HK1075832B - Use of an alpha helical apolipoprotein or hdl associated protein for the manufacture of a medicament adapted to be administered locally to prevent or treat restenosis - Google Patents

Description

Use of alpha helical apolipoprotein or HDL associated protein for preparing medicine for local administration for preventing and treating restenosis

no marking

Background

This application claims priority from US application serial No. 60/326,379 filed on 28/9/2001.

The present invention is generally in the field of methods and compositions for reducing restenosis following revascularization of diseased coronary, peripheral and cerebral arteries and stenosis or restenosis following surgical placement of bypass grafts or transplanted organs, particularly for the topical administration of substances such as apolipoprotein a-I Milano, alone or in combination with lipid agents or other cholesterol lowering or lipid modulating agents.

Angioplasty, surgery and other vascular interventions are made tricky by accelerated arterial lesions characterized by rapid cell growth into the lumen in a short period of time. This growth is often severe enough to compromise blood flow to the end organs.

As used for the superficial plaque formation (plaques) of blood vessels in atherosclerosis, vascular bypass surgery has been widely used to treat stenotic and occluded blood vessels. In bypass surgery, one or more healthy blood vessels are transplanted into a stenosed/occluded blood vessel across the site of the stenosis or occlusion to shunt blood around the stenosed or occluded blood vessel to reestablish a sufficient blood supply to the tissue whose blood supply is compromised by the stenosis or occlusion. Such procedures are often successful in revascularizing compromised tissues.

Angioplasty has evolved as an alternative treatment to bypass surgery, particularly for patients diagnosed as being in the early stages of stenosis or closure of a vessel by abnormal deposition of plaque on the wall of the vessel wall. Angioplasty generally involves introducing a catheter, usually fitted with a balloon or expandable metal mesh, into the area of a stenosed or occluded artery, and briefly expanding the balloon or mesh one or more times to compress the endovascular occlusion or plaque against the wall of the vessel's endothelium, thereby compressing the plaque and/or fragmenting the plaque and reestablishing blood flow. However, angioplasty treatment can damage the vessel, especially when the balloon is over-inflated or the mesh is over-expanded, causing many undesirable consequences such as peeling (migration) of the endothelial cell layer within the angioplasty area, dissection of the inner wall portion of the vessel from the rest of the vessel with vessel closure, or rupture of the intimal layer of the vessel.

Arterial injury in animals leads to a vascular repair process that ultimately narrows the artery. A thick new layer or neointima of smooth muscle cells and inflammatory cells grows within the vessel, encroaching on the lumen. This procedure in animals represents a procedure that occurs clinically after angioplasty, stent transplantation, organ transplantation, or bypass surgery, which greatly limits the long-term success of these techniques for treating obstructive arterial disease. Animal models of arterial injury and neointimal hyperplasia have been used to study cellular events leading to restenosis in humans to design therapeutic strategies that inhibit tissue growth in an attempt to reduce restenosis and increase long-term clinical outcome. Pigs are a particularly useful animal model for restenosis in humans.

Attempts to limit stenosis or restenosis of blood vessels following revascularization have included the use of pharmacological agents and technical approaches. There are no clinically approved drugs for preventing indications of restenosis in humans. As seen in serrouys et al, n.e.j.med.1994; 331: 489 Supper 495 and Fischman et al, N.E.J.Med.1994; 331: 496-501, a technical approach has been shown to partially reduce restenosis after coronary intervention-intravascular placement of a stent. However, the stent itself is still prone to significant restenosis in 20-30% of cases.

The increased understanding of the mechanisms underlying vascular repair has led to innovative proposals for drugs to limit accelerated arterial lesions. Circulating leukocytes, including monocytes, are known to be the very first cells recruited by blood vessels when atherosclerosis begins. When in the diseased artery wall, these cells can engulf cholesterol and other lipids, and can also produce substances that attract other cells, proliferate other cells, or degrade matrix components. Various of these secondary effects in turn promote greater intimal thickening and more severe narrowing or closure of the arterial lumen. There is no evidence that leukocytes play a similar role in restenosis following revascularization. Although leukocyte activation has been linked to restenosis in humans (Pieterma et al, Circulation 1995; 91: 1320-. This observation is a retrospective study of the use of widely effective and highly specific targeted therapies for the prevention of restenosis. Broad treatment, such as with heparin, has been limited by systemic toxicity and dose-limiting factors. Specific treatments, such as those performed by molecular strategies, do not inhibit all of the redundant cellular and molecular pathways that activate and potentiate the vascular repair process.

Ameli et al, Circulation 90 (4): 1935-41(1994) reported several epidemiological studies that showed an inverse relationship between High Density Lipoprotein (HDL) cholesterol levels and coronary heart disease, and a similar inverse relationship between HDL and restenosis following coronary angioplasty. A study was carried out to determine whether HDL directly affected neointimal formation, and the effect of recombinant apoA-I Milano (apoA-IM, a variant of human apoA-I in which Arg-173 was replaced with CyS) on intimal thickening following balloon injury in cholesterol-fed rabbits was studied. Rabbits began intravenous injection of 40mg apoA-IM attached to a phospholipid carrier every other day 5 days prior to femoral and iliac artery sac injury and continued administration for 5 days post injury (total dose of 200 mg/animal, or about 11.4 mg/kg/administration). Three weeks after the capsular injury, the intimal thickness of apoA-IM treated rabbits was significantly reduced compared to both control groups. ANOVA comparisons with both controls showed that apoA-IM also significantly reduced the intima-to-media ratio. As demonstrated by immunohistochemical studies using macrophage-specific monoclonal antibodies, there was significantly less coverage of intimal lesions by macrophages in apoA-IM treated rabbits (25.3 +/-17% vs. 59.4 +/-12.3%, P <. 005) than in vehicle-treated animals. There was no significant difference in aortic cholesterol levels between animals treated with apoA-IM and vehicle only control. Unfortunately, unlike pigs, where the lesions are more similar to humans, the results obtained in rabbits have not been predictive of human results.

Thus, there is a need for compositions and methods that promote vascular tissue healing and control vascular muscle cell proliferation (hyperplasia) to prevent restenosis of a blood vessel following angioplasty, vascular bypass surgery, organ transplantation, or vascular disease while minimizing the risk of rapid reclosure.

Accordingly, it is an object of the present invention to provide methods and compositions for reducing restenosis following revascularization of diseased coronary, peripheral and cerebral arteries, as well as stenosis or restenosis following surgical placement of bypass grafts or transplanted organs.

Another object of the present invention is to provide a simple and efficient gene transfer method.

Summary of the invention

Apolipoprotein a-I (ApoA-I), preferably a variant form such as apolipoprotein a-I Milano (ApoA-IM), alone or more preferably in combination with a lipid carrier such as a phospholipid or other drug, may be administered locally prior to or during bypass surgery, surgical implantation of grafts or transplanted organs, or angioplasty of unstable plaques on diseased coronary, peripheral and cerebral arteries. In preferred embodiments, ApoA-IM is administered using infiltrtator, an intramural release device and/or other sustained controlled release methods to administer an effective dose to the site of injury. Based on the porcine model using ApoA-IM, an effective dose for treating or preventing restenosis is 0.2 to 0.4mg ApoA-IM/kg delivered to the site of treatment, or more specifically 4 to 6mg ApoA-IM/vessel treated. The viscosity of the solution limits the upper limit of the dose for intramural administration with infiltrtator. For example, this method cannot use ApoA-IM solutions that are too viscous to pass through an INFILTRATOR pore. As demonstrated by the examples, an effective amount of 0.3 to 0.4ml of 14mg/ml ApoA-IM solution (which corresponds to a single administration of 4 to 6mg per vessel segment or about 0.2mg/kg for 25kg of each treated vessel in pigs) is preferably administered in combination with 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) in a weight ratio of about 1: 1.

In an alternative embodiment, the apolipoprotein is not provided directly, but rather a gene encoding the apolipoprotein is provided. The gene is introduced into the blood vessel in a manner similar to that used for the protein, and then the protein is expressed therein. The technique can also be used for gene delivery for the treatment or prevention of restenosis or other cardiovascular diseases.

In another embodiment, the stent is coated with apolipoprotein alone, apolipoprotein prepared with lipids, genetically engineered cells expressing apolipoprotein, naked DNA encoding apolipoprotein, or other drugs such as antiproliferative agents and then locally released to the damaged site. This embodiment also includes the combined application of the above-listed coating materials to the stent for greater efficacy. In a preferred embodiment, the system is used in combination therapy for the local release of substances such as apolipoproteins in combination with systemic antihypertensive therapy, lipid regulation and/or anticoagulant therapy. Examples of drugs that may be used include lipid modulators such as nicotinic acid, statins, and fibrates; drugs for glycemic control; anti-hypertensive agents; and substances for preventing or delaying coagulation or platelet aggregation such as those wherein the substance is aspirin, an inhibitor of IIb/IIIa, clopidogrel or heparin. The greatest benefit may be obtained using topical treatment in combination with more than one combination, e.g., using topical delivery therapy plus anticoagulation plus lipid modulation. These treatments may be initiated prior to local delivery, simultaneously with local delivery, or after local treatment. Systemic treatment is preferably initiated prior to this local delivery procedure.

Detailed description of the drawings

Figures 1-4 relate to trials in which 10 pigs were treated with a single intravenous infusion of 100mg/kg ETC-216, an apoA-IM/POPC (weight ratio of approximately 1/1) complex (n-5) or saline (n-5). The test substance was administered intravenously over about 3 hours while hypertractional percutaneous transluminal coronary angioplasty with stent deployment was performed in both coronary vessels of each animal. The dosage is based on the weight of the protein component of the complex. Animals were euthanized on day 28 (8 animals) or 29(1 animal), and then subjected to quantitative coronary angiography and intravascular ultrasound (IVUS), coronary arteries perfused and fixed for histomorphological analysis. One control animal died at day 27 and only histomorphological data was collected from that animal and used.

Fig. 1a and 1b are graphs of quantitative coronary angiography data (QCA) measured at three time points: before coronary vessel injury, immediately after coronary vessel injury and stent deployment, and before sacrifice, and is based on diameter measurements (mm) taken at the unexpanded (unexpanded) section proximal to the stent, at the proximal portion of the stent, at an average area of the entire length of the stent, at the distal portion of the stent, and at the unexpanded section distal to the stent. From these data, the maximum and minimum diameters of the expanded (held) region can be measured. QCA evaluation of luminal gain (luminal diameter after injury minus luminal diameter before injury) and luminal loss (luminal diameter after injury minus luminal diameter at follow-up observations on days 28-29) were determined at expanded and adjacent non-expanded sections. QCA data for all vessels (i.e. Right Coronary Artery (RCA) and left anterior descending artery (LAD) combined) is shown in fig. 1a and for various types of vessels (i.e. RCA or LAD) in fig. 1 b.

Fig. 2a and 2b are intravascular ultrasound (IVUS) data plots for determining the distal, medial and proximal stent and lumen area of each expanded coronary vessel prior to sacrifice (28-29 days post-surgery). The difference between these measurements is the area of the neointima. Fig. 2a is for all vessels (i.e., RCA and LAD in combination), and fig. 2b is for various types of vessels (i.e., RCA or LAD).

FIGS. 3a and 3b are histomorphological analyses of the expanded artery used to determine the Adventitial Boundary Layer (ABL), the outer elastic layer (EEL), the Inner Elastic Layer (IEL), the lumen (L), the adventitia (A), the media (M), the mean cross-sectional area of the intima (I), the intima-to-media ratio (I/M), and the lesion score. The lesion score is the mean of 36 lesion determinations obtained by measuring 12 times each of the proximal (a), medial (b) and distal (c) segments of the expanded vessel site being expanded. Lesions were scored as 0, 1, 2 or 3 points, with 0 indicating intact IEL (i.e. no lesion) and 3 indicating ruptured EEL exposed to the adventitia (i.e. the most severe lesion). Fig. 3a is the histomorphometric data for all vessels (i.e., RCA and LAD in combination), and fig. 3b is the histomorphometric data for various types of vessels (i.e., RCA or LAD).

Figure 4 shows the selective correlation between tissue morphology and IVUS variables.

Figures 5a and 5b show the histomorphometric data collected from pigs administered 0.3-0.4ml of a percutaneous transluminal coronary angioplasty procedure containing 4-6mg of protein/vessel ETC-216, apoA-IM/POPC (weight ratio about 1/1) complex (n-6 pigs) or sucrose-mannitol media (n-6 pigs) using INFILTRATOR ®, an intramural delivery device, and subsequently over-stretched with stent deployment in both coronary vessels of each animal. The dose represents the weight of the protein component of the complex. Shown is the histomorphological analysis of the expanded artery to determine the mean cross-sectional area of the Adventitia Boundary Layer (ABL), the outer elastic layer (EEL), the Inner Elastic Layer (IEL), the lumen (L), the adventitia (a), the media (M), the intima (I), the intima-to-media ratio (I/M) and the lesion score. The lesion score is the mean of 36 lesion determinations obtained by measuring 12 times each of the proximal (a), medial (b) and distal (c) segments of the dilated vessel at the dilated site. Lesions were scored as 0, 1, 2 or 3 points, with 0 indicating intact IEL (i.e. no lesion) and 3 indicating ruptured EEL exposed to the adventitia (i.e. the most severe lesion). Fig. 5a shows the histomorphometric data for all vessels (i.e., RCA, Left Circumflex (LCX), and LAD in combination), and fig. 5b shows the histomorphometric data for each type of vessel (i.e., RCA, LCX, or LAD).

Detailed description of the invention

Systems have been developed that focus on the local application of substances that can be used to treat or prevent restenosis prior to or during bypass surgery on diseased coronary, peripheral and cerebral arteries, surgical implantation of grafts or transplanted organs, or angioplasty, or stabilization of unstable plaques. The topical administration is preferably carried out using a device comprising a reservoir which slowly releases the drug over a period of time. The reservoir may be part of the device, such as a stent, or may be created by infusion into a particular tissue or organ, for example by intrapericardial or infusitrator administration. This can be accomplished with commercially available catheters.

The composition to be administered may be cholesterol-and lipid-removing substances (such as apolipoproteins in combination with phospholipids, statins, fibrates), DNA encoding such substances (e.g., DNA encoding an apolipoprotein) or other proteins such as enzymes involved in nitric oxide production, and/or drugs such as antiproliferative compounds such as rapamycin, paclitaxel, or antibodies such as tirofiban and abciximab.

Combination therapy may also be used in which a drug such as ApoA-IM is administered locally and another drug is administered systemically, e.g. systemic antihypertensive therapy, lipid regulation and/or anticoagulant therapy. Examples of drugs that can be used include lipid regulators such as nicotinic acid, statins, and fibrates; a substance for glycemic control; anti-hypertensive agents; and a substance that prevents or delays blood coagulation or platelet aggregation, such as wherein the substance is aspirin, an inhibitor of lib/IIIa, clopidogrel, or heparin.

1. Lipid modulators

Apolipoprotein formulations

Compounds that can function as HDL include synthetic HDL, which contains phospholipids such as phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, and other phospholipids in combination with HDL associated proteins such as apoA-I or variants thereof including apoAI-Milano and biologically active peptides derived therefrom, Reverse Lipid Transport (RLT) peptides, HDL associated enzymes such as paraoxonase, and apo E, formulated alone or in combination with liposomes or emulsions. HDL associated proteins as used herein include sequences present in HDL associated proteins that associate with HDL and synthetic peptides having the same linkage or action characteristics. Compounds that enhance HDL function include liposomes, where HDL shuttle from and to cells to and from the liposomes. Suitable liposome formulations are described in WO 95/23592 of the University of British Columbia.

The formulations described herein typically consist of an alpha-helical protein, such as ApoA-I, a lipid, and a carrier.

ApoA-I and ApoA-IM are representative components that may be used to treat or prevent stenosis resulting from bypass surgery, surgical implantation of grafts or organ transplants, or angioplasty of diseased coronary arteries, peripheral and cerebral arteries.

Plasma ApoA-I is a single polypeptide chain of 243 amino acids, the primary sequence of which is known (Brewer et al, biochem. Biophys. Res. Commun.80: 623-630 (1978)). ApoA-I is synthesized in cells as a 267 amino acid precursor. This preproapoprotein A-I is first processed intracellularly by N-terminal cleavage of 18 amino acids to give propapolipoproteine A-I, which is then further cleaved by 6 amino acids in plasma or lymph by specific protease activity to give apolipoprotein A-I. The main structure required for the ApoA-I molecule is believed to be the presence of a repeat unit of 11 or 22 amino acids, which is assumed to be present in an amphipathic helical configuration (Segrest et al, FEBS Lett 38: 247-. This structure is associated with the major biological activities of Apoa-I, namely lipid binding and lecithin: cholesterol Acyltransferase (LCAT) activation.

Human apolipoprotein AI-Milano (ApoA-IM) is a natural variant of ApoA-I (Weisgraber et al, J.Clin.invest 66: 901-907 (1980)). In ApoA-IM, amino acid Arg173 is replaced by amino acid Cys 173. Since ApoA-IM contains one Cys residue per polypeptide chain, it can exist in monomeric, homodimeric, or heterodimeric form. These forms are chemically interchangeable and the term ApoA-IM in this document does not differ between these forms. At the DNA level, the variant forms result from a C to T substitution in the gene sequence, i.e.the codon CGC becomes TGC, resulting in a cys-arg substitution transition at amino acid 173. However, this variant of ApoA-I is the most interesting variant, since ApoA-IM subjects are characterized by significantly reduced HDL-cholesterol levels without significantly increasing the risk of arterial disease (France schini et al, J.Clin.invest 66: 892-.

Another useful variant of ApoA-I is the Paris variant, in which arginine 151 is replaced by cysteine.

In preliminary clinical studies in experimental animals and humans (Nanjee et al, Arterioscler Thromb Vasc biol.19: 979- & 989(1999) and Eriksson et al, circulation.100: 594- & 598(1999)) it has been shown that systemic infusion of ApoA-I alone (Miyazaki et al, ariterioscler Thromb Vasc biol.15: 1882- & 1888(1995)) or HDL (Badimon et al, Lab invest.60: 455- & 461(1989) and J Clin invest.85: 1234- & 1241(1990)) can produce significant biochemical changes and also reduce the extent and severity of atherosclerotic lesions. As discussed in detail below and demonstrated by the examples below, it has now been found that it can be administered locally at the site of a lesion and can significantly reduce stenosis or restenosis.

Other HDL-related apolipoproteins with alpha helical properties may be used. Examples include Apo E, proApoA-I, ApoA-I Paris, ApoA-II, proApoA-II, ApoA-IV, ApoC-I, ApoC-II, and ApoC-III, alpha-helical sequences in these proteins, and apolipoproteins modified to contain one or more sulfhydryl groups (sulfhydrals), such as Bielicki and Oda, Biochemistry 41: 2089-2096 (2002). Additional HDL associated proteins may also be used. Examples include paraoxonase, cholesteryl ester transfer protein, LCAT and phospholipid transfer protein. The above proteins may be used alone, in combination, complexed with lipids alone or in combination. Furthermore, mixtures of complexes may also be used. Examples are the administration of a complex consisting of ApoA-I and a lipid and a complex consisting of paraoxonase and a lipid in the form of a mixture. Additional examples include complexes composed of more than one protein component. For example, a complex consisting of ApoA-I, paraoxonase and a lipid is useful.

Lipid

The lipid and ApoA-I form a complex to enhance its efficacy. The lipids are typically mixed with ApoA-I prior to administration. The apolipoprotein and lipid are mixed in an aqueous solution in appropriate proportions and can be complexed by methods well known in the art, including freeze-drying, detergent solubilization followed by dialysis, microfluidization, sonication, and homogenization. The composite efficacy may be optimized, for example, by varying pressure, ultrasonic frequency, or detergent concentration. An example of a detergent commonly used in the preparation of apolipoprotein-lipid complexes is sodium cholate.

In some cases, it may be desirable to mix the lipid and apolipoprotein prior to administration. The lipids may be in solution or may be in the form of liposomes or emulsions formed using standard techniques such as sonication or extrusion. Sonication is typically performed in an ice bath using an overhead sonication processor (tipsonifier), such as a Branson overhead sonication processor. The suspension is typically subjected to several sonication cycles. Extrusion may be performed with a biofilm extruder, such as a Lipex biofilm extruder. The pore size set in the extrusion filter can produce unilamellar liposomal vesicles of a specific size. Liposomes can also be formed by extrusion through an asymmetric ceramic filter, such as a Ceraflow Microfilter or polycarbonate filter commercially available from Norton Company, Worcester Master. or other types of polymeric materials (i.e., plastics) generally known.

In some cases, it is preferred to administer the apolipoprotein alone in a substantially lipid-free manner to treat the damaged artery. A sterile aqueous solution is added to the apolipoprotein. The apolipoprotein in this solution can be administered to treat a damaged artery. Alternatively, a lyophilized formulation of the complex may be hydrated with an aqueous solution prior to administration. In other cases, a lyophilized formulation of the complex in an aqueous solution is thawed prior to administration to the damaged blood vessel until a homogeneous solution is obtained.

Preferred lipids are phospholipids, most preferably including at least one phospholipid, typically soy phosphatidylcholine, egg phosphatidylcholine, soy phosphatidylglycerol, egg phosphatidylglycerol, palmitoyl oleoyl-phosphatidylcholine, distearoylphosphatidylcholine, or distearoylphosphatidylglycerol. Other useful phospholipid species include, for example, phosphatidylcholine, phosphatidylglycerol, sphingomyelin, phosphatidylserine, phosphatidic acid, N- (2, 3-bis (9- (Z) -octadecenyloxy)) -prop-1-yl-N, N, N-trimethylammonium chloride, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylinositol, cephalin, cardiolipin, cerebroside, dihexadecyl phosphate, dioleoylphosphatidylcholine, dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylglycerol, dioleoylphosphatidylglycerol, stearoyl-palmitoylphosphatidylcholine, di-palmitoylphosphatidylethanolamine, distearoylphosphatidylethanolamine, dimyristoyl-phosphatidylserine, sphingomyelin, phosphatidyl-1-yl-phosphatidyl-N, lysophosphatidylethanolamine, phosphatidylinositol, cephalin, cardiolipin, phosphatidylinositol, and dioleoyl-phosphatidylcholine. Lipids that do not contain phosphorus may also be used, including stearylamine, dodecylamine (dococylamine), acetyl palmitate, and fatty acid amides.

The additional lipids used are well known to the person skilled in the art and many well known sources can be cited, for example McCutcheon's Detergents and McCutcheon's Functional Materials, AlluredPublishing co, ridge wood, n.j. In general, it is desirable that the lipid is liquid crystalline at 37 ℃, 35 ℃ or 32 ℃. Liquid crystalline lipids can generally accept cholesterol more efficiently than gel-state lipids. Because patients typically have a core temperature of about 37 ℃, lipids that are liquid crystalline at 37 ℃ are typically in a liquid crystalline state during treatment.

The concentration of lipid in the formulation may vary. One of ordinary skill can vary these concentrations to optimize treatment with different lipid components or treatment for a particular patient. ApoAI is combined with lipids in a weight ratio of 1: 0.5 to 1: 3, preferably with more lipids being used for cholesterol scavenging. Preferably, a ratio of about 1: 1 is used to produce the most uniform combined population and for producing stable and reproducible batches.

Other lipid modulating drugs

The compounds may also be administered with compounds that specifically increase HDL levels (i.e., not as a by-product of lowering LDL), thereby improving the ratio of HDL cholesterol to total cholesterol, and the administration of any combination of these substances may be effective in improving the ratio of HLD to total blood cholesterol levels.

Examples of drugs include lipid regulators such as nicotinic acid, statins, and fibrates.

Anti-proliferative drug

InFILTRATOR can be used to release drugs such as antiproliferative agents like paclitaxel and topotecan (Biochemical Pharmacology, 2001; 61 (1): 119-127).

Gene delivery

In an alternative embodiment, the gene encoding the protein to be delivered may be administered instead of the protein. Gene transfer can be achieved by direct transfer of genetic material in a plasmid or viral vector, or can be delivered by transfer of genetic material in a cell or vector such as a cationic liposome. Such methods are well known in the art and are readily acceptable for use in gene-mediated toxin therapy as described herein. As with Francis et al, am.j. pharmacogenomics 1 (1): 55-66(2001), gene therapy provides a novel method for the prevention and treatment of cardiovascular disease. Technological advances in viral vector systems and the development of fusin (fusigenic) liposome vectors are important for the development of effective gene therapy strategies involving vasculature and myocardium in animal models. Gene transfer technology has been evaluated as a possibility for genetic (familial hypercholesterolemia) and acquired occlusive vascular diseases (atherosclerosis, restenosis, arterial thrombosis) as well as for the alternative treatment of cardiac disorders including heart failure, myocardial ischemia, transplant coronary sclerosis and hypertension. See also, Teiger et al, j.cardiovasc.pharmacol.33 (5): 726-732(1999).

Wolff et al, Biotechniques 11: 474-85(1991) demonstrated that injection of naked DNA into muscle can produce long-term and low expression levels of the protein encoded in the DNA sequence. Naked DNA may be administered to the smooth muscle layer and allowed to express proteins or the alpha helical region thereof for the treatment of damaged blood vessels using an intramural device such as infiltrtator ®. The transfer vector may be any nucleotide construct (e.g., a plasmid) used to deliver the gene into a cell, or may be part of a general strategy for delivering the gene, e.g., as part of a recombinant retrovirus or adenovirus (Ram et al,Cancer Res.53: 83-88, (1993)). Suitable methods for transfection have been described, including viral vectors, chemical transfectants, or physical-mechanical methods such as electroporation and direct diffusion of DNA, e.g., Wolff, J.A. et al,Science247, 1465-; and Wolff, J.A.Nature,352, 815-818, (1991). The plasmid or viral vector used herein is a substance that can transport a gene into a cell without degradation and includes a promoter that produces expression of the gene in the cell to which it is delivered. In a preferred embodiment, the vector is derived from a virus or retrovirus. Preferred viral vectors are adenovirus, adeno-associated virus, herpes virus, vaccinia virus, poliovirus, AIDS virus, neurotrophic virus (neuronic virus), Sindbis and other RNA diseasesViruses, including those with an HIV backbone. Also preferred are any virus families that can share the properties of these viruses suitable as vectors. Preferred retroviruses include murine Maloney leukemia virus, MMLV, and retroviruses expressing MMLV as a desirable property of the vector.

Retroviral vectors can carry more genetic payload, i.e., transgene or marker gene, than other viral vectors and are therefore a commonly used vector. However, it is not useful in non-proliferating cells. A retrovirus is an animal virus belonging to the family of Retroviridae, including any type, subfamily, genus, or tropism. Retroviral vectors are generally described in "retroviral vectors for gene transfer" by Verma, I.M., MICROBIOLOGY-1985, American Society for MICROBIOLOGY, p.229-232, Washington (1985). Examples of methods of gene therapy using retroviral vectors are described in US patents No. 4,868,116 and 4,980,286; PCT applications WO 90/02806 and WO 89/07136; and Mulligan, (Science 260: 926-.

Adenovirus vectors are relatively stable and easy to operate at high titers, and can be released in aerosol formulations, allowing transfection of non-dividing cells. The construction of replication-defective adenoviruses has been described (Berkner et al,J.Virology61: 1213-1220 (1987); the results of Massie et al,Mol.Cell.Biol.6: 2872 2883 (1986); Haj-Ahmad et al,J.Virology57: 267-274 (1986); the result of Davidson et al,J.Virology61: 1226-1239 (1987); zhang "Generation and identification of recombinant adenoviruses Using Liposome-mediated transfection and PCR analysis"BioTechniques15: 868-872(1993)). The benefit of using these viruses as vectors is to limit to some extent their spread to other cell types, since they can replicate within the originally infected cell, but cannot form new infectious viral particles. Has been shown to be delivered directly in vivo to the airway epithelium, hepatocytes, vascular endothelium, CNS parenchymaAnd many other tissue sites, high efficiency gene transfer can be achieved with adenovirus that has been recombined (Morsy,J.Clin.Invest.92：1580-1586(1993)；Xirshenbaum， J.Clin.Inyest.92：381-387(1993)；Roessler， J.Clin.Invest.92：1085-1092(1993)；Moullier， Nature Genetics 4：154-159(1993)；La Salle， Science259：988-990(1993)；Gomez-Foix， J.Biol.Chem.267：25129-25134(1992)；Rich， Human Gene Therapy 4：461-476(1993)；Zahner，Nature Genstics 6：75-83(1994)；Guzman， Circulation Research 73：1201-1207(1993)；Bout， Human Gene Therapy 5：3-10(1994)；Zabner，Cell 75：207-216(1993)；Caillaud， Eur.J.Neuroscience5: 1287-1291 (1993); and a group of compounds selected from the group consisting of Ragot,J.Gen.Virology 74：501-507(1993))

recombinant adenoviruses accomplish gene transduction by binding to specific cell surface receptors, upon which the virus is internalized by receptor-mediated endocytosis in the same manner as wild-type or replication-defective adenoviruses (Chardonnet and Dales,Virology 40：462-477(1970)；Brown andBurlingham， J.Virology 12：386-396(1973)；Svensson and Persson，J.Virology55: 442-449 (1985); the person of Seth et al,J.Virol.51: 650-; the person of Seth et al,Mol.Cell.Biol.4: 1528 and 1533 (1984); the Varga et al, in which,J.Virology65: 6061-6070 (1991); the Wickham et al, to,Cell73：309-319(1993))。

poxvirus vectors are large and have some sites for insertion of genes, are thermostable and can be stored at room temperature. A preferred embodiment is a viral vector designed to suppress an immune response in a host organism elicited by the viral antigen. Preferred vectors of this type will carry the coding region for interleukin 8 or 10.

The handling capacity (ability to introduce a gene) of a viral vector is higher than most of chemical or physical methods for introducing a gene into a cell. Typically, viral vectors contain an unstructured early gene, a structured late gene, an RNA polymerase III transcript, inverted terminal repeats required for replication and encapsidation, and a promoter that controls transcription and replication of the viral genome. When designed as a vector, viruses typically have one or more early genes removed and a gene or gene/promoter cassette inserted into the viral genome in place of the removed viral DNA. This type of construct can carry up to about 8kb of exogenous genetic material. The essential functions of the removed early genes are typically supplied by cell lines designed to express the gene products of the early genes in trans.

Genes inserted in viruses and retroviruses often contain promoters, and/or enhancers to help control expression of the desired gene product. A promoter is generally a DNA sequence or sequences that function when located in a relatively fixed position relative to the position at which transcription begins. Promoters comprise core elements required for the basic interaction of RNA polymerase and transcription factors, and may comprise upstream and effector elements. Preferred promoters for controlling transcription by the vector in mammalian host cells may be obtained from a variety of sources, for example, the genome of viruses such as: polyomavirus, simian virus 40(SV40), adenovirus, retrovirus, hepatitis B virus and most preferably cytomegalovirus, or a promoter from a heterologous mammal, such as the beta actin promoter. The early and late promoters of the SV40 virus are conveniently obtained as restriction fragments of SV40, which also contain the replication origin of the SV40 virus (Fiers et al,Nature,273: 113(1978)). Direct early promoter of human cytomegalovirus asHindIII E restriction fragments are readily available (Greenway, p.j. et al,Gene18: 355-360(1982)), of course, promoters derived from host cells or related species are also useful herein.

Enhancers generally refer to sequences of DNA that act at variable distances from the transcription start site and may be 5 '(Laimins, l. et al, proc.nat1. acad.sci.78: 993(1981)) or 3' (Lusky, m.l. et al, mol.cell bio.3: 1108(1983)) relative to the transcription unit. Furthermore, enhancers may be located within introns (introns) (Banerji, J.L. et al, Cell 33: 729(1983)) as well as within the coding sequence itself (Osborne, T.F. et al, mol.cell Bio.4: 1293 (1984)). It is usually 10 to 300bp in length, and it usually functions in cis form. Enhancers may function to increase transcription from a proximal promoter. Enhancers often also contain effector elements that mediate the regulation of transcription. Promoters may also contain effector elements that mediate the regulation of transcription. Enhancers often determine the regulation of gene expression. While many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, alpha-fetoprotein, and insulin), they are typically enhancers from eukaryotic cell viruses. Preferred examples are the SV40 enhancer (bp100270) on the most proximal side of the replication origin (late side), the cytomegalovirus early promoter enhancer, the polyoma enhancer located on the most proximal side of the replication origin, and adenovirus enhancers.

The promoter and/or enhancer may be specifically activated by light or a specific chemical event that may trigger its function. The system can be modulated with agents such as tetracycline and dexamethasone. There are also methods of enhancing viral vector gene expression by exposure to radiation, such as Y radiation or alkylating chemotherapeutic agents.

Preferably, the promoter and/or enhancer region acts as a constitutive promoter and/or enhancer to maximise expression of the transcribed transcription unit region. It is also preferred that the promoter and/or enhancer region is active in all eukaryotic cell types. A preferred promoter of this type is the CMV promoter (650 bases). Other preferred promoters are the SV40 promoter, cytomegalovirus (full-length promoter), and retroviral vector LTF. It has been shown that all specific regulatory elements can be cloned and used to construct expression vectors that are selectively expressed in specific cell types.

Expression vectors used in host eukaryotic cells may also contain sequences necessary for transcription termination that can affect mRNA expression. These regions are transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding tissue factor protein. The 3' untranslated region also includes a transcriptional terminal portion. Preferably, the transcription unit further comprises a polyadenylation region. One benefit of this region is that it increases the likelihood that the transcriptional unit will be processed and transported like mRNA. The identification and use of polyadenylation signals in expression constructs has been established. Preferably, homologous polyadenylation signals are used in the transgenic construct. In a preferred embodiment of the transcriptional unit, the polyadenylation region is derived from the SV40 early polyadenylation signal and consists of about 400 bases. It is also preferred that the transcribed unit comprises other standard sequences alone or in combination with the above sequences to improve expression from the construct or stability of the construct.

The viral vector may comprise a nucleic acid sequence encoding a marker product. This marker product is used to determine whether the gene has been delivered to the cell and, once delivered, is expressed. Examples of suitable markers that may be selected for mammalian cells are dihydrofolate reductase (DHFR), thymidine kinase, neomycin, the neomycin analog G418, hydromycin, and puromycin. When such selectable markers are successfully transferred into mammalian host cells, the engineered mammalian host cells can survive if placed under selective pressure.

In preferred embodiments, intramural delivery of DNA encoding ApoA-I, ApoA-IV, ApoE, paraoxonase, or the alpha-helical region within these proteins is delivered to an artery with or without lipids for treatment of damaged blood vessels.

DNA encoding many different proteins may also be delivered. For example, as Chen et al, jpn.j. pharmacol.89 (4): 327-336(2002), cardiovascular gene transfer is not only an effective technique for studying the function of specific genes in cardiovascular biology and pathology, but is also a promising strategy for treating cardiovascular disease. Since the mid-90 s of the 20 th century, Nitric Oxide Synthase (NOS), an enzyme that catalyzes the formation of Nitric Oxide (NO) from L-arginine, has received considerable attention as a potential candidate for cardiovascular gene therapy because NO plays an important and diverse role in the cardiovascular system, and biological abnormalities of NO occur during many cardiovascular diseases, including cerebral vasospasm, atherosclerosis, restenosis following angioplasty, graft vascular disease, hypertension, diabetes, impotence, and delayed wound healing. There are three NOS isoforms, endothelial (eNOS), neuronal (nNOS), and Inducible (iNOS) NOS. Three NOS isoforms have been used in cardiovascular gene transfer studies with encouraging results.

Kipshidze et al, j.am.col.cardio.39 (10): 1686-1691(2002) describes the reduction of neointima formation by intramural delivery of antisense oligonucleotides.

Turunen et al, Mol Ther 6 (3): 306(2002), describes gene therapy with nuclear-targeted 1 acZ-and TIMP-1-encoding adenoviruses that bind to peptide-motifs (HWGF) that can bind to Matrix Metalloproteinases (MMP) -2 and MMP-9. Local intravascular catheter-mediated gene transfer of HWGF-targeted TIMP-1-encoding adenovirus (AdTIMP-1(HWGF)) significantly reduced intimal thickening in rabbit aortic follicle denudation models in vivo compared to control adenovirus.

An advantage of the method disclosed herein is that it can provide delivery and release over a longer period of time at the site where treatment is needed.

Medicine for systemic treatment

Various different drugs may be administered systemically and/or locally. These drugs include substances for glycemic control; anti-hypertensive agents; anti-inflammatory agents such as steroidal anti-inflammatory agents, cyclooxygenase-2 (COX-2) inhibitors such as Celebrex, VIOXX, and cyclooxygenase inhibitors such as ibuprofen and non-steroidal anti-inflammatory agents, and substances that prevent or delay blood coagulation or platelet aggregation such as aspirin, IIb/IIIa inhibitors, clopidogrel, or heparin.

Stent coating

Stents may also be coated with apolipoproteins alone, with apolipoproteins formulated with lipids, with cells expressing apolipoproteins or other proteins, DNA encoding therapeutic proteins, or with drugs having a local effect such as paclitaxel, rapamycin, or other antiproliferative compounds. The coating then releases the drug at the lesion, plaque or area to be treated.

Pharmaceutically acceptable carrier

The pharmaceutical composition generally comprises a pharmaceutically acceptable carrier. Many pharmaceutically acceptable carriers can be used. An example is the use of sucrose-mannitol. Physiological saline is generally used as a pharmaceutically acceptable carrier. Other suitable carriers include glucose, trehalose, sucrose, sterile water, buffered water, 0.4% saline, and 0.3% glycine, and may further include glycoproteins for enhanced stability, such as albumin, apolipoproteins, lipoproteins, globulins, and the like. These compositions may be sterilized by conventional, well known sterilization techniques. The resulting aqueous solution may be packaged for use or filtered under sterile conditions and lyophilized, and the lyophilized formulation may be combined with a sterile aqueous solution prior to administration. The composition may contain pharmaceutically acceptable excipients such as pH adjusting and buffering agents, and osmotic pressure adjusting agents, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, and calcium chloride, as necessary to approximate physiological conditions.

In another embodiment, the ApoA-I is administered in the form of a gel, or a solution of a polymer that forms a gel at the site of administration. In one embodiment, calcium alginate and certain other polymers may form a malleable ionic hydrogel. For example, hydrogels can be prepared by crosslinking anionic salts of alginic acid (a carbohydrate polymer isolated from seaweed) with calcium cations, the strength of which can be increased by increasing the concentration of calcium ions or alginate. The alginate solution is mixed with ApoA-I to form an alginate suspension which can be injected directly into a patient before the suspension hardens. The suspension then hardens within a short period of time due to the physiological concentrations of calcium ions present in the body. In addition, polysaccharides which gel upon contact with monovalent cations, including bacterial polysaccharides, such as gellan gum, and plant polysaccharides, such as carrageenan, other examples of materials which can be used to form hydrogels include polyphosphazines and polyacrylates which are ionically crosslinked, or block copolymers such as Pluronics (TM) or Tetronics (TM), polyethylene oxide-polypropylene glycol block copolymers which can be crosslinked by temperature or pH, respectively Hyaluronic acid and collagen. Polymers such as polysaccharides that are very viscous liquids or thixotropic and can form gels over time through slow evolution of structure can also be used. For example, hyaluronic acid may be used which forms an injectable gel having a viscosity similar to that of a hair spray. Modified hyaluronic acid derivatives are particularly useful. Mixtures of polymers may also be used. For example, a mixture of polyethylene oxide and polyacrylic acid that gels by hydrogen bonding when mixed may be used. In one embodiment, a 5% w/w solution of polyacrylic acid may be combined with a 5% w/w mixture of polyethylene oxide (polyethylene glycol, polyethylene oxide) 100,000 to form a gel over the course of time, e.g., a gel may form rapidly within a few seconds.

Covalently crosslinkable hydrogel precursors may also be used. For example, a water-soluble polyamine, such as chitosan, can be crosslinked with a water-soluble diisothiocyanate, such as polyethylene glycol diisothiocyanate. The isothiocyanate will react with the amine to form a chemically crosslinked gel. It is also possible to use the reaction of aldehydes with amines, for example with polyethylene glycol dialdehydes. Hydroxylated water-soluble polymers may also be used.

Alternatively, polymers containing substituents that can crosslink by free radical reaction upon contact with a free radical initiator can be used. For example, polymers comprising ethylenically unsaturated groups which can be photochemically crosslinked may be used, as described in WO 93/17699. In addition, as in Matsuda et al, ASAID trans, 38: 154-157(1992), water-soluble polymers comprising cinnamoyl groups that can be photochemically crosslinked can be used.

The gel material may be applied by spraying (in an open operation) or by INFILTRATOR or a catheter (in a closed operation). In general, ApoA-I alone, in combination with lipids, or in complex with lipids, is mixed with these gels during coagulation or polymerization and then allowed to slowly diffuse out on the surface of the treated vessel.

In another embodiment, the pharmaceutically acceptable carrier is coated on the stent. The carrier may be selected to release the substance of the invention in a time-dependent manner. The carrier can be varied by the skilled artisan to obtain an optimal coating of the stent to obtain a time-dependent release of the agent of the invention from the stent.

Methods of treatment

In a main embodiment, a cholesterol lowering agent such as apolipoprotein a-I (ApoA-I), preferably a variant form such as apolipoprotein a-imilar (ApoA-IM), is administered locally alone or more preferably in combination with a lipid carrier such as a phospholipid or another drug, with a reservoir device such as INFILTRATOR ®, prior to or during bypass surgery on diseased coronary, peripheral and cerebral arteries, implantation of grafts or organ transplants by surgery, or angioplasty, or stabilization of unstable plaques, thereby administering an effective dose to the site of injury. In other embodiments, the same techniques and materials may be used to reduce the consequences of plaque rupture, including thrombosis and ischemia.

In other preferred embodiments, local treatment is initiated in conjunction with systemic treatment, for example, with substances used to reduce restenosis, reduce or prevent plaque rupture, reduce blood cholesterol, reduce atherosclerotic-damaging cholesterol, reduce blood clotting, modulate one or more blood lipids (i.e., lipid modulators), reduce inflammation, or control blood pressure. Examples of drugs that can be used include lipid regulators such as nicotinic acid, statins, and fibrates; a substance for controlling blood glucose; anti-hypertensive agents; and substances which prevent or delay blood coagulation or platelet aggregation, such as aspirin, IIb/IIIa inhibitors, clopidogrel or heparin. These additional substances are generally administered systemically at their normal therapeutic dose.

Maximal benefit can be achieved by combining local delivery therapy with one or more combinations, such as the use of local delivery plus anticoagulation plus lipid modulation. These treatments may be initiated prior to, concurrently with, or after local delivery. The systemic treatment is preferably initiated prior to the local delivery procedure.

In a preferred embodiment, the Apo A-I formulation is administered by an intramural delivery device such as InFILTRATOR available from Intravential technologies, Inc, San Diego, Calif. (now owned by Boston Scientific). Pavlides et al, cathet. cardiovasc. diagn.41 (3): 287-292(1997) describes another useful device. Other delivery means may be used with catheters that deliver the drug from a reservoir prior to angioplasty, during balloon inflation, or after inflation.

In a most preferred embodiment, the ApoA-IM formulation is administered as a single dose prior to or at the time of treatment. Treatments include angioplasty, bypass surgery of the diseased coronary, peripheral or cerebral artery, implantation of vascular stents, implantation of transplanted organs or tissues, and stabilization of plaques.

The preferred dosage is determined by experimental studies, as is done in the ApoA-IM examples below. Based on the dosage of ApoA-IM, the dosage of other apolipoproteins can be easily calculated taking into account the differences between cholesterol removal efficacy, half-life, and other relevant pharmacokinetic parameters. Alternatively, the dosage of other apolipoproteins can be readily calculated by taking into account differences in efficacy of the antioxidant, anti-inflammatory and anti-thrombotic properties of the formulation. The lipids and the amount of lipids present in the different formulations can be similarly determined on the basis of experimental data obtained from ApoA-IM.

Generally, the formulation is administered to the treatment site. The actual dose for local delivery is significantly lower than the dose required to be administered systemically to achieve the same local dose, but the local concentration is much higher than that previously achieved in studies in which ApoA-I was administered systemically. As indicated above, the preferred dosage of ApoA-IM is from 4 to 6mg ApoA-IM per vessel (up to three segments may be treated with a total dosage of about 4 to 18mg ApoA-IM), or from about 0.05 to 0.3mg ApoA-IM per kg body weight for a 70kg mammal. The preferred ratio of protein to lipid is 1: 0.5 to 1: 3, more lipid can be used for cholesterol clearance, but for stability and continuity of the formulation, a more nearly equal amount of protein to lipid is more preferred for regulatory approval. For protein to lipid ratios of formulations other than those containing apoA-IM formulations, experiments were performed at various protein to lipid ratios and the stability and continuity as well as various characteristics (such as complex size and cholesterol flux) were determined for regulatory approval.

Although a single administration has proven effective, multiple doses may be administered. For example, 4 weeks after the procedure, intravenous administration of 20mg ApoA-IM/kg body weight on days-1, 0, 1, 2, and 3 resulted in all balloon-over-inflated injured vessels exhibiting increased luminal area relative to the control group.

The invention will be further illustrated with reference to the following non-limiting examples.

Percutaneous coronary intervention is now the primary method used to increase the narrowing of the vessel lumen in patients with coronary ischemia. These procedures are performed by Percutaneous Transluminal Coronary Angioplasty (PTCA), commonly referred to as "ballooning". In most cases, this is accomplished by expanding the stent in the dilated area to increase the vessel diameter, thereby increasing blood flow and relieving ischemia. The main drawback is the vessel lumen closure after the procedure, known as restenosis. In the absence of a stent, the incidence of early postoperative rebound and thrombosis is problematic, and therefore most balloon procedures today also involve stent deployment. Although stent deployment procedures improve outcome, neointimal growth of post-operatively dilated vessels can lead to restenosis and recurrent ischemia or other coronary events, including myocardial infarction. Therefore, methods for preventing neointimal growth in vessels treated with balloons and stents are needed to improve the post-operative outcome. The pig model was selected as a suitable test system for this study. Evidence in the literature suggests that arterial dilatation and restenosis in pig models is similar to that in humans. Thus, this model can be used to evaluate therapeutic substances that may be used in the treatment of clinical restenosis.

Example 1: ETC-216, a preparation containing a complex consisting of ApoA-IM and palmitoyl-oleoyl-phosphatidylcholine (POPC), was administered intravenously in a single high dose, with effects on restenosis in Percutaneous Transluminal Coronary Angioplasty (PTCA) that is over-stretched in the coronary arteries of expanded pigs.

The purpose of this study was to determine the effect of a single intravenous drug delivery of ETC-216 on restenosis in a porcine model of vascular injury during coronary PTCA and stent placement.

Materials and methods

Laboratory animal

The pig model was chosen as a suitable experimental system for this study. Evidence in the literature suggests that arterial dilatation and restenosis in porcine models are similar to that in human restenosis. Thus, this model can be used to evaluate therapeutic substances that may be useful in treating restenosis. Animals were acclimated to the laboratory for at least 7 days and were checked for health prior to starting the study.

Animals were housed individually for feeding during the course of the test and before surgery. Animal cages were cleaned twice daily. The temperature and humidity of the animal cages (70-78F.; 30-80% RH) were monitored to maintain them at 70-80℃ and 30-80% relative humidity. The air flow in the room is sufficient to provide several exchanges per hour with 100% fresh filtered air. An automatic timing device was used to provide a 12-hour day-night cycle alternation. After surgery, the animals were allowed to recover in a recovery room and then returned to their cages. Animals were fed a meal of swine feed (Southwest farm Hog finish diet) from Newco (Rancho Cucamonga, CA) daily throughout the experiment, except that the animals were fasted overnight on the day of surgery. Animals were given ad libitum access to fresh water via an automatic water supply system.

Animals were randomly selected and divided into two study groups. When an animal died or had to be euthanized as a result of a surgical procedure or as a complication due to a surgical procedure, additional animals were designated for study.

The test substance included ETC-216, a recombinant apolipoprotein A-I Milano/1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) complex provided by Imperion Therapeutics, Inc., in the form of a solution or saline for ease of injection. The ETC-216 solution contained about 14mg/ml apoA-IM protein in a ratio of about 1: 1 protein to POPC by weight.

Intravenous administration was chosen for this study because it is the route that will be used for human clinical studies. The dosage is selected according to the animal body weight. The test substance was administered at a dose of 100mg/kg body weight. Such dosage is based on the apolipoprotein content of the complex. Thus, an average pig received approximately 3000-3500mg of drug in this study. Control animals were given saline.

Adult pigs (weighing about 30-35kg, one weighing 50kg) were used for surgery. The animals were fed and housed normally. Pigs were fasted overnight before surgery and were pretreated with oral 325mg aspirin 3 days before surgery and thereafter daily until euthanasia. Animals were administered ticlopidine (250mg) 3 days before the start of surgery and daily for 14 days after surgery. After 16 hours overnight fast, animals were fixed and given an Intramuscular (IM) injection of acepromazine (0.5mg/kg), ketamine (20mg/kg), and atropine (0.05 mg/kg); induction of anesthesia by Intravenous (IV) administration of thiopental (5-8 mg/kg); and maintained by 1-2% isoflurane after endotracheal intubation. Mechanical ventilation, arterial Blood Pressure (BP) and continuous Electrocardiogram (ECG) monitoring are performed during the procedure. Animals were given diltiazem hydrochloride * (cardizem) (120mg) daily for 2 days after surgery.

The surgical procedure involves exposing the carotid artery and then inserting an 8F sheath into the carotid artery. The animals were given tolfenpyrad (250mg IV), propranolol hydrochloride (1mg) and heparin (10,000U IV) prior to the arterial device procedure. ETC-216 or saline is administered to the animals 90 minutes prior to the start of the surgical procedure, so that the entire dose is administered within about 3 hours. An 8F AL-0.75 guide catheter was advanced to the carotid ostium under fluoroscopic guidance. After intracoronary administration of nitroglycerin (200mcg), angiography was performed to assess the size of the vessels. The injury of stent overstretching is done in the first vessel and then repeated in the second vessel. In all cases, the stent was deployed in LAD and RCA. The position of the diagonal or septal struts used as anatomical references allows the stent to be deployed in both LAD and RCA at segments averaging 2.7 to 3.0mm in diameter. All stents were deployed using a balloon that inflated 1 to 3 times to 6-8 atmospheres in 30 seconds to reach the final stent: the ratio of arteries is about 1.3: 1. Angiography is initiated to align stent positions and repeated to confirm evidence of significant "ascending" or "descending" lesions in the lesion field at the site of stent deployment. The catheter was withdrawn and the ligated carotid artery and skin incision were closed. To prevent infection, antibiotics were given to all animals at the end of the procedure. The animals recovered from anesthesia, returned to the animal feeding facility, and fed conventional diet supplemented with the drugs described above.

Angiography and IVUS measurements

Quantitative Coronary Angiography (QCA) was used to assess the mean and minimum lumen diameter at each time point, i.e. before injury, immediately after injury and at a visit of 28-29 days before euthanasia.

Definition of terms used during quantitation

Coronary angiography:

MLD-mean lumen diameter

R1 ═ reference section for proximal section (unextended)

Prox, proximal segment of a dilated artery

Mid section of expanded artery

Dist, the distal segment of the expanded artery

R2 ═ reference section for distal section (unextended)

Max ═ maximum lumen diameter throughout the expanded section

Min-the minimum lumen diameter throughout the expanded section

Lumen increment (lumen diameter after injury minus lumen diameter before injury)

Luminal loss-lumen diameter after injury minus luminal diameter at 28 days follow-up

The percent stenosis of the damaged segment was estimated using the undamaged segment as a reference. The stent area, luminal area, neointimal area,% stenosis area parameters were measured with intravascular ultrasound (IVUS).

Late luminal loss was calculated from the difference between the MLD measured immediately after the capsular injury and the MLD measured at the 28-29 day follow-up, and the remodeling index was calculated by dividing the late luminal loss by the MLD after injury.

Analysis of coronary arteries at follow-up

After 28 (n-8) or 29 (n-1) days, the animals were fasted overnight and pre-treated as above for subsequent angiography. (in a control animal treated with physiological saline that died on day 27, only histological analysis was performed). Furthermore, subsequently, for IVUS studies of each stented artery, an IVUS catheter was deployed in the stented coronary artery. The animals were then euthanized under anesthesia with 90mg/kg of IV pentobarbital and the hearts were excised after the angiotomy. Coronary arteries were perfused with saline to clear blood, then fixed perfused with 2% paraformaldehyde for 15 minutes, then impregnated with 4% paraformaldehyde in phosphate buffer (pH 7.4) for 4 hours, and finally stored in 70% ethanol. To maintain the integrity of the adventitia and perivascular tissues, the coronary arteries were carefully removed along with the adjacent tissues (adipose tissue and myocardium). For the expanded section, specific histological treatments are performed to maintain the architecture of the vessel in situ with metal struts. The tissue mass was embedded in methyl methacrylate and cut with a diamond-edged knife. Three radial cross sections containing 12 struts were cut: one from the proximal third of each stent, one from the middle third of each stent, and one from the distal third of each stent. These portions were ground to a thickness of about 50 μm, optically polished, and stained with toluidine blue (paragon stain).

Tissue morphology analysis

The following histomorphometric measurements were performed using a computer-processed imaging system (Image Pro Plus 4.0):

1. average cross-sectional area and lumen thickness (area surrounded by intima/neointima-lumen margin); neointima (the area between the lumen and the inner elastic layer, IEL, and when IEL disappears, the area between the lumen and the remnants of the media or outer elastic layer, EEL); the middle membrane (the region between the IEL and the EEL); blood vessel size (the region surrounded by the EEL but excluding the adventitial region); and adventitia (periadventitial tissue, adipose tissue and myocardium, and the region between EELs).

2. And (6) scoring the damage. To quantify the extent of vascular injury, a score based on the number and length of different wall structure ruptures was made. The degree of damage was calculated as follows:

0 ═ complete IEL

1-IEL exposed to cracking of shallow intermediate layer

IEL exposed to rupture of deeper intermediate layer (intermediate dissection layer)

Broken EEL exposed to outer membrane

Results

Five pigs treated with saline or five pigs treated with ETC-216 (four male and one female pigs per group) were evaluated for restenosis 27-29 days after treatment.

During the course of the study, blood was obtained from some but not all animals and used to measure white blood cell count, red blood cell count, hemoglobin content, hematocrit%, platelet count. In all cases where these blood variables were measured, the post-treatment values did not change appreciably compared to baseline values. I.e. they are all within the normal range.

Heart rate and blood pressure were measured as baseline in all animals at the time of study entry. Heart rate and blood pressure were also measured in most animals during surgical procedures and before sacrifice. These variables were not appreciably altered from baseline values by surgical procedures or treatments for the animals under study.

Quantitative Coronary Angiography (QCA) measurements were performed at three time points: before coronary injury, immediately after coronary injury and after stent deployment, and before sacrifice (fig. 1a and 1 b). Diameter measurements (mm) were taken at the unexpanded section proximal to the stent (R1), the proximal section of the stent (Prox), the average area of the entire length of the stent (Aver), the distal section of the stent (Dist) and the unexpanded section distal to the stent (R2). Further, the maximum diameter (Max) and the minimum diameter (Min) of the expanded region were measured. QCA evaluation of cavity gain and cavity loss was measured at the extended and adjacent non-extended sections. The maximum (luminal loss maximum index) and minimum (luminal loss minimum index) indices of the expanded vessel were determined. Fig. 1a and 1b illustrate coronary angiography data quantified for all vessels (i.e. RCA and LAD combined) or various types of vessels (i.e. RCA or LAD), respectively.

Intravascular ultrasound (IVUS) is used to measure stent and lumen area at the distal, medial and proximal regions of each of the pre-mortem dilated coronary vessels. The difference between these measurements is the neointima area. The mean of the stent, luminal and neointimal regions for each animal and segment was measured and used to measure the mean of the pooled (LAD plus RCA) or each coronary vessel (LAD or RCA) treatment group. One control-treated pig died one day before its scheduled procedure (i.e., on day 27), and therefore, histomorphometric measurements were taken only on its expanded coronary vessels. Fig. 2a and 2b illustrate intravascular ultrasound data for all blood vessels (i.e., RCA and LAD in combination) and various types of blood vessels (i.e., RCA or LAD), respectively.

Histomorphological analysis of the expanded artery was used to measure the Adventitial Boundary Layer (ABL), the outer elastic layer (EEL), the Inner Elastic Layer (IEL), the lumen (L), the adventitia (a), the media (M), the mean cross-sectional area of the intima (I), the intima-to-media ratio (I/M) and the injury score. The lesion score is the mean of 36 lesion determinations obtained by measuring 12 times each of the proximal (a), medial (b) and distal (c) sections of the dilated portion of the vessel being dilated. Lesions were scored as 0, 1, 2 or 3 points, with 0 indicating intact IEL (i.e. no lesion) and 3 indicating ruptured EEL exposed to the adventitia (i.e. the most severe lesion).

ETC-216 treatment significantly reduced the intima-to-media (I/M) ratio of coronary vessels by 32% (RCA combined with LAD). This effect is mainly due to a 38% significant reduction in the I/M ratio of LAD and a minor 22% reduction (not significant) in RCA. It should be noted that, for the lesions, the rebound was more significant for RCA (lesion score of 1.87 ± 0.54) than for LAD (lesion score of 2.57 ± 0.34). Fig. 3a and 3b illustrate histomorphological data of all blood vessels (i.e., RCA and LAD in combination) and various types of blood vessels (i.e., RCA or LAD), respectively. Figure 4 shows the selective correlation between tissue morphology and IVUS variables.

Example 2: infiltrtator intramurally delivers ETC-216, an effect of a formulation containing a complex consisting of ApoA-IM and palmitoyl-oleoyl-phosphatidylcholine (POPC), on restenosis in porcine coronary arteries following overstretched Percutaneous Transluminal Coronary Angioplasty (PTCA) with stent deployment.

Materials and methods

The experiment was carried out with adult pigs weighing 25-30 kg. The animals are fed conventional feed and fed in an animal feeding room. Pigs were fasted overnight before surgery and pre-treated with oral aspirin (325mg) 3 days before surgery and thereafter daily until euthanasia. Animals were administered ticlopidine (250mg) 3 days before the start of surgery and daily for 14 days after surgery. Diltiazem hydrochloride * (120mg) was administered to the animals daily for 2 days after surgery.

After 16 hours overnight fast, animals were fixed and given an Intramuscular (IM) injection of acepromazine (0.5mg/kg), ketamine (20mg/kg), and atropine (0.05 mg/kg); induction of anesthesia by Intravenous (IV) administration of thiopental (5-8 mg/kg); and maintained by 1-2% isoflurane after endotracheal intubation. Mechanical ventilation, arterial Blood Pressure (BP) and continuous Electrocardiogram (ECG) monitoring are performed during the procedure.

The surgical procedure involves exposing the carotid artery and then inserting an 8F sheath into the carotid artery. The animals were given tolciclib (250mg IV), propranolol hydrochloride (1mg) and heparin (8,000U IV) prior to the arterial device manipulation intervention. An 8F AL-0.75 guide catheter was advanced to the carotid ostium under fluoroscopic guidance. After intracoronary administration of nitroglycerin (200mcg), quantitative coronary angiography was performed on all three coronary arteries to assess the size of the vessels. The two largest vessels are selected for this operation. ETC-216 or sucrose-mannitol media was administered intramurally through the INFILTRATOR in this procedure prior to PTCA deployment with a stent. An infiltrtator catheter was introduced to deliver ETC-216 or sucrose-mannitol media to the coronary vessel wall at a very low dose to minimize loss of agent into the circulation. Two arteries of each animal were infiltrated with 4-6mg of ETC216 or sucrose-mannitol media, each at a dosing volume of 0.3-0.4 ml. Thus, each animal received a total dose of about 8-12mg ETC-216 at both arterial sites. Each artery is subjected to an osmotic procedure by inflating the attached balloon once to a pressure of 1.5-2 atmospheres during delivery of ETC-216 or sucrose-mannitol media prior to balloon over-inflation with stent deployment. After the infiltration procedure, the stent is precisely deployed in the infiltrated sections of LAD, RCA, and LCX using the oblique or septal branch as an anatomical reference. All stents were deployed using a balloon that inflated 1 to 3 times to 6-8 atmospheres in 30 seconds to reach the final stent: the ratio of arteries is about 1.3: 1. Angiography is initiated to align the stent position and is repeated to confirm evidence of significant "ascending" or "descending" lesions at the site of stent expansion. The catheter was withdrawn and the ligated carotid artery and skin incision were closed. To prevent infection, antibiotics were given to all animals at the end of the procedure. The animals recovered from anesthesia, returned to the animal feeding facility, and fed conventional diet supplemented with the drugs described above.

Angiography and IVUS measurements

Quantitative Coronary Angiography (QCA) was used to assess the mean and minimum lumen diameter at each time point, i.e. before injury, immediately after injury and before euthanasia at a visit of 28 days. Late luminal loss was calculated from the difference between ML measured immediately after the capsular injury and MLD measured at 28 days of follow-up, and the remodeling index was calculated by dividing the late luminal loss by the MLD after injury.

Definition of terms used during quantitation

Coronary angiography:

r1 ═ reference section for proximal section (unextended)

P ═ proximal section of expanded artery

M-middle section of expanded artery

Distal segment of expanded artery

R2 ═ reference section for distal section (unextended)

Max is the maximum lumen diameter throughout the expanded section

Min is the smallest lumen diameter throughout the expanded section

Lumen increment (lumen diameter after injury minus lumen diameter before injury)

Luminal loss-lumen diameter after injury minus luminal diameter at 28 days follow-up

The percent stenosis of the lesion was estimated using the non-lesion as a reference. Measurements of stent area, luminal area, neointimal area, stenotic area (all performed with IVUS).

Analysis of coronary arteries at follow-up

After 28 days, for subsequent angiography, animals were fasted overnight and pre-operatively treated as above. Furthermore, subsequently, for IVUS studies of each stented artery, an IVUS catheter was deployed in the stented coronary artery. The animals were then euthanized under anesthesia with 90mg/kg of IV pentobarbital and the hearts were excised after the angiotomy. Coronary arteries were perfused with saline to clear blood, then fixed perfused with 2% paraformaldehyde for 15 minutes, then impregnated with 4% paraformaldehyde in phosphate buffer (pH 7.4) for 4 hours, and finally stored in 70% ethanol. To maintain the integrity of the adventitia and perivascular tissues, the coronary arteries were carefully removed along with the adjacent tissues (adipose tissue and myocardium). For the expanded section, specific histological treatments are performed to maintain the architecture of the vessel in situ with metal struts. The tissue mass was embedded in methyl methacrylate and cut with a diamond-edged knife. Three radial cross sections containing 12 struts were cut: one from the proximal third of each stent, one from the middle third of each stent, and one from the distal third of each stent. These portions were ground to a thickness of about 50 μm, optically polished, and stained with toluidine blue (paragon stain).

Tissue morphology analysis

The following histomorphometric measurements were performed using a computer-processed imaging system (Image Pro Plus 4.0):

1. average cross-sectional area and lumen thickness (area surrounded by intima/neointima-lumen margin); neointima (the area between the lumen and the inner elastic layer, IEL, and when IEL disappears, the area between the lumen and the remnants of the media or outer elastic layer, EEL); the middle membrane (the region between the IEL and the EEL); blood vessel size (the region surrounded by the EEL but excluding the adventitial region); and adventitia (periadventitial tissue, adipose tissue and myocardium, and the region between EELs).

2. And (6) scoring the damage. To quantify the extent of vascular injury, a score based on the number and length of different wall structure ruptures was made. The degree of damage was calculated as follows:

0 ═ complete IEL

1-IEL exposed to cracking of shallow intermediate layer

IEL exposed to rupture of deeper intermediate layer (intermediate dissection layer)

Broken EEL exposed to outer membrane

Results

Two coronary arteries from fourteen pigs, respectively, were treated with sucrose-mannitol vehicle (control) or 4-6mg ETC-216 (n-7 per group). Performing Quantitative Coronary Angiography (QCA) of Left Anterior Descending (LAD), left circumflex vessels (LCX) and Right Coronary Arteries (RCA) to assess the size of each vessel; the two largest arteries are selected for this operation. In each artery, drugs were delivered intramurally by infiltrtator, followed by hyper-stenting Percutaneous Transluminal Coronary Angioplasty (PTCA) at the drug delivery site. This surgical procedure induces a vascular injury in which inflammation, neointimal hyperplasia and restenosis form. Stented arteries from all arteries were QCA prior to expansion, immediately after expansion, and at day 28 just prior to sacrifice. In addition, IVUS was used to determine stent and lumen area to estimate neointimal area just prior to sacrifice. After sacrifice, expanded arterial segments for histomorphometric measurements were obtained and evaluated for the extent of hyperstretch injury, the amount of neointimal hyperplasia, and restenosis. Coronary vessels from one medium-treated pig that died 9 days prior to their scheduled QCA and IVUS procedure were analyzed histologically only. One of the ETC-216-treated pigs was euthanized due to the wrong treatment schedule 6 days after the scheduled 28-day operation. Data from both pigs were excluded from the analysis for fair comparison.

In addition, two animals were treated with an approximately 3-fold concentration of the ETC-216 formulation delivered using INFILTRATOR. The formulation was found to be too viscous to effectively deliver the drug through the device and was found to cause damage to the balloon of the device, thereby increasing the amount of arterial injury, which limited the use of viscous solutions with the device.

During the course of the study, blood was obtained from all animals and used to measure white blood cell count, red blood cell count, hemoglobin content, hematocrit%, platelet count. In all cases where these blood variables were measured, the post-treatment values did not change appreciably compared to baseline values. I.e. they are all within the normal range. Heart rate and blood pressure were measured as baseline in all animals at the time of study entry. Heart rate and blood pressure were also measured in most animals during surgical procedures and before sacrifice. These variables were not appreciably altered by the surgical procedure or treatment compared to baseline values for the animals entering the study.

Quantitative Coronary Angiography (QCA) measurements were performed at three time points: before coronary artery injury, immediately after coronary artery injury and stent deployment, and before sacrifice. Diameter measurements (mm) were taken at the unexpanded section proximal to the stent (R1), the proximal section of the stent (Prox), the average area of the entire length of the stent (Aver), the distal section of the stent (Dist) and the unexpanded section distal to the stent (R2). Intravascular ultrasound (IVUS) is used to measure stent and lumen area at the distal, medial and proximal regions of each of the pre-mortem dilated coronary vessels. The difference between these measurements is the neointima area.

Histomorphological analysis of the expanded artery was used to measure the Adventitial Boundary Layer (ABL), the outer elastic layer (EEL), the Inner Elastic Layer (IEL), the lumen (L), the adventitia (a), the media (M), the mean cross-sectional area of the intima (I), the intima-to-media ratio (I/M) and the injury score. The lesion score is the mean of 36 lesion determinations obtained by measuring 12 times each of the proximal (a), intermediate (b) and distal (c) sections of the dilated vessel. Lesions were scored as 0, 1, 2 or 3 points, with 0 indicating intact IEL (i.e. no lesion) and 3 indicating ruptured EEL exposed to the adventitia (i.e. the most severe lesion).

Treatment with ETC-216 delivered by intramural infiltrtator significantly reduced the intima-to-media ratio of coronary vessels by 35% (LAD, LCX, and RCA combination). This effect is mainly due to the significant reduction in I/M ratio of RCA (-42%, p ═ 0.002), LCX (-38%), and LAD (-29%). Fig. 5a and 5b illustrate histomorphological data of all blood vessels (i.e., RCA, LCX, and LAD association) and various types of blood vessels (i.e., RCA or LAD), respectively.

Claims (31)

1. Use of an alpha helical apolipoprotein or HDL associated protein for the preparation of a pharmaceutical composition suitable for local use at the site of injury to a blood vessel for the prevention or treatment of stenosis or restenosis or for the stabilization of plaques, before or during bypass surgery on diseased coronary, peripheral and cerebral arteries, surgery to implant grafts or transplanted organs, or angioplasty.

2. The use according to claim 1, wherein the apolipoprotein or HDL associated protein is selected from the group consisting of ApoA-I, ApoA-I Milano, ApoA-I Paris, ApoE, proApoA-I, ApoA-II, proApoA-II, ApoA-IV, apolipoproteins modified to include one or more sulfhydryl groups, ApoC-I, ApoC-II, and ApoC-III, alpha-helical sequences in these apolipoproteins, paraoxonase, cholesteryl ester transfer protein, LCAT and phospholipid transfer protein.

3. The use according to claim 2 wherein the apolipoprotein is ApoA-IMilano.

4. The use of claim 1 wherein the apolipoprotein or HDL associated protein is used in combination with a lipid.

5. The use according to claim 4 wherein the apolipoprotein is used in combination with a lipid and HDL associated protein.

6. The use of claim 4 wherein the weight ratio of apolipoprotein to lipid is about 1: 0.5 to 1: 3.

7. The use of claim 1 wherein the pharmaceutical composition prepared using the alpha helical apolipoprotein or HDL associated protein is suitable for administration by an intraluminal osmotic device.

8. The use of claim 1 wherein the pharmaceutical composition prepared using the apolipoprotein or HDL associated protein is suitable for administration by catheter.

9. The use according to claim 1, wherein the pharmaceutical composition is prepared with the apolipoprotein in a gel.

10. Use according to claim 3, in which the ApoA-I Milano is used in combination with phospholipids in a weight ratio of about 1: 0.5 to 1: 3, at a dose of 0.05 to 0.3mg ApoA-IMilono/kg or 4 to 6mg ApoA-I Milano/vascular segment to be treated.

11. The use according to claim 1, wherein the apolipoprotein or HDL associated protein is used in a dose between 0.01mg apolipoprotein/kg and a dose of 0.4mg apolipoprotein/kg.

12. The use of claim 11 wherein the apolipoprotein is used in a dose of 0.3mg apolipoprotein/kg.

13. Use according to claim 1, wherein for the preparation of a pharmaceutical composition a nucleic acid molecule delivered by intramural infiltration is used.

14. The use according to claim 13, wherein the nucleic acid molecule encodes an alpha helical apolipoprotein or HDL associating protein.

15. The use of claim 13, wherein the nucleic acid molecule is an oligonucleotide.

16. The use as claimed in claim 1, wherein the pharmaceutical composition prepared using the alpha helical apolipoprotein or HDL associating protein is suitable for administration in a single effective dose.

17. The use according to claim 1, wherein the pharmaceutical composition prepared using the alpha helical apolipoprotein or HDL associated protein is suitable for administration in multiple doses.

18. The use of claim 1, wherein the pharmaceutical composition is suitable for use in combination with therapy with a systemically administered drug selected from the group consisting of antiproliferative compounds, anti-inflammatory compounds, antihypertensive compounds, anticoagulants, and lipid modulating agents.

19. The use according to claim 18, wherein the systemic treatment is initiated before the local treatment.

20. The use according to claim 18, wherein the lipid modulating agent is selected from the group consisting of niacin, statins, and fibrates.

21. The use according to claim 18, wherein the antiproliferative agent is selected from paclitaxel, rapamycin, tirofiban, and abciximab.

22. The use according to claim 18, wherein the anticoagulant is selected from the group consisting of aspirin, lib/IIIa inhibitors, clopidogrel, heparin and heparin fragments.

23. The use of claim 1, wherein the pharmaceutical composition is adapted for local administration by release from a catheter into the pericardial space.

24. The use according to claim 1, wherein the pharmaceutical composition is suitable for release by use of a coated stent.

25. The use according to claim 1, wherein the pharmaceutical composition is adapted to reduce plaque rupture consequences including thrombosis and ischemia.

26. A kit for treating or preventing stenosis or restenosis or stabilizing plaque prior to or during bypass surgery on diseased coronary, peripheral and cerebral arteries, surgery to implant grafts or transplanted organs, angioplasty or plaque stabilization, comprising an alpha helical apolipoprotein or HDL associating protein according to claim 1 for long term local release of an effective amount.

27. The kit of claim 26, comprising an intraluminal infiltration device.

28. The kit of claim 26 comprising a catheter in combination with a reservoir for apolipoprotein and/or lipid.

29. The kit of claim 26 comprising means for administering a nucleic acid molecule encoding an alpha helical apolipoprotein or HDL associated protein, or naked DNA encoding an alpha helical apolipoprotein or HDL associated protein.

30. The kit of claim 26, wherein the apolipoprotein or HDL associated protein is selected from the group consisting of ApoA-I, ApoA-I Milano, ApoA-I Paris, Apo E, proApoA-I, ApoA-II, proApoA-II, ApoA-IV, apolipoproteins modified to include one or more sulfhydryl groups, ApoC-I, ApoC-II, and ApoC-III, alpha-helical sequences in these apolipoproteins, paraoxonase, cholesteryl ester transfer protein, LCAT, and phospholipid transfer protein.

31. A stent coated with a substance to be released at a site to be treated selected from the group consisting of an alpha helical apolipoprotein or HDL-associated protein formulated alone or together with a lipid, cells expressing a gene encoding the alpha helical apolipoprotein or HDL-associated protein, and naked DNA encoding the alpha helical apolipoprotein or HDL-associated protein for local delivery to a lesion site.