EP1690098A2 - Substances biologiques et leurs utilisations - Google Patents

Substances biologiques et leurs utilisations

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Publication number
EP1690098A2
EP1690098A2 EP04798502A EP04798502A EP1690098A2 EP 1690098 A2 EP1690098 A2 EP 1690098A2 EP 04798502 A EP04798502 A EP 04798502A EP 04798502 A EP04798502 A EP 04798502A EP 1690098 A2 EP1690098 A2 EP 1690098A2
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EP
European Patent Office
Prior art keywords
fibrosis
tieg
smad
compound
ctgf
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EP04798502A
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German (de)
English (en)
Inventor
Nadia Abdel Wahab
Roger Maxwell Mason
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Ip2ipo Innovations Ltd
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Imperial Innovations Ltd
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Publication of EP1690098A2 publication Critical patent/EP1690098A2/fr
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    • 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
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • 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
    • 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
    • G01N33/502—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 for testing non-proliferative effects
    • G01N33/5023—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 for testing non-proliferative effects on expression patterns
    • 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
    • G01N33/502—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 for testing non-proliferative effects
    • G01N33/5041—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 for testing non-proliferative effects involving analysis of members of signalling pathways
    • 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
    • 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
    • G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
    • 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
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/71—Assays involving receptors, cell surface antigens or cell surface determinants for growth factors; for growth regulators

Definitions

  • the presently claimed invention relates to methods of identifying and/or making compounds for use in the reduction and/or prevention of fibrosis.
  • fibrosis occurs, or indeed the extent of the fibrosis, is influenced by a variety of factors, including the nature, severity and location of the injury to be healed. Fibrosis is most commonly known as scars on the surface of the skin, where it is relatively un-troublesome, except i scarring over large areas. However, fibrosis can also occur in the tissues of internal organs e.g. liver, lung and kidney. In most cases, it is fibrosis in these areas that is most serious because the specialised activity of that organ is unpaired. In the most extreme cases organ failure or death can occur because of that impahment. An example of the importance of fibrosis in the disease state is demonstrated by the occurrence of fibrosis in the kidney (diabetic nephropathy) in diabetes meUitus, a disease now reaching epidemic proportions world-wide.
  • diabetes mellitas is closely linked to a number of secondary complications, especially microvascular related complications. These complications, including the f ⁇ brotic condition nephropathy, usually develop a number of years after the onset of diabetes.
  • Diabetic nephropathy is characterised by excessive deposition of extracellular matrix proteins in the mesangium and basement membrane of the glo erulus and in the renal tubulointerstitium.
  • DN diabetic nephropathy
  • UPDS Group (1998) Lancet 352 pp. 837-853).
  • the prevalence of nephiOpathy varies according to geographical location, type of diabetes, and the length of time since diagnosis. Notwi standing influencing factors, the prevalence of diabetic nephropathy is predicted to increase in the decades ahead (Bagust A et al. (2002) Diabetes Med 19 (Suppl 4): ppl-5).
  • Diabetic nephropathy is a major cause of end-stage renal disease (11), and new therapeutic approaches are required to lknit its development.
  • the pathology of diabetic nephropathy is similar in types 1 and 2 diabetes. Both types of diabetes are associated with similar ullxastractural changes occurring in lddney glomeruli (Osterby , (1992) Diabetologica 35 pp 803- 812).
  • the glomerular basement membrane increases in thickness, and the extracellular matrix of the mesangium expands. It is expansion of the mesangium that is thought to be the main cause of reduced renal function in diabetic nephropathy (Steffes M et al. (1989) Diabetes 38 ppl077-1081).
  • Fibrotic disease is commonly associated with an imbalance in growth factors and hormones, which in turn influence the production of protein expression.
  • the abnormal protein expression in turn leads to the formation of fibrosis.
  • fibrosis is commonly nifluenced by an increase in transforming growth factor- ⁇ present in the fibrotic tissue.
  • Fibrosis is one of the largest groups of disorders for which there is no effective therapy, in part because the mechanism underlying these disorders is influenced by a variety of factors and exact cellular mechanisms have not been elucidated. Therefore, there is a lack of understanding of which, or the nature of, molecular targets that may provide targets around which anti- f ⁇ brotic therapies may be based.
  • TGF- ⁇ transforming growth factor- ⁇
  • TGF- ⁇ has a number of physiological roles including involvement in immunity and epithelial proliferation (McCartney-Francis N et al. (1998) Int. Rev. Immunol. 16 pp. 553-580). These varying physiological effects mean that TGF- ⁇ is unlikely to be a clinically advantageous target. Blocldng the actions of TGF- ⁇ may have multiple effects on the organism, causing unwanted and potentially serious side- effects.
  • Transforming growth factor- ⁇ causes .fibrosis by the direct induction of collagen and matrix synthesis. Additionally, TGF- ⁇ is also able to induce the expression of other molecules that take part in and/or influence the pathways causing fibrosis.
  • One such protein is connective tissue growth factor (CTGF), which induces proliferation, collagen synthesis and chemotaxis in mesenchymal cells (Moussad E et al. (2000) Molec Genet Metab. 71 pp.276-292).
  • CTGF CCN2
  • CCN2 is a 38 kDa secreted protein with multiple domains, encoded by an immediate-early gene and is a member of the CCN protein family (Bork et al. (1993) Febs Lett.
  • CTGF has been shown to directly bind BMP4 and TGF- ⁇ through its von Willebrand type C domam, leading to inhibition of BMP and enhancement of TGF- ⁇ signalling (Abreu et al. (2002) Nat. Cell. Biol. 4 pp. 599-604).
  • CTGF has also been shown to bind to integrins (Babic et al. (1999) Mol. Cell Biol. 19 p ⁇ .3811-3815) and it is possible that this interaction is important in mediating some of the cellular phenomena that CTGF induces.
  • CTGF is over-expressed hi a variety of fibrotic disorders, mcluding diabetic nephropathy (Wahab N et al. (2001) Biochem J. 359 pp.77-87). i fact, increasing levels of CTGF expression have been shown to correlate with increasing severity and speed of progression of diabetic nephropathy (Ito Y et al. (1998) Kidney Int. 53 pp.853-886).
  • CTGF may be a potentially useful molecular indicator of the fibrotic response.
  • CTGF has not yet been shown to directly induce renal fibrosis in vivo, but, when injected subcutaneously along with TGF- ⁇ , induces sustained dermal fibrosis in rats (Mori T et al. (1999) J. Cell. Physiol. 181 pp 153-159).
  • TGF- ⁇ directly induces CTGF expression, at least in part through elements in the CTGF promoter (Holmes A. et al. (2001) J. Biol. Chem. 276 pp.10594-10601). In fact, the control of CTGF gene expression is thought to lie chiefly at the level of transcription (reviewed in 8).
  • SMADs facilitate gene expression by acting as trans criptional co- modulators to recruit transcription factors to form an active transcriptional complex (Roberts A (1999) Microbes Infect 1 pp.1265-1273 and Wrana J (2000) Sci STKE 23 RE1). The transcription factors recruited vary depending on the gene and cell type of interest.
  • TGF- ⁇ the diversity and specificity of the biological effects of TGF- ⁇ are due, hi part, to the interaction of the general TGF- ⁇ signalling pathway with other pathways, the nature of which depends on the cell type or target gene of interest (Mulder K (2000) Growth Factor Rev 11 pp23-35).
  • TGF- ⁇ exerts its cellular effects via the Smad signalling pathway.
  • the Smad pathway provides the main signal transduction route downstream of TGF- ⁇ receptors (Massague et al (2000) Cell 103 pp. 295-309).
  • the TGF- ⁇ receptors dimerise and autophosphorylate. This in turn phosphorylates Smad 2 and Smad 3 (Itoh et al. (2000) Eur J. Biochem 267 pp.6954-6967).
  • Smad 2 and Smad 3 then form a complex with Smad 4, which translocates into the nucleus where they co-operate with other transcription factors to regulate transcription of the many TGF- ⁇ - responsive genes containing a Smad binding element (SBE) in their promoters.
  • Smad 7 Smad binding element
  • Smad 7 protein interacts with one or more of the E3-ubiquitin ligases, Smurfl and Smurf2, in the nucleus.
  • the Smad 7-Smurf complex translocates to the plasma membrane where Smurf induces ubiquitination and degradation of the TGF- ⁇ receptors (Kavsak et al (2000) Mol Cell 6 pp.1365-1375).
  • the degradation of the TGF- ⁇ receptors prevents further phosphorylation of Smad 2 and Smad 3.
  • Smad 7 provides a negative feedback response to Ihnit the effect of TGF- ⁇ .
  • Smad 7 gene has been reported to be suppressed by the transcription factor, TIEG which binds to a GC box in the proximal promoter region (Johnsen et al. (2002) Oncogene 21 pp. 5783-5790).
  • TIEG 1 and 2 are potent transcriptional repressors related to the Spl family of transcription factors. Their genes encode zinc-finger ruppel-like proteins whose over-expression mimics the effect of TGF- ⁇ hi different cell types (Cook et al. (1998) J. Biol. Chem.).
  • CTGF enhances the TGF- ⁇ signalling pathway by decreasing the availability of Smad 7, dependent on increased TIEG production in response to CTGF.
  • a method for identifying and/or making compounds for use in reducing and/or preventing fibrosis comprising the steps:
  • the method further comprises the step of isolation of a test compound which is capable of causing no change or an increase in Smad-7 expression and/or no change or a decrease in TIEG expression hi a method according to the first aspect of the invention.
  • the isolated compound may then be optionally formulated into a composition further comprising a pharmaceutically acceptable carrier, excipient and/or diluent.
  • the compound affects directly the interaction between CTGF and TIEG.
  • the compound interacts with CTGF and/or TIEG hi order to reduce and/or prevent the effect of CTGF on TIEG and/or Smad- 7 expression.
  • the compound affects indirectly the interaction between CTGF and TIEG.
  • the compound interacts with at least one fiirther compound, which in turn interacts with CTGF or TIEG in order to reduce and/or prevent the effect of CTGF on TIEG and/or Smad-7 expression.
  • a compound for use in the manufacture of a medicament for reduction and/or prevention of fibrosis characterised in that the compound reduces and/or prevents CTGF suppression of Smad 7 expression and or reduces and/or prevents induction of TIEG expression.
  • the compound is identified and/or made by the method of the first aspect of the invention.
  • the compound is at least one selected from polypeptides, antibody molecules and antisense nucleotides.
  • the compound is an antibody molecule.
  • antibody molecule shall be taken to refer to any one of an antibody, an antibody fragment, or antibody derivative. It is intended to embrace wildtype antibodies, synthetic antibodies, recombii ant antibodies or antibody hybrids, such as, but not limited to, a single-chah modified antibody molecule produced by phage-display of immunoglobulin light and/or heavy chain variable and/or constant regions, or other inmiunohiteractive molecule capable of binding to an antigen hi an hnmunoassay format that is known to those skilled in the art.
  • antibody derivative refers to any modified antibody molecule that is capable of bmding to an antigen in an immunoassay format that is known to those skilled in the art, such as a fragment of an antibody (e.g. Fab or Fv fragment), or a modified antibody molecule that is modified by the addition of one or more amino acids or other molecules to facilitate coupling the antibodies to another peptide or polypeptide, to a large carrier protein or to a solid support (e.g. the a ino acids tyrosine, lysine, glutamic acid, aspartic acid, cysteine and derivatives thereof, NH -acetyl groups or COOH-te ⁇ nrnal arnido groups, amongst others).
  • a fragment of an antibody e.g. Fab or Fv fragment
  • modified antibody molecule that is modified by the addition of one or more amino acids or other molecules to facilitate coupling the antibodies to another peptide or polypeptide, to a large carrier protein or to a solid support (e.g.
  • antisense oligonucleotides we mean single-stranded nucleic acids, which can specifically bind to a complementary nucleic acid sequence. By binding to the appropriate target sequence, an RNA-RNA, a DNA-DNA, or RNA-DNA duplex is formed. These nucleic acids are often termed “antisense” because they are complementary to the sense or coding strand of the gene. Recently, formation of a triple helix has proven possible where the oligonucleotide is bound to a DNA duplex. It was found that oligonucleotides could recognise sequences in the major groove of the DNA double helix. A triple helix was formed thereby. This suggests that it is possible to synthesise sequence-specific molecules which specifically bind double-stranded DNA via recognition of major groove hydrogen binding sites.
  • oligonucleotides By binding to the target nucleic acid, tlie above oligonucleotides can inhibit the function of the target nucleic acid. This could, for example, be a result of blocking the transcription, processing, poly (A) addition, replication, translation, or promoting inhibitory mechanisms of the cells, such as promoting RNA degradations.
  • Antisense oligonucleotides are prepared in the laboratory and then introduced into cells, for example by microinjection or uptake from the cell culture medium into the cells, or they are expressed in cells after rransfection with plasmids or retroviruses or other vectors carrying an antisense gene.
  • a compound of the second aspect of the invention for use in the treahnent and/or prevention and/or diagnosis of a fibrotic disease.
  • the compound is used in the manufacture of a medicament for the treatment and/or prevention and/or diagnosis of a fibrotic disease.
  • the fibrotic disease is one selected from diabetic nephropathy, non-diabetic kidney failure, lung fibrosis, liver fibrosis (cirrhosis), skeletal muscle fibrosis, cardiac muscle fibrosis, atherosclerosis, systemic sclerosis, scleroderrna, retinal fibrosis, radiation fibrosis, keloid scar formation and cancer-associated fibrosis
  • the disease is diabetic nephropathy.
  • a fourth aspect of the invention there is provided a method of treating and/or preventing fibrotic disease comprising amninistering a therapeutically or prophylactically effective dose, or plurality of doses, of a compound identified and/or made according to the method of the first aspect of the invention.
  • the fib otic disease is one selected from diabetic nephropathy, non-diabetic kidney fibrosis, lung fibrosis, liver fibrosis (cirrhosis), skeletal muscle fibrosis, cardiac muscle fibrosis, atherosclerosis, systemic sclerosis, scleroderma, retinal fibrosis, radiation fibrosis, keloid scar formation and cancer-associated fibrosis.
  • the disease is diabetic nephropathy.
  • HMC Serum-starved human mesangial cells
  • Serum-starved HMC were exposed to rCTGF/V5 fusion protein for different periods of time, after which cell lysates were prepared and the TIEG and ⁇ -actin levels analysed by Western blotting. A representative blot of the three independent experiments (three replicate cultures per condition per experiment) that were performed is shown.
  • Figure 4 TIEG mediates CTGF-dependent down-regulation of Smad 7 expression level.
  • Serum-starved HMC were exposed to tlie conditions indicated in the figure. After 24 h, cell lysates were prepared, and the TIEG, Smad 7, and ⁇ -actin levels analysed by Western blotting. A representative blot of the three independent experiments (three rephcate cultures per condition per experiment) that were performed is shown.
  • HMC Human mesangial cells
  • TGF- ⁇ inducible early gene (TLEG) antibodies were a gift from Dr. Steven Johnson (Mayo Foundation, Minnesota, USA).
  • Recombinant CTGF was expressed in transformed HMC and purified from the medium using Talon metal affinity resin, as reported previously (Wahab et al (2001) Biochem J. 359 pp. 77-87).
  • TGF- ⁇ was purchased from R & D Systems (Abingdon, Oxfordshire, U.K.).
  • Cells were lysed in reducing SDS-PAGE loading buffer and immediately scraped off the plate. Cell lysates were sonicated for 10 sec to shear DNA. Samples were boiled for 5 min and resolved on 4-12% gradient gels by SDS-PAGE. Proteins were transferred onto a polyvinyiidene difluoride membrane filter (Irmnobilin-P, MiUipore, Bedford, U.K.) ushig a BioRad transfer apparatus. Blots were incubated in blocking buffer containing Ix TBS, 0.1% Tween-20 with 5% (w/v) non-fat dry milk, for 1 hour.
  • Ix TBS 0.1% Tween-20 with 5% (w/v) non-fat dry milk
  • Immunodetection was performed by incubating the blots in primary antibody at the appropriate dilution in antibody dilution buffer (lx TBS, 0.1% Tween-20 with 5% BSA), overnight at 4°C. Blots were then washed 3 times with washing buffer (lx TBS, 0.1% Tween-20) and incubated with secondary horseradish peroxidase (HRP)-conjugated antibodies for 1 hour at room temperature. Bound antibodies were visualised ushig the enhanced chemiluiniiiescence reagent Luniinol (Autogen Bioclear UK Ltd, Wiltslrire, UK). Pre- stained molecular weight standards (Amersham International PLC, Amersham, UK) were used to monitor protein migration.
  • EDTA 1 mM EGTA, 0.2 mM TLCK, 1 mM N-ethyhnaleimide, 0.1 mM TPCK, and 2 nM PMSF, Sigma), 1 mM NaF, 1 mM Na 3 V0 4 ].
  • Nonidet P40 was added to a final concentration of 0.6% (v/v) and vigorously vortex-mixed for 10 seconds.
  • the nuclei were pelleted at 4°C by centrifugation for 5 min at 12000 x g.
  • the nuclear pellet was washed once with buffer A and coUected by centrifugation.
  • the pellet was then re-suspended in 500 ⁇ l of buffer B (10 rnM Hepes, pH 7.4, 1.5 mM MgC12, 450 mM NaCl, lx protease inhibitors cocktail, 1 mM NaF, ImM Na3V04, 20% (v/v) glycerol) and vortex-mixed for 15 min at 4°C.
  • the lysate was centrifuged at 12000 x g for 5 min at 4°C and the supernatant containing the nuclear proteins transferred to a fresh vial. Protein concentration was measured by Bradford assay. Extracts were stored at -70°C until further use.
  • TIEG and CTGF antisense oligonucleotide (2 ⁇ M) or a CG-matched randomised sequence oligonucleotide (negative control) was added directly to cultures 30 min prior any other treatments (Wahab et al (2002) J Am Soc Nephrol 13 pp .2437-2445).
  • the SBE4-luc reporter gene construct was transfected into 25 x 10 6 transformed human mesangial cells (THMC), by electroporation, at a concentration of 15 ⁇ g together with 5 ⁇ g of pSV- ⁇ -galactosidase control Vector (Promega, Southampton, UK), using conditions described previously (Wahab et al (2001) Biochem J. 359 pp. 77-87).
  • Equal amounts (0.5 ⁇ l) of the reverse transcription reaction (20 ⁇ l) were subjected to PCR amplification hi a 100 ⁇ l volume containing 10 ⁇ l of 10 x PCR buffer, 16 ⁇ l dNTPs (1.25 mM each), 2 mM MgC12, 0.5 ⁇ M of each specific primer and 1.25 U Amplitaq DNA polymerase (Gibco BRL).
  • Amplification was started with 5 min of denaturation at 94°C, followed by 5 27 PCR cycles for all genes except the housekeeping gene glyceraldehyde- 3-phosphate dehydrogenase (GAPDH) where 24 PCR cycles were used. Each cycle consisted of 60 seconds at 94°C, 60 seconds at 55°C and 60 seconds at 72°C The final extension was for 10 min at 72°C. GAPDH was co-amplified to aUow semi-quantitative comparison of PCR products and to l o confirm equivalent use of total RNAs .
  • GAPDH housekeeping gene glyceraldehyde- 3-phosphate dehydrogenase
  • the amount of reverse transcription reaction used for the amplification (0.5 ⁇ l) was selected as being non-saturating for the PCR product of all genes under investigation after the stated number of amplification cycles.
  • the 15 sequences of primers were designed from the published sequences of the human genes and are hsted in Table 1.
  • CTGF effect of CTGF on the activation of receptor-regulated Smads (Smad 2 and Smad 3) was investigated by incubating serum-starved HMC in the presence or absence of CTGF, TGF- ⁇ , or in the presence of both CTGF and TGF- ⁇ for 2 hours.
  • TGF- ⁇ -enhanced activity was markedly reduced in the presence of either CTGF or TIEG antisense (3-2 fold) but not with the control oligonucleotides.
  • the antisense, but not the control oligonucleotides also markedly reduced the reporter activity when both TGF- ⁇ and CTGF were present together.
  • CTGF-dependent down-regulation of Smad 7 was also studied h relation to the upregulation of the TGF- ⁇ -responsive genes, pl5INK4, PAI-1, and collagen III. Semrn-starved cells were incubated for 48 hours in the presence of different combinations of CTGF, TGF- ⁇ , CTGF+TGF- ⁇ , CTGF+TIEG antisense and control oligonucleotides and either TGF- ⁇ +TGF antisense and control oligonucleotides or TIEG antisense and control oligonucleotides. RNA was extracted and used for semi-quantitative RT-PCR analysis. Figures 6A and B demonstrate that CTGF resulted h a 60% reduction of Smad 7 expression level. However, CTGF has no significant effect on either PAI-1 or collagen III gene transcription. In contrast, pl5 expression level appears to be enhanced (5-fold) under the same conditions.
  • TGF- ⁇ resulted in induction of transcription of all four genes as they contain one or more SBE in their promoters.
  • CTGF together with TGF- ⁇ , resulted in reduction of Smad 7 (55%) and maximal induction of pi 5, PAI-1 and collagen III transcription.
  • TGF- ⁇ activity is limited by the negative feedback loop of the signalling pathway, provided by Smad 7.
  • CTGF blocks this negative feedback loop by inhibition of Smad 7 expression via TIEG, allowing continued activation of the TGF- ⁇ signalling pathway.
  • Example 2 Screening method for identifying compounds inhibitin CTGF induced fibrosis
  • Screening for compounds possessing fibrosis inhibitory properties is conducted by testing the ability of each compound to block, for example, the induction of TIEG hi HMC treated with CTGF.
  • the screening method is conducted using human mesangial cells (HMC) pre-incubated for 30 lninutes with or without the potential inhibitor. These cells are then stimulated with CTGF-V5 fusion protein (40 ng/ml) in the presence or absence of the potential inhibitor for 2 hours. After washing the cell layer with cold PBS, the cells are lysed in RIP A buffer and the lysate assayed for TIEG by ELISA.
  • NUNC microtifre plates are coated overnight at 4°C with either lysate or with standard dilutions of r-TIEG to provide a standard curve.
  • Recombinant TIEG protein is created from full length TIEG cDNA by cloning into the PcDNA 3.1/V5-His Topo vector (InVitrogen). This vector can be transfected hito a mammalian cell line to express TIEG-fusion protein.
  • the TIEG fusion protein is purified from cell lysates using probond nickel- chelating resin.
  • Anti-TIEG antibody is available from Dr. Steven Johnson (Mayo Foundation, Minnesota, USA) or can be raised in rabbits against the TIEG fusion protein using conventional methods.
  • Example 3 Pharmaceutical formulations and administration.
  • the compounds of the invention wUl normally be adrninistered orally or by any parenteral route, in the form of a pharmaceutical formulation comprising the active ingredient, optionally in the form of a non-toxic organic, or inorganic, acid, or base, addition salt, in a pharmaceutically acceptable dosage fo ⁇ n.
  • the compositions may be administered at varying doses.
  • the compounds of the invention can be adnhnistered alone but will generally be administered in admixture with a suitable pharmaceutical excipient diluent or carrier selected with regard to the intended route of administration and standard pharmaceutical practice.
  • the compounds of the invention can be administered orally, buccaUy or sublingually in the form of tablets, capsules, ovules, elixhs, solutions or suspensions, which may contain flavouring or colouring agents, for immediate-, delayed- or controlled-release applications.
  • the compounds of the invention may also be a ⁇ ninistered via intracave ⁇ iosal injection.
  • Such tablets may contain excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine, disintegrants such as starch (preferably com, potato or tapioca starch), sodium starch glycollate, croscarmellose sodium and certain complex silicates, and granulation bhiders such as polyvinylpyrxolidone, hydroxypropylmethylcellulose (HPMC), hydroxy-propylcellulose (HPC), sucrose, gelatin and acacia. Additionally, lubricating agents such as ' magnesium stearate, stearic acid, glyceryl behenate and talc may be included.
  • excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine
  • disintegrants such as starch (preferably com, potato or tapioca starch), sodium starch glycollate, croscarmellose sodium and certain complex si
  • Solid compositions of a sinrUar type may also be employed as fillers in gelatin capsules.
  • Preferred excipients hi this regard include lactose, starch, a cellulose, milk sugar or high molecular weight polyethylene glycols.
  • the compounds of the invention may be combined with various sweetening or flavouring agents, colouring matter or dyes, with emulsifying and/or suspending agents and with diluents such as water, ethanol, propylene glycol and glycerin, and combinations thereof.
  • the compounds of the invention can also be adniinistered parenteraUy, for example, intravenously, intra-arterially, intraperitoneally, intrathecaUy, intraventricularly, intrastemally, infracranially, intra-muscularly or subcutaneously, or they may be administered by infusion techniques. They are best used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood.
  • the aqueous solutions should be suitably buffered (preferably to a pH of from 3 to 9), if necessary.
  • the preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well-known to those skilled hi the art.
  • Formulations suitable for parenteral amnmisfration hi include aqueous and non- aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the ⁇ itended recipient; and aqueous and non-aqueous sterile suspensions
  • the formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, hnmediately prior to use.
  • sterile liquid carrier for example water for injections, hnmediately prior to use.
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
  • the daily dosage level of the compounds of the mvention will usually be from 1 mg/kg to 30 mg/kg.
  • the tablets or capsules of the compound of the invention may contain a dose of active compound for administration singly or two or more at a time, as appropriate.
  • the physician in any event will determine the actual dosage which will be most suitable for any individual patient and it will vary with the age, weight and response of the particular patient.
  • the above dosages are exemplary of the average case. There can, of course, be individual instances where higher or lower dosage ranges are merited and such are within the scope of this invention.
  • the compounds of the invention can also be adnhnistered hitranasally or by hihalation and are conveniently delivered in the form of a dry powder inhaler or an aerosol spray presentation from a pressurised contahier, pump, spray or nebuliser with the use of a suitable propellant, e.g. dichlorodifluoroniethane, trichlorofluoromethane, dichlorotetrafluoro- ethane, a hydrofluoraalkane such as 1,1,1,2-tetrafluoroethane (HFA 134A3 or 1,1,1,2,3,3,3-heptafluoropiOpane (HFA 227EA3), carbon dioxide or other suitable gas.
  • a suitable propellant e.g. dichlorodifluoroniethane, trichlorofluoromethane, dichlorotetrafluoro- ethane, a hydrofluoraalkane such as 1,1,1,2-tetrafluoro
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • the pressurised container, pump, spray or nebuliser may contain a solution or suspension of the active compound, e.g. ushig a mixture of ethanol and the propellant as the solvent, which may additionally contain a lubricant, e.g. sorbitan trioleate.
  • Capsules and cartridges (made, for example, from gelatin) for use in an inhaler or insufflator may be formulated to contain a powder mix of a compound of the mvention and a suitable powder base such as lactose or starch.
  • Aerosol or dry powder formulations are preferably arranged so that each metered dose or "puff delivers an appropriate dose of a compound of t he invention for delivery to the patient. It will be appreciated that the overall dahy dose with an aerosol wiU vary from patient to patient, and may be administered in a single dose or, more usually, in divided doses throughout the day.
  • the compounds of the invention can be administered in the form of a suppository or pessary, or they may be applied topicaUy in the form of a lotion, solution, cream, ointment or dusting powder.
  • the compounds of the invention may also be transdermally adrninistered, for example, by the use of a skin patch. They may also be a ⁇ ninistered by the ocular route, particularly for treating diseases of the eye.
  • the compounds of the invention can be formulated as micronised suspensions in isotonic, pH adjusted, sterile saline, or, preferably, as solutions hi isotonic, pH adjusted, sterile saline, optionally in combination with a preservative such as a benzylalkonium chloride. Alternatively, they may be formulated in an ointment such as petrolatum.
  • the compounds of the mvention can be formulated as a suitable ointment containing the active compound n o suspended or dissolved in, for example, a mixture with one or more of the following: mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene polyox ⁇ ropylene compound, emulsifying wax and water.
  • a suitable lotion or cream suspended or dissolved hi, for example, a mixture of one or more of the following: mineral oil, sorbitan monostearate, a polyethylene glycol, liquid paraffin, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2- octyldodecanol, benzyl alcohol and water.
  • Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavoured basis, usuaUy sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouth-washes comprising the active ingredient in a suitable liquid carrier.
  • oral or topical administration of the compounds of the invention is the preferred route, being the most convenient.
  • the drug may be administered parenteraUy, e.g. sublingually or buccaUy.
  • a compound of the invention is administered as a suitably acceptable formulation in accordance with normal veterinary practice and the veterinary surgeon will detemih e the dosing regimen and route of administration which will be most appropriate for a particular animal.

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Abstract

L'invention porte sur un procédé d'identification et/ou de fabrication de composés destinés à être utilisés pour réduire et/ou prévenir la fibrose, ce procédé consistant à: produire un type de cellule capable d'exprimer TIEG et/ou Smad-7; produire un composé de test; produire une quantité de CTGF ou de son équivalent fonctionnel, exposer le type de cellule au composé de test; exposer ultérieurement ou simultanément le type de cellule à CTGF ou à son équivalent fonctionnel; détecter et/ou mesurer l'expression de Smad-7 et TIEG; et comparer la quantité de Smad-7 et/ou TIEG exprimée en présence du composé de test avec la quantité de Smad-7 et/ou TIEG produite détectée et/ou mesurée en l'absence d'un composé de test; déterminer si un composé réduit et/ou prévient la fibrose en fonction d'une non modification ou d'une augmentation dans l'expression de Smad-7 et/ou d'une non modification ou d'une réduction dans l'expression de TIEG. L'invention porte également sur des composés permettant de réduire et/ou prévenir la fibrose et sur leurs utilisations.
EP04798502A 2003-11-18 2004-11-12 Substances biologiques et leurs utilisations Withdrawn EP1690098A2 (fr)

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GBGB0326778.8A GB0326778D0 (en) 2003-11-18 2003-11-18 Biological materials and uses thereof
PCT/GB2004/004781 WO2005050202A2 (fr) 2003-11-18 2004-11-12 Substances biologiques et leurs utilisations

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US8946172B2 (en) 2008-08-25 2015-02-03 Excaliard Pharmaceuticals, Inc. Method for reducing scarring during wound healing using antisense compounds directed to CTGF
KR101762734B1 (ko) * 2008-08-25 2017-07-28 엑스칼리아드 파마슈티컬즈, 인코포레이티드 결합 조직 성장 인자에 대한 안티센스 올리고뉴클레오타이드 및 그의 용도
CA2825059A1 (fr) 2011-02-02 2012-08-09 Excaliard Pharmaceuticals, Inc. Procede de traitement de cheloides ou de cicatrices hypertrophiees a l'aide de composes antisens ciblant un facteur de croissance de tissu conjonctif (ctgf)
DK2839004T3 (da) * 2012-04-18 2019-07-01 Nogra Pharma Ltd Fremgangsmåde til at behandle diabetes og/eller fremme overlevelse af langerhanske øer efter transplantation
WO2022241762A1 (fr) * 2021-05-21 2022-11-24 江苏九济华生医药科技研究院有限公司 SOUCHE CELLULAIRE POUR LA DÉTECTION DU TGFβ ET PROCÉDÉ DE DÉTECTION DU TGFβ DE HAUTE PRÉCISION

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CA2354422A1 (fr) * 1998-12-14 2000-06-22 University Of Miami Fragments de facteur de croissance du tissu conjonctif, et methodes et utilisations correspondantes
WO2001015729A1 (fr) * 1999-08-27 2001-03-08 Fibrogen, Inc. Recepteur du facteur de croissance du tissu conjonctif, agonistes et antagonistes dudit recepteur, et leurs utilisations therapeutiques et diagnostiques
GB0326780D0 (en) * 2003-11-18 2003-12-24 Imp College Innovations Ltd Biological materials and uses thereof

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CA2546397A1 (fr) 2005-06-02
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GB0326778D0 (en) 2003-12-24
WO2005050202A3 (fr) 2005-11-03
AU2004292012A1 (en) 2005-06-02
WO2005050202A2 (fr) 2005-06-02

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