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EP1699819A2 - Behandlung von koronarer oder peripherer ischämie - Google Patents

Behandlung von koronarer oder peripherer ischämie

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Publication number
EP1699819A2
EP1699819A2 EP04804488A EP04804488A EP1699819A2 EP 1699819 A2 EP1699819 A2 EP 1699819A2 EP 04804488 A EP04804488 A EP 04804488A EP 04804488 A EP04804488 A EP 04804488A EP 1699819 A2 EP1699819 A2 EP 1699819A2
Authority
EP
European Patent Office
Prior art keywords
fgf
heparin
expression vector
growth factor
molecular weight
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Withdrawn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
EP04804488A
Other languages
English (en)
French (fr)
Inventor
Andre Uzan
Alexis Caron
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.)
Aventis Pharma SA
Original Assignee
Rhone Poulenc Rorer SA
Aventis Pharma SA
Centelion SAS
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 Rhone Poulenc Rorer SA, Aventis Pharma SA, Centelion SAS filed Critical Rhone Poulenc Rorer SA
Publication of EP1699819A2 publication Critical patent/EP1699819A2/de
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/726—Glycosaminoglycans, i.e. mucopolysaccharides
    • A61K31/727—Heparin; Heparan
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1858—Platelet-derived growth factor [PDGF]
    • A61K38/1866—Vascular endothelial growth factor [VEGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • 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
    • C07K14/50—Fibroblast growth factor [FGF]

Definitions

  • the present invention relates to novel methods of treating a patient having peripheral and/or coronary ischemic syndrome and also relates to a product comprising an expression vector encoding an angiogenic growth factor and an heparin compound or a low molecular weight heparin compound, wherein the product is capable of acting in an effective manner to promote angiogenesis and arteriogenesis in skeletal and cardiac muscles.
  • Angiogenic gene therapy is a possible option aimed at improving collateral development and overcoming perfusion defects and related ischemia (6-8).
  • Therapeutic angiogenesis via the administration of nucleic acids capable of expressing an angiogenic protein either in a naked form or via liposomes or viral vectors have been investigated.
  • Protein therapy which involves delivery of the growth factor directly in the ischemic tissue constitutes an alternative option for therapeutic angiogenesis.
  • administration of several recombinant proteins, VEGF, FGF, HGF, PDGF, and TGF- ⁇ to promote collateral blood vessels has been proposed.
  • angiogenic proteins including for example recombinant human VEGF protein, recombinant human basic FGF protein (bFGF), recombinant TGF- ⁇ or PDGF proteins, promotes collateral blood vessels.
  • recombinant angiogenic proteins have been proposed for the treatment of ischemic cardiovascular diseases, Le., peripheral arterial disease or coronary arterial disease.
  • the heparins are biologically active agents of the glycosaminoglycan family, extracted from natural sources, and have valuable anticoagulant and antithrombotic properties. In particular, they are useful in the treatment of postoperative venous thromboses. Heparin compounds may be prepared with various molecular weights.
  • Standard heparin has a molecular weight ranging from 6000 to 40000 Da. The average molecular of most commercial heparin preparations is in the range of 12000 to 15000 Da.
  • standard heparin is isolated from intestinal mucus from swine of lung from cattle. Heparins may be fragmented into molecules of lower average molecular weights, that contains a preparation of a mixtures of sulfated polysaccharides, having an average molecular weight ranging from 2000 to 10 000 Da. These mixtures are produced by depolymerization and saponification of a heparin ester. They have a high antithrombotic activity and an overall anticoagulant activity lower than that of heparin.
  • the blood coagulation is based on a cascade like process where a number of proteolytic enzymes activate each other in a definite sequence; in the last stage fibrinogen is converted under the action of the proteolytic enzyme thrombin to insoluble fibrin.
  • the coagulation process comprises in fact three phases generally described as successive, even if they are intricately interrelated.
  • the first phase consists of thromboplastin formation, the phase of prothrombinase (or active thromboplastin formation).
  • the second phase consists of thrombin formation, which phase can be summarized as the conversion of the prothrombin into thrombin under the influence of the prothrombinase in the presence of ionized calcium.
  • the third phase consists of fibrin formation, wherein the blood fibrinogen is under the effect of thrombin converted into fibrin which becomes insoluble.
  • the formation of prothrombinase occurs, in the course of the thromboplastin formation step, essentially according to two different routes: the intrinsic or endogenic route, and the extrinsic or exogenic route, which end in the formation of prothrombinases of respectively plasmatic and tissue origins, both capable of activating prothrombin into active thrombin.
  • the intrinsic or endogenic route involves a large number of factors or plasmatic proenzymes capable of being successively activated (factors XII, XI, IX, VIII and X), where each activated product (factors Xlla, Xla, IXa, Villa and Xa) acts like an enzyme capable of activating the following proenzyme, the activated X factor (Xa) then taking part, notably by reaction with the V factor and a phospholipid of platelet origin, in the production of active endogenic plasmatic prothrombinase.
  • the extrinsic or exogenic system which can notably be directly dependent of a tissue lesion, calls upon a more limited number of factors and includes notably the production of tissue thromboplastin which, in combination with the VII factor, can, just as the factor Villa, convert the inactive X factor into the Xa factor.
  • the activation sequence of the prothrombin into thrombin is then substantially the same as for the intrinsic system, but the phospholipid is here of tissue and not of plasmatic origin.
  • the outcome of the coagulation process consists of the formation of an insoluble fibrin clot, intended notably to fill in the lesion at the origin of the triggering of this process, for example at the level of a blood vessel.
  • fibrinolysis intended to produce lysis of the clot, notably under the effect of plasmin, which enzyme only exists normally in the circulating blood in the form of an inactive precursor, plasminogen, the fibrin itself constituting nonetheless one of the factors capable of initiating the conversion of the inactive plasminogen into fibrinolytically active plasmin.
  • plasmin which enzyme only exists normally in the circulating blood in the form of an inactive precursor, plasminogen, the fibrin itself constituting nonetheless one of the factors capable of initiating the conversion of the inactive plasminogen into fibrinolytically active plasmin.
  • hypocoagulability exposes the host to hemorragic risks.
  • Heparin acts at several levels in cascades of successive enzymatic reactions, which are normally engaged in the course of physiological hemostasis, in any situation capable of resulting in hypercoagulability of the blood. It is more particularly capable of simultaneously depressing a large number of the coagulation factors participating to the creation and the up keeping of different forms of hypercoagulability. More precisely, heparin forms a high-affinity complex with antithrombin. The formation of antithrombin - heparin complex greatly increases the rate of inhibition of two principle procoagulant proteases, factor Xa and thrombin.
  • a heparin compound such as low molecular weight heparin (LMWH) or standard heparin may be used in combination with an expression vector encoding an angiogenic growth factor to induce a strong angiogenesis and arteriogenesis synergistic response.
  • LMWH low molecular weight heparin
  • FGF fibroblast growth factor
  • heparin is a potent inhibitor of polymerase, by competing with duplex DNA for binding to the polymerase, the Applicants have demonstrated that this was not the case with naked DNA injection in the form of an expression vector or plasmid in combination with LMWH or standard heparin, which enhances the activity of a DNA encoding an angiogenic growth factor and provides a strong synergistic angiogenic response, for example in skeletal and/or cardiac muscles.
  • Such combinations of the present invention have never been disclosed in the prior art.
  • administration of DNA encoding a n angiogenic growth factor in combination with LMWH is capable of reversing angiogenesis defects in a hypercholesterolemia or diabetes setting.
  • lipids can severely impair endothelial functions such as vasodilatation, leukocyte-endothelium-interactions, thrombosis, fibrinolysis, and endothelial cell growth (3).
  • endothelial functions such as vasodilatation, leukocyte-endothelium-interactions, thrombosis, fibrinolysis, and endothelial cell growth (3).
  • NOS nitric oxide synthase
  • LDL-C oxidized low-density lipoprotein cholesterol
  • an expression vector encoding an angiogenic growth factor such as acidic fibroblast growth factor in combination with a heparin compound such as standard heparin or LMWH is capable of efficiently reversing the defect in collateral vessels and promoting the formation of mature vessels such as arterioles in a mammalian subject suffering from hypercholesterolemia or diabetes, when administered in ischemic skeletal or cardiac muscles.
  • product comprising an expression vector encoding acidic Fibroblast Growth Factor (FGF-1) in combination with a heparin compound such as standard heparin or LMWH can be used in an effective amount to rescue angiogenesis defects of ischemic skeletal or cardiac muscles, especially in aggravated conditions such as hypercholesterolemia or diabetes.
  • FGF-1 acidic Fibroblast Growth Factor
  • a heparin compound such as standard heparin or LMWH
  • the present invention relates to compositions which have a strong angiogenic effect and is particularly selective to skeletal and cardiac muscles.
  • the invention is based in part on the fact that DNA encoding angiogenic growth factors have a strong synergistic effect in combination with a heparin compound such as low molecular weight heparin or standard heparin and induce a strong angiogenic response in skeletal and cardiac muscles.
  • the invention is not limited to the use of a DNA expression vector or naked DNA in combination with a heparin compound.
  • any agent or vector capable of promoting or causing the expression of an angiogenic growth factor in a cell or organism can be selected and used.
  • vectors such as plasmids, viruses, retroviruses, nucleic acids, cosmids, BAC, YAC, viral genomes, recombinant adenoviral vectors, other nucleic acids that integrate or reside within a cell, an adenoviral vector, an adeno-associated virus vector, lentiviral, or retroviral vector.
  • a “vector” means any nucleic acid or nucleic acid-bearing particle, cell, or organism capable of being used to transfer a nucleic acid into a host cell.
  • the term “vector” includes both viral and nonviral products and systems for introducing a nucleic acid into a cell and/or introducing a compound that causes or promotes the expression of at least one angiogenic growth factor.
  • a “vector” can be used in vitro, ex vivo, or in vivo.
  • Non-viral vectors include plasmids, cosmids, and can comprise liposomes, electrically charged lipids (cytofectins), DNA-protein complexes, and biopolymers, for example.
  • Viral vectors include adenoviruses, retroviruses, lentiviruses, adeno- associated viruses, pox viruses, baculovirus, reoviruses, vaccinia viruses, herpes simplex viruses, Epstein-Barr viruses, and recombination defective adenovirus vectors, for example.
  • Vectors can also comprise the entire genome sequence or recombinant genome sequence of a virus.
  • a vector can also comprise a portion of the genome that comprises the functional sequences for production of a virus capable of infecting, entering, or being introduced to a cell to deliver nucleic acid therein.
  • the present invention relates to a product comprising an efficient amount of an agent or expression vector or plasmid encoding an angiogenic growth factor and a heparin compound such as standard heparin or LMWH, and to the use of such product for the manufacture of a medicament for treating peripheral or cardiac ischemia.
  • the present invention also relates to a pharmaceutical composition comprising an expression vector, wherein the combination promotes angiogenesis in skeletal and cardiac muscles greater than either the vector or the heparin compound alone.
  • the present invention also relates to a novel method of promoting angiogenesis and arteriogenesis in cardiac and skeletal muscles, and to novel combinations of an expression vector encoding an angiogenic growth factor with low molecular weight heparin or standard heparin, which combination is capable of acting in a synergistic manner.
  • the present invention further relates to a novel method of treating a patient subject to peripheral ischemia or coronary ischemia, or at risk of ischemia, wherein the patient is 45 years old or older, is overweight, and/or has a high consumption of alcohol or cigarettes, and/or has atherosclerosis and/or diabetes, and/or is immobilized, comprising administering a combination of a DNA sequence encoding angiogenic factor and a heparin compound.
  • the present invention is also useful for treating a patient subject to an ischemic cardiac attack in a time period from the last 24 hours to 6 months, or up to 1, 2, or 3 years ago, comprising administering a combination of an expression vector encoding angiogenic factor and a heparin compound.
  • the present invention further provides a new method of promoting both collateral blood vessels and arterioles in ischemic skeletal or cardiac muscle tissue in a mammalian subject comprising injecting the subject with an effective amount of the product of the invention.
  • the present invention further provides a method of promoting formation of mature large conductance vessels (>150 ⁇ m collateral vessels) and small resistance arteries ( ⁇ 50 ⁇ m arterioles) in ischemia skeletal or cardiac muscle tissues in a mammalian subject by injecting the subject with an effective amount of the product of the invention.
  • a further object of the present invention is to provide a method for promoting both collateral blood vessel growth and arteriole growth in ischemic tissues, especially when endothelial function is impaired.
  • Another object of the present invention is to provide an angiogenic therapy for treating ischemic conditions such as peripheral or myocardiai ischemia, wound healing or other conditions that require neovascularization or tissue regeneration.
  • another object of the present invention is to provide a method of enhancing therapeutic angiogenesis in a patient subject of angioplasty, bypass grafting or other revascularization procedures.
  • FIG. 1 The chart corresponds to a measurement of luciferase expression after injection of Tibialis cranialis, Quadriceps femoris, and Adductores/Gracilis, in combination with (a) a daily subcutaneous injection of a saline solution during DO to D6, (b) a daily subcutaneous injection of enoxaparin at 1.5mg/kg during from DO to D6, and (c) a daily subcutaneous injection of enoxaparin at 1.5mg/kg form D1 to D6.
  • the data establishes that protein is expressed and establishes a baseline for enoxaparin treatment with a control plasmid with no angiogeneic growth factor-encoding nucleic acid.
  • the present invention provides a composition comprising an expression vector encoding an angiogenic factor and a low molecular weight heparin or standard heparin for treating a patient having coronary or peripheral ischemia.
  • the use of such combination unexpectedly provides a synergistic effect in enhancing the revascularization and perfusion by promoting angiogenesis or arteriogenesis throughout the ischemic skeletal or cardiac muscle tissues of a mammalian subject.
  • the present invention also relates to a novel method of promoting angiogenesis and arteriogenesis in cardiac and skeletal muscles.
  • subject includes, r but is not limited to, mammals, such as dogs, cats, horses, cows, pigs, rats, mice, simians, and humans.
  • the angiogenic growth factor encoding vector is typically a DNA molecule, and is generally capable of expressing the angiogenic growth factor in a cell of the individual to be treated.
  • Expression vector encoding angiogenic growth factor may comprise DNA encoding fibroblast growth factor (FGF) such as for examples, FGF-1 or acidic FGF (aFGF), FGF-2 or basic FGF (bFGF), FGF-4, FGF-5, FGF-6, and FGF-7 or any of FGF1-23. Coding sequences of such angiogenic growth factors are well known in the art.
  • FGF-1, FGF-2, FGF-4, FGF-5, FGF-6, and FGF-7 are set forth in SEQ ID NOs: 1 to 6, respectively.
  • VEGF vascular endothelial growth factor
  • genes useful in the combination of the present invention are genes encoding vascular endothelial growth factor (VEGF), such as VEGF-A (mature isoforms 206, 189, 165, 145, and 121 amino acid residues), VEGF206, VEGF189, VEGF165, VEGF145, VEGF121, VEGF120, VEGF homologues including VEGF-B, VEGF-C, VEGF-D, as well as VEGF heterodimers.
  • Other useful genes include a platelet derived growth factor (PDGF AA, AB, or BB), a hepatocyte growth factor (HGF), or an insulin growth factor (IGF-I or II).
  • PDGF AA, AB, or BB platelet derived growth factor
  • HGF hepatocyte growth factor
  • IGF-I or II insulin growth factor
  • compositions of the present invention comprise a DNA encoding the acidic fibroblast growth factor (FGF-1 or aFGF).
  • the angiogenic factor encoding sequence may be carried by a viral or non viral expression vector.
  • vector refers to a vehicle, preferably a DNA or nucleic acid molecule, which can transport the nucleic acid molecules and express the angiogenic factor that it carries.
  • the vector is a nucleic acid molecule, the nucleic acid molecules are covalently linked to the vector nucleic acid.
  • a variety of expression vectors can be used to express the angiogenic factor.
  • Such vectors include chromosomal, episomal, and virus-derived vectors, for example vectors derived from bacterial plasmids, from bacteriophage, from yeast episomes, from yeast chromosomal elements, including yeast artificial chromosomes, from viruses such as baculoviruses, papovaviruses such as SV40, Vaccinia viruses, adenoviruses, poxviruses, pseudorabies viruses, and retroviruses. Vectors may also be derived from combinations of these sources such as those derived from plasmid and bacteriophage genetic elements, e.g. cosmids and phagemids.
  • angiogenic factor is carried and expressed by a plasmid.
  • Expression plasmids are well known in the art.
  • plasmids derived from ColE1 plasmid such as pBR322, plasmid pMB1 , or plasmid pMK16 (Ausubel, Current Protocols in Molecular Biology, John Wiley and Sons, New York (1988) ⁇ 11:1.5.2) can be used and the angiogenic factor coding sequence may be cloned by transforming E. coli according to the conventional manner and screening transformants having an insert.
  • Mammalian expression vectors may be used and include pCDM ⁇ (Seed, B. Nature 329:840(1987)) and pMT2PC (Kaufman et al., EMBO J. 6:187- 195 (1987)).
  • Plasmids pCEP4 and pREP4 from Invitrogen contain the Epstein Barr virus origin of replication and nuclear antigen EBNA-1 coding region which produces high copy episomal replication without integration.
  • the expression vectors listed herein are provided by way of example only of the well-known vectors available to those of ordinary skill in the art that would be useful to express the angiogenic factors. These are found for example in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual.
  • Expression vectors or plasmids contain regulatory regions necessary for the expression of the angiogenic factor and operably linked in the vector to the angiogenic factor coding sequence and include a promoter, an initiation codon, a stop codon and a polyadenylation signal. Initiation and stop codons are generally in frame with the coding sequence of the angiogenic factor and considered to be part of a nucleotide sequence that encodes the desired angiogenic factor.
  • promoters useful to practice the present invention include but are not limited to promoters from Simian Virus 40 (SV40), Mouse Mammary Tumor Virus (MMTV) promoter, Human Immunodeficiency Virus (HIV) such as the HIV Long Terminal Repeat (LTR) promoter, Moloney virus, ALV, Cytomegalovirus (CMV) such as the CMV immediate early promoter, Epstein Barr Virus (EBV), Rous Sarcoma Virus (RSV) as well as promoters from human genes such as human alpha actin, human Myosin, human Hemoglobin, human muscle creatine and human metallothionein.
  • the regulatory sequence may provide constitutive expression in one or more host cells (i.e.
  • tissue specific or may provide for inducible expression in one or more cell types such as by temperature, nutrient additive, or exogenous factor such as a hormone or other ligand.
  • exogenous factor such as a hormone or other ligand.
  • vectors providing for constitutive and inducible expression in prokaryotic and eukaryotic hosts are well known to those of ordinary skill in the art.
  • muscle specific promoters may be used to drive the expression of the angiogenic factor and include murine or human upstream sequence of the CARP gene which is described in the US publication 2003/0039984, or the cardiac alpha actin promoter sequence as described in the international publication WO01/11064.
  • polyadenylation signals useful to practice the present invention include but are not limited to SV40 polyadenylation signals, bovine or human Growth hormone polyadenylation signals, and LTR polyadenylation signals.
  • the SV40 polyadenylation signal which is in pCEP4 plasmid (Invitrogen, San Diego Calif.), referred to as the SV40 polyadenylation signal is used.
  • other elements may also be included in the DNA molecule. Such additional elements include enhancers.
  • the enhancer may be selected from the group including but not limited to: human Actin, human Myosin, human Hemoglobin, human muscle creatine and viral enhancers such as those from SV40, RSV, and EBV enhancers, the cytomegalovirus immediate early enhancer, polyoma enhancer, adenovirus enhancers, and retrovirus LTR enhancers.
  • expression vectors can also contain sequences necessary for transcription termination and, in the transcribed region a ribosome binding site for translation.
  • the person of ordinary skill in the art would be aware of the numerous regulatory sequences that are useful in expression vectors. Such regulatory sequences are described, for example, in Sambrook et al, Molecular Cloning.
  • the angiogenic factor coding sequence can be inserted into the vector nucleic acid by well-known methodology.
  • the DNA sequence that will ultimately be expressed is joined to an expression vector by cleaving the DNA sequence and the expression vector with one or more restriction enzymes and then ligating the fragments together. Procedures for restriction enzyme digestion and ligation are well known to those of ordinary skill in the art.
  • Expression vectors generally include selectable markers that enable the selection of the subpopulation of cells that contain the recombinant vector constructs. The marker can be contained in the same vector that contains the nucleic acid molecules described herein or may be on a separate vector.
  • the expression vector is a plasmid expressing FGF-1 and is designated NV1 FGF.
  • NV1 FGF is a plasmid harboring an optimized expression cassette encoding a secreted form of human FGF-1 (sphFGF-1) inserted into an original backbone designed with conditional replication origin and called the pCOR plasmid (2).
  • the resulting plasmid is advantageously of small size of 2.4 kb and the sequence is as set forth in SEQ ID NO:7.
  • the sequence encoding sphFGF-1 is a fusion between the sequences encoding the secretion signal peptide (sp) from human fibroblast interferon and the naturally occurring truncated form of human FGF-1 from amino acids 21 to 154 (US 4,686,113; US 5,849,538). Expression of sphFGF-1 was driven by the human cytomegalovirus (CMV) immediate early enhancer/promoter (from nucleotide -522 to +72).
  • CMV human cytomegalovirus
  • the late polyadenylation signal from simian virus 40 was inserted downstream of the sphFGF-1 fusion to ensure proper and efficient transcription termination and subsequent polyadenylation of the sphFGF-1 transcript.
  • the preferred pCOR plasmid which is described in the International application WO 97/10343 and Soubrier et al. (2) is devoid of any antibiotic resistance gene. Plasmid selection relies on a suppressor transfer RNA gene in the autotrophic recipient strain. Maintenance of high copy number and strictly limited host range of the plasmid are obtained with the R6K y origin of replication.
  • the sequence coding for this protein is not usually found in bacteria but is artificially inserted into the genome of the selected host strain. Thus, the potential for unwanted dissemination is greatly limited.
  • the vector or plasmid carrying and expressing an angiogenic gene according to the present invention may be administered by any suitable means known or available to persons skilled in the art.
  • the expression vector encoding the angiogenic factor may be used in combination with a heparin compound and acts in a synergistic manner to promote growth of capillary vessels in skeletal and cardiac muscles.
  • Heparin compound may be standard heparin or low molecular weight heparin.
  • Standard or unfractionated heparin is a mucopolysaccharide with a molecular weight ranging from 6000 to 40000 Da. The average molecular of most commercial heparin preparations is in the range of 12000 to 15000 Da.
  • standard heparin is isolated from intestinal mucus from swine of lung from cattle.
  • the polymeric chain is composed of repeating disaccharide unit of D- glucosamine and uronic acid linked by 1-4 interglycosidic bond.
  • the uronic acid residue could be either glucuronic acid or L-iduronic acid.
  • heparin comprises a key structural unit which consists of a unique pentasaccharide sequence of three D-glucosamine and two uronic acid residues. Few of the hydroxyl groups on each of these monosaccharide residues are sulfated giving rise to a polymer that is highly negatively charged. Particularly, the central D- glucosamine residue contains a unique 3-O-sulfate moiety, and the first and fifth D-glucosamides of the pentasaccharide contain four sulfate groups that have been found to be critical for retaining high anticoagulant activity. Elimination of any one of them results in a dramatic reduction in the anticoagulant activity.
  • heparin exists as the anion at physiologic pH and is usually administered as the sodium salt. Twenty to fifty percent is excreted unchanged.
  • heparin polysaccharide chain is degraded in the gastric acid and is thus generally administered intravenously or subcutaneously. Heparin is generally not be given intramuscularly because of the danger of hematoma formation. Low molecular weight heparin, which is used in the combination according to the present invention, is well known in the art. LMWH and the method of preparation are described inter alia in US 5,389,618; US 4,692,435, and US 4,303,651 , European patent EP 0 040 144, and by Nenci GG (Vasc.Med, 2000; 5:251-258), which are herein incorporated by reference.
  • Low molecular weight heparin corresponds to a mucopolysaccharide fraction obtained from heparin or from fractions including heparinic constituents of molecular weights extending notably from about 2000 to 50000.
  • Processes for the fragmentation of heparin are well known in the art.
  • European Patent EP 40,144 describes the preparation of mixtures of sulfated polysaccharides of which heparin is comprised, including an ethylenic double bond at one end of their polymer chains and having a weight average molecular weight ranging from 2,000 to 10,000 daltons. These mixtures are produced by depolymerization and saponification of a heparin ester.
  • US 5,389,618 describes preparations of mixtures of polysaccharides having a weight average molecular weight less than that of heparin and comprising from 9% to 20% of polymer chains of molecular weight less than 2,000 daltons and from 5% to 20% of polymer chains of molecular weight greater than 8,000 daltons, and in which the ratio weight average molecular weight/number average molecular weight ranges from 1.3 to 1.6.
  • the process so described consists first salifying a starting material heparin in an aqueous medium by means of a long-chain quaternary ammonium salt, next esterifying the salt thus produced to form an ester having a degree of esterification ranging from 9.5% to 14%, and then depolymerizing such ester having a degree of esterification ranging from 9.5% to 14%.
  • Level of depolymerization, and molecular characteristics of the final product may be controlled by varying the degree of esterification of the heparin salt starting material.
  • the starting material, from which the mucopolysaccharide may be extracted is constituted by a heparin of conventional, injectable pharmaceutical quality, or by a crude heparin such as is obtained at the end of extraction operations for this active principle from tissues or organs of mammals, notably from intestinal mucous or from lungs, for example pork or beef.
  • the heparin starting material is a porcine heparin, and, in particular, a porcine mucosal heparin. It may be preliminarily precipitated by means of an alcohol upstream of the salification thereof.
  • the process for preparation of low molecular weight heparin may comprise the following steps.
  • the heparin salt may be prepared by the interaction of a stoichiometric excess of the corresponding salt with a heparin sodium, in an aqueous medium, at a temperature in the region of 20°C.
  • quaternary ammonium salts that are used are abenzethonium salts such as benzethonium chloride.
  • esterification is carried out.
  • the partial ester of heparin in salt form may be prepared by esterification of the long-chain quaternary ammonium salt of heparin in a chlorinated organic solvent, in the presence of a chlorine derivative. In addition, the efficiency of the reaction is increased by controlling the proportions of the various reactants and the reaction temperature and time.
  • the partial ester of heparin may be an aromatic ester.
  • the chlorine derivative may be benzyl chloride and the chlorinated solvent is either chloroform or methylene chloride.
  • the degree of esterification ranging from 9.5% to 14% may be reached by using approximately 1 part by volume of chlorine derivative per 1 part by weight of the heparin salt in 3 to 5 parts by volume of chlorinated organic solvent, and to carry out the reaction for a period of time ranging from ' 15 to 48 hours at a temperature of from 25 to 45°C.
  • the partial ester of heparin may be in the form of a sodium salt.
  • the esters so obtained may be recovered by precipitation by means of an alcohol such as, in particular, methanol, in the presence of sodium acetate. From 1 to 1.2 volumes of alcohol are used per volume of reaction medium. The degree of esterification of the ester may then be determined by high performance liquid chromatography.
  • the amount of benzyl alcohol produced by saponification of the may be measured.
  • depolymerization is carried out by contacting the ester with a strong base, such as sodium hydroxide in aqueous solution at a weight ratio base/ester ranges from 0.05 to 0.2.
  • the temperature of the reaction medium is adjusted to a value ranging from 50 to 70°C, and preferably from 55 to 65 °C, and the reaction is carried out for a period of time ranging from 30 minutes to 3 hours. It is also preferable to carry out the reaction in a medium in which the weight ratio water/ester ranges from 15 to 30.
  • the depolymerization is carried out with one part by weight of an aromatic ester of heparin as prepared in the esterification step, in salt form, the degree of esterification of which ranges from 9.5% to 14%, is admixed with from 0.08 to 0.15 part by weight of sodium hydroxide, as well as with from 20 to 30 parts by weight of water, and the resulting admixture is then maintained at a temperature of from 55 to 65°C for from 1 to 2 hours.
  • the product may then be recovered by neutralization of the reaction medium with a dilute inorganic acid, and preferably hydrochloric acid, and precipitation in the presence of an alcohol such as methanol.
  • An additional fractionation may be achieved by a supplementary step applied to each mucopolysaccharide fraction, previously redissolved in water, which step consists of adding to this aqueous solution from 1 to 2 volumes of ethanol and from 10 to 100 g/l of sodium chloride and of collecting, on the one hand, the equally active precipitate formed and, on the other hand, the content remaining dissolved in the supernatant liquor, notably by a further alcoholic precipitation, and which constitutes a fractionation product.
  • Mucopolysaccharide fractions having a ratio of Yin-Wessler/USP titers which are higher can also be obtained by gel-filtration from the fractions of the first extraction.
  • Such a solution may be passed through a gel of polyacrylamide and agarose, in bead form, ULTROGEL AcA 44, whose effective fractionating zone is situated between effective molecular weights of 4,000 to 60,000 Da.
  • ULTROGEL AcA 44 whose effective fractionating zone is situated between effective molecular weights of 4,000 to 60,000 Da.
  • the treated fractions whatever the degree of purification reached, are in the state of physiologically acceptable metallic salts, such as those of sodium, they may then be converted into mixed or simple salts containing another physiologically acceptable metal, such as calcium, by any process applicable to the salts of heparin.
  • this process consists essentially, starting, for example, from a sodium salt of heparin, of contacting the latter with a different salt of another physiologically acceptable metal, for example calcium chloride, in solution, of then proceeding with the separation of the metallic ions unbound to the heparin (for example by alcoholic precipitation or dialysis) and, to the extent that the substitution ratio reached is not sufficient, of recontacting, in solution, the mixed heparin salt obtained at the end of the first contacting, with a further amount of another salt, notably calcium chloride, according to the desired final substitution ratio.
  • another salt for example calcium chloride
  • the factions may be characterized on the one hand, by a particular affinity with regard to antithrombin III manifested by their capacity to be fixed on the latter, notably in a system comprising the contacting of the fractions with an antithrombin III fixed on a support, such as agarose, in an 0.2M NaCI, 0.05M tris-HCI buffer at pH 7.5 and, on the other hand, by Yin-Wessler and USP titers which are in the ratio (YW/USP ratio).
  • Heparin may be characterized by nuclear magnetic resonance spectra (NMR). Referring more particularly to the NMR spectrum of the compounds according to the invention for the proton ( 1 H) carried out on solutions of these compounds dissolved in deuteriated water at 35° C.
  • Standard or unfractionated heparin or LMWH that may be used in the combination according to the present invention, may have the above-indicated general properties as LMWH on the one hand, and the affinity for antithrombin III, on the other hand, these fractions having a higher molecular weight, but also containing in their structure an oligosaccharide part having the above mentioned structure.
  • Any of the above-mentioned LMWH compounds as described above or in the references cited herein can be selected for use individually, or in any combination with other LMWH compounds or other therapeutic or pharmaceutical compounds.
  • LMWH is as safe and effective as standard or unfractionated heparin and may be used in different clinical situations such as acute coronary syndromes, including myocardial infarction, heart surgery, vascular surgery, coronary and peripheral percutaneous revascularization and acute stroke (Nenci GG et al., Vase Med, 2000; 5: 251-258).
  • the combination of the present invention comprises a LMWH which is currently marketed under the name Clexane® /Lovenox ® by Aventis Pharma SA for the prevention and treatment of acute deep venous thrombosis, as well as for the prophylactic treatment of venous thromboembolic disease in moderate- or high-risk surgery, the prevention of coagulation in the extracorporeal circulation system during hemodialysis, and in combination with aspirin, for the treatment of unstable angina and of acute non-Q wave myocardial infarction.
  • Fragmin® is a low molecular weight heparin, which has been on the market since 1985 and is manufactured by Pharmacia & Upjohn.
  • Dalteparin sodium is produced through nitrous acid depolymerisation of sodium heparin from porcine intestinal mucosa. It is composed of strongly acidic sulphated polysaccaride chains with an average molecular weight of 4000-6000 and about 90% of the material within the range 2000-9000 Da.
  • heparins on the market are Fraxiparine® (nadroparin) commercialized by Sanofi, Clivarin® (reviparin) and Innohelp® (tinzaparin) commercialized by Dupont, may be used in combination with NV1 FGF according to the present invention.
  • the plasmid NV1 FGF is used in combination with enoxaparin, such as enoxaparin (Lovenox®, Clexane®), Fragmin®, or Fraxiparine®.
  • Enoxaparin is used in combination with an angiogenic growth factor encoding factor which is typically a DNA molecule, capable of expressing the angiogenic growth factor in a cell of the individual to be treated.
  • DNA encoding angiogenic growth factor may comprise DNA encoding fibroblast growth factor (FGF) such as for examples, FGF-1 or acidic FGF (aFGF), FGF-2 or basic FGF (bFGF), FGF-4, FGF-5, FGF-6, and FGF-7 or any of FGF1-22.
  • FGF fibroblast growth factor
  • enoxaparin is used in combination with the plasmid NV1 FGF at a concentration sufficient to promote a synergistic angiogenic response as shown by the formation of capillary vessels and/or mature vessels such as arterioles.
  • the combination of NV1FGF and enoxaparin is particularly potent, as it is capable of efficiently promoting therapeutic angiogenesis at a non-detectable concentration in treated muscles.
  • NV1FGF is administered at concentrations which are within a therapeutic window, thereby avoiding negative side effects due to dissemination to surrounding tissues or organs or promiscuous angiogenesis.
  • Enoxaparin may be used in the form of a composition in a pharmaceutically compatible product which may be inert or physiologically active. It is preferably used by the intravenous or subcutaneous route.
  • Sterile compositions for intravenous or subcutaneous administration are generally aqueous solutions. These compositions may also contain adjuvants, preferably selected from wetting, isotonizing, emulsifying, dispersing and stabilizing agents.
  • the sterilization can be carried out in several ways, for example, by aseptisizing filtration, by incorporating sterilizing agents into the composition, or by irradiation. They may also be prepared in the form of sterile solid compositions which may be dissolved at the time of use in sterile water or any other injectable sterile medium.
  • enoxaparin 20 mg are dissolved in a sufficient quantity of distilled water to prepare 0.2 ml of solution.
  • the doses depend on the desired effect, the duration of the treatment and the route of administration used; they are generally between 0.2 mg and 4 mg/kg per day, and preferably 1.5mg/kg daily or 0.75mg/kg twice a day, by the subcutaneous route, Le., 14 to 280 mg per day for an adult.
  • Dalteparin or Fragmin ® which is commercialized by Pharmacia & Upjohn may also be used in the synergistic combination of the present invention.
  • US patent 4,303,651 describes the structure and method of preparation of the Fragmin ® or Dalteparin.
  • the dalteparin or Fragmin ® may be prepared in several different ways.
  • One of the methods (a) comprises treatment of standard heparin with nitrous acid in dimethoxyethane as mentioned above. Said method gives this type of fragments together with a series of inactive fragments. The active fragments may then be freed from inactive elements, such as by affinity chromatography on matrix-bound antithrombin III [Hook et al., FEBS Lett. 66, 90 (1976); Hopwood et al., FEBS Lett. 63, 51 (1976); L.-O. Andersson et al., Thromb. Res. 9, 575 (1976)].
  • fragments are: (b) via periodate oxidation at low pH and low temperature; (c) via partial depolymerisation with heparinase; (d) via partial depolymerisation of heparin by esterification of carboxyl groups and subsequent alkaline ⁇ -elimination; (e) via partial depolymerisation of heparin by partial N- desulphatation and subsequent deamination with nitrous acid at a pH value of 3.9. Methods (a) and (b) are described in the examples.
  • the active fragments may contain from 14 to 18 sugar units. Structural analysis shows the same main structural components as in standard heparin, i.e.
  • the active fragments may have the structure (U-G) n -l-G-(U-G) m where n is 1 or 2 and m is 5 or 6, I is unsulphated L-iduronic acid, U is L-iduronic acid-2-O-sulphate and G is N-sulpho-D- glucosamine-6-O-sulphate.
  • a few U units may lack O-sulphate or be replaced by D-glucuronic acid and, similarly, a few G units may lack O-sulphate or be replaced by N-acetyl-D-glucosamine units.
  • Reducing or unreducing terminal units may vary with the type of method of preparation used; thus e.g., deaminitive splitting of heparin leads to the formation of 2,5-anhydro-D-mannose in reducing terminal position.
  • the active fragments may be characterized by means of physico- chemical methods, such as determination of mobility in an electric field and UV, IR and NMR spectra. However, the numerical values obtained do not give complete information, as also coagulation-inactive fragments substantially show similar characteristics.
  • the individual to be treated is typically a mammal, such as a human.
  • the individual may be one who is at risk of ishemia (such as any of the ischemic conditions mentioned herein), for example due to a genetic or environmental factor.
  • the individual may have a family history of ischemia.
  • the individual may have one or more of the following risk factors: be 45 years old or older (such as over 50, 55 or 60 years old, or older), be a smoker of cigarettes, have a high alcohol consumption, be overweight, have atherosclerosis or diabetes or be immobilized.
  • the individual may have cardiac or peripheral ischemia.
  • the individual who is treated may have had an ischemic attack in the last 24 hours, particularly an individual having a cardiac disease.
  • the individual may have had an ischemic attack more than 6 months ago, such as more than 1 , 2, 3 or more years ago, particularly an individual having a peripheral ischemia.
  • the individual may be in intensive care (typically being treated for an ischemic attack in the last 24 hours), and for example may be being given oxygen (or air enhanced in oxygen) for breathing. It is demonstrated that due to such superior characteristics in terms of synergy and potency, the combination of NV1 FGF and LMWH was particularly useful as therapeutic angiogenesis in aggravated conditions caused by hypercholesterolemia or diabetes.
  • the target tissue thus comprises in skeletal or cardiac muscle tissues suffering from or being at risk of suffering from ischemic damage which results when the tissue is deprived of an adequate supply of oxygenated blood, further aggravated in a hypercholesterolemia or diabetes setting.
  • the intramuscular or intramyocardial injection of a plasmid NV1 FGF in combination with LMWH or standard heparin may be efficiently used in a therapeutic window which is compatible with required standard of safety in gene therapy and is capable of inducing angiogenesis in an ischemic tissue further presenting an impaired endothelial function.
  • the invention also provides a product containing an agent or vector for expressing a gene encoding an angiogenic growth factor and a low molecular weight heparin or standard heparin for simultaneous, separate or sequential use in the treatment of ischemia (such as any of the ischemic conditions mentioned herein).
  • the invention provides use of agent or expression vector encoding an angiogenic growth factor in the manufacture of a medicament for treating ischemia (such as any of the ischemic conditions mentioned herein) by administering a combination of the angiogenic growth factor expressing agent and a low molecular weight heparin.
  • the invention also provides use of a low molecular weight heparin in the manufacture of a medicament for treating ischemia (such as any of the ischemic conditions mentioned herein) by administering a combination of an agent or an expression vector encoding an angiogenic growth factor and the low molecular weight heparin.
  • the DNA or vector encoding angiogenic growth factor and low molecular weight heparin may be administered separately (i.e.
  • an agent or vector for expressing a gene encoding an angiogenic growth factor will be within 1 week (such as within 1 or 2 days) of an administration of low molecular weight heparin.
  • the an agent or vector for expressing a gene encoding an angiogenic growth factor and the low molecular weight are administered at the same time (such as within 2 hours of each other) on at least 1 , 2, 3, 4, 5 or more occasions.
  • the NV1FGF plasmid is administered in a localized manner to the target skeletal or cardiac muscle tissue. While any suitable means of administering the NV1 FGF plasmid to the target tissue can be used within the context of the present invention, preferably a localized injection to the target muscle tissue is accomplished by directly injecting the NV1 FGF to the muscle using a needle or a catheter. By the term “injecting” it is meant that the NV1 FGF is forcefully introduced into the target skeletal or cardiac muscle tissue.
  • any suitable or available injection device, method, or system can be used according to the present invention. While administration of a dose of the NV1 FGF plasmid can be accomplished through a single injection to the target tissue, preferably administration of the dose is via multiple injections of NV1 FGF.
  • the multiple injections can be 2, 3, 4, 5, or more repeated injections, and preferably 5 or more injections into the ischemic muscle of a mammalian subject suffering from hypercholesterolemia or diabetes.
  • Multiple injections present an advantage over single injections in that they can be manipulated by such parameters as a specific geometry defined by the location on the target tissue where each injection is made.
  • the injection of a single dose of the NV1 FGF via multiple injections can be better controlled, and the effectiveness with which any given dose is administered may be maximized.
  • the angiogenic growth factor encoding agent or vector is administered at least more than three times at intervals of every 10 to 18 days, such as every 12 to 16 days.
  • the specific geometry of the multiple injections may be defined either in two- dimensional space, where the each application of the NV1 FGF is administered.
  • the multiple injections may be performed in or around the ischemic tissue, preferably are spaced such that the points of injection are separated by 2 or 3 cm. According to another embodiment of the present invention, each of the multiple injections is performed within about 5 to 10 minutes of each other.
  • NV1 FGF When administering the NV1 FGF to the target tissue which is affected by angiogenesis defects and wherein the endothelium function is severely impaired, it is desirable that the administration is such that the NV1 FGF is able to contact a region reasonably adjacent to the source and the terminus for the collateral blood vessel formation, as well as the area there between. Most preferably, intramuscular injection of NV1 FGF is performed into the distal thigh and distal leg muscles, and in the region close and surrounding the ischemic site. In one advantageous aspect of the present invention, a therapeutically effective dose of NV1 FGF in combination with LMWH is administered to reverse the defects in angiogenesis in a hypercholesterolemic or diabetes setting.
  • treatment is performed with increasing doses of 500 ⁇ g, 1000 ⁇ g, 2000 ⁇ g to 16000 ⁇ g of intramuscular injection of NV1 FGF. Most preferably, repeated dose injections of 2 X 500 ⁇ g , and 2 X 1000 ⁇ g.
  • NV1 FGF such as 4000 ⁇ g, 8000, 16000 or 2 x 2000 ⁇ g, 2 x 4000 ⁇ g, and 2 x 8000 ⁇ g may be administered in severe conditions of angiogenesis defects, in order to promote a sustained formation of both collateral capillary vessels and arterioles, thereby allowing to reverse angiogenesis defects due to ischemia in a mammalian subject suffering from hypercholesterolemia or diabetes.
  • the NV1 FGF desirably is administered to the target ischemic cardiac or skeletal muscle in a pharmaceutical composition, which comprises a pharmaceutically acceptable carrier and the NV1 FGF plasmid.
  • LMWH such as for example, Enoxaparin or Fraxiparin
  • Enoxaparin or Fraxiparin may be used in the form of a composition in a pharmaceutically compatible product which may be inert or physiologically active. They may preferably used by the intravenous or subcutaneous route.
  • Sterile compositions for intravenous or subcutaneous administration are generally aqueous solutions. These compositions may also contain adjuvants, preferably selected from wetting, isotonizing, emulsifying, dispersing and stabilizing agents.
  • the sterilization can be carried out in several ways, for example, by aseptisizing filtration, by incorporating sterilizing agents into the composition, or by irradiation.
  • sterile solid compositions which may be dissolved at the time of use in sterile water or any other injectable sterile medium.
  • 20 mg of LMWH are dissolved in a sufficient quantity of distilled water to prepare 0.2 ml of solution.
  • the doses depend on the desired effect, the duration of the treatment and the route of administration used; they are generally between 0.2 mg and 4 mg/kg per day by the subcutaneous route, Le., 14 to 280 mg per day for an adult with unit doses ranging from 5 to 280 mg.
  • the dose is 0.2 to 0.3 mg/kg per day.
  • the LMWH is administered over at least 20 or at least 30 days (in which days at least 1 , 2, 3 or more administrations of the angiogenic growth factor encoding factor also occur), for example so that the first and last administration of heparin (LMWH or standard heparin) are separated by at least 20 or at least 30 days.
  • Any suitable pharmaceutically acceptable carrier can be used within the context of the present invention, and such carriers are well known in the art. The choice of carrier will be determined, in part, by the particular site to which the composition is to be administered and the particular method used to administer the composition.
  • Formulations suitable for injection include aqueous and non-aqueous solutions, isotonic sterile injection solutions, which can contain anti- oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.
  • the formulations can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water, immediately prior to use.
  • Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.
  • the pharmaceutically acceptable carrier is a buffered saline solution.
  • the pharmaceutical composition comprises a solution of sodium chloride.
  • the present invention further relates to a novel method of treating a patient having peripheral and/or coronary ischemic syndrome by administering to the patient an effective amount of LMWH and an effective amount of a DNA encoding angiogenic growth factor, preferably the NV1 FGF plasmid.
  • Patients at risk include those whose suffer initial coronary or peripheral ischemic syndrome symptoms and who are therefore more likely than others who have not suffered such symptoms to experience ischemic skeletal or myocardial tissue damage.
  • the present invention also provides an angiogenic therapy for treating ischemic conditions such as peripheral or myocardial ischemia, wound healing or other conditions that require neovascularization or tissue regeneration.
  • the present invention may be useful for treating PAD (peripheral arterial disease) also designated PAOD (peripheral arterial occlusive disease) pathologies, IC (intermittent claudication) or CAD (coronary arterial diseases).
  • PAD peripheral arterial disease
  • PAOD peripheral arterial occlusive disease
  • IC intermittent claudication
  • CAD coronary arterial diseases
  • Another object of the present invention is to provide a method of enhancing therapeutic angiogenesis in a patient subject of angiogplasty, bypass grafting or other revascularization procedures by administering the combination of LMWH or standard heparin and NV1FGF as described hereinabove.
  • Still another object of the present invention is to provide a new method of stimulating and/or promoting revascularization of ischemic cardiac and skeletal muscles in a mammalian subject suffering from impaired endothelium function, by administering the combination of the invention in an amount sufficient to promote both collateral blood vessels and arterioles in ischemic skeletal or cardiac muscles. More particularly, the present invention relates to a method of stimulating and/or promoting revascularization in ischemic muscles in a hypercholesterolemia or diabetes setting, and a method for reversing angiogenesis defects in a mammalian subject in need for such treatment suffering from hypercholesterolemia or diabetes. Impaired perfusion in the hindlimb due to single or multiple large vessel occlusions is the cause of peripheral arterial disease (PAD).
  • PAD peripheral arterial disease
  • the combination of NV1 FGF and LMWH or standard heparin has been demonstrated to be particularly potent for rescuing cholesterol-induced impairment of angiogenesis in patients suffering from PAD or coronary arterial disease, by promoting the growth of both collateral vessels and arterioles.
  • the NV1 FGF in combination with LMWH or standard heparin is capable to effectively induce the formation of mature large conductance vessels (>150 ⁇ m collateral vessels) and small resistance arteries ( ⁇ 50 ⁇ m arterioles) in ischemia-injured muscles of the posterior part of the thigh, which are required to convey and to deliver blood to tissues.
  • arterioles Induction of such mature vessels such as arterioles has been evidenced to be an invaluable treatment in most severe cases where adverse angiogenesis defects are elicited by hypercholesterolemia or diabetes.
  • Outgrowth of collateral arterioles is a particularly efficient and superior therapeutic angiogenesis, as arterioles are known as mature vessels comprising a layer of endothelial cells and a mural cells formed of pericytes, and have a high capacity of these vessels compared with the capillary network (Carmeliet et al., Nat. Med., 2000; 6:389-395; Van Royen et al., Cardiovasc. Res., 2001 ;49:543-553).
  • a further object of the present invention is to provide a method of promoting angiogenesis by administering a combination of NV1 FGF and LMWH or standard heparin with the proviso that VEGF is not upregulated in the treated cells
  • various publications, patents and patent applications have been referred to. The teaching and disclosures of these publications, patents and patent applications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which the present application pertains. It is also understood and expected that variations in the principles of invention herein disclosed in an exemplary embodiment may be made by one skilled in the art and it is intended that such modifications, changes and substitutions are to be included within the scope of the present application.
  • Example 1 Animals and diets Syrian Golden hamsters of 11-12 weeks (CERJ, Le Genest St Isle, France) were used in the experiments. Animals were allowed to equilibrate in standard conditions at least 7 days before initiation of the study protocol. All animals had free access to water for the entire duration of the experiments. All animal procedures were approved by the Animal Use Committee and comply with guidelines published by the National Institute of Health (NIH publication No. 85-23, revised 1985). Hamsters under high cholesterol diet were given 20g per animal of cholesterol-enriched diet daily, made of standard chow supplemented with 3% cholesterol and 15% cocoa butter (ref.
  • Example 2 Induction of hindlimb ischemia After 35 days of LC or HC diet, animals were subjected to hindlimb ischemia, according to the following surgical procedure. Hindlimb ischemia was induced under gas anesthesia with N 2 0 (0.8 l.min “1 ), O 2 (0.4 l.min “1 ) and isofluorane (2%) according to a procedure described in other animal species (19,20). Under sterile surgical conditions, a longitudinal incision was performed on the medial thigh of the right hindlimb from the inguinal ligament to a point proximal to the patella. Through this incision, using surgical loops, the femoral artery was dissected free and its major branches were coagulated.
  • the femoral artery was completely excised from its proximal origin as a branch of the external iliac artery to the point distal where it bifurcates into the saphenous and popliteal branches (4).
  • the incision was closed in one layer with a 4.0 silk wire.
  • Example 3 Gene transfer in hindlimb skeletal muscles Saline, plasmid encoding NV1 FGF with or without enoxaparin and enoxaparin with saline was given blinded 14 days after induction of ischemia, through three 60- ⁇ L injections each in Tibialis cranialis, Adductores and Quadriceps muscles of the ischemic limb.
  • Example 4 Measures of total cholesterol, lipids and triglyceride levels in serum
  • blood was obtained from hamsters of HC/21 , HC/28, saline, NV1 FGF, enoxaparin, or NV1 FGF with enoxaparin groups by retro-orbital puncture under gas anesthesia with N 2 0 (0.8 l.min "1 ), 0 2 (0.4 l.min "1 ) and isofluorane (2%).
  • Total cholesterol and triglyceride levels in serum were determined enzymatically with commercially available kits (Olympus Diagnostica GmbH, Hamburg, Germany).
  • Example 5 Quantification of collateral vessel formation by angiography On day 21 (HC/21 group) or day 28 (LC, HC/28, saline and NV1FGF groups) after induction of ischemia, angiographic procedure was performed as follows. Immediately after injection of ⁇ 300 ⁇ l of contrast medium (0.5 g.ml "1 sulfate barium solution in water) through a catheter inserted into abdominal aorta, hamsters were sacrificed with an overdose of sodium pentobarbital.
  • contrast medium 0.5 g.ml "1 sulfate barium solution in water
  • Angiographic score was calculated as the ratio ischemic/non-ischemic percentages. In order to check the validity of the method, angiographic score was assessed in six separate age-matched hamsters not subjected to hindlimb ischemia. As expected, angiographic score calculated as the ratio right limb/left limb percentages was 1.04 + 0.18.
  • Example 6 Quantification of arteriolar formation by immunohistochemistry and typical muscle lesions induced by hindlimb ischemia through excision of the femoral artery of hypercholesterolemic hamsters Day 28 after induction of ischemia, skeletal muscles from the ischemic hindlimb were harvested and fixed in a solution of PBS-3.7 % formaline. Muscles from the non-ischemic hindlimb were sampled similarly and served as control muscles. Two transverse slices composed of different muscles (Gracilis, Semimembranosus, Adductores, Semitendinosus, Biceps femoris), were processed from the back part of each thigh. Slices were dehydrated, embedded in paraffin and 5- ⁇ m thick sections were prepared for immunohistochemistry.
  • a mouse monoclonal antibody directed against smooth muscle -actin (SMA; clone 1A4, dilution 1:200, Dako, Carpinteria, CA, USA) was used as a marker for vascular smooth muscle cells (VSMCs) since it is constitutively expressed in mature vessels.
  • SMA antibody was detected with a commercially available kit (EnvisionTM+ System/Horse Radish Peroxidase, Dako, Carpinteria, CA, USA) through an avidin- biotin-peroxidase method.
  • SMA-positive (SMA+) vessels were ranked by size (outer diameter) and arterioles with diameter ⁇ 50 ⁇ m were counted in both Adductores and Gracilis muscles.
  • Figure 2A shows a cross-section at magnification X100 of the non-ischemic controlateral muscles having no lesions, as a control.
  • Total area of Adductores and Gracilis muscles was determined to investigate the impact of ischemia on muscle volume.
  • Number of SMA+ arterioles was determined for the total muscle area. For both parameters, the ratio ischemic/non-ischemic values were then calculated. All procedures were performed by an investigator blinded to the treatment.
  • Example 7 Expression of FGF-1 after NV1FGF gene transfer in ischemic muscles 14 days after saline injection, NV1FGF gene transfer, and injection of the combination of NV1FGF and enoxaparin (i.e., 28 days after induction of ischemia) in Tibialis cranialis muscle from non-ischemic and ischemic limbs were processed as follows. FGF-1 immunohistochemistry was performed using a classical streptavidin-biotin assay used to detect FGF1 expression.
  • FGF-1 was advantageously restricted to the ischemic muscles of animals treated with NV1 FGF.
  • representative pictures magnification X100 of immunohistochemical staining with an anti-FGF-1 polyclonal antibody in muscles from the back part of the thigh (Tibialis Cranialis, Gracilis and Adductores) from non-ischemic (controlateral) non injected limbs and ischemic limbs injected with saline or with NV1FGF
  • the expression of FGF-1 could surprisingly be detected neither in the ischemic muscles of saline-treated animals, nor in non-ischemic (controlateral) muscles of saline and NV1 FGF-treated animals.
  • NV1FGF plasmid NV1 FGF which allows a slow release of the encoded FGF-1 protein within a therapeutic window sufficient to effect a sustained angiogenic response via the formation of mature blood vessels, but at a concentration which does not permit dissemination and promiscuous angiogenesis or negative side effects.
  • NV1FGF was thus proved to be particularly potent, as being capable of efficiently promoting angiogenesis at a non-detectable concentration in treated muscles, thus allowing use of concentrations of NV1FGF comprised within a therapeutic window and in conditions characterized by aggravated endothelial dysfunctions.
  • the NV1 FGF may advantageously be used as angiogenesis therapy in aggravated conditions caused by hypercholesterolemia or diabetes.
  • NV1FGF gene therapy is capable of rescuing impaired collateral formation via the increasing of arterioles.
  • the growth of >150 ⁇ m collateral vessels have been evidenced angiographically in the posterior part of the thigh, which comprises Biceps femoris, Adductores, Gracilis, Semimembranosus, and Semitendinosus muscles.
  • the Applicant has demonstrated that formation of collateral vessels was significantly stimulated into this region, 14 days after NV1 FGF gene transfer, as emphasized by angiographic score.
  • Example 8 Comparative experiments of NV1FGF gene transfer and NV1FGF in combination with enoxaparin on collateral development and arteriolar density 14 days after intramuscular administration (Le., 28 days after hindlimb ischemia) in hypercholesterolemic hamsters Combination of intramuscular NV1 FGF gene transfer with subcutaneous administration of enoxaparin 14 days after induction of hindlimb ischemia greatly improves collateral formation in the ischemic limb, when compared with saline-treated hamsters, enoxaparin, or NV1 FGF gene transfer.
  • angiographic score after administration of NV1 FGF and enoxaparin is indeed significantly higher than that of saline-treated hamsters or NV1 FGF-treated hamsters, or enoxaparin-treated hamsters.
  • SMA smooth muscle ⁇ -actin
  • a decreased area of Tibialis cranialis, Adductores and Gracilis muscles in the ischemic limb is observed in NV1 FGF & enoxaparin group.
  • Example 9 treatment of hindlimb ischemia in diabetic mice using NV1FGF and enoxaparin or standard heparin
  • angiogenic therapy with angiogenic cytokines has been proposed as an approach to treat patients with an apparent inadequacy of collateral vessels.
  • Diabetes mellitus is associated with a marked impairment in collateral formation. Therefore, an experimental model of hindlimb ischemia is used in the setting of diabetes to support the combination an anti-inflammatory or anti-coagulant agent, a low molecular weight heparin, such as enoxaparin, with NV1 FGF angiogenic gene therapy.
  • Streptozotocin selectively destroys insulin-producing beta islet cells of the pancreas providing a model of type I diabetes. Streptozotocin dissolved at 10 mg/ml in 0.1 M sodium citrate buffer (pH 5.5) is injected intraperitoneally to 8-10 week-old male C57BI/6 mice at a dose of 80 mg/kg. Mice are considered diabetic and are thus included in the study if the plasma glucose levels at 72 hours are above 250 mg/dl and remained elevated.
  • the surgical procedure is performed 4 weeks after the induction of diabetes and is done under a microscope. Skin incisions are performed at the groin of the right hindlimb overlying the iliac artery, and carried out downward. The iliac artery is then ligated proximally and distally with 4-0 silk ligatures, and excised. All accessory arteries are ligated and cut.
  • NV1 FGF plasmid solution was injected in the range of 0.4-4.0 mg/kg body weight (i.e. 10-100 ⁇ g/mouse or 5-50 ⁇ g/muscle) as a single administration in the quadriceps and/or in the tibialis cranialis muscle (volume injected: 40 ⁇ l/muscle) of the right hindlimb within the 7 days following surgery.
  • Enoxaparin is injected subcutaneously once daily for 1-2 weeks at the dose of 1.0-1.5 mg/kg, starting a few hours prior surgery, concomitantly with surgery or the day after surgery.
  • a single dose of 0.5-0.75 mg/kg enoxaparin may be administered by intravenous injection prior to the initiation of s.c. treatment.
  • Perfusion of the hindlimb is assessed non-invasively by laser Doppler imaging before surgery, immediately after surgery and weekly after surgery for 4 consecutive weeks. Ischemic (right)/normal (left) limb blood flow ratio is measured to compare the kinetics of reperfusion and the perfusion status between treatment groups.
  • a marker of angiogenesis such as CD31 may be also assessed by semi-quantitative methods such as ELISA (protein) or RT-PCR (mRNA) on muscle extracts or immunohistochemistry (protein) on muscle sections.
  • Endpoints to monitor synergistic effect - a comparison showing evidence of faster kinetics of hindlimb reperfusion in mice treated with NV1FGF + enoxparin compared to any of the single agent, or

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EP04804488A 2003-12-29 2004-12-29 Behandlung von koronarer oder peripherer ischämie Withdrawn EP1699819A2 (de)

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RU2010116271A (ru) * 2007-09-26 2011-11-10 Селджин Селльюлар Терапьютикс (Us) Ангиогенные клетки из плацентарного перфузата человека
EP2558115B1 (de) * 2010-04-16 2019-07-31 The Salk Institute for Biological Studies Verfahren zur behandlung von stoffwechselstörungen mittels fgf
WO2015061331A1 (en) 2013-10-21 2015-04-30 Salk Institute For Biological Studies Chimeric fibroblast growth factor (fgf) 2/fgf1 peptides and methods of use
CN105828833A (zh) 2013-10-21 2016-08-03 萨克生物研究学院 突变的成纤维细胞生长因子（fgf）1及使用方法
EP3368059A4 (de) 2015-10-30 2019-03-27 Salk Institute for Biological Studies Behandlung von steroid-induzierter hyperglykämie mit fibroblastenwachstumsfaktor (fgf)-1-analoga
TWI698241B (zh) * 2017-09-05 2020-07-11 國立成功大學 用於治療缺血的肝素組合物
US11542309B2 (en) 2019-07-31 2023-01-03 Salk Institute For Biological Studies Fibroblast growth factor 1 (FGF1) mutant proteins that selectively activate FGFR1B to reduce blood glucose

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US6395707B1 (en) * 1997-02-14 2002-05-28 Genentech, Inc. Methods of treatment utilizing variants of vascular endothelial cell growth factor
US7252818B2 (en) * 1998-07-24 2007-08-07 Cardiovascular Biotherapeutics, Inc. Method of producing biologically active human acidic fibroblast growth factor and its use in promoting angiogenesis
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US20050187150A1 (en) * 2001-10-31 2005-08-25 New York University Structure-based design and synthesis of FGF inhibitors and FGF modulator compounds
BRPI0410844A (pt) * 2003-06-05 2006-06-27 Centelion fator de crescimento de fibroblasto de codificação de plasmìdeo para o tratamento de defeitos angiogênicos associados com hipercolesterolemia ou diabetes

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KR20070006714A (ko) 2007-01-11
CA2550727A1 (en) 2005-07-14
WO2005063807A3 (en) 2005-09-29
JP2007536909A (ja) 2007-12-20
MXPA06007519A (es) 2007-05-23
ZA200606268B (en) 2008-04-30
AU2004309090A1 (en) 2005-07-14

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