AU2004309090A1 - Treatment of coronary or peripheral ischemia - Google Patents

Description

WO 2005/063807 PCT/EP2004/014910 TREATMENT OF CORONARY OR PERIPHERAL ISCHEMIA 5 Field of the Invention and Introduction 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 10 angiogenesis and arteriogenesis in skeletal and cardiac muscles. Background of the invention Restoration of blood perfusion in skeletal and cardiac ischemic tissues involves the complex process of angiogenesis, j., proliferation of endothelial cells, the degradation of the 15 basement membrane, the migration through the surrounding matrix, as well as the alignment, and differentiation into tube-like structures to form the walls of blood vessels thus resulting in a newly formed capillary network. Arteriogenesis, which refers to the outgrowth of collateral arterioles is also believed to be an efficient process for restoration of blood perfusion because of the high capacity of these vessels compared with the capillary network (Carmeliet et al., Nat. Med., 2000; 20 6:389-395; Van Royen et al., Cardiovasc. Res., 2001;49:543-553). Various strategies of restoring blood perfusion exist. 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 25 been investigated. Protein therapy which involves delivery of the growth factor directly in the ischemic tissue constitutes an alternative option for therapeutic angiogenesis. For example, administration of several recombinant proteins, VEGF, FGF, HGF, PDGF, and TGF-P, to promote collateral blood vessels has been proposed. 30 Animal studies have demonstrated that local administration of several different angiogenic proteins, including for example recombinant human VEGF protein, recombinant human basic FGF protein (bFGF), recombinant TGF-p or PDGF proteins, promotes collateral blood vessels. Such recombinant angiogenic proteins have been proposed for the treatment of ischemic cardiovascular diseases, ie., peripheral arterial disease or coronary arterial disease. 35 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 40 molecular of most commercial heparin preparations is in the range of 12000 to 15000 Da. On a large scale, standard heparin is isolated from intestinal mucus from swine of lung from cattle.

WO 2005/063807 PCT/EP2004/014910 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 5 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 10 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. Finally, the third phase consists of fibrin formation, wherein the blood fibrinogen is under the effect of thrombin converted into fibrin which 15 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. 20 The intrinsic or endogenic route involves a large number of factors or plasmatic proenzymes capable of being successively activated (factors XII, XI, IX, VillI and X), where each activated product (factors XIIa, 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 25 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 Vil 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 30 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. These coagulation processes normally give rise to a process, called fibrinolysis, intended 35 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. In fact, there are balanced mechanisms, according to extremely complex processes, under the dependence of opposed activator and 40 inhibitor factors. The unbalance of these mechanisms, in the sense of hypercoagulability, is then 2 WO 2005/063807 PCT/EP2004/014910 capable of resulting in thromboses. On the other hand, hypocoagulability exposes the host to hemorragic risks. The detailed mechanism of the anticoagulation activity of the heparin is now essentially known. Heparin acts at several levels in cascades of successive enzymatic reactions, which are 5 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 10 inhibition of two principle procoagulant proteases, factor Xa and thrombin. The rate of inhibition of both these enzymes by antithrombin alone is thus increased about a 1,000-fold by heparin. Accelerated inactivation of both the active forms of proteases prevents the subsequent conversion of fibrinogen to fibrin that is crucial for clot formation. Heparin thus permits the palliative effects of hypercoagulability and balance coagulation 15 fibrinolysis mechanism, when subject to a considerable disturbance, for example on the occasion of a surgical operation. It is however well known that these attempts at re-equilibration are extremely delicate and that, consequently, the administration of an elevated dose of anticoagulant -or the insufficient selectivity of the latter- for the purpose of preventing the risks of hypercoagulation, for example the appearance of post-operative thromboses, may finally lead to 20 serious hemorrhages. Summary of the Invention The Applicants have now discovered that a heparin compound such as low molecular weight heparin (LMWH) or standard heparin may be used in combination with an expression 25 vector encoding an angiogenic growth factor to induce a strong angiogenesis and arteriogenesis synergistic response. The Applicants have also shown that the composition of standard heparin and an expression vector encoding a fibroblast growth factor (FGF) results in a strong angiogenesis and arteriogenesis effect. Contrary to the general knowledge that heparin is a potent inhibitor of polymerase, by competing with duplex DNA for binding to the polymerase, the 30 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. 35 In addition, it has been surprisingly discovered that 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. High levels of lipids can severely impair endothelial functions such as vasodilatation, leukocyte-endothelium-interactions, thrombosis, fibrinolysis, and endothelial cell growth (3). Particularly, the nitric oxide synthase (NOS) system is affected by 40 those physiopathological conditions including high levels of oxidized low-density lipoprotein 3 WO 2005/063807 PCT/EP2004/014910 cholesterol (LDL-C). Vascular function in experimental models of diabetes and patients with types I and 11 diabetes is characterized by greatly impaired angiogenic functions. The Applicants have showed that an expression vector encoding an angiogenic growth factor such as acidic fibroblast growth factor in combination with a heparin compound such as 5 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. The Applicants have discovered that product comprising an expression vector encoding 10 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. The present invention relates to compositions which have a strong angiogenic effect and is particularly selective to skeletal and cardiac muscles. The 15 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, however, is not limited to the use of a DNA expression vector or naked DNA in combination with a heparin compound. Instead, any agent or vector capable of promoting 20 or causing the expression of an angiogenic growth factor in a cell or organism can be selected and used. One of skill in the art is familiar with numerous examples of these agents or vectors, including 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. Thus, 25 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 30 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 35 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 40 such as standard heparin or LMWH, and to the use of such product for the manufacture of a 4 WO 2005/063807 PCT/EP2004/014910 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. 5 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 10 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 15 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. 20 The present invention further provides a method of promoting formation of mature large conductance vessels (>150pm collateral vessels) and small resistance arteries (<50pm 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 25 collateral blood vessel growth and arteriole growth in ischemic tissues, especially when endothelial function is impaired. It is also an object of the present invention to provide 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 30 treatment suffering from hypercholesterolemia or diabetes. Another object of the present invention is to provide an angiogenic therapy for treating ischemic conditions such as peripheral or myocardial ischemia, wound healing or other conditions that require neovascularization or tissue regeneration. Still, another object of the present invention is to provide a method of enhancing 35 therapeutic angiogenesis in a patient subject of angioplasty, bypass grafting or other revascularization procedures. Brief Description of the Figures 40 Figure 1: The chart corresponds to a measurement of luciferase expression after injection of 5 WO 2005/063807 PCT/EP2004/014910 6Opg of a plasmid, such as pCOR, carrying the luciferase gene in three hamster muscles, ie., 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 5 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. Detailed Description of the invention 10 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 15 subject. The present invention also relates to a novel method of promoting angiogenesis and arteriogenesis in cardiac and skeletal muscles. The term "subject" includes,-.but is not limited to, mammals, such as dogs, cats, horses, cows, pigs, rats, mice, simians, and humans. 20 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 25 such angiogenic growth factors are well known in the art. For example, DNA coding sequences of FGF-1, FGF-2, FGF-4, FGF-5, FGF-6, and FGF-7 are set forth in SEQ ID NOs: 1 to 6, respectively. Other 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, 30 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-l or 1i). Preferred compositions of the present invention comprise a DNA encoding the acidic fibroblast growth factor (FGF-1 or aFGF). 35 The angiogenic factor encoding sequence may be carried by a viral or non viral expression vector. The term "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. When 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 40 angiogenic factor. Such vectors include chromosomal, episomal, and virus-derived vectors, for 6 WO 2005/063807 PCT/EP2004/014910 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 5 sources such as those derived from plasmid and bacteriophage genetic elements, e.g. cosmids and phagemids. Appropriate cloning and expression vectors for prokaryotic and eukaryotic hosts are described in Sambrook et al., Molecular Cloning: A Laboratory Manual. 2nd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1989). Most preferably, the angiogenic factor is carried and expressed by a plasmid. Expression 10 plasmids are well known in the art. As expression plasmids derived from ColEl 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 15 include pCDM8 (Seed, B. Nature 329:840(1987)) and pMT2PC (Kaufman et al., EMBO J. 6:187 195 (1987)). Other suitable genetic constructs can be provided with mammalian origin of replication in order to maintain the construct extrachromosomally and produce multiple copies of the construct in the cell. Plasmids pCEP4 and pREP4 from Invitrogen (San Diego, Calif.) contain the Epstein Barr virus origin of replication and nuclear antigen EBNA-1 coding region which 20 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. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 25 1989. 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 30 angiogenic factor. Examples of 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 35 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. 40 A variety of vectors providing for constitutive and inducible expression in prokaryotic and 7 WO 2005/063807 PCT/EP2004/014910 eukaryotic hosts are well known to those of ordinary skill in the art. For example, 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 5 publication WO01/ 1064. Examples of polyadenylation signals useful to practice the present invention, especially in the production of a genetic vaccine for humans, include but are not limited to SV40 polyadenylation signals, bovine or human Growth hormone polyadenylation signals, and LTR polyadenylation signals. In particular, the SV40 polyadenylation signal which is in pCEP4 plasmid 10 (Invitrogen, San Diego Calif.), referred to as the SV40 polyadenylation signal is used. In addition to the regulatory elements required for DNA expression, 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 15 SV40, RSV, and EBV enhancers, the cytomegalovirus immediate early enhancer, polyoma enhancer, adenovirus enhancers, and retrovirus LTR enhancers. In addition to containing sites for transcription initiation and control, 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 20 the numerous regulatory sequences that are useful in expression vectors. Such regulatory sequences are described, for example, in Sambrook et al, Molecular Cloning. A Laboratory Manual. 2nd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1989). The angiogenic factor coding sequence can be inserted into the vector nucleic acid by well-known methodology. Generally, the DNA sequence that will ultimately be expressed is joined 25 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 30 contained in the same vector that contains the nucleic acid molecules described herein or may be on a separate vector. Any marker that provides selection for a phenotypic trait will be effective. Where secretion of the angiogenic factor is desired, appropriate secretion signals are incorporated into the expression vector. The signal sequence can be endogenous to the peptides or heterologous to these peptides. 35 According to a most preferred embodiment, the expression vector is a plasmid expressing FGF-1 and is designated NVIFGF. NV1FGF 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 40 in SEQ ID NO:7. The sequence encoding sphFGF-1 is a fusion between the sequences encoding 8 WO 2005/063807 PCT/EP2004/014910 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). The late polyadenylation signal from simian 5 virus 40 (nucleotides 2538 to 2759 from SV40 genome, GenBank locus SV4CG; US 5,168,062) 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 10 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 15 present invention may be administered by any suitable means known or available to persons skilled in the art. According to the present invention, 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 20 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. On a large scale, standard heparin is isolated from intestinal mucus from swine of lung from cattle. 25 As shown below, 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. More precisely, 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 30 sulfated giving rise to a polymer that is highly negatively charged. Particularly, the central D glucosamine residue contains a unique 3-0-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. 9 WO 2005/063807 PCT/EP2004/014910

CH

2 SOg COO- CH 2 SO- CH2SO OH OH OS0 3 O 0 0 000 NHSO OH NHSO[ OS0 NHSO 3 Because of its highly acidic sulfate groups, heparin exists as the anion at physiologic pH and is usually administered as the sodium salt. Twenty to fifty percent is excreted unchanged. The heparin polysaccharide chain is degraded in the gastric acid and is thus generally 5 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 10 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. For example, 15 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. Other processes are also described by Johnson et al, Thrombos.Haemostas.Stuttg., 35, 586 (1976); 20 Lane et al, Thrombosis Research, 16, 651; Lasker et al, U.S. Pat. No. 3,766,167)). Mixtures of both high and low molecular weight heparins may also be used in the present invention. To this regard, 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 25 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 30 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 10 WO 2005/063807 PCT/EP2004/014910 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. Preferably, the heparin starting material is a porcine heparin, and, in particular, a porcine mucosal 5 heparin. It may be preliminarily precipitated by means of an alcohol upstream of the salification thereof. By way of example, the process for preparation of low molecular weight heparin may comprise the following steps. A preliminary step allowing the preparation of a heparin having a dermatan sulfate content of less than 2%. Then, a step of salification of the starting heparin is 10 performed. 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 200C. For example, quaternary ammonium salts that are used are abenzethonium salts such as benzethonium chloride. As a second step, esterification is carried out. The partial ester of heparin in salt form, the degree of esterification of which ranges from 9.5% to 14%, may be 15 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. 20 The degree of esterification ranging from 9.5% to 14% may be reached by using approximately I 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 450C. Alternatively, the partial ester of heparin may be in the form of a sodium salt. The esters 25 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. In particular, in the case of the benzyl ester, the amount of benzyl alcohol produced by saponification of the may be measured. 30 As a final step, 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 700C, 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 35 ratio water/ester ranges from 15 to 30. Alternatively, 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 40 then maintained at a temperature of from 55 to 650C for from 1 to 2 hours.The product may then 11 WO 2005/063807 PCT/EP2004/014910 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. It is possible to proceed with additional fractionations of the mucopolysaccharide fraction obtained at the end of the above-mentioned process, by various techniques, such as gel-filtration 5 or again selective precipitation in an aqueous-alcoholic medium of predetermined titer, in the presence of proportions also predetermined of an inorganic salt, such as sodium chloride. 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 10 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 15 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. To the extent where 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 20 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. Advantageously, it is possible to resort to the process described in French Pat. No. 73 13580 filed Apr. 13, 1973. It will be recalled that 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, 25 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. 30 The factions may be characterized on the one hand, by a particular affinity with regard to antithrombin ill manifested by their capacity to be fixed on the latter, notably in a system comprising the contacting of the fractions with an antithrombin Ill fixed on a support, such as agarose, in an 0.2M NaCl, 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 (YWIUSP ratio). 35 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 (' H) carried out on solutions of these compounds dissolved in deuteriated water at 350 C. with a radiation of 270 megahertz (MHz) there are observed as characteristic element of the spectrum, resonance signals which, for chemical displacement of the order of 4.8 and 5.2 ppm, 40 are substantially weaker than the resonance signal which is also observed for a chemical 12 WO 2005/063807 PCT/EP2004/014910 displacement of the order of 5.4 ppm (reference for the measurement of the displacements: TSP (sodium 3-trimethylsilyl propionate 2,2, 3,3-d4)). 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 5 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. 10 Several studies have shown that 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., Vasc Med, 2000; 5: 251-258). 15 According to a preferred embodiment, 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 20 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. It is an antithrombotic agent useful in the treatment and prophylaxis of thrombosis, containing dalteparin sodium with an average molecular weight of 25 5000 Da. 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. Other low molecular weight heparins on the market are Fraxiparine@ (nadroparin) 30 commercialized by Sanofi, Clivarin@ (reviparin) and Innohelp@ (tinzaparin) commercialized by Dupont, may be used in combination with NVI FGF according to the present invention. In a most preferred embodiment, the plasmid NV1FGF is used in combination with enoxaparin, such as enoxaparin (Lovenox@, Clexane@), Fragmin@, or Fraxiparine@. According to this embodiment Enoxaparin is used in combination with an angiogenic 35 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 FGFI-22. 40 According to another embodiment, enoxaparin is used in combination with the plasmid 13 WO 2005/063807 PCT/EP2004/014910 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. In addition, 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. When 5 used in combination with enoxaparin, 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 10 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 15 solid compositions which may be dissolved at the time of use in sterile water or any other injectable sterile medium. As an example of a suitable composition, 20 mg of enoxaparin 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 20 a day, by the subcutaneous route, ie., 14 to 280 mg per day for an adult. Dalteparin or Fragmine 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 25 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 IlIl [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)]. other ways of preparing fragments are: (b) via 30 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 B-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. 35 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. L-iduronosyl-2-O-sulphate (1.alpha.-4)-N-sulpho-D-glucosamine-6-O-sulphate as the dominating saccharide unit. However, the amount of unsulphated iduronic acid may be considerably higher than in the starting material. The active fragments may have the structure (U-G)n -1-G-(U-G)m where n is I or 2 and m is 5 or 6, 40 I is unsulphated L-iduronic acid, U is L-iduronic acid-2-O-sulphate and G is N-sulpho-D 14 WO 2005/063807 PCT/EP2004/014910 glucosamine-6-0-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 5 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. This depends on the fact that the biologic activity is derived from a specific sequence of the sugar residues where the 10 position of the unsulphated uronic acid is especially important. 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. Thus 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 15 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 20 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. 25 It is demonstrated that due to such superior characteristics in terms of synergy and potency, the combination of NV1FGF and LMWH was particularly useful as therapeutic angiogenesis in aggravated conditions caused by hypercholesterolemia or diabetes. The reversal of angiogenesis defects caused by attenuated blood supply regardless of its origin which is aggravated in conditions such as hypercholesterolemia or diabetes is thus 30 contemplated by the present invention. Within the context of the present invention, 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. As demonstrated in the Examples, the 35 intramuscular or intramyocardial injection of a plasmid NV1FGF 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 40 gene encoding an angiogenic growth factor and a low molecular weight heparin or standard 15 WO 2005/063807 PCT/EP2004/014910 heparin for simultaneous, separate or sequential use in the treatment of ischemia (such as any of the ischemic conditions mentioned herein). Further 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 5 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. 10 The DNA or vector encoding angiogenic growth factor and low molecular weight heparin may be administered separately (i.e. not in the form of a composition comprising both) or may be administered in the form a composition containing both of them. In the case of separate administrations at least 1, 2, 3 or more of the administrations of 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 15 an administration of low molecular weight heparin. In one embodiment 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. It is understood that administration features, such as dosage, site or timings of 20 administration which are disclosed in the context of NV1FGF plasmid, are applicable to the administration of any angiogenic growth factor encoding factor. According to one embodiment of the present invention, 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 25 context of the present invention, preferably a localized injection to the target muscle tissue is accomplished by directly injecting the NVI 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. 30 While administration of a dose of the NV1FGF plasmid can be accomplished through a single injection to the target tissue, preferably administration of the dose is via multiple injections of NV1FGF. 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 35 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 NV1FGF via multiple injections can be better controlled, and the effectiveness with which any given dose is administered may be maximized. Typically the angiogenic growth factor encoding agent or vector is administered at least 40 more than three times at intervals of every 10 to 18 days, such as every 12 to 16 days. 16 WO 2005/063807 PCT/EP2004/014910 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. 5 According to another embodiment of the present invention, each of the multiple injections is performed within about 5 to 10 minutes of each other. 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 NV1FGF is able to contact a region reasonably adjacent to the 10 source and the terminus for the collateral blood vessel formation, as well as the area there between. Most preferably, intramuscular injection of NV1FGF 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 15 NVIFGF in combination with LMWH is administered to reverse the defects in angiogenesis in a hypercholesterolemic or diabetes setting. While the effective dose will vary depending on the weight and condition of a given subject suffering from angiogenesis defects in addition to hypercholesterolemia or diabetic subject, it is considered within the skill in the art to determine the appropriate dosage for a given subject and conditions 20 According to a preferred embodiment of this aspect, treatment is performed with increasing doses of 500pg, 1000pg, 2000pg to 16000pg of intramuscular injection of NV1FGF. Most preferably, repeated dose injections of 2 X 500pg , and 2 X 1000pg. Higher doses may be used in view of the high safety of NV1FGF, such as 4000pg, 8000, 16000 or 2 x 2000pg, 2 x 4000pg, and 2 x 80OOpg may be administered in severe conditions of angiogenesis defects, in 25 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 NVI FGF desirably is administered to the target ischemic cardiac or skeletal muscle in a pharmaceutical composition, which comprises a pharmaceutically acceptable carrier and the 30 NVIFGF plasmid. According to one embodiment of the present invention, LMWH, such as for example, 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 35 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 40 or any other injectable sterile medium. As an example of a suitable composition, 20 mg of LMWH 17 WO 2005/063807 PCT/EP2004/014910 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, ie., 14 to 280 mg per day for an adult with unit doses ranging from 5 to 280 5 mg. Preferably the dose is 0.2 to 0.3 mg/kg per day. In one embodiment 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. 10 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 15 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 lyophilizedd) condition requiring only the addition of the 20 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. Preferably, the pharmaceutically acceptable carrier is a buffered saline solution. Most preferably, the pharmaceutical composition comprises a solution of sodium chloride. 25 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 NV1FGF 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 30 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 35 disease) pathologies, IC (intermittent claudication) or 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. 40 Still another object of the present invention is to provide a new method of stimulating 18 WO 2005/063807 PCT/EP2004/014910 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 5 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). At an early stage this results in discomfort in the 10 muscles of the leg with ambulation, leading at later stages to ulceration and gangrene (1). Chronic cardiovascular disorders are aggravated factors in patients who are already suffering of ischemic conditions, such as PAD, through mechanisms involving endothelium dysfunction (2, 3). Pathologies such as hypercholesterolemia, hypertension and diabetes have been investigated as possible targets for developing experimental models of PAD (4-7). Nevertheless, in such models 15 of hindlimb ischemia, a critical point is to negate a spontaneous angiogenic response to allow efficacy of any revascularization treatment. According to the present invention, the combination of NV1FGF 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 20 promoting the growth of both collateral vessels and arterioles. As shown in the following examples, the NV1 FGF in combination with LMWH or standard heparin is capable to effectively induce the formation of mature large conductance vessels (>150pm collateral vessels) and small resistance arteries (<50pm arterioles) in ischemia-injured muscles of the posterior part of the thigh, which are required to convey and to deliver blood to 25 tissues. 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 30 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 NVIFGF and LMWH or standard heparin with the proviso that 35 VEGF is not upregulated in the treated cells Throughout this application, 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. 40 It is also understood and expected that variations in the principles of invention herein 19 WO 2005/063807 PCT/EP2004/014910 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. 20 WO 2005/063807 PCT/EP2004/014910 Examples 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 5 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 10 diet daily, made of standard chow supplemented with 3% cholesterol and 15% cocoa butter (ref. 1414C, UAR, Epinay-sur-Orge, France), and were randomly allocated to four groups (i) saline group, (ii) the NV1FGF group, (iii) NV1FGF and enoxaparin, and (iv) enoxaparin and saline. Example 2: Induction of hindlimb ischemia 15 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-'), 02 (0.4 L.min-) 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. 20 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. 25 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-pL injections each in Tibialis cranialis, Adductores and Quadriceps muscles of the ischemic limb. 30 Example 4: Measures of total cholesterol, lipids and triglyceride levels in serum On days -35, -7, and +21 or +28 related to the day of surgery, blood was obtained from hamsters of HC/21, HC/28, saline, NV1FGF, enoxaparin, or NV1FGF with enoxaparin groups by retro-orbital puncture under gas anesthesia with N 2 0 (0.8 l.min"), 02 (0.4 L.min-) and isofluorane (2%). Total cholesterol and triglyceride levels in serum were determined enzymatically with 35 commercially available kits (Olympus Diagnostica GmbH, Hamburg, Germany). Cholesterol-rich diet led to a time-dependent increase both in total cholesterol and triglyceride serum levels. Serum lipids were measured at the end of experiments (i.e., 28 days post-ischemia) in all groups. 21 WO 2005/063807 PCT/EP2004/014910 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 pl of contrast medium (0.5 g.mr' sulfate barium 5 solution in water) through a catheter inserted into abdominal aorta, hamsters were sacrificed with an overdose of sodium pentobarbital. Hamsters were placed in dorsal decubitus into a radiography apparatus (model MX-20, Faxitron X-ray Corp., Wheeling, IL, USA) and post-mortem pictures of the vasculature from both limbs were collected and digitalized (software Specimen, DALSA MedOptics, Tucson, AZ, USA). This radiographic system allows visualization of vessels 10 with diameters higher than 150 pm. Pictures were analyzed off-line by an investigator blinded to the treatment, with dedicated software as previously described (5). Briefly, for both ischemic and non-ischemic limbs, the extent of collateral vessels in the posterior side of the thigh was determined as a percentage of the area analyzed. Angiographic score was calculated as the ratio ischemic/non-ischemic percentages. In order to check the 15 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 20 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 25 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-pm thick sections were prepared for immunohistochemistry. A mouse monoclonal antibody directed against smooth muscle a-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 30 expressed in mature vessels. The SMA antibody was detected with a commercially available kit (Envision

TM

+ 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 <50pm were counted in both Adductores and Gracilis muscles. These muscles have been chosen for their susceptibility to histopathological lesions in 35 our hindlimb ischemia hamster model, conversely to other muscles from the posterior side of the thigh (Semitendinosus, Semimembranosus and Biceps femoris). Typical lesions induced by excision of femoral artery are shown in Figure 2. Muscles from the back part of the thigh, i.e., Gracilis and Adductores were harvested 28 days after induction of ischemia, and 5pm thick sections of the muscles after HES staining were observed. Figures 2B 40 and 2C show the presence of mild necrosis (dashed line) and centronucleation (arrows) in 22 WO 2005/063807 PCT/EP2004/014910 ischemic muscles, respectively. 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 5 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 10 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. The incubation with a primary polyclonal anti-FGF-1 rabbit antibody (reference AB-32-NA, 1:30 dilution, R&D Systems, Abingdon, UK) was followed by incubation with a biotinylated donkey anti 15 rabbit immunoglobulin (1:200 dilution, Amersham, Buckinghamshire, UK). The immune complexes were localized using a chromogenic diaminobenzidine substrate, after adding peroxidase coupled to streptavidine. The 5-pm thick sections were counterstained with hematoxylin, dehydrated and mounted with permanent mounting media. Immunoreactive fibers were identified (brown staining) under a microscope (Axioplan 2, Zeiss, Hallbergmoos, Germany). 20 In contrast with gene therapy involving other angiogenic factors, such as VEGF or FGF-2, the inventors have evidenced that the expression of FGF-1 was advantageously restricted to the ischemic muscles of animals treated with NV1 FGF. Also, as shown by representative pictures (magnification X100) of immunohistochemical staining with an anti-FGF-1 polyclonal antibody in muscles from the back part of the thigh (Tibialis 25 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 NV1FGF-treated animals. This clearly show the superior properties of the plasmid NV1 FGF which allows a slow release of the encoded FGF-1 protein 30 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. NV1 FGF 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 35 therapeutic window and in conditions characterized by aggravated endothelial dysfunctions. Due to such superior characteristics in terms of safety and potency, the NV1 FGF may advantageously be used as angiogenesis therapy in aggravated conditions caused by hypercholesterolemia or diabetes. These results clearly demonstrate that NVIFGF gene therapy is capable of rescuing 40 impaired collateral formation via the increasing of arterioles. In effect, the growth of >150pm 23 WO 2005/063807 PCT/EP2004/014910 collateral vessels have been evidenced angiographically in the posterior part of the thigh, which comprises Biceps femoris, Adductores, Gracilis, Semimembranosus, and Semitendinosus muscles. Unexpectedly, the Applicant has demonstrated that formation of collateral vessels was significantly stimulated into this region, 14 days after NV1FGF gene transfer, as emphasized by 5 angiographic score. Though no in situ measurements of tissue oxygenation have been performed to assert that ischemia occurred in that region after femoral artery excision, the presence of histological lesions such as centronucleation, dystrophy, necrosis and inflammation was revealed. More precisely, these lesions were restricted to Adductores and Gracilis muscles whereas Biceps 10 femoris, Semimembranosus, and Semitendinosus muscles were not prone to lesions. Quantification of <50pm arterioles was therefore performed exclusively in Adductores and Gracilis muscles, just above the adductor canal. NV1FGF gene transfer increased the absolute number of arterioles into these muscles of the ischemic limb. These data thus unambiguously demonstrate the ability of NV1FGF to induce the 15 formation of mature large conductance vessels (>150pm collateral vessels) and small resistance arteries (<50pm arterioles) in ischemia-injured muscles of the posterior part of the thigh, which are required to convey and to deliver blood to tissues. Example 8: Comparative experiments of NVIFGF gene transfer and NV1FGF in 20 combination with enoxaparin on collateral development and arteriolar density 14 days after intramuscular administration (j., 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 25 the ischemic limb, when compared with saline-treated hamsters, enoxaparin, or NVIFGF gene transfer. Also, angiographic score after administration of NV1FGF and enoxaparin is indeed significantly higher than that of saline-treated hamsters or NV1FGF-treated hamsters, or enoxaparin-treated hamsters. Mature vessels labeled by smooth muscle a-actin (SMA) immunohistochemistry from 30 ischemic muscles of hamsters treated with saline and NV1FGF with or without enoxaparin and quantification of muscle area and <50pm SMA positive arterioles. A decreased area of Tibialis cranialis, Adductores and Gracilis muscles in the ischemic limb is observed in NVI FGF & enoxaparin group. 35 Example 9: treatment of hindlimb ischemia in diabetic mice using NVIFGF and enoxaparin or standard heparin The importance of angiogenesis and arteriogenesis in the development of collateral vessels is well established. Accordingly, angiogenic therapy with angiogenic cytokines has been proposed as an approach to treat patients with an apparent inadequacy of collateral vessels. 40 Diabetes mellitus is associated with a marked impairment in collateral formation. Therefore, an 24 WO 2005/063807 PCT/EP2004/014910 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. 5 Induction of diabetes: Streptozotocin selectively destroys insulin-producing beta islet cells of the pancreas providing a model of type I diabetes. Streptozotocin dissolved at 10 mg/mI in 0.1 M sodium citrate buffer (pH 5.5) is injected intraperitoneally to 8-10 week-old male C57B116 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 10 hours are above 250 mg/dl and remained elevated. Hindlimb ischemia: 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 15 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. Injection of plasmid solution: NV1FGF plasmid solution was injected in the range of 0.4-4.0 mg/kg body weight (i.e. 10-100 20 pg/mouse or 5-50 pg/muscle) as a single administration in the quadriceps and/or in the tibialis cranialis muscle (volume injected: 40pl/muscle) of the right hindlimb within the 7 days following surgery. Infection of low molecular weight heparin (enoxaparin): 25 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. 30 Endpoints to monitor therapeutic efficacy: - 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. 35 - 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: 25 WO 2005/063807 PCT/EP2004/014910 - a comparison showing evidence of faster kinetics of hindlimb reperfusion in mice treated with NVIFGF + enoxparin compared to any of the single agent, or - a comparison showing evidence of a higher level of perfusion at any given timepoint following treatment with the combination therapy versus any single agent, and/or 5 - a comparison showing evidence of a similar level of perfusion with a lower dose of NVIFGF when combined with enoxaparin compared to a given dose of NV1 FGF as a single agent. Each of the following references can be used or relied upon, in whole or in part, to make and use 10 aspects of the invention and are specifically incorporated herein by reference. In addition, the examples and description above should not be taken as a limitation on the scope of the invention and those of ordinary skill in the art can device and produce variations in the methods and compounds and combinations of compounds discussed without departing from the scope and intent of the invention. 15 References 1. Ouriel K. Peripheral arterial disease. Lancet. 2001;358:1257-1264. 2. Soubrier F, Cameron B, Manse B, Somarriba S, Dubertret C, Jaslin G, Jung G, Le Caer C, Dang D, Mouvault JM, Scherman D, Mayaux JF, Crouzet J. pCOR: a new design of plasmid 20 vectors for nonviral gene therapy. Gene Ther. 1999;6:1482-1488. 3. Dart AM, Chin-Dusting JPF. Lipids and the endothelium. Cardiovasc. Res. 1999;43:308-322. 4. Takeshita S, Isshiki T., Sato T. Increased expression of direct gene transfer into skeletal muscles observed after acute ischemic injury in rats. Lab. Invest. 1996;74:1061-1065. 5. Silvestre JS, Mallat Z, Duriez M, Tamarat R, Bureau MF, Scherman D, Duverger N, Branellec 25 D, Tedgui A, Levy BI. Antiangiogenic effect of interleukin-10 in ischemia-induced angiogenesis in mice hindlimb. Circ. Res. 2000;87:448-452. 6. Yla-Herttuala S, Martin JF. Cardiovascular gene therapy. Lancet. 2000;355:213-222. 7. Ferrara N, Alitalo K. Clinical applications of angiogenic growth factors and their inhibitors. Nat. Med. 1999;5:1359-1364. 30 8. Isner JM, Asahara T. Angiogenesis and vasculogenesis as therapeutic strategies for postnatal neovascularization. J. Clin. Invest. 1999;103:1231-1236. 26

Claims (72)

1. A product comprising an efficient amount of an expression vector encoding an 5 angiogenic growth factor and a heparin compound.

2. A product according to claim 1, wherein the heparin compound is a low molecular weight heparin or a standard heparin. 10

3. A product according to claim 1 or 2, wherein the angiogenic growth factor is a fibroblast growth factor (FGF).

4. A product according to claim 3, wherein the angiogenic growth factor is a FGF acidic or FGF-1. 15

5. A product according to claim 3, wherein the angiogenic growth factor is FGF-2 or bFGF, FGF-4, FGF-5, FGF-6, FGF-7, or any of FGF1-22.

6. A product according to claim 1 or 2, wherein the angiogenic growth factor is VEGF. 20

7. A product according to any one of claims 1 to 6, wherein the vector is a plasmid vector.

8. A product according to claim 7, wherein the plasmid vector is a conditionally replicating plasmid. 25

9. A product according to claim 8, wherein the vector is a pCOR plasmid.

10. A product according to any one of claims 1 to 9, wherein the FGF-1 or FGFa is expressed and the expression vector is NV1 FGF. 30

11. A product according to any one of claims 1 to 10, wherein the low molecular weight heparin is selected from the group consisting of enoxaparine, fraxiparine, dalteparin, ardeparin, tinzaparin and reviparin. 35

12. A product according to any one of claims 1 to 11 for simultaneous, separate or sequential use in the treatment of ischemia.

13. Use of a product according to any one of claims I to 12 for the manufacture of a medicament for treating peripheral or cardiac ischemia. 40 27 WO 2005/063807 PCT/EP2004/014910

14. Use of an expression vector encoding an angiogenic growth factor in the manufacture of a medicament for treating ischemia, wherein the expression vector encoding angiogenic growth factor is administered in combination with a heparin compound. 5

15. Use of a heparin compound in the manufacture of a medicament for treating ischemia, wherein the heparin is administered in combination with an expression vector encoding an angiogenic growth factor.

16. Use according to claim 14 or 15, wherein the heparin compound is a standard heparin or 10 a low molecular weight heparin.

17. Use according to any one of claims 13 to 16, wherein the expression vector encodes a FGF-1 or FGFa, and is a plasmid. 15

18. Use according to any one of claims 13 to 17, wherein the low molecular weight heparin is selected from the group consisting of enoxaparine, fraxiparine, dalteparin, ardeparin, tinzaparin and reviparin.

19. Use according to any one of claims 13 to 18 for the manufacture of a medicament which 20 acts by promoting mature collateral vessels in skeletal or cardiac muscle tissues of an ischemic patient.

20. Use according to any one of claims 13 to 19 for the manufacture of a medicament which acts by promoting both mature collateral vessels and arterioles in skeletal or cardiac 25 muscle tissues of an ischemic patient.

21. Use according to any one of claims 13 to 20 for the manufacture of a medicament which acts by promoting formation of mature large conductance vessels (>150pm) and small resistance arteries (<50pm arterioles) in skeletal or cardiac muscle tissues of an ischemic 30 patient.

22. Use according to any one of claims 13 to 21, wherein the ischemic patient subject to peripheral ischemia or coronary ischemia, or at risk of ischemia, and wherein the patient is 45 years old or older, is overweight, and/or has a high consumption of alcohol or 35 cigarettes, and/or has atherosclerosis and/or diabetes, and/or is immobilized.

23. Use according to any one of claims 13 to 22, wherein the ischemic patient is 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 40 encoding angiogenic factor and a low molecular weight heparin. 28 WO 2005/063807 PCT/EP2004/014910

24. Use according to any one of claims 13 to 23, wherein the low molecular weight heparin is administered subcutaneously at a dose between 0.2 and 0.4 mg/kg per day. 5

25. Use according to claim 24, wherein the low molecular weight heparin is administered subcutaneously at a dose of around 0.3 mg/kg per day.

26. Use according to any one of claims 13 to 25, wherein the low molecular weight heparin is administered over at least 20 days or at least 30 days and the DNA encoding angiogenic 10 growth factor is administered during 1, 2, 3, or more occurrences, so that first and last administration of low molecular weight heparin are separated by at least 20 or 30 days.

27. A pharmaceutical composition comprising an expression vector encoding an angiogenic growth factor and a heparin compound, wherein the combination promotes angiogenesis 15 in skeletal and cardiac muscles greater than either the vector or the heparin compound alone.

28. A pharmaceutical composition comprising an expression vector encoding an angiogenic growth factor and a heparin compound, wherein the combination promotes arteriogenesis 20 in skeletal and cardiac muscles greater than either the vector or a heparin compound alone.

29. The pharmaceutical composition of either of claims 27 or 28, wherein the heparin compound is a standard heparin or a low molecular weight heparin. 25

30. The pharmaceutical combination of either of claims 27 or 28, wherein the angiogenic growth factor is an FGF-1.

31. The pharmaceutical composition of either of claims 27 or 28, wherein the angiogenic 30 growth factor is an FGF-2 or bFGF, FGF-4, FGF-5, FGF-6, FGF-7, or any of FGF1-22.

32. The pharmaceutical composition of either of claims 27 or 28, wherein the expression vector is a viral vector. 35

33. The pharmaceutical composition of either of claims 27 or 28, wherein the expression vector is a non-viral vector.

34. The pharmaceutical composition of either of claims 27 or 28, wherein the expression vector is a plasmid. 40 29 WO 2005/063807 PCT/EP2004/014910

35. The pharmaceutical composition of either of claims 27 or 28, wherein the expression vector is pCOR.

36. The pharmaceutical composition of either of claims 27 or 28, wherein the expression 5 vector is NV1FGF.

37. The pharmaceutical compositionA of either of claims 27 or 28, wherein the low molecular weight heparin is enoxaparin, nadroparin, dalteparin, ardeparin, tinzaparin or reviparin. 10

38. A method of treating peripheral ischemia comprising administering a combination of an expression vector encoding an angiogenic factor and a heparin compound.

39. A method of treating coronary ischemia comprising administering a combination of an expression vector encoding an angiogenic factor and a heparin compound. 15

40. A 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, such treatment comprising administering a combination of an 20 expression vector encoding an angiogenic factor and a heparin compound.

41. A method of 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 a expression vector encoding an angiogenic factor and a heparin 25 compound.

42. The method of any one of claims 38 to 41, wherein the heparin compound is a standard heparin or a low molecular weight heparin. 30

43. A method of promoting mature collateral vessels in ischemic skeletal or cardiac muscle tissue in a mammalian subject in need of such treatment comprising injecting said tissues of said subject an effective amount of the product as defined in anyone of claims 1 to 11 to promote mature collateral vessels. 35

44. A method of promoting both collateral blood vessels and arterioles in ischemic skeletal or cardiac muscle tissue in a mammalian subject in need of such treatment comprising injecting said tissues of said subject with an effective amount of the product as defined in any one of claims 1 to 11, to promote both collateral blood vessels and arterioles. 40

45. A method of promoting formation of mature large conductance vessels (>150pm 30 WO 2005/063807 PCT/EP2004/014910 collateral vessels) and small resistance arteries (<50pm arterioles) in ischemia skeletal or cardiac muscle tissues in a mammalian subject in need of such treatment comprising injecting said tissues of said subject with an effective amount of the product as defined in any one of claims 1 to 11. 5

46. A method of any one of claims 38 to 45 wherein the angiogenic growth factor is FGFa or FGFI.

47. A method of any one of claims 38 to 46 wherein the heparin compound and the 10 expression vector encoding FGF-1 are administered separately or concomitantly.

48. A method according to any one of claims 38 to 47 wherein the expression vector encoding FGF-1 is injected in the skeletal or cardiac muscle tissue and the heparin compound is administered systemically. 15

49. A method according to any one of claims 38 to 48, wherein the heparin compound is administered intraperitoneally or subcutaneoulsly.

50. A method according to any one of claims 38 to 49, wherein the low molecular weight 20 heparin is administered subcutaneously at a dose between 0.2 and 0.4 mg/kg per day.

51. A method according to claim 50, wherein the low molecular weight heparin is administered subcutaneously at a dose of around 0.3 mg/kg per day. 25

52. A method according to any one of claims 38 to 51, wherein the low molecular weight heparin is administered over at least 20 days or at least 30 days and the DNA encoding angiogenic growth factor is administered during 1, 2, 3, or more occurrences, so that first and last administration of low molecular weight heparin are separated by at least 20 or 30 days. 30

53. A method according to any one of claims 38 to 52, wherein the angiogenic factor encoding gene or DNA is administered in the posterior part of the thigh and the calf.

54. A method according to any one of claims 38 to 53, wherein the angiogenic factor 35 encoding gene or DNA is administered by multiple injections around the ischemic site of said skeletal or cardiac muscle.

55. A method according to any one of claims 38 to 54, wherein the low molecular weight heparin is selected from the group consisting of enoxaparine, fraxiparine, dalteparin, 40 ardeparin, tinzaparin and reviparin. 31 WO 2005/063807 PCT/EP2004/014910

56. A method according to any one of claims 38 to 55, wherein the low molecular weight heparin is administered before the angiogenic factor encoding gene. 5

57. A method according to any one of claims 38 to 56 wherein the FGFa or FGF-1 gene or DNA sequence is carried by a non viral vector, preferably a plasmid vector.

58. A method of claim 57, wherein the plasmid vector is a conditionally replicating vector such as pCOR. 10

59. A method according to claim 57 or 58, wherein the plasmid vector is designated NV1 FGF.

60. A method of treating peripheral ischemia comprising administering a pharmaceutical 15 combination of an expression vector encoding an angiogenic growth factor and a heparin compound to a mammalian subject in need of such treatment.

61. A method of treating coronary ischemia comprising administering a pharmaceutical combination of an expression vector encoding an angiogenic growth factor and a heparin 20 compound to a mammalian subject in need of such treatment.

62. A method of promoting formation of collateral vessels of greater than 150 pm in ischemic skeletal or cardiac muscle tissue comprising administering a pharmaceutical combination of an expression vector encoding an angiogenic growth factor and a heparin compound 25 to a mammalian subject in need of such treatment.

63. A method of promoting formation of arterioles of less than 50 pm in ischemic skeletal or cardiac muscle tissue comprising administering a pharmaceutical combination of an expression vector encoding an angiogenic growth factor and a heparin compound to a 30 mammalian subject in need of such treatment.

64. The method of any one of claims 60-63, wherein the angiogenic growth factor is a FGF-1 or acidic FGF. 35

65. The method of any one of claims 60-63, wherein the angiogenic growth factor is a FGF-2 or basic FGF, FGF-4, FGF-5, FGF-6, FGF-7, or any of a FGF1-22.

66. The method of any one of claims 60-65, wherein the expression vector is a viral vector. 40

67. The method of any one of claims 60-66, wherein the expression vector is a non-viral 32 WO 2005/063807 PCT/EP2004/014910 vector.

68. The method of any one of claims 60-67, wherein the expression vector is a plasmid vector. 5

69. The method of claim 68, wherein the expression vector is pCOR.

70. The method of claims 69, wherein the expression vector is NV1 FGF. 10

71. The method of any one of claims 60 to 70, wherein the heparin compound is a standard heparin or a low molecular weight heparin.

72. The method of any one of claims 60 to 71, wherein the low molecular weight heparin is enoxaparin, nadroparin, dalteparin, ardeparin, tinzaparin or reviparin. 15 33