WO2000047235A2 - Methods of stimulating angiogenesis - Google Patents

Abstract

The invention is directed to methods for stimulating angiogenesis by in vivo intramuscular, intradermal, and/or subcutaneous administration of cationic lipid-nucleic acid complexes. By inducing angiogenesis, these compositions are used to treat ischemia, including diseases which cause or result in insufficient circulation to and perfusion of tissues, such as peripheral vascular disease (e.g., as in diabetes, atherosclerosis) and coronary artery disease.

Description

METHODS OF STIMULATING ANGIOGENESIS

FIELD OF THE INVENTION

This invention pertains generally to the induction and stimulation of angiogenesis in vivo by intramuscular, intradermal, and subcutaneous administration of cationic lipid - nucleic acid complexes. In particular, this invention pertains to the treatment of diseases which cause or result in ischemia, such as peripheral vascular disease and coronary artery disease.

INTRODUCTION

Diseases and conditions causing or involving tissue ischemia are major health concerns. Ischemia is seen, for example, in coronary artery disease (CAD) and peripheral vascular disease (PVD). It has been reported by the American Heart Association that there are about 60 million adults in the United States with cardiovascular disease, including 11 million adults with coronary heart disease.

Angina, a symptom of heart ischemia, afflicts 1.5 million adults in the United States, with about 350,000 new cases a year. It is estimated that PVD affects 30 percent of the adult population. A primary cause of PVD, atherosclerotic vascular disease, coronary heart disease (CHD), and cerebrovascular disease is diabetes mellitus. Ischemia occurs when a tissue receives an inadequate supply of blood.

For example, myocardial ischemia occurs when cardiac muscle does not receive an adequate blood supply. This can be due to occlusion or narrowing of the blood vessels, such as seen in coronary artery atherosclerosis. Treatments include surgical and pharmaceutical approaches. Surgical intervention is used to widen the narrowed lumens {e.g., balloon angioplasty) or to increase the numbers of cardiac blood vessels

(e.g., bypass surgery using grafts). Less traumatic pharmaceutical treatments act to decrease cardiac muscle demand for oxygen and nutrients or to increase the blood supply. Oxygen demand can be lowered by decreasing the contractile response of the heart to a hemodynamic load (e.g., using beta-adrenergic blockers). Cardiac blood supply can be augmented by increasing the diameter of smooth muscle- walled coronary artery vessel lumens (as with nitroglycerin or calcium channel blockers). However, these pharmaceutical treatments are inexact, transiently active, and highly prone to drug interactions and side effects. Another means to increase blood supply to an ischemic tissue is to induce the growth of blood vessels to the tissue (angiogenesis) or to increase the amount of blood bathing the tissues (increased blood perfusion). This has been accomplished by administration of angiogenic growth factors. Several angiogenic proteins have been identified, including, for example, vascular endothelial growth factors (VEGFs). VEGFs are angiogenic and permeability-inducing factors. VEGFs are important mediators of angiogenesis, as they act directly and specifically on endothelial cells. Grad (1998) Clin. Chem Lab Med. 36:379-383. In vivo, they are associated with blood vessel growth in development, wound repair (angiogenesis is a key component of the repair mechanisms triggered by tissue injury), cancer, and other diseases and conditions.

To achieve an angiogenic effect, repeated and/or long term administration of a polypeptide blood vessel growth factor, such as VEGF, would be needed. This approach, however, is typically very costly and inconvenient, as it usually requires repeated administrations by injection. Alternatively, a polypeptide blood vessel growth factor can be administered in vivo by delivering not the polypeptide itself, but instead, the nucleic acid which encodes it. Angiogenic genes have been administered in vivo intravascularly. See., e.g., Laitinen (1998) Hum. Gene Ther. 9:1481-1486; Isner (1997) Adv. Drug Deliv. Reviews 30:185-197; Giordano (1996) Nature Med. 2:534-539; Takeshita (1996) Lab. Invest. 75:487-501 ; Mc Donald, et ai, U.S. Patent No.

5,837,283 (" '283").

A novel means to stimulate angiogenesis, described for the first time in the present invention, involves administration of angiogenic factors intramuscularly (IM) and subcutaneously (SC). While polypeptide-encoding genes have been injected intramuscularly (as naked plasmid DNA or viral expression vectors), to date, no angiogenic factors have been administered directly into the muscle (IM) or into the skin (SC); see, e.g., Baumgartner (1998) Circulation 97:11 14-1123; Tsurumi (1997) Circulation 96(9 Suppl):II-II3828; Hammond, et al., U.S. Patent No. 5,792,453; and Mc Donald, et al, '283.

Considering the increasing numbers of individuals in our aging population afflicted with disease and conditions involving ischemic tissues, new treatments for ischemia that are safer and more predictable are needed. The present invention provides these needs and related advantages.

SUMMARY OF THE INVENTION

The invention provides methods for stimulating angiogenic activity in a tissue by intramuscular, intradermal, or subcutaneous administration of a pharmaceutical composition. The pharmaceutical composition comprises a pharmaceutically acceptable carrier and a cationic lipid - nucleic acid complex. The pharmaceutical composition is administered in an amount effective to induce angiogenic activity (defined below) in the tissue. In alternative embodiments, the pharmaceutical composition is administered into a skeletal muscle or a cardiac muscle; or, the subcutaneous/ intradermal administration is an intradermal injection.

In one embodiment, the pharmaceutical composition is administered in a unit dosage form. The unit dosage form can deliver between about 1 ngm to about 6 ngm, or, alternatively, about 2 ngm, of the nucleic acid-cationic lipid complex. In another embodiment, the unit dosage form of about 2 ngm of nucleic acid is administered in at least two intervals about 4 weeks apart. In alternative embodiments, the pharmaceutical composition is in the form of an injectable solution and the pharmaceutically acceptable carrier is an aqueous solution.

In one embodiment, the cationic lipid complex can have a net positive charge, and the cationic lipid can be BODAI, DOTMA, DMRIE, DOTAP, DOGS,

EDMPC, MeBOP, or DCChol. The lipid content of the cationic lipid-DNA complex formulation can be about 1.5 mM BODAI and about 1.5 mM DOPE.

In other embodiments, the nucleic acid of the cationic lipid-nucleic acid complex is DNA. The DNA can comprise a sequence that does not encode a polypeptide with biologic activity, or, it can comprise a sequence that does not encode a polypeptide with angiogenic activity. Alternatively, the DNA can comprises a sequence that encodes a polypeptide with biologic activity, including, for example, a polypeptide having an angiogenic activity, such as a vascular endothelial growth factor activity. In one embodiment, the DNA comprises a sequence encoding a vascular endothelial growth factor polypeptide, such as, for example, the polypeptide having a sequence as set forth in SEQ ID NO:2.

The present invention also provides a method for treating ischemia in a tissue, the method comprising intramuscular, intradermal, or subcutaneous administration of a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a cationic lipid -nucleic acid complex in an amount effective to treat the ischemia in the tissue. In alternative embodiments, the ischemia is caused by peripheral vascular disease, such as, for example, diabetes, atherosclerosis, coronary artery disease, and the like.

A further understanding of the nature and advantages of the present invention may be realized by reference to the remaining portions of the specification, the figures and claims.

All publications, patents and patent applications, including GenBank and ATCC library database references, as cited herein, are hereby expressly incorporated by reference for all purposes to the same extent as if fully set forth herein.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 shows the ratios of blood flow between ischemic and normal perfused gastrocnemius muscles measured at different times after surgical induction of ischemia and IM administration of cationic lipid-nucleic acid compositions of the invention at different concentrations, as explained in Example 1. Statistically significant differences between treatment groups and control are observed on weeks one and two after surgery.

Figure 2 shows a plot schematically summarizing the dynamics and the rate of restoration of the blood flow in ischemic gastrocnemius muscle after IM administration of angiogenic cationic lipid-nucleic acid compositions, as explained in Example 1. DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to pharmaceutical compositions and methods for stimulating angiogenesis in vivo, by intramuscular (IM) intradermal, and subcutaneous (SC) administration. The compositions comprise cationic lipid - nucleic acid complexes, where the nucleic acid may or may not be the same or complementary to a naturally transcribed sequence. In one embodiment, the nucleic acid is DNA encoding a polypeptide with angiogenic activity. By inducing angiogenesis, these compositions are used to treatment ischemia, including diseases which cause or result in insufficient circulation to and perfusion of tissues, such as peripheral vascular disease (e.g., as in diabetes, atherosclerosis) and coronary artery disease.

The invention is also directed to methods of stimulating angiogenesis, thereby treating ischemia, using intramuscularly, intradermally, and subcutaneously administered pharmaceutical compositions comprising cationic lipid - nucleic acid complexes. This aspect of the invention is based on the surprising discovery that angiogenesis can be induced by intramuscular (IM), intradermal, and/or subcutaneous (SQ) administration of a complex comprising cationic lipid and any nucleic acid. That is, cationic lipid-nucleic acid complexes possess an inherent ability to stimulate angiogenesis, without regard to a specific gene product, if any, encoded by the nucleic acid, when injected IM or SQ. In one embodiment, the nucleic acid is DNA. In a preferred embodiment, the DNA encodes a polypeptide which itself has angiogenic activity, thus imbuing an additive or synergistic angiogenic effect.

Definitions

To facilitate understanding the invention, a number of terms are defined below. As used herein, the term "angiogenesis" refers to stimulation or induction of an increased rate of, or de novo formation of, blood vessels, e.g., capillaries, see e.g., Folkman (1992) Nature Med. 1 :27-21. Compositions can be screened for angiogenic activity in vitro or in vivo. An exemplary in vitro capillary formation assessment uses endothelial cells imbedded in Matrigel matrix (Collaborative Research, Bedford, MA), as described by, e.g., Deramaudt (1998) J. Cell. Biochem. 68: 121-127). In vivo animal models are discussed below.

As used herein, the term "cationic lipid - nucleic acid complex" refers to a non-covalent association between a cationic lipid moiety and a nucleic acid. Typically, the positively charged lipid will associate with the negatively charged nucleic acid by charge interactions. The complex can include any number of additional constituents, such as, e.g., neutral lipids, as discussed below. Means of making these complexes are discussed below.

The term "expression cassette" refers to any recombinant expression system for the purpose of expressing a nucleic acid sequence of the invention in vitro or in vivo, constitutively or inducibly, in any cell. The term includes linear or circular expression systems. The term includes, e.g. , vectors, that remain episomal or integrate into the host cell genome. The expression cassettes can have the ability to self-replicate or not, i.e., drive only transient expression in a cell. The term includes recombinant expression cassettes which contain only the minimum elements needed for transcription of the recombinant nucleic acid, such as one which encodes a polypeptide with angiogenic activity. In one embodiment, the cationic lipid- nucleic acid complex of the invention comprises a DNA encoding a polypeptide with angiogenic activity in the form of an expression cassette. In an alternative embodiment, the nucleic acid may or may not be the same or complementary to a naturally transcribed sequence. Thus, the DNA insert in the expression cassette may be non-coding, i.e., an "empty vector."

As used herein, the term "wherein the pharmaceutical composition is administered intramuscularly or subcutaneously" incorporates the common usage of intramuscular (IM) and subcutaneous (SC), and includes any means of IM or SC administration known in the art, including, e.g., all forms of skeletal, smooth or cardiac muscle injections and subcutaneous or intradermal injections (see, e.g., Fjellner (1983) Acta Derm Venereal 63 :456-457; Ross (1997) Clin Cancer Res 3 :2191 -2196; Ciernik ( 1996) Hum Gene Ther 7:893-899; Eriksson (1998) J Surg Res 78:85-91 ).

The term "lipid" as used herein incorporates its common usage; and includes neutral, anionic, cationic and amphiphilic compositions, both naturally occurring and synthetic. The term "cationic lipid" includes any lipid with a net positive charge at neutral pH, such as under physiologic conditions. A cationic lipid can be a positively charged lipid comprising, e.g., a quaternary ammonium salt moiety. Cationic lipids can consist of a hydrophilic polar head group and lipophilic aliphatic chains. Similarly, cholesterol derivatives having a cationic polar head group can also be used, see, e.g., Farhood (1992) Biochim. Biophys. Acta 1111:239-246. The cationic lipid may be used in combination with other cationic lipids, or with neutral or anionic lipids. The cationic lipid may be in any physical form including, e.g. , liposomes, micelles, interleaved bilayers, and the like. Cationic lipids are described in further detail, below.

As used herein, the term "nucleic acid" or "nucleic acid sequence" refers to any deoxyribonucleotide or ribonucleotide sequence in, e.g., single-stranded, double- stranded or triplex form. The term encompasses nucleic acids, e.g., oligonucleotides, containing known naturally occurring nucleotides, analogues of natural nucleotides, and mixtures thereof. The term also encompasses nucleic-acid-like structures with synthetic backbones. DNA backbone analogues provided by the invention include phosphodiester, phosphorothioate, phosphorodithioate, methyl-phosphonate, phosphor-amidate, alkyl phosphotriester, sulfamate, 3'-thioacetal, methylene (methylimino), 3'-N-carbamate, morpholino carbamate, and peptide nucleic acids (PNAs); see Oligonucleotides and Analogues, a Practical Approach, edited by F. Eckstein, IRL Press at Oxford University Press (1991); Antisense Strategies, Annals of the New York Academy of Sciences, Volume 600, Eds. Baserga and Denhardt (NY AS 1992); Milligan (1993) J. Med. Chem. 36:1923-1937; Antisense Research and Applications (1993, CRC Press). PNAs contain non-ionic backbones, such as N-(2-aminoethyl) glycine units. Phosphorothioate linkages are described in WO 97/03211 ; WO 96/39154; Mata (1997) Toxicol. Appl. Pharmacol. 144:189-197. Other synthetic backbones encompasses by the term include methyl- phosphonate linkages or alternating methylphosphonate and phosphodiester linkages (Strauss-Soukup (1997) Biochemistry 36:8692-8698), and benzylphosphonate linkages

(Samstag (1996) Antisense Nucleic Acid Drug Dev. 6:153-156). As described above, the angiogenic action of the nucleic acid-cationic lipid complex is independent of whether the nucleic acid encodes any transcribable sequence, such as a naturally occurring polypeptide. In one embodiment, the nucleic acid is DNA. In a preferred embodiment, the DNA encodes a polypeptide which itself has angiogenic activity; and, can be in the form of an expression cassette, as discussed above.

The terms "ischemia," "peripheral vascular disease," "diabetes," "atherosclerosis," and "coronary artery disease" as used herein, incorporates their common usages.

As used herein, the term "pharmaceutically acceptable carrier" includes any suitable pharmaceutical excipient, including, e.g., water, saline, phosphate buffered saline, Hank's solution, Ringer's solution, dextrose/saline, glucose, lactose, or sucrose solutions, starch, cellulose, talc, gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, glycerol monostearate, dried skim milk, glycerol, propylene glycol, ethanol, and the like.

As used herein, the term "polysaccharide" or "oligosaccharide" incorporates its common usages, and includes, e.g., dextrose, glucose, lactose, mannose, mannan, and the like, as described below. As used herein, the term "wherein the DNA comprises a sequence that is not the same or complementary to a naturally transcribed DNA sequence" means that the DNA sequence cannot generate a peptide or polypeptide which is normally or naturally produced by the tissue into which it is administered. For example, the DNA sequence is not the same or complementary to a naturally transcribed DNA sequence if, when administered to a human as part of the nucleic acid-cationic lipid pharmaceutical of the invention, it cannot generate a peptide or polypeptide naturally produced by the human. For example, a reporter gene or a marker gene (e.g., for an epitope tag) would not be a naturally transcribed DNA sequence .

As used herein, the term "wherein the DNA comprises a sequence that does not encode a polypeptide with biologic activity" means the polypeptide encoded by the DNA sequence, when expressed in an animal, does not have any effect on the normal cell biology or physiology of the organisms. For example, cell growth, differentiation, apoptosis, and the like are biological activities, including, e.g., "vascular endothelial growth factor activity," "angiogenic activity," and "angiogenic growth factor activity" (described below). In contrast, the ability to act an as antigen, a reporter gene, a marker

(e.g., an epitope tag) would not be considered a biological activity. As used herein, the term "wherein the DNA comprises a sequence that does not encode a polypeptide with angiogeneic activity" means the polypeptide encoded by the DNA sequence, when expressed in an animal, does not have any effect on "vascular endothelial growth factor activity," "angiogenic activity," and "angiogenic growth factor activity" as described below.

As used herein, the terms "vascular endothelial growth factor activity," "angiogenic activity," and "angiogenic growth factor activity" include a broad range of physiologic activities that increase the amount of blood flow to a tissue, including, e.g. , increased vascular permeability, increased vascular density, endothelial cell (EC) activation, EC migration, EC proliferation, capillary formation (angiogenesis), vasculogenesis (the de novo organization of ECs into vascular structures) and neovascularization (see, e.g., Folkman (1992) supra). Angiogenic activity may include, e.g., growth factors that induce angiogenesis, or inhibitors of angiogenesis inhibitors, or factors which induce expression of endogenous growth factors (e.g., gene activators or transcriptional regulators). The terms "vascular endothelial growth factor" or "VEGF" includes growth factors which, alone or in combination with other growth factors, such as fibroblast growth factor, can initiate vascular development, angiogenesis and other angiogenic activities. VEGFs include the family of VEGF genes and their three alternatively spliced forms, including VEGF (VEGF-A); VEGF-B; VEGF-C (or VEGF-2); VEGF, ₁₅; VEGF₁₄₅; VEGF₁₂₁; VEGF₁₆₅; VEGF₁₈₉; and VEGF₂₅₆; see, e.g., Olofsson (1996)

J. Biol .Chem. 271 :19310-19317; Olofsson (1996) Proc. Natl. Acad. Sci. USA 93:2576-2581; Sugihara (1998) J. Biol. Chem. 273:3033-3038; Poltorak (1996) J. Biol. Chem. 272:7151; Beck (1997) FASEB J. 1 1 :365-373; Witzenbichler (1998) Am. J. Pathol. 153:381-394). A variety of in vivo animal models can be used to evaluate the ability of a cationic lipid-nucleic acid complex of the invention to have angiogenic activity

(in addition to the in vitro test described above, see Folkman (1992) supra). For example, neovascularization of ischemic muscle can be demonstrated by experiments in which exogenously administered cationic lipid-nucleic acid complexes of the invention augment collateral blood flow in experimentally induced mouse or rabbit hindlimb ischemia; see, e.g., Couffinhal (1998) Am. J. Pathol. 152:1667-1679; Witzenbichler (1998) supra. Pharmaceutical Compositions for Inducing Angiogenesis

This invention provides methods for inducing angiogenesis by intramuscular, intradermal, and/or subcutaneous administration of a pharmaceutical composition. The composition comprises a pharmaceutically acceptable carrier and a pharmacologically effective amount of a cationic lipid-nucleic acid complex. In one embodiment, the nucleic acid is a DNA comprising no sequences capable of generating a polypeptide, naturally occurring or otherwise. In an alternative embodiment, the nucleic acid of the complex encodes an angiogenic growth factor polypeptide.

The invention, for the first time, provides methods of using these pharmaceutical compositions to induce angiogenic activity and to treat ischemia by intramuscular (IM), intradermal, or subcutaneous (SC) administration. It is an advantage of the present invention that high levels of angiogenesis can be stimulated by IM, intradermal, or SC administration of cationic lipid - nucleic acid complexes. This is a much simpler and safer mode of administration than intravascular (IV) delivery. IM, intradermal, and SC modes of delivery are less invasive than IV. They are also more amenable to repeat administrations.

The invention can be practiced in conjunction with any appropriate method or protocol known in the art, which are well described in the scientific and patent literature.

Therefore, only a few general techniques will be described prior to discussing specific methodologies and examples relative to the novel reagents and methods of the invention.

General Techniques

The nucleic acids used in the cationic-lipid complexes of this invention, whether RNA, cDNA, genomic DNA, oligonucleotides, or hybrids thereof, maybe isolated from a variety of sources, genetically engineered, amplified, and/or expressed recombinantly. Any recombinant expression system can be used, including, e.g.. bacterial, yeast, insect or mammalian systems. Alternatively, these nucleic acids can be chemically synthesized in vitro (see definition of nucleic acids). Techniques for the manipulation of nucleic acids, such as, e.g. , subcloning into expression vectors, labeling probes, sequencing DNA, DNA hybridization are described in the scientific and patent literature, see e.g., Sambrook, ed., MOLECULAR CLONING: A LABORATORY MANUAL (2ND ED.), Vols. 1-3,

Cold Spring Harbor Laboratory, (1989) ("Sambrook"); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausubel, ed. John Wiley & Sons, Inc., New York (1997) ("Ausubel"); and, LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULAR BIOLOGY: HYBRIDIZATION WITH NUCLEIC ACID PROBES, Part I. Theory and Nucleic Acid Preparation, Tijssen, ed. Elsevier, N.Y. (1993) ("Tijssen"). Product information from manufacturers of biological reagents and experimental equipment also provide information regarding known biological methods. Such manufacturers include the SIGMA chemical company (Saint Louis, MO), R&D systems (Minneapolis, MN), Pharmacia Biotech (Piscataway, NJ), Clontech Laboratories, Inc. (Palo Alto, CA), Aldrich Chemical Company (Milwaukee, Wl), GIBCO BRL Life Technologies, Inc. (Gaithersburg, MD), Fluka Chemica-Biochemika Analytika (Fluka Chemie AG, Buchs, Switzerland), Applied Biosystems (Foster City, CA), as well as many other commercial sources known to one of skill.

The nucleic acids of the invention can also be generated or quantitated using amplification techniques. Suitable amplification methods include, but are not limited to : polymerase chain reaction, PCR (PCR PROTOCOLS , A GUIDE TO METHODS AND APPLICATIONS, ed. Innis, Academic Press, N.Y. (1990) and PCR STRATEGIES (1995), ed. Innis, Academic Press, Inc., N.Y. (Innis )), ligase chain reaction (LCR) (Wu (1989) Genomics 4:560; Landegren (1988) Science 241 : 1077; Barringer (1990) Gene 89:117); transcription amplification (Kwoh (1989) Proc. Natl. Acad. Sci. USA, 86:1173); and, self- sustained sequence replication (Guatelli (1990) Proc. Natl. Acad. Sci. USA, 87:1874); Q Beta replicase amplification (Smith (1997) J. Clin. Microbiol. 35:1477-1491, automated

Q-beta replicase amplification assay; Burg (1996) Mol. Cell. Probes 10:257-271 ) and other RNA polymerase mediated techniques (e.g. , NASB A, Cangene, Mississauga, Ontario); see also Berger (1987) Methods Enzymol. 152:307-316, Sambrook, Ausubel, Mullis (1987) U.S. Patent Nos. 4,683,195, and 4,683,202; Arnheim (1990) C&EN 36-47; Lomell J. Clin. Chem., 35:1826 (1989); Van Brunt (1990) Biotechnology, 8:291-294; Wu (1989)

Gene 4:560; Sooknanan (1995) Biotechnology 13:563-564. Methods for cloning in vitro amplified nucleic acids are described in Wallace, U.S. Pat. No. 5,426,039.

Nucleic acids and lipids are analyzed and quantified by any of a number of general means well known to those of skill in the art. These include, e.g., analytical biochemical methods such as NMR, spectrophotometry, radiography, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), and hyperdiffusion chromatography, various immunological methods, such as fluid or gel precipitin reactions, immunodiffusion (single or double), immunoelectrophoresis, radioimmunoassays (RIAs), enzyme-linked immunosorbent assays (ELISAs), immuno- fluorescent assays, Southern analysis, Northern analysis, dot- blot analysis, gel electrophoresis (e.g., SDS-PAGE), RT-PCR, quantitative PCR, other nucleic acid or target or signal amplification methods, radiolabeling, scintillation counting, and affinity chromatography.

Nucleic Acids Encoding Angiogenic Polypeptides In one embodiment, the invention is directed to pharmaceutical compositions of cationic lipid - nucleic acids and methods of inducing angiogenesis and treating ischemia by administering these compositions IM, intradermally, or SC, where the nucleic acid encodes no polypeptide, or, encodes a polypeptide which itself has angiogenic activity. Because the cationic lipid - nucleic acid complex alone induces angiogenic activity, in vivo synthesis of an angiogenic polypeptide only generates an additive or synergistic effect.

Any single or combination of nucleic acids encoding angiogenic activity- inducing polypeptides can be incorporated into the lipid-nucleic acid complex of the invention. In one embodiment, the nucleic acid encodes a vascular endothelial growth factor (VEGF) which, alone or in combination with other exogenously or endogenously derived growth factors, such as fibroblast growth factor, can initiate vascular development, increased vascular permeability (VEGF is also called "vascular permeability factor," see, e.g. , Hippenstiel ( 1998) Am. J. Physiol.274: L678-L684), endothelial cell (EC) activation, EC migration, EC proliferation, capillary formation (angiogenesis), vasculogenesis, and neovascularization. VEGF includes several isoforms (including alternatively spliced forms): VEGF (VEGF-A), VEGF-B, VEGF-C (or VEGF-2) and VEGF-D (see, e.g., Beck (1997) FASEB J. 11 :365-373). When expressed in vivo (or in vitro models, as described above), VEGF promotes angiogenesis (see, e.g., Witzenbichler (1998) supra). The role of VEGF in therapeutic angiogenesis has been demonstrated by experiments in which exogenously administered VEGF augments collateral blood flow in animals and patients with experimentally induced hindlimb or myocardial ischemia. Human VEGF nucleic acid coding sequences and mR A have been described, e.g. , by Herold (1997) Cardiovasc. Pathobiol.2 : 88-96 (see GenBank Accession No. AF024710); Matsuda, et al., Japanese patent JP 1997173075-A 1, 08-JUL- 1997 (see GenBank Accession No. E13332); Achen (1998) Proc. Natl. Acad. Sci. USA 95:548-553 (see GenBank Accession No. AJ000185); Olofsson (1996) J. Biol. Chem.

271 : 19310-19317 (see GenBank Accession No. U52819); Leung (1989) Science 246: 1306-1309 (see GenBank Accession No. M32977); Tischer, et. al., United States Patent No. (USPN) 5,219,739; Robinson, G.S., USPN 5,661,135; Chen, et. al., USPN 5,073,492. Other angiogenic protein-encoding nucleic acids can be used in the cationic lipid-nucleic acid complexes of the invention, including, e.g., any member of the family of fibroblast growth factors (FGFs); see, e.g., Chen (1997) Proc. Assoc. Am. Physicians 109:351 -361 ; Harada, et al., ( 1994) J. Clin. Invest. 94:623-630; Thomas, et. al., U.S. Patent No. 5,409,897; Slavin (1995) Cell Biol. Intl. 19:431-444. Other angiogenic protein-encoding nucleic acids that can be used also include angiopoietin- 1 , angiopoietin-

2, del-1, monocyte chemotactic protein-1, see, e.g., Maisonpierre (1997) Science 277:55-60; Koblizek (1998) Curr. Biol. 8:529-532.

In one embodiment of the invention, nucleic acids encoding polypeptides (including, e.g., angiogenic activity-inducing polypeptides, gene activation peptides that upregulate VEGF, VEGF-R, etc., such as trα«s-acting transcriptional activators, e.g., zinc finger proteins that upregulate VEGF) are used; and in an alternative embodiment, they are incorporated in expression cassettes, as described above. In these embodiments, the polypeptide's coding sequence is operably linked to a promoter. This complex can also be incorporated into a expression vector or plasmid or the like; see, e.g., Hammond, et al., USPN 5,792,453, which incorporates transgenes encoding angiogenic proteins into recombinant adenovirus vectors for in vivo administration to induce angiogenesis.

The promoters and vectors used in this invention can be isolated from natural sources, obtained from such sources as ATCC or GenBank libraries, or prepared by synthetic or recombinant methods, as described herein. Typical expression systems contain, in addition to coding sequence, transcription and translation terminators, polyadenylation sequences, transcription and translation initiation sequences, and transcriptional regulatory elements, e.g., promoters and enhancers. The expression systems optionally contain at least one independent terminator sequence, sequences permitting replication of the cassette both in vitro and in vivo, e.g., eukaryotes or prokaryotes, or a combination thereof, (e.g., shuttle vectors). They may further include selection markers for the selected expression system, e.g., prokaryotic or eukaryotic systems.

Cationic Lipid - Nucleic Acid Complexes

The invention provides methods for inducing angiogenesis comprising administration IM, intraderrnally, or SC of a cationic lipid - nucleic acid complex.

Cationic lipids have been shown to mediate intracellular delivery of plasmid DNA (Feigner (1987) Proc. Natl. Acad. Sci. USA 84:7413-7416), and mR A (Malone (1989) Proc. Natl. Acad. Sci. USA 86:6077-6081). Use of lipids complexed with nucleic acids, such as DNA, is described, e.g., by Zhu (1993) Science 261:209-211; Vigneron (1996) Proc. Natl. Acad. Sci. USA 93:9682-9686; Hofland (1996) Proc. Natl. Acad. Sci. USA

93:7305-7309; Alton (1993) Nat. Genet. 5:135-142; von der Leyen (1995) Proc. Natl. Acad. Sci. USA 92:1137-1141. For a review of liposomes in gene therapy, see Lasic and Templeton (1996) Adv. Drug Deliv. Rev. 20:221-266. Cationic Lipids Cationic lipid carriers contain a positively charged lipid. The liposomes may have a single lipid bilayer (unilamellar) or more than one bilayer (multilamellar). They are generally categorized according to size, where those having diameters up to about 50 to 80 nm are termed "small" and those greater than about 80 to 1000 nm, or larger, are termed "large." Thus liposomes are typically referred to as large unilamellar vesicles (LUVs), multilamellar vesicles (MLVs) or small unilamellar vesicles (SUVs). Methods of producing cationic liposomes are known in the art. See, e.g., Liposome Technology (CFC Press, NY 1984); Liposomes, Ortro (Marcel Schher, 1987); Methods Biochem Anal. 33:337462 (1988).

Cationic lipids that can be incorporated in the cationic lipid-nucleic acid complexes of the invention include, e.g., imidazolinium derivatives (WO 95/14380), guanidine derivatives (WO 95/14381), phosphatidyl choline derivatives (WO 95/35301), and piperazine derivatives (WO 95/14651). Additional examples of cationic lipids that may be used in the present invention include DOTIM (also called BODAI) (see, e.g., Solodin (1995) Biochem.34:13537-13544); DDAB (see, e.g., Rose (1991) BioTechniques 10:520-525); DOTMA (also called N-[ l -(2,3-dioleoyloxy)propyl-N,N, N-trimethylammonium chloride, see, e.g., Song (1998) Biochim. Biophys. Acta 1372:141-150, and U.S. PatentNo.5,550,289); DOTAP (see, e.g., Eibl and Wooley (1979)

Biophys. Chem. 10:261-271); DMRIE (see, e.g., Feigner (1994) J. Biol. Chem. 269:2550-2561 ; Huang (1998) Chem. Biol. 5:345-354); EDMPC (see, e.g., Avanti Polar Lipids, Alabaster, AL); DCChol (see, e.g., Gau and Huang (1991) Biochem. Biophys. Res. Comm. 179:280-285); DOGS (see, e.g., Behr (1989) Proc. Natl. Acad. Sci. USA 86:6982-6986; Meyer (1998) J. Biol. Chem. 273: 15621-15627); MBOP (also called

MeBOP) (see, e.g., WO 95/14651); and, those described in WO 97/00241. See also: Wang (1998) J. Med. Chem. 41 :2207-2215, describing the synthesis of long chain alkyl acyl carnitine esters as biodegradable cationic lipids for use in gene delivery; Stegmann (1997) Biochim Biophys Acta. 1325:71-79; Liu (1996) Pharm. Res. 13:1856-1860; Templeton (1997) Nat. Biotechnol. 15:647-652. In addition, cationic lipid carriers having more than one cationic lipid species may be used to produce complexes according to the method of the present invention.

Non-Cationic Lipids

Non-cationic lipids can also be used in the complexes of the invention. For example, cationic lipid carriers can also contain a neutral lipid. If present, the neutral lipid is usually in approximately equimolar amounts with the cationic lipid. Cationic lipid and non-cationic lipids can, however, also be used in various proportions other than equimolar amounts. The neutral lipid is helpful in maintaining a stable lipid bilayer in liposomes, and can significantly affect transfection efficiency. Usually, the cationic lipid and non-cationic lipid will be prepared as liposomes by methods known in the art. Generally, the lipids are dried to a film and resuspended in an aqueous solution. The resulting liposomes may be further reduced in size by sonication or extrusion through a membrane of fixed pore size. In one embodiment, the liposomes are rehydrated in 5% dextrose in water and heated to 50°C for 6 hours. They are then extruded through a filter. Another useful neutral lipid is cholesterol, see, e.g., Liu (1997) Nat. Biotech. (15): 167-173. The effect of cholesterol on liposomes in vivo is described in Semple (1996) Biochem. 35:2521-2525. Other useful neutral lipids include, e.g., DLPE and DiPPE, see, e.g., U.S. Patent Application Serial No. (USSN) 09/054,769. The role of non-cationic, helper lipids in cationic lipid-mediated gene delivery is described, e.g., in Feigner (1994) J. Biol. Chem. 269:2550-2561, describing improved transfection using DOPE; Hui (1996) Biophys. J. 71 : 590-599. Other Complex Substituents

The lipid-nucleic acid complexes of the invention can include other compositions, such as polysaccharides or peptides or proteins, e.g., to enhance cellular uptake, endosomal release or nuclear transport. Examples include, e.g., polyamines, carbohydrates, synthetic polycationic polymers, polylysine, polyarginine, protamine, polybrene, histone, cationic dendrimer, and synthetic polypeptides. See, e.g., WO 96/22765. See, e.g., Sugimoto, et al., U.S. Patent No. 5,759,572, describing liposomes with oligosaccharides on the surface.

Synthesis of Lipid-Nucleic Acid Complexes

Typically, complexes are prepared by adding one solution to the other, i.e. nucleic acid to the cationic liposomes, or cationic liposomes to nucleic acid, with constant stirring. For in vivo uses, it is desirable to prevent the formation of macroaggregates or precipitation during the complexation process. Thus, for complexes having a net positive charge, the nucleic acid is added to the liposome suspension; for complexes having a net negative charge, the liposomes are added to the nucleic acid solution.

The liposomes are typically prepared in low ionic strength solutions, such as 5% dextrose in water. The nucleic acid is also typically prepared in a low ionic strength solution to prevent interference by additional ions with the lipid complexation process.

A low-ionic strength solution means a solution having a conductivity less than about 35 mS, preferably less than about 10 mS, and most preferably less than about 1 mS. Desirably, the DNA solution will contain no salts. Typically, the DNA is in a low ionic strength solution, such as about 5% dextrose in 5 mM Tris-HCl (pH 8.0). The nucleic acid-cationic lipid complexes of the invention can also be prepared using a reduced-volume, dual feed stream process. It involves the collision of two feed streams (nucleic acid and lipid) in a minimal volume, and the exit of the complex stream away from the site of interaction.

Alternatively, the mixture can be flowed through a static mixer to ensure complete mixing of the nucleic acid and lipid. Static mixers are advantageous because substantially complete mixing can be obtained while minimizing shear of the nucleic acid.

"Static mixer" refers to any flow through device which provides enough contact time between two or more liquids to allow substantially complete mixing of the liquids. Typically, static mixers contain an internal helical structure which allows the liquids to come in contact in an opposing rotational flow and causes them to mix in a turbulent or laminar flow. Such mixers are described, e.g., in U.S. Patent No. 3,286,922.

A method of preparing cationic lipid-nucleic acid complexes by first forming lipid micelles in the presence of detergent is described in WO 96/37194. Methods of preparing DNA-lipid complexes using polyethylene glycol-phospho lipid conjugates and polyamines are described in Hong (1997) FEBS Lett. 400:233-237. A number of analytical methods are known for characterizing the cationic lipid-nucleic acid complexes used in the methods of the invention. Visual inspection may provide initial information as to aggregation of the complexes. Spectrophotometric analysis may be used to measure the optical density, giving information as to the aggregated status of the complexes; surface charge may be determined by measuring zeta potential; agarose gel electrophoresis may be utilized to examine the amounts and physical condition of the polynucleotide molecules in the complexes; particle sizing may be performed using commercially available instruments; HPLC analysis will give additional information as to resulting component ratios; and dextrose or sucrose gradients may be used to analyze the composition and heterogeneity of complexes formed. The final cationic lipid-nucleic acid complex can be also analyzed by, e.g. , using thin-layer chromatography, as described, e.g., in Brailoiu (1994) Biomed. Chromatogr. 8: 193-195.

Cationic Lipid-Nucleic Acid Pharmaceuticals as Formulations

The cationic lipid-nucleic acid compositions of the invention can be formulated as pharmaceuticals for administration IM, intradermally, or SQ in a variety of ways. Details on techniques for formulation and administration are well described in the scientific and patent literature, see, e.g., the latest edition of "Remington's Pharmaceutical Sciences" (Maack Publishing Co, Easton PA). See also, Lasic and Templeton (1996) Adv. Drug Deliv. Rev. 20: 221-266 and references cited therein. The ratios of each component in the cationic lipid-nucleic acid complexes, final concentrations, buffer solutions, and the like can be readily optimized by the skilled artisan, taking into consideration the mode of delivery (IM, intradermal, or SQ administration), the anatomical site of delivery, the ischemic condition or disease treated, the condition and age of the patient, and the like. This can be done, e.g. , by testing for the levels of angiogenic stimulation, or levels of gene expression if a nucleic acid comprising a polypeptide coding sequence is used, using any of the well-known, art-accepted animal models relating to in vivo stimulation of angiogenesis and angiogeneic activity, and in vivo gene expression.

If testing for levels of gene expression in an appropriate animal model, e.g., mouse or rabbit, any number of reporter genes can be used, such as CAT, lacZ, alkaline phosphates, luciferase(Altschmied (1997)5rø?ec (n!r_?weΛ' 1997 Sep;23(3):436-438), green fluorescent protein, and the like. The optimized formulation is then used to deliver the angiogenic gene of interest, as described above.

Aqueous Solutions for Parenteral Administration Aqueous solutions are appropriate for injection and, in particular, for intramuscular, intradermal, or subcutaneous injection. Examples of aqueous solutions that can be used as pharmaceutically acceptable carriers in formulations include, e.g., water, saline, phosphate buffered saline, Hank's solution, Ringer's solution, dextrose/saline, glucose solutions and the like. The formulations can contain pharmaceutically acceptable auxilliary substances to enhance stability, deliverability or solubility, such as buffering agents, tonicity adjusting agents, wetting agents, detergents and the like. Additives can also include additional active ingredients such as bactericidal agents, or stabilizers. For example, the solution can contain sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate or triethanolamine oleate. These compositions can be sterilized by conventional, well-known sterilization techniques, or can be sterile filtered. The resulting aqueous solutions can be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile aqueous solution prior to administration (or, administered as a powder).

The cationic lipid-nucleic acid complexes of the invention are administered to induce angiogenesis and angiogenic activity (including increase blood perfusion of a tissue) to treat ischemia in a tissue. In one embodiment, the methods of invention comprise administering a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a cationic lipid-nucleic acid complex in an amount effective to treat the ischemia in the tissue. In a preferced embodiment, the angiogenic activity is induced in muscle tissue. The site of administration of the pharmaceutical composition is primarily determined by the location of the ischemic pathogenesis, which in turn determines preferred anatomical site(s) for inducing angiogenesis and angiogenic activity. For example, in one embodiment, the invention provides for treating myocardial ischemia, such as ischemia caused by coronary artery disease, including atherosclerosis and myocardial infarction. For this indication, the compositions of the invention can be administered by intra-cardiac muscle (intramuscular) injection; see, e.g., Kaplitt (1996) Ann. Thorac. Surg. 62: 1669-1676.

In another embodiment, the invention provides for treating ischemia caused by peripheral vascular disease, such as atherosclerosis or diabetes. For these indications, the compositions of the invention can be administered locally by intramuscular or subcutaneous administration, in the general vicinity, where angiogenesis is desired. See, e.g., Suzuki (1998) Hum. Gene Ther. 9:1223-1231.

The methods of the invention stimulate angiogenesis and angiogenic activity to treat ischemia. The amount of cationic lipid-nucleic acid complexes adequate to accomplish this is defined as a "pharmacologically effective amount" or a

"therapeutically effective dose." The dosage schedule and amounts effective for this use, i.e., the "dosing regimen," will depend upon a variety of factors, including the stage of the disease or condition, the site and severity of the disease or condition, the general state of the patient's health, the patient's physical status, age, whether the pharmaceutical is administered IM, intradermally, or SC, the which muscle or where in or under the skin the pharmaceutical is administered, and the like. In calculating the dosage regimen for a patient, the mode of administration also is taken into consideration.

The dosage regimen must also take into consideration the pharmacokinetics, i.e., the cationic lipid-nucleic acid complexes' rate of absorption, bioavailability, metabolism, clearance, and the like (see, e.g., the latest Remington's edition, supra).

Single or multiple administrations of the cationic lipid-nucleic acid complexes can be carried out with dose levels and pattern being selected by the treating physician. In any event, the pharmaceutical formulations should provide a quantity of a cationic-lipid-nucleic acid complex sufficient to treat the patient effectively. The total effective amount of a cationic lipid-nucleic acid complex of the present invention can be administered to a subject as a single dose, or can be administered using a fractionated treatment protocol, in which the multiple doses are administered over a more prolonged period of time. A typical dosing regimen would be from about one to about five doses of cationic lipid-nucleic acid complex formulation, where each dose is from about 0.5 ngm DNA to about 2 ngm DNA. Each dose may be given as a single injection, or, preferably, as multiple injections into the area where stimulation of angiogenesis is desired, such as, e.g., an ischemic muscle. For example, if a dose of about 2 ngm DNA is given, at least two doses will generally be required, typically at about 4 week intervals.

Diagnosis and Treatment Ischemic Diseases and Conditions

Detecting PVD can be relatively easy and inexpensive, and based either on subjectively supplied historical information or upon physical examination findings, e.g., ankle-brachial index (ABI). The ABI is a marker for increased risk for systemic vascular disease. An abnormally low ABI is associated with systemic vascular disease. Epidemiology of and risk factors for PVD are in similar to coronary heart disease. PVD is uncommon until middle age and then increases dramatically. Prevalence of PVD is slightly higher in men than women, yet this tends to diminish with age. Cigarette smoking is probably the most important risk factor for PVD. Hypertension, hypercholesterolemia, and diabetes are also risk factors for the development of symptomatic PVD. Patients who have vascular disease in one organ system often exhibit evidence of vascular disease elsewhere. Presence of coronary artery disease among patients with symptomatic PVD is between two and four times higher than those without PVD, and PVD is also associated with cerebrovascular disease. Other risk factors for PVD include hypertension; low levels of high-density lipoprotein cholesterol; and high levels of triglycerides, apolipoprotein B, lipoprotein(a), homocysteine, fibrinogen and blood viscosity (see, e.g., Criqui (1997)

Vase. Med. 2:221-226).

Diabetes mellitus and particularly non-insulin-dependent diabetes mellitus (NIDDM) increases the risk for all manifestations of atherosclerotic vascular disease; coronary heart disease; cerebrovascular disease; and peripheral vascular disease. NIDDM is known to be associated with several adverse cardiovascular risk factors, including: hypertension; obesity; central obesity; hyperinsulinemia; and serum lipid and lipoprotein abnormalities, characterized mainly by elevated serum total triglycerides and low high-density lipoprotein cholesterol.

It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.

EXAMPLES

The following examples are offered to illustrate, but not to limit the claimed invention.

Example 1: Induction of Angiogenic Activity In vivo by Intramuscular Administration The following example demonstrates that a cationic lipid-nucleic acid complex of the invention after intramuscular (IM) injection can effectively induce angiogenic activity in the muscle in an art-accepted animal model for angiogenesis.

A lipid-DNA formulation was synthesized using standard techniques, as described above: 1.5 mg/ml DNA, 1.5 mM BODAI (also called DOTIM)/l .5 mM DOPE, 5% w/v dextrose, approx. 3 mM Tris-HCl, pH 8.0. In one sample, as the nucleic acid component, an expression plasmid containing VEGF coding sequence was included in the complex. In a second sample, as the nucleic acid component, a gene for human placental alkaline phosphatase (HPAP) was included. Concentrations of the complexes were based on the DNA content. They were 250 (VEGF250), 500 (VEGF500), 750 (VEGF750), and 1000 (VEGF 1000) μg per rabbit for the VEGF plasmid. HPAP complexes were dosed at

250 and 1000 μg per rabbit (CONTROL250 and CONTROLIOOO). There were five animals per treatment group.

New Zealand White rabbits were anesthetized. A longitudinal incision was performed on the right leg, from inguinal ligament area to just proximal to the patella. The femoral artery and all branches were exposed and dissected free along its entire length, including the popliteal and saphenous arteries. The external iliac artery as well as all other branch arteries were ligated. The femoral artery was excised to the point where it bifurcates into the saphenous and popliteal arteries. The wound was closed with sutures and the animal was replaced in a cage. Analgesics were administered postoperatively as required.

Two days later, the rabbits were lightly anesthetized. Aliquots of lipid- nucleic acid complex, described above, were injected intramuscularly into four sites on one side of and adjacent to the original incision. The volume of complex at each site was 0.5 ml to give a total injection volume of 2 ml. The animals were returned to their cages and allowed to recover.

To evaluate the induction of angiogenic activity, blood flow measurements were made at various times after administration; at days 6 (one week, see Figure 1), 13 (week 2), 20 (week 3), and 27 (week 4). To prepare the animals for blood flow measurements, radiolabeled microspheres ( 15 um diameter) were injected as a single bolus injection into the circulation of the rabbit. A few minutes later muscles (gastrocnemus and adductor on both the injured leg and the normal contralateral leg) were dissected out, harvested, and weighted. Dissected muscles were homogenized and aliquots of the homogenates were analyzed with a scintillation counter to obtain cpm. The data were normalized to total cpm per muscle. Figure 1 shows the data, plotted as the ratio of blood flow of the right

(ischemic) leg to the left leg (control, or normal contralateral limb). The results show that in this model system, the animal eventually recovers in 3 to 4 weeks by itself; seen as saline-treated legs returning to same blood flow as uninjured legs by 3 to 4 weeks.

However, IM administration of cationic lipid-nucleic acid complexes of the invention significantly accelerate recovery in the injured muscle tissue. Blood flow in animals treated with the cationic lipid-DNA formulation containing VEGF-coding sequence at the 1000 μg dose (VEGF 1000) recovered at a faster rate than animals treated with saline or the cationic lipid-DNA formulation containing alkaline phosphatase

(HPAP)-coding sequence controls (CONTROL250 and CONTROLIOOO), and faster than other doses of VEGF. At week 1 , animals treated with VEGF 1000 had blood flow 112% higher than the blood flow in animals treated with saline (p=0.0007), while VEGF250, 500, and

750 ug dose, as well as HPAP groups were not different from saline.

At week 2 after treatment, the blood flow in the VEGFlOOO-treated group was 70% higher than in the group treated with saline (p=0.0001) and 24% higher than in the HPAP control groups. However, the HPAP treatment group had a blood flow 28% higher than the saline-treated group (p=0.002) on week 2, while the blood flow in

VEGF250, 500, and 750 ug dose remained similar to saline.

Figure 2 is a plot showing VEGF1000, the combined 250 μg and 1000 μg

HPAP (CONTROL250 and CONTROLIOOO in Fig. 1, "Plasm/lipom" in Fig. 2) plasmid complexes and saline control at weeks one through four. It is a simplified presentation of the data shown in Figure 1, described above. It shows only the treatment groups that are statistically significantly different from saline control.

Therefore, both formulations - the cationic lipid DNA formulation containing VEGF-coding sequence at the 1000 μg dose and the cationic lipid DNA formulation containing alkaline phosphatase-coding sequence accelerate the rate of recovery of blood flow in ischemic tissue at different time points. The VEGF 1000 is most effective at one week after treatment. The HPAP formulations are effective at week 2 after treatment, probably having an additive effect on the improvement of blood flow. Example 2: Induction of Angiogenic Activity In vivo

The following example demonstrates that a lipid-nucleic acid complex of the invention can effectively induce angiogenic activity in vivo after intradermal and/or subcutaneous administration. A lipid-DNA formulation was synthesized using standard techniques, as described above: The formulation included 2.0 mg/ml DNA, 2.0 mM BODAI (also called DOTIM)/2.0 mM DOPE, 5% w/v dextrose, approximately 5.0 mM Tris-HCL, pH 7.1. In one sample, as the nucleic acid component, an expression plasmid containing a non-coding vector (designated C 192-75.6) was included in the complex. In a second sample, a vehicle control (5% dextrose in water - D5W) was substituted for the lipid-DNA component.

Concentrations of the complexes were based on the DNA content.

A single dose volume of 90ul of C192-75.6 (180 ug DNA) or D5W was delivered to rabbits at three, 1 cm. diameter intradennal/ subcutaneous injection sites on a weekly or biweekly basis (270ul total/rabbit/weekly or biweekly injection). Injections were administered into the dorsal skin of the back of rabbits lightly sedated with acetyl promazine. Injections were made with tuberculin syringes and 27 G needles. Although the injections were intended for intradermal delivery, because rabbit skin is so thin, there was some extension into the underlying subcutaneous area.

One group of rabbits (Group 7) and controls (Group 10) received weekly injections for 6 weeks and were sacrificed 2 days following the sixth injection. Another group of rabbits (Group 8) and controls (Group 11 ) received weekly injections for 6 weeks and were sacrificed weeks following the sixth injection. Another group of rabbits (Group 9) and controls (Group 12) received injections every other week (biweekly) for 8 weeks (4 injection intervals) and were sacrificed 2 days following the fourth injection. Skin injection sites were tagged such that sites could be positively identified at necropsy.

Injection sites were harvested at necropsy and fixed in 10% neutral buffered formalin for histological evaluation. Histologic sections were prepared from paraffin-embedded tissues and stained with hematoxylin and eosin.

Distinct neovascular formation (angiogenesis) was recognizable approximately 1 week following intradermal delivery of lipid:DNA complexes. These newly formed vessels were present in the mid- to deep dermis and were confined to focal clusters of inflammatory cells consisting predominately of histiocytes and lymphocytes. The vessels within these foci were morphologically consistent with capillaries and frequently contained intraluminal erythrocytes. One to multiple cross-sections of capillaries were contained within most inflammatory foci. Neovascular elements persisted within these foci as long as inflammatory cells were present. Neovascularization was not evident in areas lacking inflammatory cell accumulations.

In contrast, rabbits receiving intradermal injections of D5W (control groups) lacked inflammatory cell accumulations and lacked any new vessel growth. Similarly rabbits receiving intradermal lipid:DNA complexes that had complete resolution of dermal inflammation lacked neovascular elements. Such resolution typically occurred in intradermal sites injected with lipid:DNA complexes six to eight weeks prior to histologic evaluation.

Claims

We claim:

1. A method for stimulating angiogenic activity in a tissue, the method comprising administering a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a cationic lipid - nucleic acid complex in an amount effective to induce angiogenic activity in the tissue, wherein the pharmaceutical composition is administered intramuscularly intradermally, or subcutaneously.

2. The method of claim 1, wherein the pharmaceutical composition is in a unit dosage form.

3. The method claim 2, wherein the unit dosage form delivers between about 1 ngm to about 6 ngm of the nucleic acid.

4. The method claim 3, wherein the unit dosage form delivers about 2 ngm of the nucleic acid.

5. The method claim 4, wherein a unit dosage form of about 2 ngm of nucleic acid is administered in at least two intervals, wherein the intervals are about 4 weeks apart.

6. The method of claim 1, wherein the pharmaceutical composition is in the form of an injectable solution.

7. The method of claim 1, wherein the pharmaceutically acceptable carrier is an aqueous solution.

8. The method of claim 1, wherein the cationic lipid complex has a net positive charge.

9. The method of claim 8, wherein the cationic lipid is selected from the group consisting of BODAI, DOTMA, DMRIE, DOTAP, DOGS, EDMPC, MeBOP, and DCChol.

10. The method of claim 8, wherein the lipid content of a cationic lipid-DNA complex formulation of about 1.5 mM BODAI and about 1.5 mM DOPE.

11. The method of claim 1 , wherein the pharmaceutical composition is administered into a skeletal muscle.

12. The method of claim 1, wherein the pharmaceutical composition is administered into a cardiac muscle.

13. The method of claim 1, wherein the nucleic acid is DNA.

14. The method of claim 13, wherein the DNA comprises a sequence that does not encode a polypeptide with biologic activity.

15. The method of claim 13, wherein the DNA comprises a sequence that does not encode a polypeptide with angiogenic activity.

16. The method of claim 13, wherein the DNA comprises a sequence that encodes a polypeptide with biologic activity.

17. The method of claim 16, wherein the polypeptide has angiogenic activity

18. The method of claim 16, wherein the polypeptide has vascular endothelial growth factor activity.

19. The method of claim 18, wherein the polypeptide is a vascular endothelial growth factor.

20. The method of claim 19, wherein the polypeptide has a sequence as set forth in SEQ ID NO:2.

21. The method of claim 1, wherein the intramuscular injection is into a skeletal muscle.

22. The method of claim 1, wherein the intramuscular injection is into a cardiac muscle.

23. The method of claim 1, wherein the administration is an intradermal injection.

24. The method of claim 1, wherein the administration is a subcutaneous injection.

25. A method for treating ischemia in a tissue, the method comprising administering a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a cationic lipid -nucleic acid complex in an amount effective to treat the ischemia in the tissue, wherein the pharmaceutical composition is administered intramuscularly, intradermally, or subcutaneously.

26. The method of claim 25, wherein the ischemia is caused by peripheral vascular disease.

27. The method of claim 26, wherein the peripheral vascular disease is caused by diabetes.

28. The method of claim 25, wherein the ischemia is caused by atherosclerosis.

29. The method of claim 25, wherein the ischemia is caused by coronary artery disease.