by coadministration with (a) a proprotein convertase inhibitor.
The invention also provides the use of (a) proprotein convertase inhibitor in the manufacture of a medicament for use in the treatment of a fibrotic disorder by
coadministration with (b) a further antifibrotic agent.
The invention further provides the use of (b) a further antifibrotic agent in the manufacture of a medicament for use in the treatment of a fibrotic disorder by
coadministration with (a) a proprotein convertase inhibitor. The invention further provides the use of (a) proprotein convertase inhibitor and (b) a further antifibrotic agent in the manufacture of a medicament for use in the treatment of a fibrotic disorder.
The invention also provides a method of treating a fibrotic disorder which method comprises administering to a subject in need of such treatment therapeutically effective amounts of (a) a proprotein convertase inhibitor and (b) a further antifibrotic agent.
The invention also provides a kit of parts comprising a proprotein convertase inhibitor together with instructions for simultaneous, concurrent, separate or sequential use in combination with a further antifibrotic agent, for the treatment of a fibrotic disorder.
The invention also provides a kit of parts comprising a further antifibrotic agent together with instructions for simultaneous, concurrent, separate or sequential use in combination with a proprotein convertase inhibitor, for the treatment of a fibrotic disorder.
The invention also provides a kit of parts comprising a proprotein convertase inhibitor, a further antifibrotic agent, and instructions for their simultaneous, concurrent, separate or sequential use for the treatment of a fibrotic disorder.
The proprotein convertase inhibitor employed in these "combination" aspects of the invention may be any of the proprotein convertase inhibitors described herein.
The further antifibrotic agent may be any known antifibrotic agent. An example of a known antifibrotic agent is 5-methyl-l-phenylpyridin-2-one, which is also known as pirfenidone. Accordingly, in one embodiment, the further antifibrotic agent is 5-methyl-l- phenylpyridin-2-one (i.e. pirfenidone) or a pharmaceutically acceptable salt thereof.
Other classes of antifibrotic agents that may be used in combination with a proprotein convertase inhibitor include Endothelin antagonists, Endothelin receptor inhibitors (e.g. Bosentan), Transforming growth factor beta (TGFbeta) antagonists, TGFbeta receptor inhibitors, Platelet-derived growth factor (PDGF) antagonists, PDGF receptor inhibitors, chemokine antagonists, chemokine receptor inhibitors, cytokine antagonists, cytokine receptor inhibitors, N-acetylcysteine, prednisone, azathioprine, and tyrosine kinase inhibitors. Accordingly, in one embodiment, the further antifibrotic agent is an Endothelin antagonist, an Endothelin receptor inhibitor (e.g. Bosentan), a Transforming growth factor beta (TGFbeta) antagonist, a TGFbeta receptor inhibitor, a Platelet-derived growth factor (PDGF) antagonist, a PDGF receptor inhibitor, a chemokine antagonist, a chemokine receptor inhibitor, a cytokine antagonist, a cytokine receptor inhibitor, N- acetylcysteine, prednisone, azathioprine, or a tyrosine kinase inhibitor. Another class of antifibrotic agent that can be employed in combination with the proprotein convertase inhibitor in the treatment of a fibrotic disorder is a PDE6D inhibitor. Accordingly, in one embodiment, the further antifibrotic agent is a PDE6D inhibitor.
PDE6D inhibitors are well known in the art. However, particularly preferred classes of PDE6D inhibitor that may be employed in combination with the proprotein convertase inhibitors in the treatment of fibrotic disorders is described, for instance, in EP 2 698 367 Al; in Zimmermann, G. et al. "Small molecule inhibition of the KRAS-PDE5 interaction impairs oncogenic KRAS signalling." Nature 497, 638-642 (2013); and in Zimmermann, G. et al. "Structure Guided Design and Kinetic Analysis of Highly Potent Benzimidazole Inhibitors Targeting the PDE5 Prenyl Binding Site." J. Med. Chem. 57, 5435-5448 (2014).
Such PDE6D inhibitor compounds, and their utility in the treatment of fibrotic disorders, are disclosed in the priority application, UK patent application no. 1602641.1, which was filed on 15 February 2016. The contents of UK patent application no.
1602641.1 are incorporated herein by reference in their entirety.
Accordingly, in one embodiment, the further antifibrotic agent is a PDE6D inhibitor which is a compound of formula (X)
Figure imgf000066_0001
wherein
R1 is -R4, -CH2R4, -CH2OR4, -CH(OH)R4, -CH=CH-C(0)R4 or -C2H4-NHC(0)R4; R2 is -C(CH3)=CH2, -CH2-CH(CH3)2, -C≡CH, -C(0)NH-Rn,
Figure imgf000066_0002
Figure imgf000066_0003
-C(0)N(Ru)C(0)OC(CH3)3, -CH2OH, -CH2 H2, -CH2CH=CH2, -CH=CH2 or
-C(0)OCH3;
R3 is -H, -CH3,
Figure imgf000067_0001
-C2H5, -CH2OH, -C(0)OCH3, -cyclo-CeHn or -Ph, or R3 together with R5 is the residue - H-;
R4 is -CH3, -C2H5, -C3H7, -CH(CH3)2, -C4H9, -CH2-CH(CH3)2,
-CH(CH3)-C2H5, -C(CH3)3, -CsHn, -CH(CH3)-C3H7, -CH2-CH(CH3)-C2H5,
-CH(CH3)-CH(CH3)2, -C(CH3)2-C2H5, -CH2-C(CH3)3, -CH(C2H5)2, -C2H4-CH(CH3)2, -C6Hi3, -C3H6-CH(CH3)2, -C2H4-CH(CH3)-C2H5, -CH(CH3)-C4H9,
-CH2-CH(CH3)-C3H7, -CH(CH3)-CH2-CH(CH3)2 -CH(CH3)-CH(CH3)-C2H5, -CH2-CH(CH3)-CH(CH3)2, -CH2-C(CH3)2-C2H5, -C(CH3)2-C3H7,
-C(CH3)2-CH(CH3)2, -C2H4-C(CH3)3, -CH2-CH(C2H5)2, -CH(CH3)-C(CH3)3,
-C(C2H5)3, -CH2-C(C2H5)3, -C3H6-CH(CH3 R5,
Figure imgf000067_0002
R5, R5' and R5" are independently selected from -H, -OCH3, -OC2H5,
-C(0) HR6, -OR6, -N(CH3)2, -N(C2H5)2, -C(0)R7, -CH2-CH=CH2, -OH, - H2, -F, -CI, -Br, -I, -CH3, -C2H5, -COOH and -C(0)OR6;
R6 is -CH2-R7, -C2H4-R7, -C3H6-R7,
Figure imgf000068_0001
or
R is -H, -CH3, -CH=CH2, -CH2COOH, -CH2-CH=CH2, -NHR , -N(CH3)R8, -N(C2H5)R8, -CH2CO HC3H6(OC H4)3CH2 HR8,
Figure imgf000068_0002
R8 and R8' are independently selected fr m -H, -C02C(CH3)3,
Figure imgf000068_0003
and
R9 and R10 are independently selected from -H, -F, -Br, -CI, -I, -CN, -OH, -OCH3, -OC2H5, -CH3 and -C2H5;
R11 is -CH2-R12 or -C2H4-R12; and
R12 is -Ph or -OH;
or a pharmaceutically acceptable salt thereof.
The compound of formula (X) may be selected from 2-(4-(Allyloxy)phenyl)-l- benzyl- lH-benzo[d]imidazole, 2-(4-(Allyloxy)phenyl)-l-(pyridin-2-ylmethyl)-lH- benzo[d]imidazole, 2-(4-(Allyloxy)phenyl)- 1 -(2-methylbenzyl)- lH-benzo[d]imidazole, 2- (4-(Allyloxy)phenyl)-l-(3-methylbenzyl)-lH-benzo[d]imidazole, 2-(4-(Allyloxy)phenyl)-
1- (4-fluorobenzyl)-lH-benzo[d]imidazole, 4-(l -Benzyl- lH-benzo[d]imidazol-2-yl)phenol, 4-(l-(Pyridin-2-ylmethyl)-lH-benzo[d]imidazol-2-yl)phenol, 4-(l-(4-Fluorobenzyl)-lH- benzo[d]imidazol-2-yl)phenol, 4-(l-(2-Methylbenzyl)-lH-benzo[d]imidazol-2-yl)phenol, 4-(l-(3-Methylbenzyl)-lH-benzo[d]imidazol-2-yl)phenol, N-Benzyl-2-cyclohexyl-2-(2- phenyl- lH-benzo[d]imidazol- 1 -yl)acetamide, N-Benzyl-2-(2-phenyl- 1H- benzo[d]imidazol-l-yl)-2-(piperidin-4-yl)acetamide, 2-Cyclohexyl-2-(2-phenyl-lH- benzo[d]imidazol- 1 -yl)ethanol, Methyl 2-(2-phenyl- lH-benzo[d]imidazol- 1 -yl)acetate, Methyl 2-(2-phenyl- lH-benzo[d]imidazol- 1 -yl)pent-4-enoate, 2-(2-Phenyl- 1H- benzo[d]imidazol- 1 -yl)pent-4-en- 1 -ol, 2-(2 -Phenyl- lH-benzo[d]imidazol- 1 -yl)ethanol, 2- Phenyl- 1 -( 1 -(piperidin-4-yl)-2-(4-( 1 -(pyridin-2-ylmethyl)- lH-benzo[d]imidazol-2- yl)phenoxy)ethyl)-lH-benzo[d]imidazole, l-(3-Methylbenzyl)-2-(4-(2-(2-phenyl-lH- benzo[d]imidazol- 1 -yl)-2-(piperidin-4-yl)ethoxy)phenyl)- lHbenzo[d]imidazole, 1 -(4- Fluorobenzyl)-2-(4-(2-(2-phenyl-lH-benzo[d]imidazol-l-yl)-2-(piperidin-4- yl)ethoxy)phenyl)-lHbenzo[d]imidazole, l-(2-Methylbenzyl)-2-(4-(2-(2-phenyl-lH- benzo[d]imidazol- 1 -yl)-2-(piperidin-4-yl)ethoxy)phenyl)- lHbenzo[d]imidazole, Ethyl 1 - (l-benzyl-lH-benzo[d]imidazol-2-yl)piperidine-4-carboxylate, Ethyl l-(l-(2-chloro-6- fluorobenzyl)-lH-benzo[d]imidazol-2-yl)piperidine-4-carboxylate, Ethyl 1-(1 -(thiophen-3 - ylmethyl)-lH-benzo[d]imidazol-2-yl)piperidine-4-carboxylate, 1-(1 -(Thiophen-3 - ylmethyl)- lH-benzo[d]imidazol-2-yl)piperidine-4-carboxylic acid, 1 -( 1 -Benzyl- 1H- benzo[d]imidazol-2-yl)piperidine-4-carboxylic acid, l-(l-(2-Chloro-6-fluorobenzyl)-lH- benzo[d]imidazol-2-yl)piperidine-4-carboxylic acid, 2-Cyclohexyl-2-(2-phenyl-lH- benzo[d]imidazol- 1 -yl)ethyl 1 -( 1 -benzyl- lH-benzo[d]imidazol-2-yl)piperidine-4- carboxylate, 2-Cyclohexyl-2-(2-phenyl- lH-benzo[d]imidazol- 1 -yl)ethyl 1 -( 1 -(2-chloro-6- fluorobenzyl)-lH-benzo[d]imidazol-2-yl)piperidine-4-carboxylate, 2-Cyclohexyl-2-(2- phenyl- lH-benzo[d]imidazol- 1 -yl)ethyl 1 -( 1 -(thiophen-3 -ylmethyl)- lH-benzo[d]imidazol-
2- yl)piperidine-4-carboxylate, 2-(2-Phenyl-lH-benzo[d]imidazol-l-yl)-2-(piperidin-4- yl)ethyl l-(l-(2-chloro-6-fluorobenzyl)-lH-benzo[d]imidazol-2-yl)piperidine-4- carboxylate, 2-(2 -Phenyl- lH-benzo[d]imidazol- 1 -yl)-2-(piperidin-4-yl)ethyl 4-( 1 -benzyl- lH-benzo[d]imidazol-2-yl)piperazine-l-carboxylate, (,S)-tert-Butyl 4-(l-(2-(3- cyanophenyl)- lH-benzo[d]imidazol- 1 -yl)-2-( 1 -( 1 -(thiophen-3 -ylmethyl)- lHbenzo[d]imidazol-2-yl)piperidine-4-carbonyloxy)ethyl)piperidine-l-carboxylate, 1- Benzyl-2-phenyl-lH-benzo[d]imidazole, 6-Pyiridyl-5,6-dihydrobenzo[4-5]imidazo[l,2- c]quinazoline, 1 -Benzyl-2-(4-(2-(2-phenyl- lH-benzo[d]imidazol- 1 -yl)pent-4- enyloxy)phenyl)-lH-benzo[d]imidazole, l-Benzyl-2-(4-(2-cyclohexyl-2-(2-phenyl-lH- benzo[d]imidazol- 1 -yl)ethoxy)phenyl)- lH-benzo[d]imidazole, 4-(4-( 1 -Benzyl- 1H- benzo[d]imidazol-2-yl)phenoxy)-3 -(2-phenyl- lH-benzo[d]imidazol- 1 -yl)butanoic acid, 1 - Benzyl-2-(4-(2-(2-phenyl- 1 H-benzo[d]imidazol- 1 -yl)ethoxy)phenyl)- 1H- benzo[d]imidazole, N-(18-(4-(l-Benzyl-lH-benzo[d]imidazol-2-yl)phenoxy)-15-oxo-17- (2-phenyl-lH-benzo[d]imidazol-l-yl)-4,7, 10-trioxa-14-azaoctadecyl)-5-((3aS,4S,6aR)-2- oxohexahydro-lH-thieno[3,4-d]imidazol-4-yl)pentanamide, tert-Butyl-4-(2-(4-(l-benzyl- lH-benzo[d]imidazol-2-yl)phenoxy)- 1 -(2-phenyl- lH-benzo[d]imidazol- 1 - yl)ethyl)piperidine- 1 -carboxylate, (^-tert-Butyl 4-(2-(4-( 1 -benzyl- lH-benzo[d]imidazol-2- yl)phenoxy)- 1 -(2-phenyl- lH-benzo[d]imidazol- 1 -yl)ethyl)piperidine- 1 -carboxylate, 1 - Benzyl-2-(4-(2-(2-phenyl-lH-benzo[d]imidazol-l-yl)-2-(piperidin-4-yl)ethoxy)phenyl)- lH-benzo[d]imidazole, (S)- 1 -Benzyl-2-(4-(2-(2-phenyl- lH-benzo[d]imidazol- 1 -yl)-2- (piperidin-4-yl)ethoxy)phenyl)-lH-benzo[d]imidazole (deltarasin), (R)-l-Benzyl-2-(4-(2- (2-phenyl- lH-benzo[d]imidazol- 1 -yl)-2-(piperidin-4-yl)ethoxy)phenyl)- 1H- benzo[d]imidazole, N-(2-(2 -Phenyl- lH-benzo[d]imidazol- 1 -yl)-2-(piperidin-4-yl)ethyl)- 1 - (l-(thiophen-3-ylmethyl)-lHbenzo[d]imidazol-2-yl)piperidine-4-carboxamide, 2-(2- Phenyl- lH-benzo[d]imidazol- 1 -yl)-2-(piperidin-4-yl)ethyl 1 -( 1 -benzyl- 1H- benzo[d]imidazol-2-yl)piperidine-4-carboxylate, (S)-2-(2-Phenyl-lH-benzo[d]imidazol-l- yl)-2-(piperidin-4-yl)ethyl l-(l-(thiophen-3-ylmethyl)-lHbenzo[d]imidazol-2- yl)piperidine-4-carboxylate, and (R)-2-(2 -Phenyl- lH-benzo[d]imidazol-l-yl)-2-(piperidin- 4-yl)ethyl l-(l-(thiophen-3-ylmethyl)-lHbenzo[d]imidazol-2-yl)piperidine-4-carboxylate.
The further antifibrotic agent may for instance be a PDE6D inhibitor of the following formula (Xa) which is deltarasin:
Figure imgf000070_0001
(Xa)
or a pharmaceutically acceptable salt thereof. Alternatively, the further antifibrotic agent may be a PDE6D inhibitor which is a compound of formula (II):
Figure imgf000071_0001
wherein
R is OH, OR20, H2, NHR20 or R21R22;
R20 is unsubstituted or subsitituted C1-20 alkyl, unsubstituted or substituted aryl, unsubstituted or substituted C3-20 heteroaryl, unsubstituted or substituted C3-25 cycloalkyl, unsubstituted or substituted C3-2o heterocyclyl, or -PEG-Biotin, wherein said Ci-20 alkyl is optionally interrupted by one or more N(R'), O, S or arylene groups wherein R' is H, Ci-6 alkyl or aryl; and
R21 and R22 are independently selected from unsubstituted or substituted Ci-20 alkyl, unsubstituted or substituted aryl, unsubstituted or substituted C3-20 heteroaryl, unsubstituted or substituted C3-25 cycloalkyl, unsubstituted or substituted C3-2o heterocyclyl, and -PEG-Biotin, wherein said Ci-20 alkyl is optionally interrupted by one or more N(R'), O, S or arylene groups;
provided that R21 and R22, may together form an unsubstituted or substituted Ci-20 alkylene group, wherein said Ci-20 alkylene is optionally interrupted by N(R'), O or S wherein R' is H, Ci-6 alkyl or aryl;
or a pharmaceutically acceptable salt thereof.
Usually, in the compound of formula (II), R is OH, NHR20 or R21R22; R20 is -CH2-
CH2-N(CH3)2, -CH2-(CH2-CH2-0)3-CH3 or -PEG-Biotin; and R21 and R22 together form
Figure imgf000071_0002
As a further alternative, the further antifibrotic agent may be a PDE6D inhibitor which is a compound of formula (III)
Figure imgf000071_0003
wherein
R1 is H, unsubstituted or substituted aryl, unsubstituted or substituted C3-10 cycloalkyl, unsubstituted or substituted C3-7 heterocyclyl, unsubstituted or substituted C3-10 heteroaryl, and unsubstituted or substituted Ci-10 alkyl; and
R2 is H or unsubstituted or substituted Ci-10 alkyl;
or a pharmaceutically acceptable salt thereof.
Usually, in the compound of formula (III), R1 is H, unsubstituted or substituted phenyl, or unsubstituted or substituted C3-10 cycloalkyl; and R2 is H or unsubstituted or substituted Ci-6 alkyl. More typically, R2 is zso-propyl and R1 is phenyl, cyclopropyl or cyclopentyl.
In one embodiment, the proprotein convertase inhibitor is a compound of formula
(la):
Figure imgf000072_0001
or a pharmaceutically acceptable salt thereof; and
the further antifibrotic compound is a PDE6D inhibitor of the following formula:
Figure imgf000072_0002
or a pharmaceutically acceptable salt thereof.
In another embodiment, the proprotein convertase inhibitor comprises an
oligonucleotide which specifically hybridises to a part of SEQ ID NO: 5 (human PCSK 3 mRNA) or any isoform thereof; and
the further antifibrotic compound is a PDE6D inhibitor of the following formula:
Figure imgf000073_0001
or a pharmaceutically acceptable salt thereof.
In yet another embodiment, the proprotein convertase inhibitor comprises an oligonucleotide which comprises 50 or fewer consecutive nucleotides from (a) SEQ ID NO: 6 (human PCSK 3 cDNA) or any isoform thereof or (b) a variant sequence which has at least 95% homology to SEQ ID NO: 6 or any isoform thereof based on nucleotide identity over the entire sequence; and
the further antifibrotic compound is a PDE6D inhibitor of the following formula:
Figure imgf000073_0002
or a pharmaceutically acceptable salt thereof.
In yet another embodiment, the proprotein convertase inhibitor comprises an oligonucleotide which is (a) any one of SEQ ID NOs: 19, 20, 21 and 22, or (b) a variant sequence which has at least 95% homology to any one of SEQ ID NOs: 19, 20, 21 and 22 or any isoform thereof based on nucleotide identity over the entire sequence; and
the further antifibrotic compound is a PDE6D inhibitor of the following formula:
Figure imgf000073_0003
or a pharmaceutically acceptable salt thereof.
For instance, the proprotein convertase inhibitor may comprise an oligonucleotide which is any one of SEQ ID NOs: 19, 20, 21 and 22; and
the further antifibrotic compound is a PDE6D inhibitor of the following formula:
Figure imgf000074_0001
or a pharmaceutically acceptable salt thereof.
The following disclosure regarding the administration and dosage of the proprotein convertase inhibitor applies equally to the further antifibrotic compound when a further antifibrotic compound is employed.
A proprotein convertase inhibitor, for use in accordance with the present invention, can be administered in a variety of dosage forms, for example orally such as in the form of tablets, capsules, sugar- or film-coated tablets, liquid solutions or suspensions or parenterally, for example intramuscularly, intravenously or subcutaneously. The compound may therefore be given by injection or infusion.
The proprotein convertase inhibitor may be presented for administration in a liposome. Thus, the compound may be encapsulated or entrapped in the liposome and then administered to the patient to be treated. Active ingredients encapsulated by liposomes may reduce toxicity, increase efficacy, or both. Notably, liposomes are thought to interact with cells by stable absorption, endocytosis, lipid transfer, and fusion (R.B. Egerdie et al., 1989, J. Urol. 142:390).
The dosage depends on a variety of factors including the age, weight and condition of the patient and the route of administration. Daily dosages can vary within wide limits and will be adjusted to the individual requirements in each particular case. Typically, however, the dosage adopted for each route of administration when a compound is administered alone to adult humans is 0.0001 to 50 mg/kg, most commonly in the range of 0.001 to 10 mg/kg, body weight, for instance 0.01 to 1 mg/kg. Such a dosage may be given, for example, from 1 to 5 times daily. For intravenous injection a suitable daily dose is from 0.0001 to 1 mg/kg body weight, preferably from 0.0001 to 0.1 mg/kg body weight. A daily dosage can be administered as a single dosage or according to a divided dose schedule.
Typically a dose to treat human patients may range from about 0.1 mg to about 1000 mg of a compound for use in accordance with the invention, more typically from about 10 mg to about 1000 mg of a compound for use in accordance with the invention. A typical dose may be about 100 mg to about 300 mg of the compound. A dose may be administered once a day (QID), twice per day (BID), or more frequently, depending on the pharmacokinetic and pharmacodynamic properties, including absorption, distribution, metabolism, and excretion of the particular compound. In addition, toxicity factors may influence the dosage and administration regimen. When administered orally, the pill, capsule, or tablet may be ingested daily or less frequently for a specified period of time. The regimen may be repeated for a number of cycles of therapy.
A proprotein convertase inhibitor is formulated for use as a pharmaceutical composition also comprising a pharmaceutically acceptable carrier or diluent. The compositions are typically prepared following conventional methods and are administered in a pharmaceutically suitable form. The proprotein convertase inhibitor compound may be administered in any conventional form, for instance as follows:
A) Orally, for example, as tablets, coated tablets, dragees, troches, lozenges, aqueous or oily suspensions, liquid solutions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs. Compositions intended for oral use may be prepared according to any method known in the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavouring agents, colouring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations.
Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. These excipients may be for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, dextrose, saccharose, cellulose, corn starch, potato starch, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, maize starch, alginic acid, alginates or sodium starch glycolate; binding agents, for example starch, gelatin or acacia; lubricating agents, for example silica, magnesium or calcium stearate, stearic acid or talc; effervescing mixtures; dyestuffs, sweeteners, wetting agents such as lecithin, polysorbates or lauryl sulphate. The tablets may be uncoated or they may be coated by known techniques to delay disintegration and adsorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed. Such preparations may be manufactured in a known manner, for example by means of mixing, granulating, tableting, sugar coating or film coating processes.
Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is present as such, or mixed with water or an oil medium, for example, peanut oil, liquid paraffin, or olive oil.
Aqueous suspensions contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example, sodium carboxymethylcellulose, methylcellulose,
hydroxypropylmethyl-cellulose, sodium alginate, polyvinylpyrrolidone gum tragacanth and gum acacia; dispersing or wetting agents may be naturally-occurring phosphatides, for example lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides for example polyoxyethylene sorbitan monooleate.
The said aqueous suspensions may also contain one or more preservatives, for example, ethyl or n-propyl p-hydroxybenzoate, one or more colouring agents, such as sucrose or saccharin.
Oily suspension may be formulated by suspending the active ingredient in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol.
Sweetening agents, such as those set forth above, and flavouring agents may be added to provide a palatable oral preparation. These compositions may be preserved by this addition of an antioxidant such as ascorbic acid. Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, a suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, for example sweetening, flavouring and colouring agents, may also be present.
Pharmaceutical compositions for use in accordance with the invention may also be in the form of oil-in-water emulsions. The oily phase may be a vegetable oil, for example olive oil or arachis oils, or a mineral oil, for example liquid paraffin or mixtures of these. Suitable emulsifying agents may be naturally-occurring gums, for example gum acacia or gum tragacanth, naturally occuring phosphatides, for example soy bean lecithin, and esters or partial esters derived from fatty acids an hexitol anhydrides, for example sorbitan mono- oleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxy ethylene sorbitan monooleate. The emulsion may also contain sweetening and flavouring agents. Syrups and elixirs may be formulated with sweetening agents, for example glycerol, sorbitol or sucrose. In particular a syrup for diabetic patients can contain as carriers only products, for example sorbitol, which do not metabolise to glucose or which only metabolise a very small amount to glucose.
Such formulations may also contain a demulcent, a preservative and flavouring and coloring agents;
B) Parenterally, either subcutaneously, or intravenously, or intramuscularly, or intrasternally, or by infusion techniques, in the form of sterile injectable aqueous or oleaginous suspensions. This suspension may be formulated according to the known art using those suitable dispersing of wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic paternally-acceptable diluent or solvent, for example as a solution in 1,3 -butane diol.
Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition fatty acids such as oleic acid find use in the preparation of injectables;
C) By inhalation, in the form of aerosols or solutions for nebulizers; D) Rectally, in the form of suppositories prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperature but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Such materials are cocoa butter and poly-ethylene glycols; E) Topically, in the form of creams, ointments, jellies, collyriums, solutions or suspensions.
F) Vaginally, in the form of pessaries, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the active ingredient such carriers as are known in the art to be appropriate.
The invention also provides an in vitro method of inhibiting the formation of collagen matrix, the method comprising contacting an in vitro sample comprising collagen- secreting cells with an amount of a proprotein convertase inhibitor effective to inhibit the formation of collagen matrix by the cells. The proprotein convertase inhibitor is usually as further defined herein. Thus, it is usually a compound of formula (I), or of formula (la), as defined herein, or a prodrug or pharmaceutically acceptable salt thereof.
It is a finding of the invention that proprotein convertase inhibitors can reduce procollagen secretion in vitro. Thus, typically, inhibiting the formation of collagen matrix involves inhibiting the secretion of procollagen, and thus the in vitro method typically comprises contacting said sample with an amount of the proprotein convertase inhibitor effective to inhibit the secretion of procollagen by the cells.
It is a further finding of the invention that, advantageously, the proprotein convertase inhibitor does not significantly affect fibronectin secretion.
Thus, in another aspect the invention provides an in vitro method of inhibiting the formation of collagen matrix without inhibiting fibronectin secretion, the method comprising contacting an in vitro sample comprising collagen-secreting cells with an amount of a proprotein convertase inhibitor effective to inhibit the formation of collagen matrix by the cells without inhibiting fibronectin secretion.
Usually, in this aspect of the invention, the in vitro method comprises contacting said sample with an amount of a proprotein convertase inhibitor effective to inhibit the formation of collagen matrix by the cells without inhibiting the secretion by the cells of fibronectin. Typically, for example, it comprises contacting said sample with an amount of a proprotein convertase inhibitor effective to inhibit the formation of collagen matrix by the cells without altering the secretion by the cells of fibronectin.
Again, inhibiting the formation of collagen matrix typically involves inhibiting the secretion of procollagen, and the in vitro method of this aspect of the invention may therefore comprise contacting said sample with an amount of the proprotein convertase inhibitor that is effective to inhibit secretion by the cells of procollagen without inhibiting fibronectin secretion. The in vitro method may for instance comprise contacting said sample with an amount of a proprotein convertase inhibitor which amount is effective to inhibit the secretion by the cells of procollagen without altering the secretion by the cells of fibronectin.
The term "collagen-secreting cells", as used herein, refers to cells that are capable of secreting collagen. Such cells are well known to the skilled person. The collagen- secreting cells employed in the in vitro method of the invention typically comprise fibroblasts.
The cells employed in the in vitro method may be any kind of collagen-secreting cell; many are known to the skilled person, for instance: dermal fibroblasts, lung fibroblasts, liver fibroblasts, renal fibroblasts, myofibroblasts and cardiac fibroblasts. The collagen-secreting cells are preferably human.
In one embodiment, dermal fibroblasts are employed.
An amount of a proprotein convertase inhibitor which is effective to inhibit the formation of collagen matrix by the cells may be easily determined by the skilled person.
Often, however, the amount of proprotein convertase inhibitor which is effective to inhibit the formation of collagen matrix by the cells is in the micromolar range, for instance from 0.01 μΜ to 500 μΜ, or for example from 0.1 to 50 μΜ, for instance from 0.5 to 25 μΜ.
The invention also provides a novel assay method for identifying molecules that affect collagen secretion in vitro, and thus for identifying antifibrotic agents.
Accordingly, the invention provides a method of identifying an agent as being capable of inhibiting collagen matrix formation, which method comprises (a) contacting an agent with an in vitro sample comprising collagen- secreting cells in the absence of exogenous transforming growth factor (TGF) and (b) measuring the amount of collagen matrix formed by the cells and thereby determining whether or not the agent is capable of inhibiting collagen matrix formation.
The agent used in the method of the invention may be any of the types discussed above. The agent may be a compound or an oligonucleotide as defined above. The agent is contacted with an in vitro sample comprising collagen-secreting cells. Such samples and cells are defined above. The cells are preferably human cells.
The cells are preferably fibroblasts. The fibroblasts may be any of those discussed above. The cells are preferably normal human dermal fibroblast ( HDF). The cells are preferably not WI-38 human lung fibroblasts. NHDF cells provide better morphological contrast for quantitative imaging as compared to WI-38 lung fibroblasts. The agent is contacted with the in vitro sample comprising collagen-secreting cells in the absence of exogenous TGF. Exogenous TGF is TGF that is added to the in vitro sample, such as to the culture medium, comprising the cells. In other words, exogenous TGF is TGF that is not produced by the cells themselves. The culture medium added to the cells does not contain exogenous TGF.
The TGF is preferably human TGF. The TGF is preferably TGFp, such as TGFpi, TGFP2 or TGFP3. The agent is preferably contacted with the in vitro sample comprising collagen-secreting cells in the absence of human TGFpi .
The contacting may be carried out in any suitable volume. Typical volumes range from about ΙΟμΙ to about 1ml, preferably from about 50μ1 to about 500μ1, more preferably from about ΙΟΟμΙ to about 200μ1. Typically, the length of time for which the cells are contacted with the agent is from about 1 day to about 5 days, such as about 2 days, about 3 days or about 4 days.
The cells may be contacted with the agent at any suitable temperature. The suitable temperature is typically in the same range as the normal body temperature of the human or animal from which the cells are derived. Typically, the incubation is carried out at a fixed temperature between about 4°C and about 38°C, preferably at about 37°C.
Techniques for culturing cells are well known to a person skilled in the art. The cells are typically cultured under standard conditions of 37°C, 5% CO2 in medium supplemented with serum. The cells, such as fibroblasts, are preferably conditioned before they are contacted with the agent. Fibroblasts are preferably conditioned with an optimal conditioned medium (Fibroblast Growth Medium, Lonza CC-3131) supplemented with insulin and fibroblast growth factor (FGF) before they are contacted with the agent. The optimal conditioned medium preferably comprises 2% fetal bovine serum (FBS). The cells, such as fibroblasts, may be conditioned for at least about one day.
The cells may be contacted with the agent in any culture medium. The culture medium preferably comprises Ficoll PM-400. For instance, the culture medium preferably comprises Ficoll PM-400 and ascorbic acid. The cells, such as fibroblasts, are preferably contacted with the agent in Fibroblast Growth Medium (Lonza CC-3131) supplemented with insulin, FGF, 0.5% FBS, ascorbic acid and Ficoll PM400.
The method may be carried out using any number of agents. For instance, the method may be carried out using 1, 2, 5, 10, 15, 20, 30, 40, 50 , 100, 150, 200, 300, 500 or more agents. The method is preferably carried out using 6, 12, 24, 48, 96 or 384 or 1526 agents. This allows high-throughput screening of agents. The method may be carried out using any number of in vitro samples. For instance, the method may be carried out using 1, 2, 5, 10, 15, 20, 30, 40, 50 , 100, 150, 200, 300, 500 or more samples. The method is preferably carried out using 6, 12, 24, 48, 96 or 384 or 1526 samples. This allows high-throughput screening of agents.
The number of samples and agents may be the same. For instance, each sample may be contacted with a different agent. Alternatively, the number of samples and agents may be different. For instance, each agent may be contacted with two or more samples.
The cells are preferably captured or immobilized on a surface. Any method of immobilizing or capturing the cells can be used. The cells may be immobilized or captured on the surface using Fc receptors, capture antibodies, avidimbiotin, lectins, polymers or any other capture chemicals. Fibroblasts may naturally adhere to the surface.
The samples are typically present in wells. The samples are preferably present in the wells of a flat plate. The samples are more preferably present in the wells of a standard 96 or 384 well plate. Such plates are commercially available Fisher scientific, VWR suppliers, Nunc, Starstedt or Falcon. The cells are preferably immobilized or captured on a surface of one or more, preferably all, of the wells. Any method of immobilizing or capturing the cells can be used. The cells may be immobilized or captured on a surface of the well(s) by coating the surface with Fc receptors, capture antibodies, avidimbiotin, lectins, polymers or any other capture chemicals.
The wells typically have a capacity of from about 25 μΐ to about 250μ1, from about
30μ1 to about 200μ1, from about 40μ1 to about 150μ1 or from about 50 to ΙΟΟμΙ.
Collagen matrix formation may be measured using standard techniques, such as those disclosed in the Examples. Collagen matrix formation is preferably measured by immunofluorescence. It may for instance measured using dyes, e.g. Sirius dye picrosirius red or Sirius Red F3BA. The Sircol collagen assay may be employed, or for instance, enzyme linked immunoassays, gas chromatography, mass spectrometry, polyacrylamide gel electrophoresis following stable metabolic labelling with radiolabelled glycine and proline, or followed by silver staining, or by western blotting with an antibody, among other methods; see, for instance: Chen, C. Z. and M. Raghunath (2009); Fibrogenesis Tissue Repair 2(1): 7.
The agent is capable of inhibiting collagen matrix formation if the cells produce less collagen matrix {i.e. a decreased amount of collagen matrix) in the presence of the agent than in the absence of the agent. The method preferably comprises comparing the amount of collagen matrix produced by the cells in the presence of the agent with the amount of collagen produced by the cells in the absence of the agent. In one embodiment, the amount of collagen matrix produced in the absence of the agent (i.e. the control value) is obtained separately from the method of the invention. For instance, the control value may be obtained beforehand and recorded, for instance on a computer. The control value is typically used for comparison purposes in method of the invention. The control value may be used for multiple repetitions of the method of the invention. The control value is preferably obtained under the same conditions, such as cell number, cell type and culture conditions, under which the method of the invention is carried out.
In another embodiment, the amount of collagen matrix produced in the absence of the agent (i.e. the control value) is obtained at the same time as carrying out the method of the invention (i.e. using one or more control samples of cells which are not contacted with the agent). This is straightforward to do if the test samples and control sample(s) are present in the wells of a standard 96 or 384 well plate. The test and control samples are then assayed using the same conditions.
The agent may decrease or inhibit the collagen matrix formation by any amount, such as by at least 5%, at least 10%, at least 20%, at least 30%>, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90%. The agent may completely inhibit collagen matrix formation.
The agent is not capable of inhibiting collagen matrix formation if the cells produce the same amount of collagen matrix in the presence of the agent as in the absence of the agent. The agent is not capable of inhibiting collagen matrix formation if the cells produce more collagen matrix (i.e. an increased amount of collagen matrix) in the presence of the agent than in the absence of the agent.
The present invention is further illustrated in the Examples which follow:
EXAMPLES
Proprotein convertases (PCSKs) cleave target peptides in the secretory pathway, at the cell surface and in the extra cellular matrix (Seidah, N. G. & Prat, A. The biology and therapeutic targeting of the proprotein convertases. 1-17 (2012). doi: 10.1038/nrd3699). The inventors discovered using bioinformatics that there is a substantial intersect between targets that are cleaved by PCSKs and candidate mediators of fibrosis. These include: ADAM 12, EDN1, ITGAV, NOTCH, OSM, PDGFA & PDGFB. The inventors also bioinformatically determined that PCSKs are responsible for the cleavage and activation of proteins that biochemically play a role in collagen secretion. These include BMP1/TLL1 enzymes, which are essential for the maturation of procollagen to collagen. This led the inventors to hypothesise that PCSK inhibition may be effective in inhibiting collagen matrix formation.
US2011/0059896 indicates that the commercially-available proprotein convertase inhibitors decanoyl-Arg-Val-Lys-Arg-CMK (Cayman Chemical Item Number 14965; CAS 150113-99-8) and hexa-D-arginine (Calbiochem® catalogue number 344931; CAS 673202-67-0) would block TGF-β activation, and it was suggested on this basis that these two compounds would be effective in reducing scarring and fibrosis. However, no direct evidence of this was provided.
Commercially available chemical inhibitors of proprotein convertases are:
a) decanoyl-RVKR-CMK: an irreversible inhibitor (Garten, W. et al. Processing of viral glycoproteins by the subtili sin-like endoprotease furin and its inhibition by specific peptidylchloroalkylketones. Biochimie 76, 217-225, 1994), active against FURIN, PCSK1, PCSK2, PCSK5, PCSK6 and PCSK7 at Ki<2 nM (Jean, F. et al. alphal- Antitrypsin Portland, a bioengineered serpin highly selective for furin: application as an antipathogenic agent. Proc. Natl. Acad. Sci. U.S.A. 95, 7293-7298, 1998);
b) hexa-D-arginine amide (Ki=7.6 nM against FURIN; Cameron, A., Appel, J., Houghten, R. A. & Lindberg, I. Polyarginines Are Potent Furin Inhibitors. Journal of Biological Chemistry 275, 36741-36749; 2000; and Kacprzak, M. M. et al. Inhibition of Furin by Polyarginine-containing Peptides: Nanomolar Inhibition By Nona-D-Arginine. Journal of Biological Chemistry 279, 36788-36794, 2004);
c) Calbiochem 537076 (p-guanidinomethyl-phenylacetyl-Arg-Val-Arg-4- ami dinobenzyl amide; structure shown below) which has a high degree of cell permeability, and is highly active against FURIN, PCSK4, PCSK5, and PCSK6 at Ki < 17 pM (Becker, G. L. et al. Highly potent inhibitors of proprotein convertase furin as potential drugs for treatment of infectious diseases. Journal of Biological Chemistry 287, 21992-22003, 2012).
Figure imgf000084_0001
Calbiochem 537076
In experiments performed by the inventors, it was found:
a) that decanoyl-RVKR-CMK, and hexa-D-arginine amide showed little activity against collagen matrix formation. In contrast, proprotein convertase inhibitor Calbiochem 537076 showed potent activity in inhibiting collagen matrix secretion in vitro,
b) Calbiochem 537076 does not have significant effect on fibronectin secretion in vitro. c) Calbiochem 537076 reduces procollagen type I C-peptide secretion in vitro. This implies that Calbiochem 537076 affects secretion of procollagen.
Example 1: Modified scar-in-a-iar assay and evaluation of Calbiochem 537076 (and decanoyl-RVKR-CMK and hexa-D-arginine) Figure 6: A miniaturized "Scar-in-a-Jar" assay.
The "Scar-in-a-Jar" assay is a well-validated in vitro assay that uses WI-38 human embryonic lung fibroblasts, macromolecular crowding, and exogenous TGFB 1 to achieve rapid deposition of cross-linked collagen in vitro (Chen, C. et al. The Scar-in-a-Jar:
studying potential antifibrotic compounds from the epigenetic to extracellular level in a single well. British Journal of Pharmacology 158, 1196-1209 (2009); Chen, C.Z. & Raghunath, M. Focus on collagen: in vitro systems to study fibrogenesis and antifibrosis _ state of the art. Fibrogenesis Tissue Repair 2, 7 (2009); Lareu, R.R. et al. Collagen matrix deposition is dramatically enhanced in vitro when crowded with charged macromolecules: The biological relevance of the excluded volume effect. FEBS Letters 581, 2709-2714
(2007)). As human cardiac fibroblasts are difficult to obtain in sufficient numbers for high- throughput screening purposes, we elected to develop an assay of collagen deposition using juvenile normal human dermal fibroblasts (J HDF) as a plentiful supply at a consistent passage number can be purchased from PromoCell and validated batches reserved. As an in vitro model for cardiac fibrosis the use of human dermal fibroblasts is appropriate as they not only produce the 3 key cardiac profibrotic peptides (TGFB
(Mansbndge, J.N. et al. Growth factors secreted by fibroblasts: role in healing diabetic foot ulcers. Diabetes ObesMetab 1, 265-79 (1999); Jutley, J.K., Wood, E.J. & Cunliffe, W.J. Influence of retinoic acid and TGF-beta on dermal fibroblast proliferation and collagen production in monolayer cultures and dermal equivalents. Matrix 13, 235-41 (1993)), PDGF (Ivarsson, M., McWhirter, A., Borg, T.K. & Rubin, K. Type I collagen synthesis in cultured human fibroblasts: regulation by cell spreading, platelet-derived growth factor and interactions with collagen fibers. Matrix Biol. 16, 409-425 (1998); Mansbridge, J.N. et al. Growth factors secreted by fibroblasts: role in healing diabetic foot ulcers. Diabetes Obes Metab 1, 265-79 (1999)) and EDN1 (Shi- Wen, X. et al. Fibroblast matrix gene expression and connective tissue remodeling: role of endothelin-1. J Invest Dermatol 116, 417-25 (2001); Kawaguchi, Y. et al. Increased endothelin-1 production in fibroblasts derived from patients with systemic sclerosis. Ann Rheum Dis 53, 506-10 (1994))) endogenously, but also they respond by increasing type I collagen secretion.
The published in vitro collagen matrix formation assay (Chen, C. Z. & Raghunath, M. Focus on collagen: in vitro systems to study fibrogenesis and antifibrosis - state of the art. Fibrogenesis Tissue Repair 2, 7, 2009) was modified and miniaturised as follows:
The normal human dermal fibroblast (NUDF) was used for the assay, instead of the WI-38 human lung fibroblasts that were used in the paper by Chen, C. Z. et al. It was found that NUDF cells provide better morphological contrast for quantitative imaging as compared to what is shown in the paper.
Due to the difference in cell type (cell size), the macromolecular crowding conditions were re-optimized accordingly. In Chen, C. Z. et al, either a mixture of neutral 70 and 400 Ficoll™, or dextran sulphate, was used for
macromolecular crowding, whereas it was found here that NUDF cells work best when cultured in 37.5mg/ml Ficoll PM-400 alone.
iii) The addition of active TGFb 1 in the assay was omitted to ensure that any
involvement of TGFb 1 in the process was entirely endogenous.
iv) Chen et al. seeded their fibroblasts on 24-well plates at 50,000 cells per well in
10% FBS. The present inventors miniaturized the assay into 96-well format so that it is compatible with high-throughput screening with various established compound libraries. The miniaturization also allows comprehensive and systematic quantification of collagen signals using the Operetta for high- throughput imaging. Z-factors of 0.5 to 1.0 have been obtained, which is considered to be an indication for an excellent assay.
v) An optimal conditioned medium (Fibroblast Growth Medium, Lonza CC-
3131), supplemented with insulin and FGF, was used for culturing FDF cells.
Thus, in the modified version of the published "Scar-in-a-Jar" assay (Chen, C. et al. The Scar-in-a-Jar: studying potential antifibrotic compounds from the epigenetic to
extracellular level in a single well. British Journal of Pharmacology 158, 1196-1209 (2009)) (Figure 6, 1), we a) used j HDFs; b) omitted the addition of exogenous TGFB1 (used in the original assay) to make the assay dependent on endogenous TGFB; and c) miniaturized the assay to 96 well plates making it more cost effective for large-scale screening. We obtained a z'factor=0.77, indicating that the assay is robust for high- throughput screening (see Figure 2). j- HDF (C-12300, Promo) were seeded on 96-well plates at 6,000 cells per well in fibroblast basal medium (FGM, CC-3132, Lonza) containing 2% fetal bovine serum and cultured under 5% CO2 at 37°C. After 24h, the medium was changed to FGM containing 0.5% FBS, ΙΟΟμΜ L-ascorbic acid 2-phosphate (A8960, Sigma) with Ficoll 400 at 25mg/ml (F4375, Sigma). After 3 days of biophysical crowding, cells were fixed in ice-cold methanol for 10 mins, blocked in 3% BSA for 30 mins, and incubated in 1/500 mouse monoclonal anti-collagen type 1 (C2456, Sigma) and 1/500 rabbit polyclonal anti-fibronectin (F3648, Sigma) overnight. Subsequently, goat anti-mouse IgG-Alexa488 and goat anti-rabbit IgG-Alexa647 (A-11001 and A-21245, Life Technologies) were used at 1/400 for 1.5h. Signals of collagen type 1 (COL-1) and fibronectin were imaged on an Operetta System (Perkin Elmer) and quantified using the Harmony software (Perkin Elmer). In the cases of small molecules, the treatments were added at the same time as Ficoll 400. For RNAi silencing, fibroblasts were reverse transfected into 10,000 cells with Dharmacon SMARTpool ON-TARGET plus siRNA (GE Healthcare Dharmacon Inc.) using Lipofectamine RNAiMAX (Life Technologies) as per manufacturers' instructions. In these assays cell number and fibronectin secretion were used as markers of cellular health - i.e. excluding small molecules that for instance block the secretory pathway. We tested commercially available PCSK inhibitors using a miniaturized "Scar-in-a-Jar" assay (Figure 6) to determine their efficacy in suppressing reticular collagen production (Figure 1). PCSK inhibitors are typically peptidomimetics that mimic substrate structure. Commercially available inhibitors include dec-RVKR-CMK (Garten, W. et al. Processing of viral glycoproteins by the subtili sin-like endoprotease furin and its inhibition by specific peptidylchloroalkylketones. Biochimie 76, 217-225 (1994)); hexa-D-arginine amide (Cameron, A., Appel, J., Houghten, R.A. & Lindberg, I. Polyarginines Are Potent Furin Inhibitors. Journal of Biological Chemistry 275, 36741-36749 (2000); Kacprzak, M.M. et al. Inhibition of Furin by Polyarginine-containing Peptides: NANOMOLAR INHIBITION BY NONA-D- ARGININE. Journal of Biological Chemistry 279, 36788-36794 (2004)); and ^-guanidinomethyl-phenylacetyl-Arg-Val-Arg-4-amidinobenzylamide (Calbiochem 537076; Becker, G.L. et al. Highly potent inhibitors of proprotein convertase furin as potential drugs for treatment of infectious diseases. Journal of Biological Chemistry 287, 21992-22003 (2012)). dec-RVKR-CMK is an irreversible inhibitor (Garten, W. et al. Processing of viral glycoproteins by the subtili sin-like endoprotease furin and its inhibition by specific peptidylchloroalkylketones. Biochimie 76, 217-225 (1994)), active against FURIN, PCSK1, PCSK2, PCSK5, PCSK6 and PCSK7 at Ki<2 nM (Jean, F. et al. alphal- Antitrypsin Portland, a bioengineered serpin highly selective for furin: application as an antipathogenic agent. Proc. Natl. Acad. Sci. U.S.A. 95, 7293-7298 (1998)). Hexa-D- arginine amide is a competitive inhibitor, with a Ki=7.6 nM against FURIN (Cameron, A., Appel, J., Houghten, R.A. & Lindberg, I. Polyarginines Are Potent Furin Inhibitors.
Journal of Biological Chemistry 275, 36741-36749 (2000); Kacprzak, M.M. et al.
Inhibition of Furin by Polyarginine-containing Peptides: NANOMOLAR INHIBITION BY NONA-D- ARGININE. Journal of Biological Chemistry 279, 36788-36794 (2004)). Calbiochem 537076 has a higher degree of cell permeability, and is highly active against FURIN, PCSK4, PCSK5, and PCSK6 at Ki < 17 pM (Becker, G.L. et al. Highly potent inhibitors of proprotein convertase furin as potential drugs for treatment of infectious diseases. Journal of Biological Chemistry 287, 21992-22003 (2012)). We found that Calbiochem 537076 markedly inhibits reticular collagen formation (Figure 1, 2) while two other proprotein convertase inhibitors - dec-RVKR-cmk and hexa-D- arginine amide - had a smaller effect. This is possibly due to the -1000 fold excess inhibitory activity of Calbiochem 537076 in comparison to the other two compounds. Calbiochem 537076 has an IC50 of 2.17 μΜ (Figure 2). This is substantially greater than its published Ki on FURIN (< 17 pM (Becker, G.L. et al. Highly potent inhibitors of proprotein convertase furin as potential drugs for treatment of infectious diseases. Journal of Biological Chemistry 287, 21992-22003 (2012))). A possible reason for this discrepancy is that FURIN functions not only extracellularly but also intracellularly in the secretory pathway (Bass, J., Turck, C, Rouard, M. & Steiner, D.F. Furin-mediated processing in the early secretory pathway: sequential cleavage and degradation of misfolded insulin receptors. Proc. Natl. Acad. Sci. U.S.A. 97, 11905-11909 (2000)), and intracellular uptake of Calbiochem 537076 may be limited. Figure 1 Calbiochem 537076 (Calbiochem 537076) specifically inhibits extracellular collagen levels
Scar-in-a-jar assay was carried out as per Figure 6 on three different passages of juvenile NHDF (j-NHDF) treated with 0, 1.25, 2.5, 5, 10, or 20 μΜ Calbiochem
537076. [a] Immunocytochemistry of collagen type 1 (COL-1) and fibronectin, and nuclear staining with Hoechst as imaged on the Operetta, [b-d] Graphs show
quantification from image analysis of total COL-1 divided by total fibronectin (b), total COL-1 divided by cell number (c), and total fibronectin divided by cell number (d).
Data were expressed as mean ±SEM, with n = 2. Two-way ANOVA was used to evaluate the effect of passage number (P < 0.0001, P < 0.0001, P < 0.0001) and the concentration of Calbiochem 537076 (P < 0.0001, P < 0.0001, P = 0.0001) in (b), (c), and (d) respectively.
Figure 2 Calbiochem 537076 inhibits extracellular collagen in a dosage dependent manner
Three commercially available PCSK inhibitors were tested on the scar-in-a-jar assay, including 'Calbiochem 537076' (Calbiochem 537076, 'PCi'), dec-RVKR-cmk (Cayman 14965, 'Fil'), and hexa-D-arginine amide (Calbiochem 344931, 'Fill'). To generate a dose response curve, 12 data points in quarter log interval were included, with the highest concentration being 50 μΜ (5 x 10"5 M) down to 0.158 μΜ (5 x 10"7 5 M). All treatments contained 1% DMSO. The graph shows quantification from image analysis of total collagen divided by total fibronectin. A dose response curve was fitted by a nonlinear sigmoidal dose response function to a variable slope without constraints, based on which an IC50 for each drug was determined. In this experiment, j- HDF at passage 5 was used. Data were expressed as mean±SEM, with n = 4. The z '-factor (used as a quality metric of dynamic range during optimisation (Birmingham, A. et al. Statistical methods for analysis of high-throughput RNA interference screens. Nature Methods 6, 569-575 (2009))) for each data point was calculated as χ=1-(3(σρ+ση))/|μρ - μη|, where p and n denotes positive (dose) and negative (concentration at 0) controls respectively, z ' > 0.5 indicates that it is robust for high-throughput screening (Birmingham, A. et al. Statistical methods for analysis of high-throughput RNA interference screens. Nature Methods 6, 569-575 (2009)). z'-factors in the figure are indicated as: *, 0 < z ' < 0.5; **, 0.5 < z ' < 1. We have obtained a manual z'-factor of 0.77 at 5 μΜ for the "Scar-in-a-Jar" assay, using
Calbiochem 537076.
Example 2: Anti-FURIN siRNA suppresses type I collagen deposition and
transcription
To validate the role of PCSKs in type I collagen deposition using a genetic approach, we knocked them down with siRNA. We first validated each siRNA by transfection followed by qPCR to determine the extent of knockdown (Figure 3). Next we determined the effect of individual and combinatorial PCSK knockdown on collagen deposition in the miniaturized Scar-in-a-Jar assay. This showed that knockdown of FURIN dramatically reduced the ratio of extracellular collagen to fibronectin (Figure 4). Knockdown of PCSK5 or PCSK6 did not have this effect on collagen deposition, despite effectively knocking down activity of the target gene. As profibrotic peptides (TGFB, EDNl, PDGF) function, in part, by activating collagen type I (COL1 Al) transcription, we next determined the effect of FURIN knockdown on collagen isoform transcription in jNHDF cells in the Scar-in-a-Jar assay using RT-QPCR for COL1A1, COL2A1, and COL3A1. This showed that anti-FURIN siRNA suppressed COL1 Al but not COL2A1 or COL3A1 mRNA expression (Figure 5). Our results indicate for the first time that FURIN plays a key role in collagen deposition and transcription. As FURIN expression is increased in the failing heart (Ichiki, T. et al. Differential expression of the pro-natriuretic peptide convertases corin and furin in experimental heart failure and atrial fibrosis. Am J Physiol Regullntegr Comp Physiol 304, R102-9 (2013)), our results indicate that FURIN is a therapeutic target in collagen deposition and cardiac fibrosis. Its safety as a target is supported by the fact that PCSK and FURIN inhibitors explored in mice (reviewed in Couture, F., D'Anjou, F. & Day, R. On the cutting edge of proprotein convertase pharmacology: from molecular concepts to clinical applications. Biomol Concepts 2, 421- 438 (2011)) are not associated with significant reported toxicity (Bassi, D.E. et al.
Proprotein convertase inhibition results in decreased skin cell proliferation, tumorigenesis, and metastasis. Neoplasia 12, 516-526 (2010); Remacle, A.G. et al. Selective and potent furin inhibitors protect cells from anthrax without significant toxicity. International Journal of Biochemistry and Cell Biology 42, 987-995 (2010); Hajdin, K.A., Valentina, Niggli, F.K., Schafer, B.W. & Bernasconi, M. Furin Targeted Drug Delivery for Treatment of Rhabdomyosarcoma in a Mouse Model. PLoS ONE 5, el0445 (2010); Shiryaev, S.A. et al. Targeting Host Cell Furin Proprotein Convertases as a Therapeutic Strategy against Bacterial Toxins and Viral Pathogens. Journal of Biological Chemistry 282, 20847-20853 (2007); Sarac, M.S., Cameron, A. & Lindberg, I. The Furin Inhibitor Hexa-D-Arginine Blocks the Activation of Pseudomonas aeruginosa Exotoxin A In Vivo. Infect. Immun. 70, 7136-7139 (2002). Levesque, C. et al. PACE4 inhibitors and their peptidomimetic analogs block prostate cancer tumor progression through quiescence induction, increased apoptosis and impaired neovascularisation. Oncotarget 6, 3680-93 (2015)). Moreover, although zygotic FURIN knockout is embryonic lethal due to its role in embryonic development, inducible knockout of FURIN in adult mice has no apparent effect (Roebroek, A.J. et al. Limited redundancy of the proprotein convertase furin in mouse liver. J Biol Chem 279, 53442-50 (2004)).
Figure 3 Validation of siRNA against individual PCSKs
Normal human dermal fibroblasts (NHDF) from juvenile foreskin (j-NHDF, C-12300, Promo) were seeded on 96-well plates at 10,000 cells per well, and reverse transfected with Dharmacon SMARTpool OTP siRNA: L-005882 FURIN siRNA (SEQ ID NOs: 19, 20, 21 and 22), L-005987 PCSK5 siRNA (SEQ ID NOs: 23, 24, 25 and 26), L-005983 PCSK6 siRNA (SEQ ID NOs: 27, 28, 29 and 30), or D-001810 non-targeting control (SEQ ID NOs: 31, 32, 33 and 34). We used Dharmacon SMARTPool siRNAs as they minimize off-target effects by using subthreshold concentrations of 4 independent siRNAs targeting a single gene, and strand modifications to reduce the off-target effects of the sense strand, and seed-region modification to minimize off target effects from the antisense strand. In each case, a total of 48nM siRNA (alone, or in combination with the other two genes) and 0.4μ1 Lipofectamine RNAiMAX transfection reagent was used as per manufacturer's instructions. Whole RNA was extracted using Qiagen RNeasy kit, and transcribed into cDNA using QuantiTect reverse transcription kit. Gene expression was determined by qPCR using Taqman gene expression assays for FURIN (Hs00965485_gl) (a), PCSK5 (Hs00196400_ml) (b), and PCSK6 (Hs00159844_ml) (c). Data were normalised to HPRT (Hs99999909_m 1 ), and presented using the 2Λ(-ΔΔΟΤ) method, with the NTC control normalised to 100 and expressed as mean ±SEM. For each combination, 3 transfection replicates were pooled, and 3 technical replicates were performed for qPCR. NTC, non- targeting control. Figure 4 Knockdown of FURIN (PCSK3) inhibits extracellular collagen
PCSK3 (FURIN), PCSK5, and PCSK6 were knocked down in j-NHDF cells as per Figure 3, and scar-in-a-jar assay was carried out. Graphs show quantification from image analysis of (a) total collagen divided by cell number, (b) total fibronectin divided by cell number, and (c) total collagen divided by total fibronectin. To eliminate background signals, only pixels that were above a certain threshold were included in the quantification. Data expressed as mean ±SEM, with n = 3. The z '-factor for each data point was calculated based on χ=1-(3(σρ+ση))/|μρ - μη|, where p and n denotes positive (siRNA) and negative (non-targeting control, NTC) controls respectively. *, 0 < z ' < 0.5; **, 0.5 < z ' < 1.
Figure 5 Knockdown of FURIN suppresses COL1A1 niRNA expression
Normal human dermal fibroblasts (NHDF) from juvenile foreskin (j-NHDF, C-12300, Promo) were seeded on 6-well plates at 300,000 per well, and reverse transfected with Dharmacon SMARTpool OTP siRNA: L-005882 FURIN siRNA (SEQ ID NOs: 19, 20, 21 and 22) or D-001810 non-targeting control (SEQ ID NOs: 31, 32, 33 and 34). In each case, a total of 1392nM siRNA and 70 μΐ Lipofectamine RNAiMAX transfection reagent was used as per manufacturer's instructions. Whole RNA was extracted using Qiagen RNeasy kit, and transcribed into cDNA using QuantiTect reverse transcription kit. Gene expression was determined by qPCR using Taqman gene expression assays for FURIN
(Hs00965485_gl), COL1A1 (Hs00164004_ml), COL1A2 (Hs00164099_ml), and COL3A1 (Hs00943809_ml). Data were normalised to HPRT (Hs99999909_ml), and presented using the 2Α(-ΔΔΟΤ) method, with the NTC control normalised to 100 and expressed as mean ±SEM. 3 technical replicates were performed for qPCR. Example 3: Tablet composition
Tablets, each weighing 0.15 g and containing 25 mg of a proprotein convertase inhibitor, for use in accordance with the invention, are manufactured as follows:
Composition for 10,000 tablets
Active compound: a proprotein convertase inhibitor (250 g)
Lactose (800 g)
Corn starch (415g)
Talc powder (30 g)
Magnesium stearate (5 g)
The proprotein convertase inhibitor, lactose and half of the corn starch are mixed. The mixture is then forced through a sieve 0.5 mm mesh size. Corn starch (10 g) is suspended in warm water (90 ml). The resulting paste is used to granulate the powder. The granulate is dried and broken up into small fragments on a sieve of 1.4 mm mesh size. The remaining quantity of starch, talc and magnesium is added, carefully mixed and processed into tablets.
Example 4: Injectable Formulation
Formulation A
Active compound: a proprotein convertase inhibitor 200 mg
Hydrochloric Acid Solution 0.1M or
Sodium Hydroxide Solution 0.1M q.s. to pH 4.0 to 7.0
Sterile water q.s. to 10 ml
The proprotein convertase inhibitor, for use in accordance with the invention, is dissolved in most of the water (35° to 40° C) and the pH adjusted to between 4.0 and 7.0 with the hydrochloric acid or the sodium hydroxide as appropriate. The batch is then made up to volume with water and filtered through a sterile micropore filter into a sterile 10 ml amber glass vial (type 1) and sealed with sterile closures and overseals.
Formulation B
Active Compound: proprotein convertase inhibitor 125 mg
Sterile, Pyrogen-free, pH 7 Phosphate
Buffer, q.s. to 25 ml
Active compound 200 mg
Benzyl Alcohol 0.10 g
Glycofurol 75 1.45 g
Water for injection q.s to 3.00 ml
The proprotein convertase inhibitor is dissolved in the glycofurol. The benzyl alcohol is then added and dissolved, and water added to 3 ml. The mixture is then filtered through a sterile micropore filter and sealed in sterile 3 ml glass vials (type 1).
Example 5: Syrup Formulation
Active compound (proprotein convertase inhibitor) 250 mg
Sorbitol Solution 1.50 g
Glycerol 2.00 g
Sodium benzoate 0.005 g
Flavour 0.0125 ml
Purified Water q.s. to 5.00 ml
The proprotein convertase inhibitor, for use in accordance with the invention, is dissolved in a mixture of the glycerol and most of the purified water. An aqueous solution of the sodium benzoate is then added to the solution, followed by addition of the sorbitol solution and finally the flavour. The volume is made up with purified water and mixed well. DESCRIPTION OF THE SEQUENCES
SEQ ID NOs: 1, 3, 5, 7, 9, 1 1, 13, 15 and 17 set forth human PCSKl, 2, 3, 4, 5, 6, 7, 8 and 9 mRNA respectively.
SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16 and 18 set forth human PCSKl, 2, 3, 4, 5, 6, 7, 8 and 9 cDNA respectively.
SEQ ID NO: 19 sets forth anti- human PCSK 3 (FURIN) siRNA no. 1.
SEQ ID NO: 20 sets forth anti- human PCSK 3 (FURIN) siRNA no. 2.
SEQ ID NO: 21 sets forth anti- human PCSK 3 (FURIN) siRNA no. 3.
SEQ ID NO: 22 sets forth anti- human PCSK 3 (FURIN) siRNA no. 4.
SEQ ID NO: 23 sets forth anti- human PCSK 5 siRNA no. 1.
SEQ ID NO: 24 sets forth anti- human PCSK 5 siRNA no. 2.
SEQ ID NO: 25 sets forth anti- human PCSK 5 siRNA no. 3.
SEQ ID NO: 26 sets forth anti- human PCSK 5 siRNA no. 4.
SEQ ID NO: 27 sets forth anti- human PCSK 6 siRNA no. 1.
SEQ ID NO: 28 sets forth anti- human PCSK 6 siRNA no. 2.
SEQ ID NO: 29 sets forth anti- human PCSK 6 siRNA no. 3.
SEQ ID NO: 30 sets forth anti- human PCSK 6 siRNA no. 4.
SEQ ID NO: 31 sets forth non-targeting control siRNA no. 1.
SEQ ID NO: 32 sets forth non-targeting control siRNA no. 2.
SEQ ID NO: 33 sets forth non-targeting control siRNA no. 3.
SEQ ID NO: 34 sets forth non-targeting control siRNA no. 4.
SEQ ID NO: 35 sets forth the commercially-available proprotein convertase inhibitor decanoyl-Arg-Val-Lys-Arg-CMK.

Claims

1. A proprotein convertase inhibitor for use in the treatment of a fibrotic disorder, wherein the proprotein convertase inhibitor is a compound of formula (I)
P5-P4-P3-P2-P1 (I) wherein:
PI is an arginine mimetic selected from the following structures:
Figure imgf000095_0001
Figure imgf000095_0002
wherein
Ri is selected from H, OH, 0-CH3, H2, 0-C(0)-CH3 and -C(0)-0-(CH2)m-CH3 wherein m is an integer from 1 to 5;
R2 is H or an amidino group;
n is equal to 3, 4 or 5; and
s is equal to 2 or 3;
P2, P3 and P4 are independently selected from amino acid residues and imino acid residues; and
P5 is an N-terminal modification of the amino or imino acid P4, wherein P5 is selected from the following groups:
-H, -C(0)-R7, -C(0)-X-R7,
a pyroglutamyl residue or another amino or imino acid residue,
-S(0)2-R7,
Ci-10 alkyl which is unsubstituted or substituted with a halogen, NH2, guanidino, C(0)OH or C(0)OR5 group,
a sphingosine acylated with a bifunctional group, having the following structure:
Figure imgf000096_0001
a sphingosyl-phosphorylcholine acylated with a bifunctional group, having the following structure:
Figure imgf000097_0001
wherein
z is an integer from 2 to 20;
X is O, CH2 or H;
R5 is H or unsubstituted or substituted C1-3 alkyl; and
R7 is:
(i) unsubstituted or substituted C1-24 alkyl, or unsubstituted or substituted C2-24 alkenyl,
(ii) unsubstituted or substituted aryl, unsubstituted or substituted heteroaryl, unsubstituted or substituted aralkyl, unsubstituted or substituted heteroaralkyl or unsubstituted or substituted C3-20 cycloalkyl,
wherein the heteroaryl ring in said heteroaryl and in said heteroaralkyl contains from 1 to 3 heteroatoms selected from N, S and O,
and wherein the substituent or substituents which may be present at R7 are independently selected from - H2, -CH2- H2, -amidino, -hydroxyamidino, -guanidino, - CH2-guanidino, methylguanidino, -halogen, -CI, -Br, -I, -CN, -CF3, aryl, C1-3 alkyl, C3-2o cycloalkyl, C1-3 alkoxy, and -C(0)0-R5 wherein R5 is H or unsubstituted or substituted C1-3 alkyl, or
(iii) a cholesterol group;
or a prodrug thereof, or a pharmaceutically acceptable salt thereof.
2. A proprotein convertase inhibitor as claimed in claim 1 wherein PI in the compound of formula (I) is selected from groups of the following structures:
Figure imgf000098_0001
Ri is selected from H, OH, 0-CH3, H2, 0-C(0)-CH3 and -C(0)-0-(CH2)m-CH3 wherein m is an integer from 1 to 5;
R2 is H or an amidino group; and
n is equal to 3, 4 or 5.
3. A proprotein convertase inhibitor as claimed in claim 1 or claim 2 wherein P2 in the compound of formula I) is selected from groups of the following structures
Figure imgf000098_0002
wherein:
p is an integer from 0 to 5;
R3 is amino, guanidino, methylguanidino, -O-guanidino, unsubstituted or substituted aryl, or unsubstituted or substituted heteroaryl, wherein the heteroaryl ring contains from 1 to 3 heteroatoms selected from N, S and O, and wherein the or each substituent on the substituted aryl ring and on the substituted heteroaryl ring is independently selected from - H2, -CH2- H2, -amidino, -hydroxyamidino, -guanidino, - CH2-guanidino, -halogen, -CI, -Br, -I, -CN, -CF3, Ci-3 alkyl, Ci-3 alkoxy and -COOR5, wherein R5 is H or Ci-3 alkyl; and
R4 is -H, -OH or -0-(CH2)q-R6 wherein q is an integer from 2 to 4 and R5 is an amino or guanidino group.
4. A proprotein convertase inhibitor as claimed in any one of claims 1 to 3 wherein P3 in the compound of formula (I) is selected from groups having the following structures
Figure imgf000099_0001
Figure imgf000100_0001
5. A proprotein convertase inhibitor as claimed in any one of the preceding claims wherein P4 in the compound of formula (I) is a selected from groups of the following structures:
Figure imgf000100_0002
p is an integer from 0 to 5;
R.3 is amino, guanidino, methylguanidino, C1-3 alkyl, -O-guanidino,
-N(H)C(0)OCH2Ph, unsubstituted or substituted aryl or unsubstituted or substituted heteroaryl, wherein the heteroaryl ring contains from 1 to 3 heteroatoms selected from N, S and O, and wherein the or each substituent on the substituted aryl ring and on the substituted heteroaryl ring is independently selected from - H2, -CH2- H2, -amidino, - hydroxyamidino, -guanidino, -CH2-guanidino, -halogen, -CI, -Br, -I, -CN, -CF3, C1-3 alkyl, Ci-3 alkoxy and -COOR5, wherein R5 is H or C1-3 alkyl; and
R4 is -H, -OH or -0-(CH2)q-R6 wherein q is an integer from 2 to 4 and R5 is an amino or guanidino group.
6. A proprotein convertase inhibitor as claimed in any one of the preceding claims wherein P5 in the compound of formula (I) is selected from H, -CO-R7, -CO-X-R7 and - SO2-R7, wherein R7 and X are as defined in claim 1.
7. A proprotein convertase inhibitor as claimed in claim 6 wherein P5 is:
H;
-C(0)-CH2-R7 wherein R7 is unsubstituted or substituted phenyl;
-C(0)-R7 wherein R7 is either unsubstituted or substituted C1-24 alkyl or
unsubstituted or substituted C2-24 alkenyl;
-C(0)-0-R7 wherein R7 is either unsubstituted or substituted phenyl or
unsubstituted or substituted C1-24 alkyl; or
S(0)2-R7 wherein R7 is unsubstituted or substituted phenyl,
wherein the substituent or substituents which may be present at R7 are
independently selected from - H2, -CH2- H2, -amidino, -hydroxyamidino, -guanidino, - CH2-guanidino, methylguanidino, -halogen, -CI, -Br, -I, -CN, -CF3, aryl, C1-3 alkyl, C3-20 cycloalkyl, C1-3 alkoxy, and -C(0)0-R5 wherein R5 is H or unsubstituted or substituted C1-3 alkyl.
8. A proprotein convertase inhibitor as claimed in any one of the preceding claims which is a compound having any one of the following structures
Figure imgf000101_0001
Figure imgf000102_0001
Figure imgf000103_0001
Figure imgf000104_0001
Figure imgf000105_0001
Figure imgf000106_0001
Figure imgf000107_0001
or a prodrug thereof, or a pharmaceutically acceptable salt thereof.
9. A proprotein convertase inhibitor as claimed in any one of the preceding claims which is a compound of formula (la)
Figure imgf000107_0002
or a prodrug thereof or a pharmaceutically acceptable salt thereof.
10. A proprotein convertase inhibitor for use in the treatment of a fibrotic disorder, wherein the inhibitor comprises an oligonucleotide which specifically hybridises to a part of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15 or 17 (human PCSK 1 to 9 mRNA respectively) or any isoform thereof.
11. A proprotein convertase inhibitor as claimed in claim 10, wherein the inhibitor comprises an oligonucleotide which specifically hybridises to a part of SEQ ID NO: 5, 9 or 11 (human PCSK 3, 5 or 6 mRNA respectively) or any isoform thereof.
12. A proprotein convertase inhibitor as claimed in claim 10 or claim 11, wherein the inhibitor comprises an oligonucleotide which specifically hybridises to a part of SEQ ID NO: 5 (human PCSK 3 mRNA) or any isoform thereof.
13. A proprotein convertase inhibitor for use in the treatment of a fibrotic disorder, wherein the inhibitor comprises an oligonucleotide which comprises 50 or fewer consecutive nucleotides from (a) SEQ ID NO: 2, 3, 6, 8, 10, 12, 14, 16 or 18 (human PCSK 1 to 9 cDNA respectively) or any isoform thereof or (b) a variant sequence which has at least 95% homology to SEQ ID NO: 2, 3, 6, 8, 10, 12, 14, 16 or 18 or any isoform thereof based on nucleotide identity over the entire sequence.
14. A proprotein convertase inhibitor as claimed in claim 13, wherein the inhibitor comprises an oligonucleotide which comprises 50 or fewer consecutive nucleotides from (a) SEQ ID NO: 6, 10 or 12 (human PCSK 3, 5 or 6 cDNA respectively) or any isoform thereof or (b) a variant sequence which has at least 95% homology to SEQ ID NO: 6, 10 or 12 or any isoform thereof based on nucleotide identity over the entire sequence.
15. A proprotein convertase inhibitor as claimed in claim 13 or claim 14, wherein the inhibitor comprises an oligonucleotide which comprises 50 or fewer consecutive nucleotides from (a) SEQ ID NO: 6 (human PCSK 3 cDNA) or any isoform thereof or (b) a variant sequence which has at least 95% homology to SEQ ID NO: 6 or any isoform thereof based on nucleotide identity over the entire sequence.
16. A proprotein convertase inhibitor as claimed in any one of claims 10 to 15, wherein the inhibitor comprises an oligonucleotide which is (a) any one of SEQ ID NOs: 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30, or (b) a variant sequence which has at least 95% homology to any one of SEQ ID NOs: 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30 or any isoform thereof based on nucleotide identity over the entire sequence.
17. A proprotein convertase inhibitor as claimed in claim 16, wherein the inhibitor comprises an oligonucleotide which is (a) any one of SEQ ID NOs: 19, 20, 21 and 22, or (b) a variant sequence which has at least 95% homology to any one of SEQ ID NOs: 19, 20, 21 and 22 or any isoform thereof based on nucleotide identity over the entire sequence.
18. A proprotein convertase inhibitor as claimed in claim 17, wherein the inhibitor comprises an oligonucleotide which is any one of SEQ ID NOs: 19, 20, 21 and 22.
19. A proprotein convertase inhibitor for use in the treatment of a fibrotic disorder by inhibiting the formation of collagen matrix without inhibiting fibronectin secretion.
20. A proprotein convertase inhibitor as claimed in claim 19 which is for use in the treatment of said fibrotic disorder by inhibiting procollagen secretion without inhibiting fibronectin secretion.
21. A proprotein convertase inhibitor as claimed in claim 19 or claim 20 which is a compound as defined in any one of claims 1 to 9, a prodrug thereof, or a pharmaceutically acceptable salt thereof, or which is an inhibitor as defined in any one of claims 10 to 18.
22. A proprotein convertase inhibitor as claimed in any one of the preceding claims wherein the fibrotic disorder is selected from atrial fibrillation; cardiac diastolic
dysfunction; heart failure; chronic heart failure (CHF); cardiomyopathy; cardiovascular fibrosis; myocardial fibrosis; myocardial hibernation; fibrosis following myocardial infarction; dermal fibrosis; a fibrotic skin disease; scleroderma; nephrogenic fibrosing dermopathy; mixed connective tissue disease; scleromyxedema; scleredema; eosinophilic fasciitis; a fibrotic skin disease caused by exposure to a chemical or physical agent; liver fibrosis; cirrhosis; cirrhosis of the liver; Crohn's Disease (CD); progressive kidney disease; glomerulonephritis; renal fibrosis; pulmonary fibrosis; idiopathic pulmonary fibrosis; systemic sclerosis; central nervous system fibrosis following a stroke or following a neurodegenerative disorder; proliferative vitreoretinopathy (PVR); restenosis; and arthritis.
23. A proprotein convertase inhibitor as claimed in any one of the preceding claims which is for use in said treatment of said fibrotic disorder by coadministration with a further antifibrotic agent.
24. A combination comprising (a) a proprotein convertase inhibitor and (b) a further antifibrotic agent, which combination is for use in the treatment of a fibrotic disorder.
25. A combination according to claim 24 wherein the proprotein convertase inhibitor and the further antifibrotic agent are for separate, simultaneous, concomitant or sequential administration in the treatment of the fibrotic disorder.
26. A proprotein convertase inhibitor as claimed in claim 23 or a combination as claimed in claim 24 or claim 25 wherein the further antifibrotic agent is a PDE6D inhibitor, pirfenidone, a pharmaceutically acceptable salt of pirfenidone, an Endothelin antagonist, an Endothelin receptor inhibitor (e.g. Bosentan), a Transforming growth factor beta (TGFbeta) antagonist, a TGFbeta receptor inhibitor, a Platelet-derived growth factor (PDGF) antagonist, a PDGF receptor inhibitor, a chemokine antagonist, a chemokine receptor inhibitor, a cytokine antagonist, a cytokine receptor inhibitor, N-acetylcysteine, prednisone, azathioprine, or a tyrosine kinase inhibitor.
27. A proprotein convertase inhibitor as claimed in claim 23 or claim 26 or a combination according to any one of claims 24 to 26 wherein the proprotein convertase inhibitor is a compound of formula (la):
Figure imgf000110_0001
or a prodrug thereof or a pharmaceutically acceptable salt thereof; and the further antifibrotic agent is a PDE6D inhibitor of the following formula
Figure imgf000111_0001
or a pharmaceutically acceptable salt thereof.
28. A proprotein convertase inhibitor as claimed in claim 23 or claim 26 or a combination according to any one of claims 24 to 26 wherein:
the proprotein convertase inhibitor is as defined in any one of claims 12, 15, 17 and
18; and
the further antifibrotic agent is a PDE6D inhibitor of the following formula
Figure imgf000111_0002
or a pharmaceutically acceptable salt thereof.
29. A composition for use in the treatment of a fibrotic disorder, which composition comprises a proprotein convertase inhibitor as defined in any one of claims 1 to 18 and a pharmaceutically acceptable carrier or diluent.
30. A composition for use in the treatment of a fibrotic disorder by inhibiting the formation of collagen matrix without inhibiting fibronectin secretion, which composition comprises a proprotein convertase inhibitor and a pharmaceutically acceptable carrier or diluent.
31. A composition as claimed in claim 29 or claim 30 which additionally comprises a further antifibrotic agent as defined in any one of claims 23 to 27.
32. Use of a proprotein convertase inhibitor as defined in any one of claims 1 to 18 in the manufacture of a medicament for use in the treatment of a fibrotic disorder.
33. Use of a proprotein convertase inhibitor in the manufacture of a medicament for use in the treatment of a fibrotic disorder by inhibiting collagen matrix formation without inhibiting fibronectin secretion.
34. A method of treating a fibrotic disorder, which method comprises administering to a subject in need of such treatment a therapeutically effective amount of a proprotein convertase inhibitor as defined in any one of claims 1 to 18.
35. A method of treating a fibrotic disorder in a subject by inhibiting collagen matrix formation without inhibiting fibronectin secretion, which method comprises administering to a subject in need of such treatment an amount of a proprotein convertase inhibitor effective to inhibit collagen matrix formation in the subject without inhibiting fibronectin secretion.
36. An in vitro method of inhibiting the formation of collagen matrix, the method comprising contacting an in vitro sample comprising collagen-secreting cells with an amount of a proprotein convertase inhibitor as defined in any one of claims 1 to 18 effective to inhibit the formation of collagen matrix by the cells.
37. An in vitro method of inhibiting the formation of collagen matrix without inhibiting fibronectin secretion, the method comprising contacting an in vitro sample comprising collagen-secreting cells with an amount of a proprotein convertase inhibitor effective to inhibit the formation of collagen matrix by the cells without inhibiting fibronectin secretion.
38. An in vitro method according to claim 36 or claim 37 wherein the collagen- secreting cells comprise fibroblasts.
39. A composition as claimed in claim 30, the use according to claim 33 or a method according to claim 35 or claim 37, wherein the proprotein convertase inhibitor is as defined in any one of claims 1 to 18.
40. A method of identifying an agent as being capable of inhibiting collagen matrix formation, which method comprises (a) contacting an agent with an in vitro sample comprising collagen-secreting cells in the absence of exogenous transforming growth factor (TGF) and (b) measuring the amount of collagen matrix formed by the cells and thereby determining whether or not the agent is capable of inhibiting collagen matrix formation.
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