EP1066323A1 - Behandlung und verhinderung von vaskulären krankheiten - Google Patents

Behandlung und verhinderung von vaskulären krankheiten

Info

Publication number
EP1066323A1
EP1066323A1 EP99915041A EP99915041A EP1066323A1 EP 1066323 A1 EP1066323 A1 EP 1066323A1 EP 99915041 A EP99915041 A EP 99915041A EP 99915041 A EP99915041 A EP 99915041A EP 1066323 A1 EP1066323 A1 EP 1066323A1
Authority
EP
European Patent Office
Prior art keywords
seq
tgfβ
nucleic acid
group
activity
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP99915041A
Other languages
English (en)
French (fr)
Inventor
Brian William Grinnell
Laura Jean Myers
Mark Alan Richardson
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Eli Lilly and Co
Original Assignee
Eli Lilly and Co
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Eli Lilly and Co filed Critical Eli Lilly and Co
Publication of EP1066323A1 publication Critical patent/EP1066323A1/de
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/495—Transforming growth factor [TGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the vascular endothelium constitutes a major organ that functions as a regulator of blood coagulation, inflammation, and in the exchange of fluids and mediators between the intravascular compartment and parenchyma tissues. As such, the proper function of the endothelium is critical to overall homeostasis.
  • a dysfunction of the endothelium resulting from an alteration in the expression of important surface molecules can result in coagulation defects, local and systemic vascular inflammation, and enhancement in the progression and rupture of atherosclerotic plaque. These effects can further result in conditions including myocardial infarction, deep venous thrombosis, disseminated intravascular thrombosis, and stroke .
  • Certain cell surface proteins are altered in response to a vascular injury or insult and can be used as markers of a dysfunctional endothelium. Two such factors are plasminogen activator inhibitor 1 (PAI-1) and thrombomodulin (TM)
  • PAI-1 plays a critical role in the fibrinolytic system by reducing the endogenous ability to remove fibrin by inhibiting plasminogen activators such as tissue type plasminogen activator (tPA) .
  • plasminogen activators such as tissue type plasminogen activator (tPA) .
  • tPA tissue type plasminogen activator
  • PAI-1 can be produced in a variety of tissues, substantial levels are secreted by the vascular endothelial cell. Since PAI-1 can be increased in - 2
  • endothelial cells in response to certain stimuli, including cytokines, it contributes to dysfunction of the endothelium and the attendant problems.
  • TM plays a critical role in maintaining vessel anticoagulant activity as a cofactor for thrombin-catalyzed activation of protein C, the major endogenous antithrombotic factor.
  • protein C the major endogenous antithrombotic factor.
  • the surface anticoagulant responses can be impaired in states of endothelial dysfunction.
  • TM levels become suppressed in cytokine activated endothelium.
  • TGF ⁇ transforming growth factor beta
  • Smads family of related gene products that function downstream of the receptors of the TGF ⁇ family. Upon activation, several of the Smads translocate directly to the nucleus where they activate transcription. The Smads have been implicated in the regulation of cell growth and proliferation, development and differentiation of cell types, and in endothelial cell response to shear stress.
  • Smadl is a bone morphogenetic protein (BMP) signal transducer
  • Smad2 and Smad3 are TGF ⁇ and activin signal transducers
  • Smad4 also known as DPC4
  • Smad6 SEQ ID NO: 2
  • Smad7 SEQ ID NO: 4
  • Smad7 directly interferes with TGF ⁇ mediated activation of Smad2 by preventing phosphorylation, interaction with Smad4, and nuclear accumulation.
  • proteins such as Smad6 and Smad7, which molecules can be used to treat or affect cells, tissues, animals (e.g., humans) for various diseases or pathologies affected by such proteins .
  • Smad6 and Smad7 polypeptides or agents that modulate their expression and/or activity in the endothelium can be used to treat conditions such as, but not limited to, sepsis, injuries involving major tissue damage and trauma, systemic inflammatory response syndrome, sepsis syndrome, septic shock and multiple organ dysfunction syndrome (including DIC) , as well as myocardial infarction, deep venous thrombosis, disseminated intravascular thrombosis, atherosclerotic plaque rupture, and associated sequela.
  • Smad6 and Smad7 polypeptides or other molecules that modulate their expression and/or activity can be used to prevent stroke. Further, because of the critical role of fibrin in tumor cell biology, Smad6 and Smad7 polypeptides or molecules that modulate their expression and/or activity can be used as anti-metastatic agents.
  • Smad6 polypeptide has been determined to increase expression of TGF ⁇ .
  • Smad6-induced TGF ⁇ expression can be inhibited by inhibitors of protein kinase C (PKC) .
  • PDC protein kinase C
  • the current invention provides methods for treating or preventing at least one aspect of at least one vascular disease; the method comprising administering to a patient in • 4 -
  • TGF ⁇ regulatable activities include but are not limited to an induction of plasminogen activator inhibitor- 1 expression, an increase in plasminogen activator inhibitor- 1 secretion, a suppression of thrombomodulin activity, an increase in TGF ⁇ secretion, and a decrease in TGF ⁇ secretion.
  • the current invention provides in one aspect a method for treating or preventing a disease in which TGF ⁇ is responsible for inducing cellular effects that lead to at least one disease state; the method comprising administering to a patient in need of such treatment, a compound that modulates expression of, or the activity of the protein product encoded by a nucleic acid molecule selected from the group consisting of SEQ ID NO:l (encoding at least one Smad6 protein), SEQ ID NO: 3 (encoding at least one Smad7 protein) , and a nucleic acid molecule that is complementary to SEQ ID NO : 1 or 3.
  • the current invention also provides methods for identifying compounds that modulate anticoagulant and fibrinolytic functions in the vasculature endothelium; the method comprising contacting at least one cell or an organism with a compound which modulates expression or activity of PAI-I and TM.
  • methods for identifying compounds such as polypeptides or organic molecules, that modulate the expression or activity of Smad6 or Smad7 (e.g., proteins or polypeptides encoded by SEQ ID NO : 1 and 2 , respectively) , which in turn modulate expression or activity of anticoagulant and fibrinolytic functions, namely PAI-I and TM, in the vasculature endothelium.
  • the present invention also provides a method for modulating TGF ⁇ regulatable activities comprising administering to a patient in need of such treatment, a • 5-
  • Preferred protein products for use in the current invention include SEQ ID NO: 2 and SEQ ID NO: 4.
  • a nucleic acid molecule having at least 70% identity to the specified sequences are preferred for use in the present invention; identity of at least 95% is especially preferred.
  • Preferred TGF ⁇ regulatable activities include but are not limited to an induction of plasminogen activator inhibitor-1 expression, an increase in plasminogen activator inhibitor-1 secretion, a suppression of thrombomodulin activity, an increase in TGF ⁇ secretion, and a decrease in TGF ⁇ secretion.
  • the present invention also provides a method for modulating TGF ⁇ regulatable activities comprising administering to a cell or cells, a protein product encoded by a nucleic acid molecule having at least 70, 75, 80, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to a member selected from the group consisting of at least 30 nucleotides of at least one of SEQ ID NO:l, SEQ ID NO: 3, and a nucleic acid molecule that is complementary to SEQ ID NO : 1 or SEQ ID NO : 3.
  • Preferred protein products for use in the current invention include SEQ ID NO : 2 and SEQ ID NO : 4.
  • TGF ⁇ regulatable activities include but are not limited to an induction of plasminogen activator inhibitor-1 expression, an increase in plasminogen activator inhibitor-1 secretion, a suppression of thrombomodulin activity, an increase in TGF ⁇ secretion, and a decrease in TGF ⁇ secretion.
  • the present invention relates to a pharmaceutical formulation
  • a pharmaceutical formulation comprising as an active ingredient a Smad6 or Smad7 polypeptide, associated with one or more pharmaceutically acceptable carriers, excipients, or diluents thereof.
  • Preferred protein products for use in the current invention include SEQ ID NO: 2 and SEQ ID NO: 4.
  • the current invention also provides a method for modulating TGF ⁇ regulatable activities comprising administering to at least one cell, an organism, or a patient in need of such treatment, an antisense nucleic acid molecule having a nucleotide sequence complementary to a contiguous sequence of mRNA transcribed from a gene selected from the group consisting of SEQ ID NO:l and SEQ ID NO: 3, wherein said antisense nucleic acid molecule hybridizes to said contiguous sequence such that translation of said mRNA is inhibited. It is preferred if the contiguous sequence includes at least fifteen nucleotides.
  • Preferred nucleic acid molecules for use in the current invention include SEQ ID NO: 5 and SEQ ID NO: 7.
  • Preferred TGF ⁇ regulatable activities include but are not limited to an induction of plasminogen activator inhibitor-1 expression, an increase in plasminogen activator inhibitor-1 secretion, a suppression of thrombomodulin activity, an increase in TGF ⁇ secretion, and a decrease in TGF ⁇ secretion.
  • the invention also provides methods for the identification of compounds that modulate a TGF ⁇ regulatable activity comprising administering to at least one cell or an organism a compound that modulates expression of, or the activity of the protein product of, a nucleic acid molecule selected from the group consisting of SEQ ID NO:l, SEQ ID NO: 3, and fragments thereof.
  • Preferred TGF ⁇ regulatable activities include but are not limited to an induction of - 7 -
  • the present invention also provides methods for the identification of compounds that modulate a TGF ⁇ regulatable activity comprising administering to at least one cell, or an organism a compound that results in an induction of plasminogen activator inhibitor-1 expression, an increase in plasminogen activator inhibitor-1 secretion, a suppression of thrombomodulin activity, an increase in TGF ⁇ secretion, or a decrease in TGF ⁇ secretion.
  • complementary refers to the capacity of purine and pyrimidine nucleotides to associate through hydrogen bonding to form double stranded nucleic acid molecules.
  • the following base pairs are related by complementarity: guanine and cytosine; adenine and thymine; and adenine and uracil.
  • complementary means that the aforementioned relationship applies to substantially all base pairs comprising two single-stranded nucleic acid molecules over the entire length of said molecules.
  • Partially complementary refers to the aforementioned relationship in which one of two single-stranded nucleic acid molecules is shorter in length than the other such that a portion of one of the molecules remains single-stranded.
  • conservative substitution or “conservative amino acid substitution” refers to a replacement of one or more amino acid residue (s) in a parent polypeptide as stipulated by Table 1.
  • fragment thereof refers to a fragment, piece, or sub-region of a nucleic acid or polypeptide molecule whose sequence is disclosed herein, such that the fragment comprises 5 or more amino acids, or 10 or more nucleotides that are contiguous in the parent polypeptide or nucleic acid molecule. "Fragment thereof” may or may not retain biological activity. A fragment of a polypeptide disclosed herein could be used as an antigen to raise a specific antibody against the parent polypeptide molecule.
  • fragment thereof refers to 10 or more contiguous nucleotides, derived from a parent nucleic acid.
  • the term also encompasses the complementary sequence. For example if the fragment entails the sequence 5' -AGCTAG-3 ' , then “fragment thereof” would also include the complementary sequence, 3'- TCGATC-5' .
  • functional fragment refers to a region, or fragment of a full length polypeptide, or sequence of amino acids that, for example, comprises an active site, or any other motif, relating to biological function. Functional fragments are capable of providing a substantially similar biological activity as a polypeptide disclosed herein, in vivo or in vitro, viz . the capacity to modulate cell proliferation. Functional fragments may be produced by cloning technology, or as the natural products of alternative splicing mechanisms.
  • polypeptides that are related to the Smad6 or Smad7 polypeptides used in the present invention, said functionally related polypeptides constituting modifications of said Smad6 or Smad7 polypeptides, in which conservative amino acid changes are present as natural polymorphic variants of the polypeptides disclosed herein.
  • Conservative amino acid substitutions and modifications may also be - 9 -
  • N in a nucleic acid sequence refers to adenine ("A”), guanine (“G”), cytosine ("C”), thymine (T”), or uracil ( "U” ) ; "Z” designates an unknown amino acid residue .
  • plasmid refers to an extrachromosomal genetic element.
  • the plasmids disclosed herein are commercially available, publicly available on an unrestricted basis, or can be constructed from readily available plasmids in accordance with published procedures.
  • a “primer” is a nucleic acid fragment which functions as an initiating substrate for enzymatic or synthetic elongation of, for example, a nucleic acid molecule .
  • promoter refers to a nucleic acid sequence that directs transcription, for example, of DNA to RNA.
  • An inducible promoter is one that is regulatable by environmental signals, such as carbon source, heat, or metal ions, for example.
  • a constitutive promoter generally operates at a constant level and is not regulatable .
  • protein and “polypeptide” are used interchangeably herein and are intended to mean a biopolymer comprising a plurality of amino acid residues covalently bound in peptide linkage .
  • recombinant DNA cloning vector refers to any autonomously replicating agent, including, but not limited to, plasmids and phages, comprising a DNA molecule to which one or more additional DNA segments can or have been incorporated.
  • recombinant DNA expression vector or "expression vector” as used herein refers to any recombinant DNA cloning vector, for example a plasmid or phage, in which ⁇ 10 -
  • a promoter and other regulatory elements are present thereby enabling transcription of an inserted DNA, which may encode a protein.
  • substantially pure used in reference to a polypeptide, means substantial separation from other cellular and non-cellular molecules, including other protein molecules.
  • a substantially pure preparation would be about at least 85% pure; preferably about at least 95% pure.
  • a "substantially pure” protein can be prepared by a variety of techniques, well known to the skilled artisan, including, for example, the IMAC protein purification method.
  • vector refers to a nucleic acid compound used for introducing exogenous or endogenous DNA into host cells.
  • a vector comprises a nucleotide sequence which may encode one or more protein molecules. Plasmids, cosmids, viruses, and bacteriophages, in the natural state or which have undergone recombinant engineering, are examples of commonly used vectors.
  • nucleic acid molecule as set forth in SEQ ID NO:l encodes a polypeptide (SEQ ID NO: 2) that mediates the intracellular signal induced by the binding of TGF ⁇ to specific cell surface receptors.
  • the current invention arises from the discovery that Smad6 and Smad7 mediate the expression of anticoagulant and fibrinolytic functions in the vascular endothelium, as well as modulation of TGF ⁇ secretion. Specifically, the over-expression of Smad6 increases the secretion of PAI-1 - 11 -
  • TM measured by the ability of cells to support thrombin-mediated activation of protein C
  • Smad6 inhibites the expression of TM (measured by the ability of cells to support thrombin-mediated activation of protein C) , similar to the direct affect of TGF ⁇ itself.
  • the inhibition of Smad6 by antisense decreases TGF ⁇ -induced PAI-1 levels/activity and increases TGF ⁇ - induced suppression of TM.
  • the effects of Smad6 on these coagulation markers can be antagonized by Smad7.
  • Smad6 acts as a feedback amplifier of the pathway by up-regulating the secretion of TGF ⁇ l and TGF ⁇ 3. Protein kinase C and inhibitors thereof also modulate the activity of Smad6.
  • Other effects of inhibiting Smad6 expression and activity include blocking suppression of thrombomodulin, which in turn leads to an increase in activated protein C.
  • Smad6 is predominately expressed in atherosclerotic lesions, but not in the normal endothelium, whereas Smad7 is predominately expressed in the normal endothelium but not in atherosclerotic lesions. Strikingly, Smad6 is also highly over-expressed in diseased heart, where TGF ⁇ is known to play a critical role in congestive heart failure .
  • Smad7 inhibits type I receptor kinase signaling by acting as intracellular antagonist of the type I receptor domain. Smad7 interacts directly with the activated type I receptor, thereby blocking Smad2 phosphorylation, Smad2 association with Smad4 , and nuclear accumulation of the complex.
  • agents that suppress the level and/or function of Smad6, or increase the level and/or function of Smad7 would be useful agents to increase anticoagulant or - 12 -
  • Compounds that modulate expression of, or the activity of the protein product of, a nucleic acid molecule molecule selected from the group consisting of SEQ ID NO:l, SEQ ID NO: 3, and fragments or compounds having at least 90% identity to SEQ ID NO: 2, SEQ ID NO: 4, or fragments thereof can be used for the manufacture of a medicament for the treatment and/or prevention of diseases, including but not limited to, myocardial infarction, congestive heart failure, dilated cardiomyopathy, deep venous thrombosis, disseminated intravascular thrombosis, stroke, sepsis, injuries involving major tissue damage and trauma, systemic inflammatory response syndrome, sepsis syndrome, septic shock, multiple organ dysfunction syndrome (including DIC) , atherosclerotic plaque rupture, and associated sequela arising therefrom.
  • diseases including but not limited to, myocardial infarction, congestive heart failure, dilated cardiomyopathy, deep venous thrombosis, dissemin
  • PAI-1 and TM are particularly useful in the treatment of hypercoagulable states and disseminated intravascular coagulation associated with systemic inflammatory responses, sepsis, burns, obstetrical complications, and trauma.
  • Smad6 or Smad7 polypeptide For therapeutic use in preventing or treating vasculature disease an effective amount of Smad6 or Smad7 polypeptide is administered to an organism in need thereof in a dose between about 0.001 and 10,000 ⁇ g/kg body weight.
  • Smad6 or Smad7 can be administered in a single daily dose or in multiple doses per day. The amount per administration will be determined by the physician and depend on such factors as the nature and - 13 -
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising as the active agent a Smad6 or Smad7 polypeptide or fragment thereof, or a pharmaceutically acceptable non-toxic salt thereof, and a pharmaceutically acceptable solid or liquid carrier.
  • compounds comprising Smad6 or Smad7 can be admixed with conventional pharmaceutical carriers and excipients, and used in the form of tablets, capsules, elixirs, suspensions, syrups, wafers, and the like.
  • the compositions comprising Smad6 or Smad7 will contain from about 0.1% to 90% by weight of the active compound, and more generally from about 10% to 30%.
  • compositions may contain common carriers and excipients such as corn starch or gelatin, lactose, sucrose, microcrystalline cellulose, kaolin, mannitol, dicalcium phosphate, sodium chloride, and alginic acid.
  • the compounds can be formulated for oral or parenteral administration .
  • the Smad6 or Smad7 polypeptide is administered in commonly used intravenous fluid (s) and administered by infusion.
  • Such fluids for example, physiological saline, Ringer's solution or 5% dextrose solution can be used.
  • a sterile formulation preferably a suitable soluble salt form of the Smad6 or Smad7 polypeptide, for example SEQ ID NO: 2 or SEQ ID NO: 4, such as the hydrochloride salt
  • a pharmaceutical diluent such as pyrogen- free water (distilled) , physiological saline or 5% glucose solution.
  • a suitable insoluble form of the compound may be prepared and administered as a suspension in an aqueous base or a pharmaceutically acceptable oil base, e.g. an ester of a long chain fatty acid such as ethyl oleate. - 14 -
  • proteins used in the present invention can be synthesized by a number of different methods, such as chemical methods well known in the art, including solid phase protein synthesis or recombinant methods. Both methods are described in U.S.
  • Patent 4,617,189 entirely incorporated herein by reference.
  • proteins may be synthesized by solid-phase methodology utilizing an Applied Biosystems 43OA protein synthesizer (Applied Biosystems, Foster City, CA) and synthesis cycles supplied by Applied Biosystems.
  • the proteins used in the present invention can also be produced by recombinant DNA methods using a cloned or other Smad6 or Smad7 nucleic acid template . Recombinant methods are preferred if a high yield is desired.
  • Smad6 or Smad7 gene can be carried out in a variety of suitable host cells, well known to those skilled in the art.
  • the Smad6 or Smad7 gene or fragments thereof e.g. SEQ ID NOS: 1 or 3
  • SEQ ID NOS: 1 or 3 can be introduced into host cells by any suitable means well known to those skilled in the art .
  • chromosomal integration of the cloned genes is possible, it is preferred that the gene or fragment thereof be cloned into a suitable extra-chromosomally maintained expression vector so that the coding region of the Smad6 or Smad7 gene is operably-linked to a constitutive or inducible promoter .
  • Smad6 or Smad7 polypeptide are: a) constructing a natural, synthetic or semi-synthetic DNA encoding a Smad6 or Smad7 polypeptide; ⁇ 15 -
  • Procaryotes may be employed in the production of recombinant Smad6 or Smad7 polypeptide.
  • Escherichia coli K12 strain 294 ATCC No. 318466
  • Other strains of E. coli, bacilli such as Bacillus subtilis, enterobacteriaceae such as Salmonella typhimurium or Serratia marcescens , various Pseudomonas species and other bacteria, such as Streptomyces, may also be employed as host cells in the cloning and expression of the recombinant proteins of this invention.
  • Promoter sequences suitable for driving the expression of genes in procaryotes include ⁇ -lactamase (e.g. vector pGX2907 (ATCC 39344) which contains a replicon and ⁇ - lactamase gene), lactose systems (Chang et al . , Nature
  • Hybrid promoters such as the tac promoter (isolatable from plasmid pDR540 (ATCC-37282) ) are also suitable. Still other bacterial promoters, whose nucleotide sequences are generally known, may be ligated to DNA encoding the protein of the instant invention, using linkers or adapters to supply any required restriction sites. Promoters for use in bacterial systems also will contain a Shine-Dalgarno sequence operably-linked to the DNA encoding the desired polyproteins . These examples are illustrative rather than limiting.
  • the proteins used in this invention may be synthesized either by direct expression or as a fusion protein comprising the protein of interest as a translational fusion with another protein or proteins .
  • a glutathione-S-transferase (GST) -Smad6 or Smad7 fusion protein can be synthesized essentially as described in Smith & Johnson, Gene, 61 , 31, 1988, herein incorporated by reference .
  • Fusion partners can be removed by enzymatic or chemical cleavage. It is often observed in the production of certain proteins in recombinant systems that expression as a fusion protein prolongs the lifespan, increases the yield of the desired protein, or provides a convenient means of purifying the protein.
  • amphibian expression systems such as frog oocytes, and mammalian cell systems can be used in the production of recombinant Smad6 or Smad7 polypeptides.
  • the choice of a particular host cell depends to some extent on the particular expression vector used.
  • Exemplary mammalian host cells suitable for use in the present invention include HepG-2 (ATCC HB 8065) , CV-1 (ATCC CCL 70), LC-MK2 (ATCC CCL 7.1), 3T3 (ATCC CCL 92), CHO-Kl (ATCC CCL 61), HeLa (ATCC CCL 2), RPMI8226 (ATCC CCL 155) , H4IIEC3 (ATCC CCL 1600) , C127I (ATCC CCL 1616) , HS- Sultan (ATCC CCL 1884), and BHK-21 (ATCC CCL 10), for example.
  • HepG-2 ATCC HB 8065
  • CV-1 ATCC CCL 70
  • LC-MK2 ATCC CCL 7.1
  • 3T3 ATCC CCL 92
  • CHO-Kl ATCC CCL 61
  • HeLa ATCC CCL 2
  • RPMI8226 ATCC CCL 1505
  • H4IIEC3 ATCC CCL 1600
  • C127I AT
  • the pSV2- type vectors comprise segments of the simian virus 40 (SV40) genome required for transcription and polyadenylation.
  • SV40 simian virus 40
  • a large number of plasmid pSV2-type vectors have been constructed, such as pSV2-gpt, pSV2-neo, pSV2-dhfr, pSV2- hyg, and pSV2- -globin, in which the SV40 promoter drives transcription of an inserted gene.
  • These vectors are widely available from sources such as the American Type Culture Collection (ATCC) , 12301 Parklawn Drive, Rockville, Maryland, 20852, or the Northern Regional Research Laboratory (NRRL) , 1815 N. University Street, Peoria, Illinois, 61604.
  • ATCC American Type Culture Collection
  • NRRL Northern Regional Research Laboratory
  • Promoters suitable for expression in mammalian cells include the SV40 late promoter, promoters from eukaryotic genes, such as, for example, the estrogen- inducible chicken ovalbumin gene, the interferon genes, the glucocorticoid-inducible tyrosine aminotransferase gene, the thymidine kinase gene promoter, and the promoters of the major early and late adenovirus genes.
  • eukaryotic genes such as, for example, the estrogen- inducible chicken ovalbumin gene, the interferon genes, the glucocorticoid-inducible tyrosine aminotransferase gene, the thymidine kinase gene promoter, and the promoters of the major early and late adenovirus genes.
  • eukaryotic genes such as, for example, the estrogen- inducible chicken ovalbumin gene, the interferon genes, the glucocorticoid-inducible tyros
  • Plasmid pRSVcat (ATCC 37152) comprises portions of a long terminal repeat of the Rous Sarcoma virus, a virus known to infect chickens and other host cells .
  • This long terminal repeat contains a promoter which is suitable for use in the vectors of this invention.
  • the plasmid pMSVi (NRRL B-15929) comprises the long terminal repeats of the Murine Sarcoma virus, a virus known to infect mouse and other host cells.
  • the mouse metallothionein promoter has also been well characterized for use in eukaryotic host cells and is suitable for use in the present invention. This promoter is present in the plasmid pdBPV-MMTneo (ATCC 37224) which can serve as the starting material for the construction of other plasmids.
  • Transfection of mammalian cells with vectors can be performed by a plurality of well-known processes including, but not limited to, protoplast fusion, calcium phosphate co-precipitation, electroporation and the like. See, e . g. , Maniatis et al . , supra . Some viruses also make appropriate vectors.
  • Examples include the adenoviruses, the adeno-associated viruses, vaccinia viruses, herpes viruses, baculoviruses, and rous sarcoma viruses, as described in U.S. Patent 4,775,624, entirely incorporated herein by reference.
  • Eucaryotic microorganisms such as yeast and other fungi are also suitable host cells.
  • the yeast Saccharomyces cerevisiae is the preferred eucaryotic microorganism.
  • Other yeasts such as Kluyveromyces lactis and Pichia pastor is are also suitable.
  • the plasmid YRp7 ATCC-40053
  • Plasmid YRp7 contains the TRP1 gene which provides a selectable marker for use in a trpl auxotrophic mutant . - 19 -
  • An expression vector carrying a cloned or isolated or endogenous Smad6 or Smad7 genomic or coding sequence or fragment thereof (e.g., SEQ ID NO: 1 or 3) is transformed or transfected into a suitable host cell using standard methods.
  • Cells that contain the vector are propagated under conditions suitable for expression of a recombinant Smad6 or Smad7 polypeptide.
  • suitable growth conditions would incorporate the appropriate inducer.
  • the recombinantly- produced protein may be purified from cellular extracts of transformed cells by any suitable means.
  • a Smad6 or Smad7 gene or fragment thereof is modified at the 5 ' end to incorporate several histidine codons .
  • This modification produces an "histidine tag" at the amino terminus of the encoded protein, that enables a single- or few-step protein purification method [i.e. "immobilized metal ion affinity chromatography” (IMAC)], as described in U.S. Patent 4,569,794, entirely incorporated by reference.
  • IMAC immobilized metal ion affinity chromatography
  • nucleic acids including those disclosed herein, could be generated using a conventional DNA synthesizing apparatus, such as the Applied Biosystems Model 380A or 380B DNA synthesizers (Applied Biosystems, Inc., 850 Lincoln Center Drive, Foster City, CA 94404) using phosphoramidite chemistry, thereafter -20-
  • phosphotriester chemistry may be employed to synthesize the nucleic acids of this invention. ⁇ See, e . g. , M.J. Gait, ed., Oligonucleotide Synthesis, A Practical Approach, (1984) ) .
  • RNA or cDNA preparation e.g., a cDNA library
  • suitable oligonucleotide primers complementary to SEQ ID NO: 1, SEQ ID NO: 3, or to a sub-region therein, for example are prepared as described in U.S. Patent No. 4,889,818, hereby incorporated by reference.
  • Other suitable protocols for the PCR are disclosed in PCR Protocols: A Guide to Method and Applications, Ed. Michael A. Innis et al . , Academic Press, Inc. (1990) .
  • any region of the Smad6 or Smad7 gene(s) can be targeted for amplification such that full or partial length gene sequences may be produced.
  • ribonucleic acids used in the present invention may be prepared using polynucleotide synthetic methods discussed supra, or they may be prepared enzymatically, for example, using RNA polymerase to transcribe a Smad6 or Smad7 DNA template.
  • RNA polymerase from the bacteriophage T7 or the bacteriophage SP6. These RNA polymerases are highly specific, requiring the insertion of bacteriophage-specific sequences at the 5 ' end of the template to be transcribed. See, Maniatis et al . , supra .
  • This invention also requires nucleic acids, RNA or DNA, that are complementary to Smad6 or Smad7 genes or fragments thereof, for example, SEQ ID NOS : 1 or 3. - 21 -
  • nucleic acid vectors are those which comprise DNA, for example, SEQ ID NOS: 1 or 3 or a subregion therein.
  • cloning vector or expression vector depends upon a number of factors including the availability of restriction enzyme sites, the type of host cell into which the vector is to be transfected or transformed, the purpose of the transfection or transformation (e.g., stable transformation as an extrachromosomal element, or integration into the host chromosome) , the presence or absence of readily assayable or selectable markers (e.g., antibiotic resistance and metabolic markers of one type and another) , and the number of copies of the gene desired in the host cell .
  • readily assayable or selectable markers e.g., antibiotic resistance and metabolic markers of one type and another
  • Vectors suitable to carry the nucleic acids used in the present invention comprise RNA viruses, DNA viruses, lytic bacteriophages, lysogenic bacteriophages, stable bacteriophages, plasmids, viroids, and the like.
  • the most preferred vectors are plasmids.
  • an expression vector When preparing an expression vector the skilled artisan understands that there are many variables to be considered, for example, whether to use a constitutive or inducible promoter. The practitioner also understands that the amount of nucleic acid or protein to be produced dictates, in part, the selection of the expression system. Regarding promoter sequences, inducible promoters are preferred because they enable high level, regulatable expression of an operably-linked gene. The skilled artisan will recognize a number of suitable promoters that respond to a variety of inducers, for example, carbon source, metal ions, and heat. Other relevant considerations regarding an expression vector include whether to include sequences for -22 -
  • a sequence encoding a signal protein e.g., SEQ ID NO: 5
  • a sequence encoding a signal protein e.g., SEQ ID NO: 5
  • the present invention may require constructing a recombinant host cell capable of expressing the proteins used in the invention, said method comprising transforming or otherwise introducing into a host cell a recombinant DNA vector that comprises an isolated DNA sequence that encodes a protein of this invention.
  • a suitable host cell is any eucaryotic cell that can accomodate high level expression of an exogenously introduced gene or protein, and that will incorporate said protein into its membrane structure.
  • Vectors for expression are those which comprise a Smad6 or Smad7 gene or fragment thereof, e.g. SEQ ID NO: 1, SEQ ID NO: 3, or suitable subregion therein.
  • Transformed host cells may be cultured under conditions well known to skilled artisans such that a protein used in the present invention is expressed, thereby producing a recombinant Smad6 or Smad7 polypeptide in the recombinant host cell .
  • Embodiments of a Smad6 and Smad7 DNA sequence are disclosed herein by SEQ ID NOS: 1 and 3, respectively.
  • SEQ ID NOS: 1 and 3 SEQ ID NOS: 1 and 3
  • numerous "silent" substitutions of nucleotide base pairs could be introduced into the sequences identified herein without altering the identity of the encoded amino acid(s) or protein or protein product .
  • Amino acids in a Smad6 or Smad7 polypeptide used in the present invention that are essential for function can be identified by methods known in the art, such as site- directed mutagenesis or alanine- scanning mutagenesis (Cunningham and Wells, Science 244:1081-1085 (1989)). The latter procedure introduces single alanine mutations at -23-
  • a Smad6 or Smad7 polypeptide can further comprise a polypeptide encoded by 4-300 contiguous amino acids of SEQ ID NO: 2 or SEQ ID NO: 4, or any range or value therein.
  • proteins that are functionally related to Smad6 or Smad7 are also contemplated by the present invention.
  • proteins that are functionally related to SEQ ID NO: 2 may be produced by conservative amino acid substitutions, deletions, or insertions, at one or more amino acid positions within Smad6 or Smad7, in accordance with Table 1 presented herein.
  • Smad6 or Smad7 polypeptides made in accordance with Table 1 are generally expected to retain the biological activity of the parent molecule based on art recognized substitutability of the amino acids specified in Table 1 (See e.g. M. Dayhoff, In Atlas of Protein Sequence and Structure, Vol. 5, Supp. 3, pgs 345-352, 1978) . Smad6 functionality is easily tested, for example, in an assay such as that described in Example IB herein.
  • Fragments of the proteins used in the present invention may be generated by any number of suitable techniques, including chemical synthesis of any portion of Smad6 or Smad7 (e.g. SEQ ID NO: 2 or SEQ ID NO: 4), proteolytic digestion of said proteins, or most preferably, by recombinant DNA mutagenesis techniques, well known to the skilled artisan. See. e.g. K. Struhl, "Reverse biochemistry: Methods and applications for synthesizing yeast proteins in vitro, " Meth . Enzymol . 194, 520-535.
  • a nested set of deletion mutations are introduced into a gene encoding Smad6 or Smad7 (e.g. SEQ ID NOS: 1 or 3) , or gene fragment thereof such that varying -25-
  • the protein coding region is deleted, either from the amino terminal end, or from the carboxyl end of the protein molecule.
  • This method can also be used to create internal fragments of the intact protein in which both carboxyl and amino terminal ends are removed.
  • nucleases can be used to create such deletions, for example Bal31, or in the case of a single stranded nucleic acid molecule, mung bean nuclease.
  • the Smad6 or Smad7 gene be cloned into a single-stranded cloning vector, such as bacteriophage M13, or equivalent. If desired, the resulting deletion fragments can be subcloned into any suitable vector for propagation and expression in any suitable host cell.
  • Functional fragments of the Smad6 or Smad7 polypeptide of this invention may be produced as described above, preferably using cloning techniques to engineer smaller versions of the Smad6 or Smad7 gene, lacking sequence from the 5' end, the 3' end, from both ends, or from an internal site. Smaller fragments of the genes or gene fragments of this invention can be used as a template to produce the encoded proteins .
  • Smad6 or Smad7 gene used in the present invention could be obtained by a plurality of recombinant DNA techniques including, for example, hybridization, polymerase chain reaction (PCR) amplification, or de novo DNA synthesis.
  • PCR polymerase chain reaction
  • de novo DNA synthesis See e.g., T. Maniatis et al . Molecular Cloning: A Laboratory Manual, 2d Ed. Chap. 18 (1989)).
  • a suitable vector such as a plasmid or phage for propagation in procaryotic or eucaryotic cells. [See e . g. Maniatis et al . Supra] . Suitable cloning vectors are well known and are widely available . ⁇ 26 -
  • the Smad6 or Smad7 gene, or fragments thereof, can be isolated from any tissue in which said gene is expressed.
  • mRNA is isolated from a suitable tissue that expresses Smad6 or Smad7, and first strand cDNA synthesis is carried out .
  • Second round DNA synthesis can be carried out for the production of the second strand.
  • the double-stranded cDNA can be cloned into any suitable vector, for example, a plasmid, thereby forming a cDNA library.
  • Oligonucleotide primers targeted to any suitable region of the sequences disclosed herein can be used for PCR amplification of Smad6 or Smad7 genes. See e.g.
  • the PCR amplification comprises template DNA, suitable enzymes, primers, and buffers, and is conveniently carried out in a DNA Thermal Cycler (Perkin Elmer Cetus, Norwalk, CT) .
  • a positive result is determined by detecting an appropriately- sized DNA fragment following agarose gel electrophoresis .
  • the present invention is also further described by reference to the following examples, which are not intended to limit the invention, but are to provide more specific, non-limiting embodiments, which support the full scope of the present invention as described herein.
  • the cells were maintained in DMEM/F-12 (3:1), a medium comprised of a 3:1 v/v mixture of Dulbecco's Modified Eagle's Medium and Ham's nutrient mixture F-12.
  • the basal medium was supplemented with 10 nM selenium, 50 ⁇ M 2-aminoethanol, 20 mM HEPES, 50 ⁇ g/ml gentamicin, and 5% fetal bovine serum (FBS) .
  • the PAI-1 basal promoter driving the expression of the CAT indicator gene (pOCAT2336) and a TGF ⁇ hyper-responsive PAI-l/TRE promoter construct (3TP-lux) driving the expression of the luciferase indicator gene were used.
  • Smad6/Ha and Smad7/Ha vectors were constructed by inserting the Smad6 coding sequence into the BamHI/Xhol sites of HA Tag expression vector pN8e23/3xFlu2 and the Smad7 coding sequence into the EcoRl/Xhol site of pN8e23/5xFlu2, a standard expression vectors for epitope tagging.
  • ECV304 cells were seeded in 6-well plates to 80% confluence. DNA was transfected at a concentration of 1 ⁇ g per well for pOCAT2336 and 3TP-lux and 5 ⁇ g per well for Smad6 and Samd7 with lipofectin reagent (Gibco/Life Technologies, Gaithersburg, MD) . Expressed CAT protein from the PAI-1 basal promoter construct was determined using a CAT ELISA kit (Boehringer Mannheim; Indianapolis, IN) . Chemiluminescence resulting from expression of the luciferase gene was determined as a measure of the effect on the PAI-l/TRE promoter. The plates were read kinetically and data expressed in terms of promoter activity relative to control (Table 2) . -28-
  • thrombomodulin anticoagulant activity was done in confluent cultures of SVHA-1 cells. The cultures were washed once with Hank's Basal Salt Solution to remove serum proteins and incubated with serum-free medium (DMEM/F-12 medium, 20 mM-HEPES, pH 7.5, 50 mg/ml gentamicin, 1 ⁇ g/ml human transferrin and 1 ⁇ g/ml bovine insulin) containing 400 nM recombinant human protein (made according to techniques as set forth in U.S. Patent No. 4,981,952) and 10 nM human thrombin (Sigma; St. Louis, MO).
  • serum- free medium DMEM/F-12 medium, 20 mM-HEPES, pH 7.5, 50 mg/ml gentamicin, 1 ⁇ g/ml human transferrin and 1 ⁇ g/ml bovine insulin
  • the amount of activated protein C generated was determined by adding chromogenic substrate (S2366) (Chromogenix, Molndal, Sweden) to a final concentration of 0.75 mM, and measuring the change in absorbance units/minute - 29 -
  • Results are expressed in terms of maximal response to TGF ⁇ (Table 4) .
  • the amount of activated protein C generated was directly related to the level of surface TM.
  • the over-expression of Smad6 resulted in a suppression of TM activity on the surface of the cells, to approximately 50% that observed with TGF ⁇ .
  • the oligonucleotides used in antisense experiments were synthesized using phosphorothioates and C-5 propyne pyrimidines .
  • Antisense oligodeoxynucleotides were designed to hybridize to the region of the Smad6 or the Smad7 mRNA encompassing the initial ATG.
  • the antisense oligodeoxynucleotide for Smad6, was 5' -GGTTTGCCCATTCTGGACAT- 3' (SEQ ID NO: 5) .
  • the sequence of the sense strand control oligodeoxynucleotide for Smad6, was 5' -ATGTCCAGAATGGGCAAACC- 3' (SEQ ID NO: 6) .
  • the antisense oligodeoxynucleotides for Smad7 was 5' -GATCGTTTGGTCCTGAACAT-3' (SEQ ID NO: 7).
  • the sense strand control oligodeoxynucleotide for Smad7 was 5' -ATGTTCAGGACCAAACGATC-3' (SEQ ID NO: 8).
  • PAI-1 levels were assayed 16 hours later from the culture supernatant using a commercially available PAI-1 ELISA kit (American Diagnostica Inc., Greenwich, CT) .
  • Cell surface TM levels were assayed in SVHA-1 cells indirectly by measuring the ability of the cell surface TM to activate human protein C.
  • antisense to Smad6 blocked the TGF ⁇ -dependent suppression of TM surface levels, as measured by the ability of the cells to support the thrombin-dependent activation of human protein C.
  • antisense to Smad6 suppressed the TGF ⁇ -dependent activation of PAI-1.
  • Smad6 and Smad 7 were examined in multiple samples of normal and diseased human arterial endothelium using immunohistochemical and in situ hybridization techniques. Table 7 reports the percentage of such samples that were positive for expression for the indicated gene .
  • Zinc formalin fixed paraffin embedded tissues were microtomed at 5 ⁇ m and placed in an oven overnight at 60°C.
  • the hybridization protocol followed was the Super Sensitive mRNA Detection System Kit from Biogenex. Briefly, slides were deparaffinized in xylene and rehydrated through graded alcohols to water (95%, 70% alcohols and water had RNAse block (Biogenex) added) . Tissues were then digested with proteinase K solution (kit component) at room temperature for 15 minutes. After rinsing in TBS (pH 9.5) with RNAse block, the sections were dehydrated through graded alcohols and dried at room temperature.
  • the fluorescein labeled riboprobes (both sense and anti-sense) were diluted in the kit's hybridization solution and added to the tissue and covered with a RNAse-free coverslip. Control slides had only hybridization solution (no probe) added. Hybridization was performed in a humidified thermal cycler for 10 minutes at 95°C and then 2 hours at 37°C. After several Tris washes to remove the coverslips and hybridization solution, the slides were then placed on the autostainer and the hybridized probes detected using an anti-fluorescein primary antibody, a BCIP/NBT substrate, and a nuclear fast red counterstain.
  • Smad6 gene expression was observed to be significantly increased in 13 of 14 samples attained from the endothelium of atherosclerotic lesions . Smad6 was not found to be consistently expressed in normal, non-diseased vessels. In contrast, Smad7 was not consistently found • 34 -
  • Plaque CD34 is a marker of endothelial cells
  • Smad gene expression was also determined in myocytes from hearts of normal patients as compared to those with congestive heart failure by means of immunohistochemical and in si tu hybridization (conducted as in Example 3) . These experiments demonstrated that Smad6 is highly over-expressed in the myocytes of hearts of patients with congestive heart failure. Because of the well described role of TGF ⁇ in promoting myocyte hypertrophy and dysfunction, agents that suppress Smad6 and thus TGF ⁇ secretion, including kinase inhibitors, have a positive benefit in the prevention and treatment of heart failure. The ability of Smad7 to antagonize the induction of TGF ⁇ by Smad6 indicates that ⁇ 35 -
  • agents that increase the function or level of Smad7 are useful in the treatment of heart failure.
  • TGF ⁇ 3 promoter construct driving CAT expression (1 ⁇ g) was co-transfected into ECV304 cells with Smad6 (5 ⁇ g)/pcineo vector (5 ⁇ g) , Smad7 (5 ⁇ g)/pcineo vector (5 ⁇ g) , or both. Controls were co-transfected with 1 ⁇ g promoter and 10 ⁇ g pcineo vector (Promega, Madison, WI) .
  • Transfection was initiated by plating ECV304 cells at 3 x 10 5 cells per well in a 6-well plate with DMEM/F12 media in 5% fetal bovine serum (FBS) . The cells were allowed to attach overnight at 37°C.
  • FBS fetal bovine serum
  • lipofectin Twenty ⁇ l lipofectin were diluted with serum-free medium to a total volume of 200 ⁇ l per transfection and placed at room temperature for 30 to 60 minutes. Eleven ⁇ g DNA was diluted with 200 ⁇ l of serum-free medium and mixed with the lipofectin solution. The resulting reagent mixture was incubated at room temperature for 15 minutes.
  • the medium was aspirated from the culture plates, and the cultures were washed twice with PBS . Two ml of medium were added per well, and the cells were incubated at
  • the cells were washed twice with phosphate buffered saline (PBS) , and serum free DMEM medium containing 100 ⁇ g/ml Conn's fractionated bovine serum albumin (BSA) and 2 ng/ml TGF ⁇ 3 (R&D Systems) - 36 -
  • PBS phosphate buffered saline
  • BSA Conn's fractionated bovine serum albumin
  • TGF ⁇ 3 R&D Systems
  • Example 1 Lysates were normalized in BCA assay measuring total protein concentration .
  • TGF ⁇ RII receptor R&D systems
  • Latent TGF ⁇ 3 was activated by adding 0.1 ml IN HCL to 0.5 ml supernatant, and incubating 10 minutes at room temperature. The mixture was neutralized with 0.1 ml 1.2 N NaOH and 0.5 M Hepes . The blocking mixture was removed from the prepared plates, and samples were added at 200 ⁇ l per well . Standard TGF ⁇ 3 , serially diluted from 2 ng/ml to 0.016 ng/ml in PBS 3% BSA, was added at 200 ⁇ l per well. The plates were incubated for two hours and washed as before.
  • Rabbit anti-TGF ⁇ 3 (Santa Cruz catalog# SC-082) was prepared at 1 ⁇ g/ml in PBS 3% BSA, added to the plates at 100 ⁇ l per well, incubated for one hour at room temp., and washed as before.
  • Goat anti-Rabbit IgG alkaline phosphate conjugate at 1/250 dilution in PBS 3% goat serum was added to the plates at 100 ⁇ l per well, incubated one hour, and washed as before.
  • One PnPP tablet (Sigma) was prepared in 3 ml H 2 0, added at 100 ⁇ l per well. The plates were incubated for 20-30 minutes and read at OD 405 nm. -37-
  • Latent TGF ⁇ l was activated as described for TGF ⁇ 3 and assayed according to directions in a commercial TGF ⁇ l ELISA kit (R&D Systems) .
  • results show that in addition to the role of Smads in the signaling of TGF ⁇ , Smad6 induces TGF promoters and the secretion of TGF ⁇ .
  • Smad6 As shown in Table 8 (promoter) and Table 9 (secretion) , Smad6 induced both the promoter activity and the secretion of TGF ⁇ 3 by around three fold. (Similar results were observed specifically for TGF ⁇ l as well) .
  • Smad7 had no significant affect on TGF ⁇ promoter activity or secretion, but could block the increase in TGF ⁇ secretion observed with Smad6.
  • Smad6 to increase endogenous TGF ⁇ secretion and promoter activity could be blocked by the addition of a kinase inhibitor 24 hours prior to the assay.
  • Staurosporin inhibited Smad6-induced TGF ⁇ 3 promoter activity, as measured by a decrease in CAT expression, and the PKC specific inhibitor decreased the endogenous secretion of Smad6-induced TGF ⁇ 3 measured directly (Table 10) .

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pain & Pain Management (AREA)
  • Biochemistry (AREA)
  • Rheumatology (AREA)
  • Biophysics (AREA)
  • Zoology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Genetics & Genomics (AREA)
  • Toxicology (AREA)
  • Diabetes (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Vascular Medicine (AREA)
  • Cardiology (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Peptides Or Proteins (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
EP99915041A 1998-03-27 1999-03-25 Behandlung und verhinderung von vaskulären krankheiten Withdrawn EP1066323A1 (de)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US7968198P 1998-03-27 1998-03-27
US79681P 1998-03-27
PCT/US1999/006595 WO1999050296A1 (en) 1998-03-27 1999-03-25 Treatment and prevention of vascular disease

Publications (1)

Publication Number Publication Date
EP1066323A1 true EP1066323A1 (de) 2001-01-10

Family

ID=22152123

Family Applications (1)

Application Number Title Priority Date Filing Date
EP99915041A Withdrawn EP1066323A1 (de) 1998-03-27 1999-03-25 Behandlung und verhinderung von vaskulären krankheiten

Country Status (7)

Country Link
EP (1) EP1066323A1 (de)
JP (1) JP2002509711A (de)
AU (1) AU3365299A (de)
CA (1) CA2324476A1 (de)
HU (1) HUP0101731A2 (de)
IL (1) IL138692A0 (de)
WO (1) WO1999050296A1 (de)

Families Citing this family (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6566131B1 (en) 2000-10-04 2003-05-20 Isis Pharmaceuticals, Inc. Antisense modulation of Smad6 expression
WO2001011013A2 (en) * 1999-08-04 2001-02-15 Uab Research Foundation The interaction of smad6 with hox proteins and uses thereof
US6159697A (en) * 2000-01-19 2000-12-12 Isis Pharmaceuticals, Inc. Antisense modulation of Smad7 expression
ITRM20030149A1 (it) 2003-04-02 2004-10-03 Giuliani Spa Oligonucleotidi (odn) antisenso per smad7 e loro usi in campo medico
JP5602365B2 (ja) 2005-10-25 2014-10-08 ザ・ジョンズ・ホプキンス・ユニバーシティ マルファン症候群及び関連疾患を治療するための方法及び組成物。
EP1998810A4 (de) 2006-03-13 2009-09-02 Univ Johns Hopkins Erhöhung der endothelialen thromboresistenz
CN105457016B (zh) * 2010-09-22 2022-06-24 科罗拉多大学董事会 Smad7的治疗应用
WO2012090997A1 (ja) 2010-12-27 2012-07-05 京都府公立大学法人 ｉＰＳ細胞とその製造法
KR101359886B1 (ko) * 2011-12-28 2014-02-12 성균관대학교산학협력단 스매드 6번(Ｓｍａｄ６)으로부터 유래된 펩타이드를 유효성분으로 포함하는 패혈증 또는 패혈성 쇼크의 치료용 조성물
EP2799537B1 (de) 2011-12-28 2021-09-22 Kyoto Prefectural Public University Corporation Normalisierung der züchtung von hornhautendothelzellen
WO2014138670A1 (en) 2013-03-08 2014-09-12 The Regents Of The University Of Colorado, A Body Corporate Ptd-smad7 therapeutics
EP3804760A1 (de) 2013-10-31 2021-04-14 Kyoto Prefectural Public University Corporation Therapeutikum für erkrankungen im zusammenhang mit endoplasmatischem retikulumszelltod im hornhautendothel
US10882903B2 (en) 2015-05-18 2021-01-05 Arizona Board Of Regents On Behalf Of The University Of Arizona Methods and compositions for treating an alphavirus infection

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9950296A1 *

Also Published As

Publication number Publication date
WO1999050296A1 (en) 1999-10-07
IL138692A0 (en) 2001-10-31
CA2324476A1 (en) 1999-10-07
JP2002509711A (ja) 2002-04-02
AU3365299A (en) 1999-10-18
HUP0101731A2 (hu) 2001-09-28

Similar Documents

Publication Publication Date Title
US5928939A (en) Vascular endothelial growth factor-b and dna coding therefor
US5607918A (en) Vascular endothelial growth factor-B and DNA coding therefor
JP5108797B2 (ja) 色素上皮誘導因子：ｐｅｄｆ遺伝子の特性評価、ゲノム構成および配列
Roy et al. Fibronectin overexpression in retinal microvessels of patients with diabetes.
JP2003517275A (ja) 哺乳動物ｖｅｇｆレセプター−２に結合し、これを活性化するオルフウイルスｎｚ２由来の血管内皮細胞増殖因子様タンパク質
JP2002532068A (ja) 血管新生阻害タンパク質に結合するタンパク質およびそれを使用した組成物および方法
EP1066323A1 (de) Behandlung und verhinderung von vaskulären krankheiten
US6180602B1 (en) Human novel cDNA, TGF-beta superfamily protein encoded thereby and the use of immunosuppressive agent
JP2002525086A (ja) 血小板由来増殖因子ｃ、それをコードするｄｎａ、及びその利用法
JP2003327542A (ja) インターフェロン−γの産生を誘導するポリペプチド
US6326466B1 (en) Double-stranded RNA dependent protein kinase derived peptides to promote proliferation of cells and tissues in a controlled manner
JPH11510054A (ja) モルフォゲンアナログを同定するための方法および組成物
WO1996008260A1 (en) MUTANT ACTIVATED Gsα* AND ADENYLYL CYCLASE 2 FOR USE AS THERAPEUTIC AGENTS
US20020164687A1 (en) Platelet-derived growth factor C, DNA coding therefor, and uses thereof
JP2002526099A (ja) Ｎｌｋ１−相互作用タンパク質
JPH11510387A (ja) モルフォゲンアナログを同定するための方法および組成物
WO1999062925A1 (en) Angiopoietin related gene sequence scarface 1
WO2000059940A2 (en) Platelet-derived growth factor related gene and protein
IL139873A (en) Angiostatin-1-binding protein (1 - ABP), its sequence, related molecules and their use in drug production
AU2003261514A1 (en) Treatment and Prevention of Vascular Disease
JP2004521605A (ja) 細胞接着および創傷治癒のための方法
CA2188423A1 (en) Factor interacting with nuclear proteins
MXPA00009355A (en) Treatment and prevention of vascular disease
JP2770926B2 (ja) 毛管内皮細胞プロテアーゼ合成、ｄｎａ合成および遊走を促進することの出来るヒト胎盤血管形成誘導因子
ZA200600914B (en) Methods and compositions for treating disorders of the extracellular matrix

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20000926

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU NL PT SE

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20031001