KR20070006714A - Treatment of coronary or peripheral ischemia - Google Patents

Abstract

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 a heparin compound, wherein the product is capable of acting in a synergistic manner to promote angiogenesis and arteriogenesis in skeletal and cardiac muscles. ® KIPO & WIPO 2007

Description

Coronary or Peripheral Ischemia Treatment {TREATMENT OF CORONARY OR PERIPHERAL ISCHEMIA}

Field and Introduction of the Invention

The present invention provides a novel method of treatment for patients with peripheral and / or coronary ischemic syndrome and also includes an expression vector encoding an angiogenic growth factor and a heparin compound or a low molecular weight heparin compound, thereby creating angiogenesis and side vessel growth in skeletal muscle and myocardium. It relates to products that can act in an effective way to promote arteriogenesis.

Background of the Invention

Restoration of blood perfusion in skeletal and cardiac ischemic tissues leads to a complex process of angiogenesis, ie endothelial cell proliferation, basal membrane degradation, migration through the surrounding matrix, as well as the formation of capillary networks by alignment and differentiation into tubular structures to form vascular walls. Include. Side vessel growth, which indicates the growth of the arterial artery, is also thought to be an effective process for the restoration of blood perfusion due to the larger vascular capacity compared to capillary networks (Carmeliet et al., Nat. Med., 2000; 6: 389-395; Van Royen et al., Cardiovasc. Res., 2001; 49: 543-553).

Various strategies exist for restoring blood perfusion. Angiogenic gene therapy is one of the possible treatment strategies to improve lateral development and overcome perfusion deficiency and associated ischemia (6-8). Treatment of angiogenesis is under study by administering liposomes or viral vectors, or by administering nucleic acids capable of expressing angiogenic proteins in naked form.

Protein therapy, which involves delivering growth factors directly to ischemic tissue, is an alternative to angiogenesis treatment. For example, it has been proposed to administer several recombinant proteins, VEGF, FGF, HGF, PDGF and TGF-β, to promote side vessels.

Animal testing has demonstrated that side vessels can be promoted by topical administration of several different angiogenic proteins including, for example, recombinant human VEGF protein, recombinant human basic FGF protein (bFGF), recombinant TGF-β or PDGF protein. Such recombinant angiogenic proteins have been proposed for the treatment of ischemic cardiovascular disease, ie peripheral arterial disease or coronary artery disease.

Heparin is a biologically active agent of the glycosaminoglycan family extracted from natural products and has useful anticoagulant and antithrombotic properties. In particular, they are useful for the treatment of postoperative venous thrombi. Heparin compounds can be prepared in various molecular weights.

Standard heparin has a molecular weight between 6000 and 40000 Da. Most of the commercially available heparin formulations have an average molecular weight of 12000 to 15000 Da. Standard heparin is isolated on a large scale from swine intestinal mucosa or from bovine lungs.

Heparin can be broken down into low average molecular weight molecules with an average molecular weight of 2000 to 10000 Da containing a mixture formulation of sulfated polysaccharides. These mixtures are prepared by depolymerization and saponification of heparin esters. They have high antithrombotic activity and total anticoagulant activity is lower than heparin.

Blood coagulation interacts with a number of proteolytic enzymes in a certain order; The last step is based on the cascade process in which fibrinogen is converted to insoluble fibrin under the action of the proteolytic enzyme thrombin. The coagulation process actually involves three steps, usually described in series, even in complex interrelated cases. The first step consists of the prothrombinase step (or active thromboplastin formation) of thromboplastin formation. The second step consists of a thrombin formation step that can be summarized as the conversion of prothrombin to thrombin under the influence of prothrombinase in the presence of ionized calcium. Finally, the third step consists of the fibrin formation step, where blood fibrinogen is converted to insoluble fibrin under the influence of thrombin.

Once prothrombinase is formed, during the process of thromboplastin formation, it is necessary to establish that of the plasma and tissue origin capable of activating prothrombin to active thrombin, respectively, according to two essentially different pathways: intrinsic or endogenous pathways and external or exogenous pathways. Prothrombinase is formed.

There are a number of factors or protoplasmic enzyme precursors (factors XII, XI, IX, VIII and X) that can be activated sequentially in the intrinsic or endogenous pathway, in which each activation product (factors XIIa, XIa, IXa, VIIIa and Xa) is involved. ) Acts similarly to enzymes that can be activated along the enzyme precursor, followed by activation of factor X (Xa), which is primarily involved in the production of active endogenous plasma prothrombinase by reacting with phospholipids of factor V and platelet origin. Done. Obviously, external or exogenous systems that may be directly involved in tissue lesions involve a more limited number of factors, mainly tissue thromboplastin, which, like factor VIIIa, can bind factor VII to convert inactive factor X into factor Xa. Involves producing. The order of activating prothrombin to thrombin is substantially the same as the native system, but phospholipids are of tissue origin and not of plasma origin.

The coagulation process results in an insoluble fibrin clot that fills the lesion at the origin of the process, such as the vascular level.

These coagulation processes generally result in a process called fibrinolysis, which will provide a clot lysate, apparently under the influence of plasmin (which is an enzyme usually present only in the circulating blood in the form of plasminogen, an inert precursor). It itself produces one of the factors that can initiate the conversion of inactive plasminogen to fibrinolytic active plasmin. Indeed, the mechanism is balanced by a highly complex process depending on the counter activator and inhibitor. The concept of overcoagulation can cause a thrombus if this mechanism's equilibrium is broken. Excessive coagulation, on the other hand, exposes the host to bleeding risk.

Currently, a detailed underlying mechanism for the anticoagulant activity of heparin is known. Heparin acts at varying levels on a series of enzymatic reaction cascades that normally participate in physiological hemostatic processes in any situation that can result in excessive coagulation of blood. More particularly, heparin can attenuate a number of coagulation factors that participate in and maintain different types of overcoagulation. More specifically, heparin forms a high affinity complex with antithrombin. Antithrombin-heparin complex formation increases the rate of inhibition of two major coagulation promoting proteases, Factor Xa and thrombin. The rate of inhibition of these two enzymes by antithrombin alone is increased by about 1,000-fold with heparin. Inactivation of both active forms of the protease inhibits the subsequent process of converting fibrinogen into fibrin, which is critical for clot formation.

Thus, heparin, when subjected to significant impairments, for example in the case of surgical operations, provides a balance of hypercoagulation-mitigating effects and coagulation-fibrinolysis mechanisms. However, upon rebalancing this action is attenuated, leading to severe bleeding if the dose of the anticoagulant is increased or the selectivity is insufficient for the purpose of preventing excessive coagulation risk such as postoperative thrombus development.

Summary of the Invention

We have found that when used in combination with expression vectors encoding angiogenic growth factors, heparin compounds such as low molecular weight heparin (LMWH) or standard heparin can provide potent synergism for potent angiogenesis and side vessel growth. We also found that compositions of expression vectors encoding standard heparin and fibroblast growth factor (FGF) have potent angiogenesis and side vessel growth effects. Contrary to the general idea that heparin is a potent inhibitor of polymerase by competing with double-stranded DNA for polymerase binding, we believe that this situation leads to injection of naked DNA in the form of expression vectors or plasmids combined with LMWH or standard heparin. It did not occur in the case, it was demonstrated to increase the activity of the DNA encoding the angiogenic growth factor, such as to provide a potent synergistic angiogenic response in skeletal muscle and / or myocardium. Such combinations of the invention have never been disclosed in the prior art.

It has also been surprisingly found that administration of DNA encoding angiogenic growth factors with LMWH in accordance with the present invention may reverse angiogenesis deficiency in hypercholesterolemia or diabetic symptoms. High levels of lipids significantly impair endothelial function such as vasodilation, leukocyte-endothelial interaction, thrombosis, fibrinolysis and endothelial cell growth (3). In particular, nitric oxide synthase (NOS) systems may be affected by physiological pathologies, including high levels of oxidized low density lipoprotein cholesterol (LDL-C). Experimental models of diabetes and vascular function in patients with type I and II diabetes are characterized by significant angiogenic dysfunctions.

The present inventors have found that mammals suffering from hypercholesterolemia or diabetes when expression vectors encoding angiogenic growth factors such as acidic fibroblast growth factor are administered to ischemic skeletal or cardiac muscle in combination with standard heparin or heparin compounds such as LMWH. It has been shown that it can promote the formation of mature blood vessels such as arterioles in the subject and effectively reverse the side vessel deficiency.

The inventors have found that a composition comprising an expression vector encoding acidic fibroblast growth factor (FGF-1) together with a heparin compound such as standard heparin or LMWH is particularly effective for ischemic skeletal muscle or myocardium in aggravating symptoms such as hypercholesterolemia or diabetes. It has been found that it can be used in an effective amount to rescue formation deficiency. The present invention relates to a composition having a strong angiogenic effect and particularly selective for skeletal muscle and myocardium. The present invention is based in part on the fact that DNA encoding angiogenic growth factors has a potent synergistic effect when combined with heparin compounds such as low molecular weight heparin or standard heparin and provides a potent angiogenic response in skeletal muscle and myocardium.

However, the present invention 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 can be selected and used that can cause or promote the expression of angiogenic growth factors in cells or organisms. Those skilled in the art will appreciate plasmids, viruses, retroviruses, nucleic acids, cosmids, BACs, YACs, viral genomes, recombinant adenovirus vectors, other nucleic acids integrated or present in cells, adenovirus vectors, adeno-associated virus vectors, lentiviruses or retroviruses. Examples of many of these agents or vectors are known, including vectors such as vectors. Thus, a "vector" includes a particle, cell or organism having any nucleic acid or nucleic acid that can be used to introduce the nucleic acid into a host cell. The term "vector" includes both viral and nonviral products and systems for introducing compounds that introduce nucleic acids into cells and / or cause or promote the expression of at least one angiogenic growth factor. "Vector" can be used in vitro, ex vivo or in vivo. Non-viral vectors include plasmids, cosmids, and can include, for example, liposomes, electrically charged lipids (cytofectin), DNA-protein complexes, and biopolymers. Viral vectors are, for example, adenoviruses, retroviruses, lentiviruses, adeno-associated viruses, pox viruses, baculoviruses, leoviruses, vaccinia viruses, herpes simplex viruses, Epstein-Barr viruses and recombinant deficient adenosine. Include viral vectors. The vector may also include the entire genome sequence of the virus or its recombinant genomic sequence. The vector may also comprise a portion of the genome comprising a functional sequence that produces a virus that can be infected, introduced or introduced into a cell to deliver a nucleic acid.

FIELD OF THE INVENTION The present invention relates to medicaments or expression vectors or plasmids encoding angiogenic growth factors and products comprising an effective amount of heparin compounds, such as standard heparin or LMWH, and their use for the preparation of medicaments for treating peripheral or cardiac ischemia.

The present invention also relates to a pharmaceutical composition comprising an expression vector that promotes angiogenesis more in skeletal muscle and myocardium than in the case of the vector or heparin compound alone.

The present invention also relates to novel methods of promoting angiogenesis and side vessel growth in myocardium and skeletal muscle and to novel combinations of low molecular weight heparin or standard heparin with expression vectors encoding angiogenic growth factors that can act synergistically.

The present invention also relates to a novel method for treating a patient with a peripheral ischemic or coronary ischemic condition, or at risk of ischemia, comprising administering a combination of a heparin compound with a DNA sequence encoding an angiogenic factor, wherein The patient is at least 45 years old, overweight and / or high in alcohol or tobacco consumption, and / or has atherosclerosis and / or diabetes and / or is unable to move. The invention is also characterized by administering a combination of an expression vector encoding an angiogenic factor and a heparin compound to a patient who has developed an ischemic heart attack in the last 24 hours to 6 months or up to 1, 2 or 3 years. It is useful for treatment.

The present invention also provides a novel method for promoting both ischemic skeletal muscle or myocardial tissue side vessels and arterioles, in which an effective amount of the product of the invention is injected into a mammalian subject.

The present invention is also characterized by injecting an effective amount of a product of the invention into a mammalian subject, wherein the large mature conducting blood vessel (> 150 μm side vessels) and small resistance artery (<150 μm) of ischemic skeletal muscle or myocardial tissue in the subject. A novel method of promoting the formation of 50 μm arterioles) is provided.

Another object of the present invention is to provide a method for promoting both side vessel growth and arterial growth in ischemic tissues, especially when endothelial function is impaired.

Providing a method of stimulating and / or promoting angioplasty of ischemic muscle in hypercholesterolemia or diabetic symptoms and reversing angiogenesis deficiency in a mammalian subject suffering from hypercholesterolemia or diabetes. It is also an object of the present invention.

Another object of the present invention is to provide angiogenesis therapy for the treatment of peripheral or myocardial ischemia, wound healing or other symptoms requiring angiogenesis or tissue regeneration.

In addition, another object of the present invention is to provide a method for enhancing therapeutic angiogenesis in a patient subject of angioplasty, circuit transplantation or other angioplasty surgery.

1 is a daily subcutaneous injection of saline from (a) DO to D6 in three hamster muscles, namely Tibialis cranialis, quadriceps (Quadriceps femoris), and Adductores / Gracilis, (b) DO 60 μg, such as pCOR with luciferase gene, with daily subcutaneous injection of enoxaparin at 1.5 mg / kg for D6 and (c) daily subcutaneous injection of enoxaparin at 1.5 mg / kg with D1 to D6 After inject | pouring the plasmid of, it is the chart which measured luciferase expression. The data confirmed that the protein was expressed and established a baseline for enoxaparin treatment with a control plasmid that did not have a nucleic acid encoding vascular growth factor.

The present invention provides a composition for treating a coronary artery or peripheral ischemic patient comprising an expression vector encoding angiogenic factors and low molecular weight heparin or standard heparin. The use of such combinations unexpectedly promotes angiogenesis or side vessel growth throughout ischemic skeletal muscle or myocardial tissue of a mammalian subject, providing a synergistic effect on vascular remodeling and perfusion enhancement.

The invention also relates to a novel method of promoting angiogenesis and side vessel growth in myocardium and skeletal muscle.

The term "subject" includes, but is not limited to, mammals such as dogs, cats, horses, cattle, pigs, rats, mice, monkeys, and humans.

Vectors encoding angiogenic growth factors are typically DNA molecules and generally can express angiogenic growth factors in the cells of the individual to be treated. Expression vectors encoding angiogenic growth factors are fibroblast growth factor (FGF), such as FGF-1 or acidic FGF (aFGF), FGF-2 or basic FGF (bFGF), FGF-4, FGF-5, FGF-6 And DNA encoding any one of FGF-7 or FGF1-23. Coding sequences of such angiogenic growth factors are known to those skilled in the art. For example, the DNA coding sequences of FGF-1, FGF-2, FGF-4, FGF-5, FGF-6 and FGF-7 are shown as SEQ ID NOs: 1-6, respectively. Other genes useful in the combinations of the invention include vascular endothelial growth factors (VEGF) such as VEGF-A (mature isoform 206, 189, 165, 145 and 121 amino acid residues), VEGF206, VEGF189, VEGF165, VEGF145, VEGF homologs including VEGF121, VEGF120, VEGF-B, VEGF-C, VEGF-D and genes encoding VEGF heterodimers. Other useful genes include platelet derived growth factors (PDGF AA, AB, or BB), hepatocyte growth factor (HGF) or insulin growth factor (IGF-I or II). Preferred compositions of the invention include DNA encoding acidic fibroblast growth factor (FGF-1 or aFGF).

Angiogenic factor coding sequences may be accompanied by viral or nonviral expression vectors. The term "vector" refers to a vehicle, preferably DNA or nucleic acid molecule, capable of carrying and expressing angiogenic factors accompanying it. If the vector is a nucleic acid molecule, the nucleic acid molecule is covalently linked to the vector nucleic acid. Various expression vectors can be used to express angiogenic factors. Such vectors include chromosomal, episomal and viral derived vectors such as yeast chromosomal elements, including bacterial plasmids, bacteriophages, yeast episomes, yeast artificial chromosomes, viruses such as baculoviruses, papovaviruses such as SV40, vaccinia virus, Vectors derived from adenoviruses, poxviruses, Pseudorabies viruses and retroviruses. Vectors can also be derived from combinations of these products, such as those derived from plasmids and bacteriophage genetic elements such as cosmids and phagemids. Cloning and expression vectors suitable 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 accompanied and expressed by the plasmid. Expression plasmids are known in the art. Expression plasmids derived from ColE1 plasmids can be used, such as pBR322, plasmid pMB1 or plasmid pMK16 (Ausubel, Current Protocols in Molecular Biology, John Wiley and Sons, New York (1988) XII: 1.5.2), and angiogenic factors The sequence encoding can be cloned by transforming E. coli in a conventional manner and screening for transformants with inserts. Mammalian expression vectors can be used and include pCDM8 (Seed, B. Nature 329: 840 (1987)) and pMT2PC (Kaufman et al., EMBO J. 6: 187-195 (1987)). Other suitable genetic constructs of mammalian replication origin can be provided to maintain the construct extrachromosomally and produce multiple copies of the intracellular construct. Plasmids pCEP4 and pREP4 (San Diego, Calif) from Invitrogen contain a replication and nuclear antigen EBNA-1 coding region of Epstein Barr virus origin that produces high copy episomal replication without integration. The expression vectors listed herein are provided solely for the purpose of illustrating well known vectors available to those of skill in the art, which may be useful for expressing angiogenic factors. These are described in Sambrook, J., Eritsh, EF, and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989. Expression vectors or plasmids contain regulatory regions necessary for the expression of angiogenic factors, operably bind to vectors for angiogenic factor coding sequences, and include promoters, start codons, stop codons, and polyadenylation signals.

Initiation and stop codons are generally constructed with the coding sequence of the angiogenic factor and are considered to be part of the nucleotide sequence encoding the desired angiogenic factor.

Examples of promoters useful in practicing the present invention include monkey virus 40 (SC 40), mouse breast tumor virus (MMTV) promoter, human immunodeficiency virus (HIV) such as HIV Long Terminal Repeat (LTR) promoter, molony virus, ALV, Human genes such as human alpha actin, human myosin, human hemoglobin, humans as well as promoters from cytomegalovirus (CMV), such as CMV immediate early promoter, Epstein Barr virus (EBV), Raus sarcoma virus (RSV) And promoters from muscle creatine and human metallothionein. Regulatory sequences may provide structural expression to one or more host cells (ie, tissue specific) or may provide inducible expression to one or more cell types by temperature, nutritional additives or external factors such as hormones or other ligands, and the like. have. Various vectors that provide structural and inducible expression in eukaryotic and prokaryotic hosts are well known to those skilled in the art. For example, muscle specific promoters can be used to induce the expression of angiogenic factors and include the mouse or human upstream sequences of the CARP gene disclosed in US 2003/0039984, or in International Publication No. W0 01/11064. The disclosed cardiac alpha actin promoter sequence.

Particularly in the preparation of human genetic vaccines, examples of polyadenylation signals useful in practicing the present invention include, but are not limited to, SV40 polyadenylation signals, bovine or human growth hormone polyadenylation signals, and LTR polyadenylation signals. It is not limited. In particular, the SV40 polyadenylation signal (Invitrogen, San Diego Calif.) In the pCEP4 plasmid, 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 an enhancer. Enhancers are selected from the group including human actin, human myosin, human hemoglobin, human muscle creatine and virus enhancers such as SV40, RSV, and EBV enhancer, cytomegalovirus modulator, polyoma enhancer, adenovirus enhancer and retrovirus LTR enhancer. It may be, but is not limited to these.

In addition to containing transcription initiation and regulatory sites, expression vectors may also include ribosomal binding sites for transcription in the sequences and transcription regions necessary for transcription termination. Those skilled in the art will know a number of regulatory sequences useful for expression vectors. Such regulatory sequences are described, eg, in Sambrook et al., Molecular Cloning. A Laboratory Manual. 2nd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1989).

Sequences encoding angiogenic factors can be inserted into vector nucleic acids by well known methods. In general, the DNA sequence and the expression vector are cleaved with one or more restriction enzymes and the fragments ligated together to bind the DNA sequence to be finally expressed to the expression vector. Restriction enzyme digestion and ligation are well known to those skilled in the art.

Expression vectors generally comprise a selection marker that allows selection of a subpopulation of cells containing the recombinant vector construct. The marker may be contained in the same vector containing the nucleic acid molecule disclosed herein or may be on a separate vector. Any marker that provides selectivity to phenotypic properties will be effective.

When aiming for the secretion of angiogenic factors, an appropriate secretion signal is introduced into the expression vector. The signal sequence may be endogenous to the peptides or may be heterologous to these peptides.

According to the most preferred aspect, the expression vector is a plasmid that expresses FGF-1 and is designated NV1FGF. NV1FGF is a plasmid harvested from an optimal expression cassette encoding the secreted form of human FGF-1 (sphFGF-1) inserted into the original backbone designed for conditional replication origin, referred to as COR plasmid (2). The resulting plasmid is advantageously of small size of 2.4 kb and the sequence is represented by SEQ ID NO: 7. The sequence encoding sphFGF-1 is fused between the sequence encoding human FGF-1 in naturally occurring nodular form from amino acids 21-154 and secretory signal peptide (sp) from human fibroblast interferon (US 4,686,113; US 5,849,538). ). Expression of sphFGF-1 was derived from human cytomegalovirus (CMV) best enhancer / promoter (nucleotides -522- + 72). The post polyadenylation signal from monkey virus 40 (nucleotides 2538 to 2759 from the SV40 genome, gene bank site SV4CG; US 5,168,062) was inserted downstream of the sphFGF-1 fusion to confirm proper and effective transcription termination, followed by sphFGF-1 transcription. Was polyadenylation. Preferred pCOR plasmids described in International Application WO 97/10343 and in Soubrier et al. (2) have no antibiotic resistance genes. Plasmid selection depends on inhibitor transcription RNA genes in autotrophic receptor strains. Replication of R6Kγ origin yielded a strictly limited host range of plasmids and maintained high copy numbers. Sequences encoding such proteins are not common to bacteria but are artificially inserted into the genome of a selected host strain. Thus, the possibility of unwanted seeding is greatly limited.

Vectors or plasmids carrying and expressing angiogenic genes according to the present invention may be administered by any suitable means known to or available to those skilled in the art.

According to the present invention, expression vectors encoding angiogenic factors can be used in combination with heparin compounds and act synergistically to promote the growth of capillaries in skeletal muscle and myocardium. Heparin compounds can be standard heparin or low molecular weight heparin.

Standard or unfractionated heparin is mucopolysaccharide with a molecular weight of 6000 to 40000 Da. Most of the commercially available heparin formulations have an average molecular weight ranging from 12000 to 15000 Da. Standard heparin is isolated on a large scale from swine intestinal mucosa or from bovine lungs.

As described below, the polymer chain consists of disaccharide repeat units of D-glucosamine and euonic acid linked by 1-4 interglycosidic bonds. The euronic acid residue may be glucuronic acid or L-iduronic acid. More specifically, heparin comprises a major structural unit consisting of the unique pentasaccharide sequence of three D-glucosamine and two euonic acid residues. Hydroxyl groups on each of these monosaccharide residues are rarely sulfated resulting in highly negatively charged polymers. In particular, the central D-glucosamine residue comprises a unique 3-0-sulfate moiety, and the first and fifth D-glucosamides of pentasaccharide have four sulfate groups that have been found to be critical for high anticoagulant activity retention. Excluding any one of them significantly reduced anticoagulant activity.

Due to its high acid sulphate group, heparin is present as an anion at physiological pH and is generally administered as a sodium salt. 20 to 50% is discharged unchanged. Heparin polysaccharide chains degrade in gastric acid and are therefore usually administered intravenously or subcutaneously. Heparin is generally not administered intramuscularly due to the risk of hematoma formation.

Low molecular weight heparin used in the formulations according to the invention is known in the art. US 5,389,618 to which LMWH and methods of manufacture are incorporated herein by reference; US 4,692,435 and US 4,303,651, European Patent EP 0 040 144 and Vasc. Med, 2000; 5: 251-258 by Nenci GG.

Low molecular weight heparin corresponds to a mucopolysaccharide fraction obtained from heparin or a fraction comprising a heparin component having a molecular weight of about 2000 to 50000.

Heparin grinding processes are well known in the art. For example, European Patent EP 40,144 discloses the preparation of sulfated polysaccharide mixtures in which heparin comprises an ethylene double bond at one end of its polymer chain and has a weight average molecular weight of 2,000 to 10,000 Daltons. These mixtures are prepared by depolymerization and saponification of heparin esters. Other methods are also described in Johnson et al. Thrombos. Haemostas. Stuttg., 35, 586 (1976); Lane et al., Thrombosis Research, 16, 651; US Pat. No. 3,766,167 to Lasker et al.

Mixtures of both high molecular weight and low molecular weight heparin can also be used in the present invention. In this regard, US Pat. No. 5,389,618 includes 9% to 20% of the polymer chains having a lower weight average molecular weight than heparin, a molecular chain of less than 2,000 Daltons, and 5% to 20% of the polymer chains having a molecular weight greater than 8,000 Daltons. Polysaccharide mixture formulations having a number average molecular weight ratio of 1.3 to 1.6 have been described. The process described first comprises first starting the heparin as a long chain quaternary ammonium salt in an aqueous medium, followed by esterification of the obtained salt to form an ester having an esterification degree of 9.5% to 14%, followed by an esterification degree of 9.5% to Depolymerization of 14% ester. The degree of depolymerization and the molecular properties of the final product can be controlled by varying the degree of esterification of the heparin salt starting material.

Starting materials from which mucopolysaccharides can be extracted are obtained from conventional injectable pharmaceutical quality heparin or mammalian tissues or organs, mainly by the extraction process according to the principle of action according to the above principle of action of the intestinal membranes or lungs of pigs or cattle. It is composed of the same crude heparin. Preliminary precipitation may be by alcohol upstream means of saponification.

For example, the manufacturing process of low molecular weight heparin may include the following steps. A preliminary step that can provide heparin with a dermatan sulfate content of less than 2%. Thereafter, the saponification step of starting heparin is performed. The heparin salt can be prepared by interacting a stoichiometric excess of the corresponding salt with heparin sodium at an temperature of 20 ° C. in an aqueous medium. For example, the quaternary ammonium salts used are abenzetonium salts such as benzetonium chloride. In a second step, esterification is carried out. Partial esters of heparin in salt form having an esterification degree of 9.5% to 14% can be prepared by esterifying the long chain quaternary ammonium salt of heparin in the presence of a chlorine derivative in a chlorinated organic solvent. In addition, it is possible to increase the reaction efficiency by adjusting the ratio and reaction temperature and time of the various reactants. The partial ester of heparin may be an aromatic ester. The chlorine derivative may be benzyl chloride and the chlorinated solvent may be chloroform or methylene chloride. An esterification degree of 9.5% to 14% can be achieved by using about 1 part by volume of chlorine derivatives per 1 part by weight of chlorinated organic solvent and 1 to 5 parts by weight of heparin salt at 25 to 45 ° C. for 15 to 48 hours. .

In addition, the partial esters of heparin may be in the form of sodium salts. The obtained ester can be recovered by precipitation with alcohol, in particular methanol, in the presence of sodium acetate. 1 to 1.2 volumes of alcohol are used per volume of reaction medium. The degree of esterification of the ester can then be determined by high performance liquid chromatography. In particular, in the case of benzyl esters, the amount of benzyl alcohol produced by saponification can be measured.

As a final step, the depolymerization is carried out by contacting the ester with a strong base such as sodium hydroxide in aqueous solution with a weight ratio of 0.05 to 0.2 of base / ester. The temperature of the reaction medium is adjusted to 50 to 70 ° C, preferably 55 to 65 ° C and the reaction is carried out for 30 minutes to 3 hours. It is also preferred to carry out the reaction in a medium having a weight ratio of water / ester of from 15 to 30.

Alternatively, the depolymerization is prepared in the esterification step, and 1 part by weight of an aromatic of salt form heparin having an esterification degree of 9.5% to 14% is mixed with 0.08 to 0.15 parts by weight of sodium hydroxide and 20 to 30 parts by weight of water, and the resulting mixture is Is maintained at 55 to 65 ° C. for 1 to 2 hours. The reaction medium can then be neutralized with dilute inorganic acid, preferably hydrochloric acid, and precipitated in the presence of an alcohol such as methanol to recover the product.

The mucopolysaccharide fractions obtained in the above-mentioned methods are further fractionated by various techniques such as gel-filtration or selective reprecipitation in a predetermined amount of aqueous-alcohol medium and also in the presence of a certain amount of certain inorganic salts such as sodium chloride. It is possible to make it.

Further fractionation can be carried out as a supplementary step applied to each mucopolysaccharide fraction previously re-dissolved in water, which is added to an aqueous solution of 1 to 2 volumes of ethanol and 10 to 100 g / l sodium chloride, Collection of the equivalent activity formed and the residual contents dissolved in the supernatant, in particular by further alcohol precipitation (fractionation product composition).

Gel-filtration of the fractions of the primary extraction can also yield higher mucopolysaccharide fractions with higher Yin-Wessler / USP titration ratios. This solution can be passed through bead form of polyacrylamide and agarose gel, ULTROGEL AcA 44, with an effective fractional zone located between effective molecular weights of 4,000 to 60,000 Da.

Regardless of the degree of purification, once the treated fractions are in a physiologically acceptable metal salt state, such as sodium salt, they may be mixed or simple containing other physiologically acceptable metals such as calcium by any method applicable to the heparin salt. Can be converted to salts. Advantageously, the method described in French Patent No. 73 13580 filed April 13, 1973 can be applied. This process consists essentially of, for example, starting with the sodium salt of heparin and contacting it with a different salt of another physiologically acceptable metal, such as calcium chloride, in solution and separating metal ions that do not bind heparin (e.g. alcohol precipitation). Or by dialysis), the substitutional heparin salt obtained after the first contact is in contact with other salts according to the desired final substitution ratio in the solution.

The fraction, on the one hand, comprises contacting the fraction with antithrombin III immobilized on a support such as agarose in 0.2 M NaCl, 0.05 M Tris-HCl buffer (pH 7.5), in particular in the system Can be characterized by specific affinity for antithrombin III, and, on the other hand, by Yin-Wessler and USP titrations of specific ratios (YW / USP ratios).

Heparin can be characterized by nuclear magnetic resonance spectra (NMR). More specifically, NMR spectra of the compounds according to the invention for protons ( ¹ H) are performed on these compound solutions dissolved in deuterium water at 35 ° C. Resonance signals for 4.8 and 5.2 ppm chemical substitutions, substantially weaker than the 5.4 ppm resonance signals also observed for chemical substitutions when using 270 MHz (MHz) radiation, were observed as specific elements of the spectrum (substitution criteria) : TSP (sodium 3-trimethylsilyl propionate 2,2, 3,3-d ₄ )).

Standard or unfractionated heparin or LMWH which can be used in the formulations according to the invention can have the general properties described above for LMWH on the one hand and affinity for anti thrombin III on the other hand, and these fractions have a high molecular weight. And also include in its structure the oligosaccharide moiety of the abovementioned structure. Any of the above mentioned LMWH compounds described above or described in the references can be selected or used individually or in combination with other LMWH compounds or other therapeutic or pharmaceutical compounds.

Several studies have shown that LMWH is safe and effective with standard or unfractionated heparin and can be used in different clinical situations such as acute coronary syndrome, cardiac surgery, vascular surgery, coronary and peripheral percutaneous angioplasty and acute stroke, including myocardial infarction. Has been demonstrated (Nenci GG et al., Vasc Med, 2000; 5: 251-258).

According to a preferred aspect, the combinations of the present invention not only prevent or treat acute deep vein thrombosis, but also prophylactically treat vascular thromboembolic diseases during moderate or high risk surgery and prevent aggregation in the extracorporeal circulation during hemodialysis. And commercially available LMWH under the trade name Clexane ^® / Lovenox ^® by Aventis Pharma SA for the purpose of treating unstable angina and acute non-Q wave myocardial infarction when combined with aspirin.

Fragmin ^® is a low molecular weight heparin manufactured by Pharmacia & Upjohn and marketed since 1985. It is an antithrombotic agent useful for the treatment and prevention of thrombosis containing dalteparin sodium with an average molecular weight of 5000 Da. Dalteparin sodium is prepared by nitrite depolymerization of sodium heparin from porcine urinary tract membranes. It consists of strongly acidated sulfated polysaccharide chains with an average molecular weight of 4000-6000 and about 90% of the material ranging from 2000-9000 Da.

Which is commercialized by Sanofi as other low molecular weight heparin in the commercially available Fraxiparine ^® (parin in Oxnard), ^® a Innohelp commercialized by Dupont (tin Jaffa Lin) and Clivarin ^® (Levy parin), these and NV1FGF according to the invention It can be used in combination.

In the most preferred embodiment, the plasmid NV1FGF is used to enoxaparin, for example combined with enoxaparin ^{(Lovenox ®, Clexane ®),} Fragmin ® or Fraxiparine ^®.

According to this aspect, enoxaparin is typically combined with an angiogenic growth factor coding factor capable of expressing angiogenic growth factor in the cells of the individual to be treated as a DNA molecule. DNA encoding angiogenic growth factors may be fibroblast growth factor (FGF) such as FGF-1 or acidic FGF (aFGF), FGF-2 or basic FGF (bFGF), FGF-4, FGF-5, FGF-6 and DNA encoding any of FGF-7 or FGF1-22.

According to another aspect, enoxaparin is used in combination with plasmid NV1FGF at a concentration sufficient to promote synergistic angiogenic responses, as shown by the formation of capillary vessels and / or mature vessels such as arterioles. In addition, the combination of NV1FGF with enoxaparin is particularly effective as it can efficiently promote therapeutic angiogenesis at undetectable concentrations in the treated muscles. When combined with enoxaparin, NV1FGF can be administered at concentrations within the treatment window to avoid adverse side effects caused by surrounding tissues or organs or interspersed angiogenics.

Enoxaparin can 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 intravenous or subcutaneous route. Sterile compositions for intravenous or subcutaneous administration are generally aqueous solutions. These compositions may also comprise an adjuvant, preferably selected from wetting agents, isotonic agents, emulsifiers, dispersants and stabilizers. Sterilization can be performed by several methods, for example, aseptic filtration, introduction of sterilant into the composition or by irradiation. They can also be prepared in the form of sterile solid compositions that can be dissolved in sterile water or any other injectable sterile medium in use. In one example of a suitable composition, 0.2 ml of a solution is prepared by dissolving 20 mg of enoxaparin in a sufficient amount of distilled water. Dosage depends on the desired effect, duration of treatment and route of administration utilized; They are generally administered 0.2 mg to 4 mg / kg per day, preferably 1.5 mg / kg twice a day or 0.75 mg / kg twice daily, i.e. 14 to 280 mg per day in adults.

The dalte parin or Fragmin ^® commercialized by Pharmacia & Upjohn, may also be used in the synergistic combination of the present invention. US Patent No. 4,303,651 discloses illustrated the structure and manufacturing method of Fragmin ^® or dalte parin. Dalteparin or Fragmin ^® can be prepared in several different ways. One of these methods includes the steps of (a) treating standard heparin with nitrous acid in the above-mentioned dimethoxyethane. This method provides fragments of this type with a series of inert fragments. Inactive elements from the active fragments can then be removed, for example, by affinity chromatography on matrix-bound antithrombin III [Hook et al., FEBS Lett. 66, 90 (1976); Hopwood et al., FEBS Lett. 63, 51 (1976); L.-O. Andersson et al., Thromb. Res. 9, 575 (1976). Other methods of preparing fragments include (b) piodate oxidation at low pH and low temperature; (c) partial depolymerization by heparanase; (d) partial depolymerization of heparin by esterification of carboxyl groups and subsequent alkali β-removal; (e) partial depolymerization of heparin by partial N-desulfation and subsequent deamidation with nitrous acid at pH 3.9. Methods (a) and (b) are illustrated in the examples.

The active fragment may comprise 14 to 18 sugar units. Structural analysis was performed with the same major structural components as in standard heparin, i.e. L-eduurosyl-2-O-sulfate- (1.alpha.-4) -N-sulfo-D-glucosamine-6-O -Represents sulfate. However, the amount of unsulfated eduuronic acid may be much higher than in the starting material. The active fragment has the structure (UG) _n -IG- (UG) _m , where n is 1 or 2, m is 5 or 6, I is unsulfated L-duuronic acid, and U is L- Duronic acid-2-O-sulfate, and G is N-sulfo-D-glucosamine-6-O-sulfate. Some U units may not have 0-sulfate or may be replaced by D-glucuronic acid, and likewise, some G units may not have 0-sulfate, or by N-acetyl-D-glucosamine unit Can be replaced. The reducing or non-reducing end unit may vary depending on the type of preparation method used, ie 2,5-anhydro-D- at the reducing end site, for example according to the deaminitive splitting of heparin. Mannose is formed. Active fragments can be characterized by physicochemical methods such as measurement of mobility in the electric field and UV, IR and NMR spectra. However, because the aggregate-inactive fragments also exhibit similar properties, the obtained values are not complete information. This is based on the fact that biological activity derives from the particular sequence of sugar residues, where the position of the unsulfated euroonic acid is of particular interest.

The individual to be treated is typically a mammal, such as a human. The individual may be at risk for ischemia (eg, ischemic symptoms as exemplified herein) due to, for example, genetic or environmental factors. Thus, an individual may have an ischemic family history. The individual may have one or more of the following risk factors: 45 or older (eg 50, 55 or 60 or older), smoker, excessive alcohol consumption, overweight, atherosclerosis or diabetes or inoperable condition. The subject may have a heart or peripheral ischemia.

The individual to be treated may have developed an ischemic attack within the last 24 hours, or is particularly a person suffering from heart disease. The individual may have had an ischemic attack 6 months ago, such as 1, 2, 3 years or earlier, or is an individual who has in particular peripheral ischemia.

The individual may be in a condition where, for example, artificial oxygen (or oxygen enriched air) is injected in an intensive care unit (typically being treated with an ischemic attack within the last 24 hours).

Due to their excellent properties in terms of synergy and efficacy, the combination of NV1FGF and LMWH is particularly useful for treating angiogenesis, especially in conditions of exacerbation caused by hypercholesterolemia or diabetes.

Therefore, reversal of angiogenesis deficiency caused by attenuated blood is considered by the present invention regardless of its inception which exacerbates symptoms such as hypercholesterolemia or diabetes.

In the present invention, the target tissue is of skeletal muscle or myocardial tissue which is in an ischemic injury state or at risk of pain, whereby hypercholesterolemia or diabetic symptoms are worsened if the proper supply of oxygenated blood to the tissue is interrupted. As demonstrated in the Examples, intramuscular or intramyocardial infusion of plasmid NV1FGF in combination with LMWH or standard heparin can be effectively used in therapeutic windows that meet the stability criteria required by gene therapy, and in blood vessels in endothelial insufficiency ischemic tissue. Can induce formation.

The invention also provides a low molecular weight heparin and an agent or vector that expresses a gene encoding angiogenic growth factors for use simultaneously, separately or in a sequential manner for the treatment of ischemia (such as any of the ischemic symptoms mentioned herein). Or a product containing standard heparin. In addition, the present invention provides an angiogenic growth factor in the manufacture of a medicament for treating ischemia (eg, any of the ischemic symptoms mentioned herein) by administering a combination of an agent encoding angiogenic growth factor and a low molecular weight heparin. Provided is the use of a medicament or an expression vector encoding a. The invention also provides a low molecular weight in the manufacture of a medicament for treating ischemia (such as any of the ischemic symptoms referred to herein) by administering a combination of an agent or expression vector encoding an angiogenic growth factor and low molecular weight heparin. Provides the use of heparin.

DNA or vectors encoding angiogenic growth factors and low molecular weight heparin may be administered separately (ie, not in the form of a composition comprising both) or in the form of a composition comprising both. When administered separately, agents or vectors expressing genes encoding angiogenic growth factors will be administered at least one, two, three or more times in a week (eg 1 or 2 days) after low molecular weight heparin. In one example, agents or vectors and genes expressing genes encoding angiogenic growth factors and low molecular weight heparin are administered at least one, two, three, four, five or more times simultaneously (eg, within two hours relative to each other).

It is to be understood that administration characteristics, such as dosage, site of administration or timing, associated with the NV1FGF plasmid are applicable to the administration of any factor encoding angiogenic growth factors.

According to one aspect of the invention, the NV1FGF plasmid is administered in a local manner to the target skeletal muscle or myocardial tissue. Any means suitable for administering the NV1FGF plasmid to the target tissue may be used in the present invention, but for topical injection into the target muscle tissue, it is preferred to inject NV1FGF directly into the muscle using a needle or catheter.

The term "injection" means forcing the introduction of NV1FGF into target skeletal muscle or myocardial tissue. Any suitable or available apparatus, method or system may be used in accordance with the present invention.

Administration of NV1FGF plasmid administration may be done in a single injection into the target tissue, but multiple injections of NV1FGF are preferred. Multiple infusions may be repeated infusions of 2, 3, 4, 5 or more, preferably 5 or more injections into the hull tissue of a mammalian subject suffering from hypercholesterolemia or diabetes. Multiple injections have been shown to be advantageous over single injections in that each injection can be performed with parameters such as a specific shape specific to the location on the target tissue to be performed. Injecting a single dose of NV1FGF in multiple infusions may be easier to control and may maximize the effect of any given dose.

Typically, agents or vectors encoding angiogenic growth factors are administered at least three times every 10 to 18 days, such as every 12 to 16 days.

The particular shape of the multiple infusions can be defined by the two-dimensional space where each NV1FGF is administered. Multiple infusions can be done in or around ischemic tissue, with the infusion point preferably located 2 or 3 cm apart.

According to another aspect of the invention, each multiple injection is done within about 5 to 10 minutes of each other.

If NV1FGF is administered to target tissues that are significantly affected by angiogenesis deficiency and severely impaired endothelial function, it is desirable to administer NV1FGF to contact the region between, as well as the region substantially adjacent to, the origin and end of side vessel growth formation. .

Most preferably, intramuscular administration of NV1FGF is done in the peripheral region adjacent to the ischemic site in the distal thigh and distal leg muscles.

In an advantageous aspect of the present invention, a therapeutically effective dose of a combination of NV1FGF and LMWH is administered such that the angiogenic deficiency is reversed in hypercholesterolemic or diabetic symptoms. Effective dosages will depend on the weight and symptoms of subjects suffering from angiogenesis deficiency in addition to high cholesterol or diabetic subjects, and those skilled in the art will be able to determine appropriate dosages for a given subject and condition.

According to a preferred aspect of this aspect, the treatment is performed by increasing the amount of NV1FGF injected intramuscularly to 500 μg, 1000 μg, 2000 μg to 16000 μg. Most preferred are repeated doses of 2 × 500 μg and 2 × 1000 μg. 4000 μg, 8000 μg, 16000 μg in consideration of the high stability of NV1FGF to promote the constant formation of the side capillary vessels and arterioles to reverse the angiogenic deficiency caused by ischemia in mammalian subjects suffering from hypercholesterolemia or diabetes. Or higher doses of 2 × 2000 μg, 2 × 4000 μg and 2 × 8000 μg can be used for the severe symptoms of angiogenic deficiency.

NV1FGF is preferably administered to the target ischemic myocardium or skeletal muscle in a pharmaceutical composition comprising a pharmaceutically acceptable carrier and NV1FGF plasmid.

In accordance with one aspect of the present invention, LMWH, for example enoxaparin or Fraxiparin, can be used in the form of a composition in an inert or physiologically active pharmaceutical compatible product. They are preferably used by intravenous or subcutaneous route. Sterile compositions for intravenous or subcutaneous administration are generally aqueous solutions. These compositions may also include an adjuvant selected from wetting agents, isotonic agents, emulsifiers, dispersants and stabilizers. Sterilization can be carried out in several ways, for example by aseptic filtration, by introducing sterilant into the composition or by irradiation. They can also be prepared in the form of sterile solid compositions that can be dissolved in sterile water or any other injectable sterile medium in use. In one example of a suitable composition, 0.2 mg solution is prepared by dissolving 20 mg of LMWH in a sufficient amount of distilled water.

Dosage depends on the desired effect, duration of treatment and route of administration utilized; They are generally administered between 14 and 280 mg per day in the subcutaneous route 0.2 mg to 4 mg / kg per day, ie in adult dosages of 5 to 280 mg. Preferably, 0.2 to 0.3 mg / kg is administered per day. In one example, the LMWH is administered for at least 20 or at least 30 days, such that the first and last administration of heparin (LMWH or standard heparin) is at least 20 or at least 30 days apart, during which the factor encoding angiogenic growth factor is also at least 1 More than 2 times, 3 times).

Any suitable pharmaceutically acceptable carrier can be used in the present invention, and such carriers are well known in the art. The choice of carrier will depend in part on the particular site where the composition is to be administered and the particular method by which the composition is to be administered. Formulations suitable for injection include aqueous and non-aqueous solutions, antioxidants, buffers, bacteriostatic agents and isotonic sterile infusions which may contain solutes that may render the formulation isotonic with the blood of the subject receptor, and suspending agents, solubilizers , Aqueous and non-aqueous sterile suspensions which may include thickening agents, stabilizers and preservatives. The formulations may be present in unit doses or in multiple doses in closed containers, such as ampoules and vials, and may be stored in lyophilized (freeze-dried) state requiring only the addition of a sterile liquid carrier such as water immediately before use. Temporary infusions and suspensions can be prepared from sterile powders, granules and tablets of a known type. Preferably, the pharmaceutically acceptable carrier is buffered saline. Most preferably, the pharmaceutical composition comprises sodium chloride solution.

The invention also relates to a novel method of treating patients with peripheral and / or coronary ischemic syndromes by administering to the patient an effective amount of LMWH and a DNA encoding an effective amount of angiogenic growth factor, preferably the NV1FGF plasmid. . Risk patients include individuals who have symptoms of early coronary or peripheral ischemic symptoms that are more likely to damage ischemic skeletal muscle or myocardial tissue than others who do not have these symptoms.

The present invention also provides angiogenesis therapies for treating ischemic symptoms such as peripheral or myocardial ischemia, wound healing or other conditions requiring angiogenesis or tissue regeneration. The present invention may be useful for treating PAD (Peripheral Arterial Disease), IC (Intermittent claudication) or CAD (Coronary Artery Disease), also referred to as PAOD (Peripheral Arterial Obstructive Disease) pathology.

It is another object of the present invention to provide a method for enhancing therapeutic angiogenesis in a patient subject of angioplasty, circuit transplantation or other angioplasty surgery by administering LMWH or a combination of standard heparin and NV1FGF as described above.

Another object of the present invention is to administer the combination of the present invention in an amount sufficient to promote both the side vessels and the arterioles in ischemic skeletal muscle or myocardium, thereby revascularizing the ischemic myocardium and skeletal muscle in a mammalian subject suffering from endothelial dysfunction. To provide a method of stimulating and / or promoting formation. More specifically, the present invention provides a method of stimulating and / or promoting vascular remodeling of ischemic muscles in hypercholesterolemia or diabetic symptoms and a method for reversing angiogenesis deficiency in mammalian subjects requiring hypercholesterolemia or diabetes treatment. It is about.

Perfusion damage to the hind limbs caused by single or multiple large vessel occlusion is a factor in peripheral arterial disease (PAD). In the early stages, this is a degree of discomfort in the walking leg muscles, leading to ulceration and necrosis in the late stages (1). Chronic cardiovascular disease is aggravating in patients already suffering from ischemic symptoms such as PAD through mechanisms involving endothelial impairment (2, 3). Pathologies such as hypercholesterolemia, hypertension and diabetes are under investigation as possible targets for the developmental experimental model of PAD (4-7). Nevertheless, in this hind limb ischemia model, an important point is that any vascular remodeling treatment does not induce a spontaneous angiogenic response to be efficient.

In accordance with the present invention, combinations of NV1FGF and LMWH or standard heparin have proven to be particularly effective in promoting the growth of the side vessels and arterioles in patients suffering from PAD or coronary artery disease to rescue angiogenic damage caused by cholesterol.

As shown in the examples below, NV1FGF in combination with LMWH or standard heparin is used to treat large mature conducting vessels (> 150 μm side vessels) and small in ischemic damaged femoral posterior muscles that require the transport and delivery of blood to tissues. Resistance artery (<50 μm arterioles) formation can be efficiently induced. Induction of such mature blood vessels, such as the arterioles, has proven to be a very useful treatment in most severe cases where harmful angiogenesis deficiency is caused by hypercholesterolemia or diabetes.

Arterioles are known as mature vessels that contain a wall of wall cells composed of endothelial and perivascular cells, and because of the larger vascular capacity compared to capillary networks, lateral arterial growth is particularly effective and excellent therapeutic angiogenesis (Carmeliet et al. , Nat. Med., 2000; 6: 389-395; Van Royen et al., Cardiovasc. Res., 2001; 49: 543-553).

Another object of the present invention is to provide a method for promoting angiogenesis by administering a combination of NV1FGF and LMWH or standard heparin, provided that VEGF should not be upregulated in treated cells.

Throughout this application, various documents, patents and patent applications have been cited. The teachings and descriptions of these documents, patents and patent applications are incorporated by reference for the purpose of describing in more detail the context of the industry with which the present application relates.

In addition, it is to be understood that the principles of the invention disclosed herein, by way of example, may be modified by those skilled in the art, and such modifications, changes, and substitutions are intended to be included within the scope of this application.

Example 1: Animals and Diet

An 11-12 week old Syrian golden hamster (CERJ, Le Genest St Isle, France) was used for the experiment. Animals were equilibrated at standard conditions at least 7 days before the start of the experiment. All animals were given free access to water for the entire duration of the experiment. All animal procedures were approved by the Animal Use Committee and followed the guidelines published by the National Institutes of Health (NIH Publication No. 85-23, revised 1985).

Hamsters with a high cholesterol diet were given 20 g of a cholesterol-rich diet per animal, consisting of a standard solid diet with 3% cholesterol and 15% cocoa butter daily (1414C, UAR, Epinay-sur-Orge, France), Randomly divided into four groups: (i) saline group, (ii) NV1FGF group, (iii) NV1FGF and enoxaparin, and (iv) enoxaparin and saline.

Example 2: Induced Hind Leg Ischemia

After 35 days of LC or HC diet, hind limb ischemia was induced in animals according to the following surgical procedures: N ₂ 0 (0.8 L.min ⁻¹ ), 0 ₂ (0.4 L.min ⁻¹ ) and isofluorane (2%) Anesthesia was induced and ischemia was induced in the hind limbs following the procedures of other animal species (19, 20). Under sterile surgical conditions, the right hind limb was dissected longitudinally from the inguinal ligament to the patella. Through this incision, the femoral artery was freely incised using a surgical loop and its main branch coagulated. The femoral artery was completely incised from the proximal start of the outer iliac artery branch to the bifurcated distal point of the dizziness and popliteal branches (4). The incision was closed one layer with 4.0 silk sutures.

Example 3: Gene introduction into hindlimb skeletal muscle

14 days after ischemia induction, plasmid encoding NV1FGF with or without saline, enoxaparin, and saline and enoxaparin were injected three times with 60 μl each into the forearm, anterior and quadriceps muscles of the ischemic leg. It was.

Example 4 Determination of Total Cholesterol, Lipid and Triglyceride Levels in Serum

N ₂ 0 (0.8) from hamsters of the NV1FGF group administered in combination with HC / 21, HC / 28, saline, NV1FGF, enoxaparin, or enoxaparin on days of surgery-related -35, -7 and +21 or +28. Blood was collected by puncturing the orbital posterior anesthesia (L.min- ¹ ), 0 ₂ (0.4 L.min- ¹ ) and isofluorane (2%). Total cholesterol and triglyceride levels in serum were measured enzymatically using a commercial kit (Olympus Diagnostica GmbH, Hamburg, Germany). Cholesterol rich diets raised both total cholesterol and triglyceride serum levels in a time dependent manner. Serum lipids were measured in all groups at the end of the experiment (ie 28 days after ischemia).

Example 5: Quantification of Side Blood Vessel Formation by Angiography

On day 21 (HC / 21 group) or day 28 (LC, HC / 28, saline and NV1FGF group) after ischemia induction, angiography was performed according to the following procedure.

Approximately 300 μl of contrast agent (0.5 g.mol ⁻¹ barium sulfate aqueous solution) was injected through a catheter inserted into the abdominal aorta and immediately hamstered the hamster by overdose with sodium pentobarbital. Hamsters were placed on a radiograph (model MX-20, Faxitron X-ray Corp., Wheeling, IL, USA), and post-vascular images of both legs were collected and digitalized (software Specimen, DALSA MedOptics, Tucson, AZ, USA). This radiographic system visualizes blood vessels larger than 150 μm in diameter. An investigator who did not participate in the process (blind) analyzed the photo offline using known public software (5).

Briefly, the degree of lateral side vessels in the posterior thigh for both ischemic and non-ischemic legs was determined as a percentage of the analytical area. Angiography scores are expressed as the percentage of ischemia / non ischemia. To verify the validity of the method, angiography scores were evaluated on six different hamsters of age matched that did not cause hind limb ischemia. As expected, the angiography score calculated as the percentage of right leg / left leg was 1.04 ± 0.18.

Example 6: Quantification of Arterial Formation by Immunohistochemistry and Typical Muscle Lesions Induced by Hind Limb Ischemia through Femoral Artery Incision of High Cholesterol Hamsters

28 days after ischemia induction, skeletal muscle from the ischemic hind limbs was collected and immobilized in PBS-3.7% formalin solution. Skeletal muscle from the non-ischemic hind limb was similarly sampled and used as a control muscle. Two transverse slices were obtained from different muscles at the back of each femoral (cubicus, semimembranosus, vowel, semitendinosus, biceps femoris). After the sulace was dehydrated and impregnated with paraffin, 5 μm thick sections were prepared for immunohistochemistry. Because of its structural expression in mature blood vessels, mouse monoclonal antibodies against smooth muscle α-actin (SMA; clone 1A4, dilution 1: 200, Dako, Carpinteria, CA, USA) were used as vascular smooth muscle cell (VSMC) markers. . SMA antibodies were detected with a commercial kit (Envision ^™ + System / Carrot Peroxidase, Dako, Carpinteria, CA, USA) by the avidin-biotin-peroxidase method. SMA-positive (SMA +) vessels were sorted by size (outer diameter) and counted arterioles of diameter <50 μm in both the anterior and the anterior biceps.

Unlike the other muscles from the posterior femoral (semicondylar, apex, and biceps), the muscles were selected to determine their susceptibility to histopathological lesions in the hind limb ischemic hamster model.

Typical lesions due to femoral artery extraction are shown in FIG. 2. Muscles from the posterior thigh, namely the vowels and the cephalospinal muscles, were collected 28 days after ischemia induction, and 5 μm-thick muscle sections were observed after HES staining. 2B and 2C show the presence of moderate necrosis (solid line) and central nucleation (arrow) in ischemic muscle, respectively. 2A is a 100-fold magnification of the cross section of the non-ischemic opposite muscle as a control. The ischemic effect on muscle volume was investigated by measuring the total area of the anterior and bicuspid biceps. The number of SMA + arterioles against total muscle area was measured. The ratio of ischemic / non-ischemic values for both parameters was then calculated. All procedures were performed by investigators who did not participate in the treatment.

Example 7: FGF-1 expression after introduction of NV1FGF gene into ischemic muscle

After 14 days of saline injection, NV1FGF gene introduction and injection of NV1FGF and enoxaparin combination into the forearm bone from the ischemic and ischemic leg (ie, 28 days after ischemia induction) were performed as follows. FGF-1 immunohistochemistry was performed using conventional streptavidin-biotin assays to detect FGF1 expression. After incubation with primary polyclonal anti-FGF-1 rabbit antibody (reference AB-32-NA, 1:30 dilution, R & D Systems, Abingdon, UK), followed by biotinylated donkey anti-rabbit immunoglobulin (1: 200 dilution, Amersham, Buckinghamshire, UK). After the addition of peroxidase bound to streptavidin, the colored complex diaminobenzidine was used to localize the immune complex. Sections 5 μm thick were counterstained with hematoxylin, dehydrated and sampled with permanent encapsulant. Immunoreactive fibers were identified under microscope (Axioplan 2, Zeiss, Hallbergmoos, Germany) (brown staining).

We have demonstrated that, unlike gene therapy involving other angiogenic factors such as VEGF or FGF-2, expression of FGF-1 is advantageously limited to ischemic muscles of animals treated with NV1FGF.

In addition, immunohistochemical staining of anti-FGF-1 polyclonal antibodies in the femoral posterior muscles (forebones, vowels and biceps ganglia) from non-ischemic (opposite) non-implanted legs and saline or ischemic legs injected with NV1FGF. As exemplified by a representative photograph (100-fold magnification), expression of FGF-1 was surprisingly not detected in ischemic muscles of saline-treated animals nor in ischemic (opposite) muscles of animals treated with saline and NV1FGF. This is apparently sufficient to provide a sustained angiogenic response through the formation of mature blood vessels, while allowing plasmid NV1FGF to slowly release the FGF-1 protein encoded in the treatment window at a concentration that does not allow sowing and irregular angiogenesis or adverse side effects. It shows excellent characteristics. Thus, NV1FGF is particularly potent because the concentration of NV1FGF contained in the treatment window can be used for symptoms characterized by endothelial cell dysfunction exacerbated by being able to efficiently promote angiogenesis at non-detected concentrations in treated muscles. Proved to be Due to this property excellent in stability and efficacy, NV1FGF can be advantageously used as angiogenesis therapy for the worsening symptoms caused by hypercholesterolemia or diabetes.

These results clearly demonstrate that NV1FGF gene therapy can increase the arterioles to rescue impaired side vessel formation. Indeed, angiography has demonstrated> 150 μm side vessel growth in the posterior thigh, including the biceps, biceps, biceps, diaphragm and semiphysiolisus. Unexpectedly, we demonstrated that 14 days after the NV1FGF gene introduction, side vessel formation was significantly promoted in this region, as highlighted by angiography scores.

In situ measurements of tissue oxygenation were not performed to confirm that ischemia occurred in the region after femoral artery extraction, but the presence of histological lesions such as central nucleation, axis of regression, necrosis and inflammation appeared. More specifically, these lesions are confined to the vowel and biceps triceps, whereas the biceps femoral, semiapecular, and semiconjunctival muscles are less likely to appear. Accordingly, quantification of the <50 μm arterioles was performed directly on the vertebral muscle only for the lateral muscles and the cephalic muscle. Introduction of the NV1FGF gene increased the absolute number of arterioles in these ischemic leg muscles.

Thus, these data clearly show that large mature conducting vessels (> 150 μm side vessels) and small resistance arteries (<50 μm arteries) in the posterior femoral muscle where NV1FGF is damaged by ischemia and require transport and delivery of blood to tissues. ) Demonstrates the ability to induce formation.

Example 8 Comparative Study of NV1FGF Gene Introduction and Effects of NV1FGF in Combination with Enoxaparin on Lateral Development and Arterial Density 14 Days After Intramuscular Administration in High Cholesterol Hamsters

Subcutaneous administration of enoxaparin at 14 days after induction of hind limb ischemia and the introduction of the NV1FGF gene intramuscularly significantly improved lateral vessel formation in the ischemic leg compared to the introduction of saline-treated hamsters, enoxaparin or NV1FGF genes. In addition, angiography scores after administration of NV1FGF and enoxaparin were significantly higher than that of either saline or NV1FGF treated hamsters, or enoxaparin treated hamsters.

Mature blood vessels were labeled by smooth αactin (SMA) immunohistochemistry from ischemic muscles of hamsters treated with NV1FGF with or without saline and enoxaparin, and quantified muscle regions and <50 μm SMA positive arterioles.

In the ischemic limbs of the NV1FGF and Enoxaparin groups, the areas of the tibialis anterior muscles, vowel muscles, and biceps tendon decreased.

Example 9 Method of Treating Hind Limb Ischemia in Diabetic Mice Using NV1FGF and Enoxaparin or Standard Heparin

The importance of angiogenesis and lateral blood vessel growth in lateral blood vessel development has already been established. Thus, angiogenesis therapy with angiogenic cytokines presents a solution in patients with side vessel apparent failure. Diabetes entails significant damage of side blood vessel growth. Thus, hind limb ischemia experimental models have been used in the diabetic environment to support the combination of anti-inflammatory or anticoagulants, low molecular weight heparins such as enoxaparin and NV1FGF angiogenic gene therapy.

Induced diabetes :

Streptozotocin selectively destroys the insulin-producing beta islet cells of the pancreas to provide a type I diabetes model. Streptozotocin dissolved at 10 mg / ml in 0.1 M sodium citrate buffer (pH 5.5) was intraperitoneally injected at a dose of 80 mg / kg to 8-10 week old male C57BI / 6 mice. At 72 hours, if plasma glucose levels were higher than 250 mg / dl and kept elevated, mice were considered to have diabetes and used in the experiment.

Hind limb ischemia :

Surgical operation was performed under the microscope 4 weeks after diabetes induction. A skin incision was made downward from the inguinal area of the right hind limb above the hip artery. Subsequently, the hip artery was ligated close and wide using a 4-0 silk suture. The secondary artery was ligated and cut.

Plasmid solution injection :

NV1FGF plasmid solution was added to the quadriceps and / or forearm muscle (injection volume: 40 μl / muscle) of the right hind limb within 0.4-4.0 mg / kg body weight (ie 10-100 μg / mouse or 5-50 within 7 days post-surgery). Μg / mouse) in single dose mode.

Low molecular weight heparin (enoxaparin) injection :

Enoxaparin was injected subcutaneously once a day at a dose of 1.0-1.5 mg / kg during or several hours before surgery. A single dose of 0.5-0.75 mg of enoxaparin per kg may be administered by intravenous injection prior to subcutaneous treatment.

End points for monitoring treatment efficacy :

Hind limb perfusion was assessed for non-invasiveness by laser Doppler imaging every week before surgery, immediately after surgery, and every four weeks after surgery. Ischemia (right) / normal (left) leg blood flow ratios were measured to compare perfusion status and reperfusion kinetics between treatment groups.

Angiogenesis markers such as CD31 can also be assessed by semiquantitative methods such as ELISA (protein) or RT-PCR (mRNA) for muscle extracts, or immunohistochemistry (protein) for muscle incisions.

Endpoints to monitor synergy :

Evidence of rapid kinetics of hind limb reperfusion in mice treated with NV1FGF + enoxaparin compared to any single agent or

Evidence and / or evidence of high levels of reperfusion at any given end time after treatment with combination therapy for any single agent

Evidence that when NV1FGF in combination with enoxaparin exhibits similar levels of reperfusion at a lower dose compared to a given dose of NV1FGF as a single agent.

Each of the following references may be used to construct and use the features of the invention, or may be incorporated in whole or in part thereof, in particular incorporated herein by reference. In addition, the above examples and description should not be understood as limiting the scope of the invention, and those skilled in the art can modify the methods, compounds and combinations of the compounds discussed without departing from the scope and object of the invention.

Reference

Ouriel K. Peripheral arterial desease. 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 vectors for nonvirus gene therapy. Gene Ther. 1999; 6: 1482-1488.

Dart AM, Chin-Dusting JPF. Lipids and theendothelium. Cardiovasc. Res. 1999; 43: 308-322.

Takeshita S, Isshiki T., Sato T. Increased expression of direct gene transfer into skeletal muscle observed after acute ischemic injury in rats. Lab. Invest. 1996; 74: 1061-1065.

Silvestre JS, Mallat Z, Duriez M, Tamarat R, Bureau MF, Scherman D, Duverger N, Branellec D, Tedgui A, Levy BI. Antiangiogenic effect of interleukin-10 in ischemic-induced angiogenesis in mice hindlimb. Circ. Res. 2000; 87: 448-452.

6. Ylae-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.

8. Isner JM, Asahara T. Angiogenisis and vasculogenesis as therapeutic strategies for postnatal neovascularization. J. Clin. Invest. 1999; 103: 1231-1236.

                         SEQUENCE LISTING <110> CENTELION       AVENTIS PHARMA SA <120> TREATMENT OF CORONARY OR PERIPHERAL ISCHEMIA <130> GC03005 <140> NOT YET KNOWN <141> 2004-12-27 <150> US60 / 532,993 <151> 2003-12-29 <160> 7 <170> PatentIn version 3.1 <210> 1 <211> 405 <212> DNA <213> Homo sapiens <400> 1 aattacaaga agcccaaact cctctactgt agcaacgggg gccacttcct gaggatcctt 60 ccggatggca cagtggatgg gacaagggac aggagcgacc agcacattca gctgcagctc 120 agtgcggaaa gcgtggggga ggtgtatata aagagtaccg agactggcca gtacttggcc 180 atggacaccg acgggctttt atacggctca cagacaccaa atgaggaatg tttgttcctg 240 gaaaggctgg aggagaacca ttacaacacc tatatatcca agaagcatgc agagaagaat 300 tggtttgttg gcctcaagaa gaatgggagc tgcaaacgcg gtcctcggac tcactatggc 360 cagaaagcaa tcttgtttct ccccctgcca gtctcttctg attaa 405 <210> 2 <211> 867 <212> DNA <213> Homo sapiens <400> 2 ctggtgggtg tcgggggtgg agatgtagaa gatgtgacgc cgcggcccgg cgggtgccag 60 attagcggac gcgctgcccg cggttgcaac gggatcccgg gcgctgcagc ttgggaggcg 120 gctctcccca ggcggcgtcc gcggagacac ccatccgtga accccaggtc ccgggccgcc 180 ggctcgccgc gcaccagggg ccggcggaca gaagagcggc cgagcggctc gaggctgggg 240 gaccgcgggc gcggccgcgc gctgccgggc gggaggctgg ggggccgggg ccggggccgt 300 gccccggagc gggtcggagg ccggggccgg ggccggggga cggcggctcc ccgcgcggct 360 ccagcggctc ggggatcccg gccgggcccc gcagggacca tggcagccgg gagcatcacc 420 acgctgcccg ccttgcccga ggatggcggc agcggcgcct tcccgcccgg ccacttcaag 480 gaccccaagc ggctgtactg caaaaacggg ggcttcttcc tgcgcatcca ccccgacggc 540 cgagttgacg gggtccggga gaagagcgac cctcacatca agctacaact tcaagcagaa 600 gagagaggag ttgtgtctat caaaggagtg tgtgctaacc gttacctggc tatgaaggaa 660 gatggaagat tactggcttc taaatgtgtt acggatgagt gtttcttttt tgaacgattg 720 gaatctaata actacaatac ttaccggtca aggaaataca ccagttggta tgtggcactg 780 aaacgaactg ggcagtataa acttggatcc aaaacaggac ctgggcagaa agctatactt 840 tttcttccaa tgtctgctaa gagctga 867 <210> 3 <211> 621 <212> DNA <213> Homo sapiens <400> 3 atgtcggggc ccgggacggc cgcggtagcg ctgctcccgg cggtcctgct ggccttgctg 60 gcgccctggg cgggccgagg gggcgccgcc gcacccactg cacccaacgg cacgctggag 120 gccgagctgg agcgccgctg ggagagcctg gtggcgctct cgttggcgcg cctgccggtg 180 gcagcgcagc ccaaggaggc ggccgtccag agcggcgccg gcgactacct gctgggcatc 240 aagcggctgc ggcggctcta ctgcaacgtg ggcatcggct tccacctcca ggcgctcccc 300 gacggccgca tcggcggcgc gcacgcggac acccgcgaca gcctgctgga gctctcgccc 360 gtggagcggg gcgtggtgag catcttcggc gtggccagcc ggttcttcgt ggccatgagc 420 agcaagggca agctctatgg ctcgcccttc ttcaccgatg agtgcacgtt caaggagatt 480 ctccttccca acaactacaa cgcctacgag tcctacaagt accccggcat gttcatcgcc 540 ctgagcaaga atgggaagac caagaagggg aaccgagtgt cgcccaccat gaaggtcacc 600 cacttcctcc ccaggctgtg a 621 <210> 4 <211> 807 <212> DNA <213> Homo sapiens <400> 4 atgagcttgt ccttcctcct cctcctcttc ttcagccacc tgatcctcag cgcctgggct 60 cacggggaga agcgtctcgc ccccaaaggg caacccggac ccgctgccac tgataggaac 120 cctataggct ccagcagcag acagagcagc agtagcgcta tgtcttcctc ttctgcctcc 180 tcctcccccg cagcttctct gggcagccaa ggaagtggct tggagcagag cagtttccag 240 tggagcccct cggggcgccg gaccggcagc ctctactgca gagtgggcat cggtttccat 300 ctgcagatct acccggatgg caaagtcaat ggatcccacg aagccaatat gttaagtgtt 360 ttggaaatat ttgctgtgtc tcaggggatt gtaggaatac gaggagtttt cagcaacaaa 420 tttttagcga tgtcaaaaaa aggaaaactc catgcaagtg ccaagttcac agatgactgc 480 aagttcaggg agcgttttca agaaaatagc tataatacct atgcctcagc aatacataga 540 actgaaaaaa cagggcggga gtggtatgtt gccctgaata aaagaggaaa agccaaacga 600 gggtgcagcc cccgggttaa accccagcat atctctaccc attttcttcc aagattcaag 660 cagtcggagc agccagaact ttctttcacg gttactgttc ctgaaaagaa aaatccacct 720 agccctatca agtcaaagat tcccctttct gcacctcgga aaaataccaa ctcagtgaaa 780 tacagactca agtttcgctt tggataa 807 <210> 5 <211> 744 <212> DNA <213> Homo sapiens <400> 5 tttagggcca ttaattctga ccacgtgcct gagaggcaag gtggatggcc ctgggacaga 60 aactgttcat cactatgtcc cggggagcag gacgtctgca gggcacgctg tgggctctcg 120 tcttcctagg catcctagtg ggcatggtgg tgccctcgcc tgcaggcacc cgtgccaaca 180 acacgctgct ggactcgagg ggctggggca ccctgctgtc caggtctcgc gcggggctag 240 ctggagagat tgccggggtg aactgggaaa gtggctattt ggtggggatc aagcggcagc 300 ggaggctcta ctgcaacgtg ggcatcggct ttcacctcca ggtgctcccc gacggccgga 360 tcagcgggac ccacgaggag aacccctaca gcctgctgga aatttccact gtggagcgag 420 gcgtggtgag tctctttgga gtgagaagtg ccctcttcgt tgccatgaac agtaaaggaa 480 gattgtacgc aacgcccagc ttccaagaag aatgcaagtt cagagaaacc ctcctgccca 540 acaattacaa tgcctacgag tcagacttgt accaagggac ctacattgcc ctgagcaaat 600 acggacgggt aaagcggggc agcaaggtgt ccccgatcat gactgtcact catttccttc 660 ccaggatcta aggacccaca aaagaaggct tacagattta aagcatcatc tgttcgattg 720 aaattttgca ccagcgaaga attc 744 <210> 6 <211> 585 <212> DNA <213> Homo sapiens <400> 6 atgcacaaat ggatactgac atggatcctg ccaactttgc tctacagatc atgctttcac 60 attatctgtc tagtgggtac tatatcttta gcttgcaatg acatgactcc agagcaaatg 120 gctacaaatg tgaactgttc cagccctgag cgacacacaa gaagttatga ttacatggaa 180 ggaggggata taagagtgag aagactcttc tgtcgaacac agtggtacct gaggatcgat 240 aaaagaggca aagtaaaagg gacccaagag atgaagaata attacaatat catggaaatc 300 aggacagtgg cagttggaat tgtggcaatc aaaggggtgg aaagtgaatt ctatcttgca 360 atgaacaagg aaggaaaact ctatgcaaag aaagaatgca atgaagattg taacttcaaa 420 gaactaattc tggaaaacca ttacaacaca tatgcatcag ctaaatggac acacaacgga 480 ggggaaatgt ttgttgcctt aaatcaaaag gggattcctg taagaggaaa aaaaacgaag 540 aaagaacaaa aaacagccca ctttcttcct atggcaataa cttaa 585 <210> 7 <211> 2420 <212> DNA <213> Homo sapiens <400> 7 gatctgaaga tcagcagttc aacctgttga tagtacgtac taagctctca tgtttcacgt 60 actaagctct catgtttaac gtactaagct ctcatgttta acgaactaaa ccctcatggc 120 taacgtacta agctctcatg gctaacgtac taagctctca tgtttcacgt actaagctct 180 catgtttgaa caataaaatt aatataaatc agcaacttaa atagcctcta aggttttaag 240 ttttataaga aaaaaaagaa tatataaggc ttttaaagct tttaaggttt aacggttgtg 300 gacaacaagc cagggatgta acgcactgag aagcccttag agcctctcaa agcaattttg 360 agtgacacag gaacacttaa cggctgacat gggaattcta gtaaatgccc gttacataac 420 ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa 480 tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt 540 atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc 600 ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat 660 gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc 720 ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacgggga tttccaagtc 780 tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa 840 aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg 900 tctatataag cagagctcgt ttagtgaacc gtcagatcgc ctggagacgc catccacgct 960 gttttgacct ccatagaaga caccgggacc gatccagcct ccgtctagag ccttcgaagc 1020 ttgccatgac caacaagtgt ctcctccaaa ttgctctcct gttgtgcttc tccactacag 1080 ctctttccat gaattacaag aagcccaaac tcctctactg tagcaacggg ggccacttcc 1140 tgaggatcct tccggatggc acagtggatg ggacaaggga caggagcgac cagcacattc 1200 agctgcagct cagtgcggaa agcgtggggg aggtgtatat aaagagtacc gagactggcc 1260 agtacttggc catggacacc gacgggcttt tatacggctc acagacacca aatgaggaat 1320 gtttgttcct ggaaaggctg gaggagaacc attacaacac ctatatatcc aagaagcatg 1380 cagagaagaa ttggtttgtt ggcctcaaga agaatgggag ctgcaaacgc ggtcctcgga 1440 ctcactatgg ccagaaagca atcttgtttc tccccctgcc agtctcttct gattaactcg 1500 agcatgcatc taggcagaca tgataagata cattgatgag tttggacaaa ccacaactag 1560 aatgcagtga aaaaaatgct ttatttgtga aatttgtgat gctattgctt tatttgtaac 1620 cattataagc tgcaataaac aagttaacaa caacaattgc attcatttta tgtttcaggt 1680 tcagggggag gtgtgggagg ttttttaaag caagtaaaac ctctacaaat gtggtagccc 1740 gggcgcgcag atctgtcatg atgatcattg caattggatc catatatagg gcccgggtta 1800 taattacctc aggtcgacgc gtctgcagaa gcttaaaaaa aatccttagc tttcgctaag 1860 gatctgcagt gcccggactc ggaatcgaac caaggacacg gggatttaga atcccctgct 1920 ctaccgactg agctatccgg gcgcgttaca agtattacac aaagtttttt atgttgagaa 1980 tatttttttg atggggcgac ctgcaggtcg gggcacaact caatttgcgg gtactgatta 2040 ccgcagcaaa gaccttaccc cgaaaaaatc caggctgctg gctgacacga tttctgcggt 2100 ttatctcgat ggctacgagg gcagacagta agtggattta ccataatccc ttaattgtac 2160 gcaccgctaa aacgcgttca gcgcgatcac ggcagcagac aggtaaaaat ggcaacaaac 2220 cacccgaaaa actgccgcga tcgcgcctga taaattttaa ccgtatgaat acctatgcaa 2280 ccagagggta caggccacat tacccccact taatccactg aagctgccat ttttcatggt 2340 ttcaccatcc cagcgaaggg ccatccagcg tgcgttcctg tatttccgac ggatccggcc 2400 acgatgcgtc cggcgtagag 2420

Claims (72)

A product comprising an effective amount of an expression vector encoding an angiogenic growth factor and a heparin compound. The product of claim 1, wherein the heparin compound is low molecular weight heparin or standard heparin. The product of claim 1 or 2, wherein the angiogenic growth factor is fibroblast growth factor (FGF). The product of claim 3, wherein the angiogenic growth factor is acidic FGF or FGF-1. The product of claim 3, wherein the angiogenic growth factor is any of FGF-2 or bFGF, FGF-4, FGF-5, FGF-6, FGF-7, or FGF1-22. The product of claim 1 or 2, wherein the angiogenic growth factor is VEGF. The product of claim 1, wherein the vector is a plasmid vector. 8. The product of claim 7, wherein the plasmid vector is a conditional replicating plasmid. The product of claim 8, wherein the vector is a pCOR plasmid. The product of claim 1, wherein FGF-1 or FGFa is expressed and the expression vector is NV1FGF. The product according to claim 1, wherein the low molecular weight heparin is selected from the group consisting of enoxaparin, proxiparin, dalteparin, adeparin, tinzaparin and leviparin. The product according to claim 1, which is used simultaneously, separately or sequentially for the treatment of ischemia. Use of a product according to any one of claims 1 to 12 for the manufacture of a medicament for the treatment of peripheral or cardiac ischemia. Use of an expression vector encoding an angiogenic growth factor administered in combination with a heparin compound for the manufacture of a medicament for the treatment of ischemia. Use of a heparin compound administered in combination with an expression vector encoding angiogenic growth factor for the manufacture of a medicament for the treatment of ischemia. Use according to claim 14 or 15, wherein the heparin compound is standard heparin or low molecular weight heparin. The use according to any one of claims 13 to 16, wherein the expression vector encodes FGF-1 or FGFa and is a plasmid. 18. The use according to any one of claims 13 to 17, wherein the low molecular weight heparin is selected from the group consisting of enoxaparin, proxiparin, dalteparin, adeparin, tinzaparin and leviparin. Use according to any one of claims 13 to 18 for the manufacture of a medicament that acts to promote mature side vessels in skeletal muscle or myocardial tissue of an ischemic patient. 20. The use according to any one of claims 13 to 19, for the manufacture of a medicament that acts to promote both mature side vessels and arterioles in skeletal muscle or myocardial tissue of an ischemic patient. 21. An agent according to any one of claims 13 to 20, which acts to promote the formation of large mature conducting blood vessels (> 150 μm) and small resistance arteries (<50 μm arteries) in skeletal muscle or myocardial tissue of an ischemic patient. Use for manufacturing 22. The method according to any one of claims 13 to 21, wherein the ischemic patient is in peripheral ischemia or coronary ischemia or at risk of ischemia, is overweight 45 years of age, and / or has high alcohol or tobacco consumption and / or atherosclerosis. And / or a condition suffering from diabetes and / or immobile. 23. The expression vector of any one of claims 13-22, wherein the ischemic patient has developed an ischemic heart attack in the last 24 hours to 6 months, or up to 1, 2 or 3 years ago and encodes an angiogenic factor. And combination administration of low molecular weight heparin compounds. The use according to any one of claims 13 to 23, wherein the low molecular weight heparin is administered subcutaneously at a dosage of 0.2 to 0.4 mg / kg per day. The use of claim 24, wherein the low molecular weight heparin is administered subcutaneously at a dosage of about 0.3 mg / kg per day. The method of claim 13, wherein the low molecular weight heparin is administered for at least 20 days or at least 30 days and the time interval between the initial and final administration of low molecular weight heparin is at least 20 or 30 days. At least one, two, three or more administrations of DNA encoding angiogenic growth factors. A pharmaceutical composition comprising an expression vector encoding an angiogenic growth factor and a heparin compound, wherein the combination thereof promotes angiogenesis more in skeletal muscle and myocardium than in the case of the vector or heparin compound alone. A pharmaceutical composition comprising an expression vector encoding an angiogenic growth factor and a heparin compound, the combination of which promotes atherosclerosis in skeletal muscle and myocardium more than the vector or heparin compound alone. The pharmaceutical composition according to claim 27 or 28, wherein the heparin compound is standard heparin or low molecular weight heparin. The pharmaceutical composition of claim 27 or 28, wherein the angiogenic growth factor is FGF-1. The pharmaceutical composition of claim 27 or 28, wherein the angiogenic growth factor is any one of FGF-2 or bFGF, FGF-4, FGF-5, FGF-6, FGF-7 or FGF1-22. The pharmaceutical composition of claim 27 or 28, wherein the expression vector is a viral vector. The pharmaceutical composition of claim 27 or 28, wherein the expression vector is a non-viral vector. The pharmaceutical composition of claim 27 or 28, wherein the expression vector is a plasmid. The pharmaceutical composition of claim 27 or 28, wherein the expression vector is pCOR. The pharmaceutical composition of claim 27 or 28, wherein the expression vector is NV1FGF. 29. The pharmaceutical composition of claim 27 or 28, wherein the low molecular weight heparin is enoxaparin, nadroparin, dalteparin, adeparin, tinzaparin or leviparin. A method of treating peripheral ischemia comprising administering a combination of an expression vector encoding an angiogenic factor and a heparin compound. A method of treating coronary ischemia comprising administering a combination of an expression vector encoding an angiogenic factor and a heparin compound. A method of treating a patient in peripheral ischemia or coronary ischemia, or at risk of ischemia, comprising administering a combination of an expression vector encoding an angiogenic factor and a heparin compound, wherein the patient is overweight 45 years old and / or Alcohol or tobacco consumption is high and / or suffers from atherosclerosis and / or diabetes and / or is unable to move. A method of treating a patient who has developed an ischemic heart attack for a period of the last 24 hours to 6 months, or up to 1, 2 or 3 years, comprising administering a combination of an expression vector encoding an angiogenic factor and a heparin compound. 42. The method of any one of claims 38-41, wherein the heparin compound is standard heparin or low molecular weight heparin. A method for promoting mature side vessels of ischemic skeletal muscle or myocardial tissue in a mammalian subject in need of treatment, wherein the effective amount of the product as defined in any one of claims 1 to 11 is promoted to promote mature side vessels. Injecting into the tissue of the subject. 12. A method of promoting both the side vessels and the arterioles of ischemic skeletal muscle or myocardial tissue in a mammalian subject in need of treatment, the product defined in any one of claims 1-11 for promoting both the side vessels and the arteries. Injecting an effective amount of an into said tissue of said subject. A method for promoting the formation of large mature conducting blood vessels (> 150 μm side vessels) and small resistance arteries (<50 μm arterioles) of ischemic skeletal muscle or myocardial tissue in a mammalian subject in need thereof. Injecting an effective amount of a product as defined in any one of the preceding claims into said tissue of said subject. 46. The method of any one of claims 38-45, wherein the angiogenic growth factor is FGFa or FGF1. 47. The method of any one of claims 38-46, wherein the heparin compound and the expression vector encoding FGF-1 are administered separately or simultaneously. 48. The method of any one of claims 38-47, wherein the expression vector encoding FGF-1 is injected into skeletal muscle or myocardial tissue and the heparin compound is administered systemically. 49. The method of any one of claims 38-48, wherein the heparin compound is administered intraperitoneally or subcutaneously. 50. The method of any one of claims 38-49, wherein the low molecular weight heparin is administered subcutaneously at a dosage of 0.2 to 0.4 mg / kg per day. 51. The method of claim 50, wherein the low molecular weight heparin is administered subcutaneously at a dosage of about 0.3 mg / kg per day. 52. The method of any one of claims 38-51, wherein the low molecular weight heparin is administered for at least 20 days or at least 30 days and the time interval between the initial and final administration of low molecular weight heparin is at least 20 or 30 days. How to administer DNA encoding angiogenic growth factor 1, 2, 3 or more times 53. The method of any one of claims 38-52, wherein the gene or DNA encoding the angiogenic factor is administered posterior to the thigh and the lower thigh. 54. The method of any one of claims 38-53, wherein the gene or DNA encoding the angiogenic factor is administered by multiple infusions around the ischemic portion of skeletal muscle or myocardium. 55. The method of any one of claims 38-54, wherein the low molecular weight heparin is selected from the group consisting of enoxaparin, proxiparin, dalteparin, adeparin, tinzaparin and leviparin. The method of any one of claims 38-55, wherein the low molecular weight heparin is administered prior to the gene encoding the angiogenic factor. The method of any one of claims 38-56, wherein the FGFa or FGF-1 gene or DNA sequence is carried by a nonviral vector, preferably a plasmid vector. The method of claim 57, wherein the plasmid vector is a conditional replication vector such as pCOR. The method of claim 57 or 58, wherein the plasmid vector is a vector named NV1FGF. A method of treating peripheral ischemia comprising administering a pharmaceutical combination of an heparin compound and an expression vector encoding angiogenic growth factors to a mammalian subject in need thereof. A method of treating coronary ischemia comprising administering to a mammalian subject in need thereof a pharmaceutical combination of an heparin compound and an expression vector encoding angiogenic growth factors. A method of promoting the formation of side vessels larger than 150 μm in ischemic skeletal muscle or myocardial tissue, comprising administering a pharmaceutical combination of an expression vector encoding angiogenic growth factor and a heparin compound to a mammalian subject in need of treatment. . A method of promoting the formation of arterioles of less than 50 μm in ischemic skeletal muscle or myocardial tissue, comprising administering a pharmaceutical combination of an heparin compound and an expression vector encoding angiogenic growth factors to a mammalian subject in need thereof. 64. The method of any one of claims 60-63, wherein the angiogenic growth factor is FGF-1 or acidic FGF. 64. The method of any one of claims 60-63, wherein the angiogenic growth factor is any of FGF-2 or basic FGF, FGF-4, FGF-5, FGF-6, FGF-7 or FGF1-22. . 66. The method of any one of claims 60-65, wherein the expression vector is a viral vector. 67. The method of any one of claims 60-66, wherein the expression vector is a nonviral vector. The method of any one of claims 60-67, wherein the expression vector is a plasmid vector. The method of claim 68, wherein the expression vector is pCOR. The method of claim 69, wherein the expression vector is NV1FGF. The method of any one of claims 60-70, wherein the heparin compound is standard heparin or low molecular weight heparin. 72. The method of any one of claims 60-71, wherein the low molecular weight heparin is enoxaparin, nadroparin, dalteparin, adeparin, tinzaparin or leviparin.

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