WO2014049574A2 - Methods for the stabilization of arterial plaque for the treatment of ischemic stroke and peripheral artery disease - Google Patents

Abstract

The present invention relates generally to medical methods. More particularly, the present invention relates to medical methods for delivering in a targeted and reproducible manner, lipid binding agents to the adventitial tissue adjacent to a lipid rich necrotic core in an artery for the prevention and treatment of ischemic stroke and peripheral artery disease. Under the guidance of an external ultrasound probe, an echogenic needle is percutaneously placed in the adventitial layer of an artery whereby a sufficient amount of a lipid binding agent is delivered to rapidly stabilize the lipid rich necrotic core of the targeted artery. Such delivery amount and location are sufficient to at least partially delipidate the lipid rich necrotic core.

Description

TITLE OF INVENTION

Methods for the Stabilization of Arterial Plaque for the Treatment of Ischemic Stroke and

Peripheral Artery Disease

CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] The present application claims the benefit of the following United States provisional application: No. 61/744,503, filed September 28, 2012 which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0001] Field of the Invention [0002] The present invention relates generally to medical methods. More particularly, the present invention relates to medical methods for delivering in a targeted and reproducible manner, lipid binding agents to the adventitial tissue surrounding a blood vessel containing an accumulation of lipid rich plaque as a component of vascular disease and more specifically in the treatment and prevention of ischemic stroke and peripheral artery disease.

[0003] Vascular disease consists of a broad group of diseases of the heart and blood vessels. One of the most common vascular diseases stems from the progression of atherosclerosis. Atherosclerosis occurs as a result of a complex cycle of events that range from the infiltration of monoctytes across the endothelial lining of a blood vessel, the differentiation of monocytes into macrophages, the oxidation and uptake of oxidized LDL by macrophages, the upregulation of certain adhesion molecules and the conversion of macrophages into foam cells. This complex process results in the accumulation of different types of plaque ranging from cholesterol and other lipid based plaques to fibrous and fibro-calcific plaques. The latter representing a phenotype that tends to cause vessel stenosis, but also tends to be more stable and less prone to rupture. Approximately 70% of ischemic events are caused by a plaque rupture. This includes thrombo-embolic myocardial infarctions as well as embolic ischemic strokes. [0004] Upon rupture of a plaque lesion, the lipid rich plaque often spills out into the blood flow conduit. This can result in a partial or complete blockage of blood flow or an embolic clot that can travel to a more distal and smaller diameter vessel causing a complete or partial occlusion. Plaque ruptures occur when a stable plaque phenotype becomes acutely unstable. The conversion from stable to unstable of a plaque lesion is an inflammation mediated process which largely impacts the lipid based plaques in the lipid rich necrotic cores of arterial blood vessels. The lipid rich core consists largely of macrophage and foam cells.

[0005] Carotid artery disease consists of a narrowing of the common, internal and or external right or left carotid arteries due to the buildup of plaque within the artery wall usually at the bifurcation of the common carotid artery. Upon rupture of a carotid plaque, an embolic clot can travel downstream and occlude a smaller distal cerebral vessel causing an acute ischemic stroke or transient ischemic attack. The presence of a lipid rich necrotic core is a marker for increased risk of plaque rupture along with an increased density of adventitial vasa vasorum and intra plaque neovascularization. These latter two markers can be readily assessed using contrast enhanced ultrasound. Once a patient has been deemed at high risk for a rupture of the carotid artery, the invasive treatments consist of carotid endarterectomy or carotid artery stenting. Both of these treatments have their benefits and risks. For those patients that have already experienced an ischemic event where the carotid artery is the culprit lesion, the risk of a recurrent event at or adjacent to the same location is substantially elevated for the following 3 - 4 weeks. As such the current standard of care is to perform a surgical endarterectomy whereby the entire tunica media layer is removed. The procedure is designed to reduce the risk of a recurrent stroke yet the procedure itself carries the risk of causing an ischemic event. There is no treatment that can effectively reduce the risk of a recurrent event during the high risk period immediately following an ischemic event where the carotid artery is the culprit lesion.

[0006] Peripheral artery disease consists of a broad range of plaque formation within one or more of the peripheral arteries. For the purposes of this invention, the formation of plaque is contained at least at the bifurcation of the common femoral artery into the superficial femoral artery due to the disturbed laminar flow at the bifurcation. The underlying pathology of the superficial artery region, however is distinct from the carotid artery in that the lipid rich plaques that occur within the superficial femoral artery do not rupture in the manner that the carotid arteries do. As such the targeting of the bifurcation of the superficial femoral artery needs to be able to stabilize the lipid rich plaque to prevent the progression of vessel calcification and flow limiting stenosis. [0007] The most commonly administered drug to treat vascular disease is statins. Statins work to reduce the levels of circulating low density lipoprotein thereby reducing the amount of oxidized LDL present in the arterial wall. When administered over long periods of time, statins can be effective at reducing the risk of an ischemic event, however they are not effective at rapidly stabilizing the underlying lipid based plaque that is responsible for the acute ischemic event.

[0008] One particular lipid binding agent is high density lipoprotein (HDL). HDL has a known effect on removing cholesterol from arterial walls in a process known as reverse cholesterol transport. The primary protein in HDL is known as ApoA-1. ApoA-1 is capable of binding directly to macrophages and removing cholesterol from the core of the cell. HDL infusions have been clinically evaluated with intravenous administration and found to reduce the size of atheroma volume over a 4 week period. The doses administered in these limited clinical studies were in the range of 20 mg/kg to 80 mg/kg. The dosing used in these studies presents a significant manufacturing challenge.

[0009] The local catheter based delivery of HDL based therapeutics has been evaluated as a treatment for restenosis as HDL has also been shown to possess anti-inflammatory properties that may be useful in the reduction of restenosis. Because the introduction of catheter into the arterial system is an invasive procedure, these studies utilized a single administration to the site of stenosis. The local uptake via intramural infusion is typically less than 2% of the total injectate volume. The single administration and very low tissue uptake creates a challenge for the rapid stabilization of arterial plaque.

[0010] Transluminal catheter based delivery to the perivascular region of a blood vessel provides enhanced tissue uptake of a therapeutic over catheter based intramural infusion. This delivery technique however is limited by the required surgical access techniques required to introduce the injection catheter into the relevant blood vessel. This limitation significantly reduces the frequency of and timing of drug regimens that can be administered. Additionally while catheter based, local delivery of lipid binding agents can be anticipated from the prior art, the use of a catheter to locally deliver a drug to a carotid or peripheral artery containing a lipid rich necrotic core is extremely dangerous due to the potential for dislodging plaque contained along the treatment area. A safer alternative for drug delivery is lacking in the prior art.

Description of the Background Art

[0011] The following references are pertinent to the use of local delivery of therapeutics to treat vascular disease: Soma MR, Donetti E, Parolini C, Sirtori CR, Fumagalli R, Franceschini G. Recombinant

100 apolipoprotein A-l Milano dimer inhibits carotid intimal thickening induced by perivascular manipulation in rabbits. Circ Res. 1995;76:405-11. Chiesa G, Monteggia E, Marchesi M, et al. Recombinant apolipoprotein A-l(Milano) infusion into rabbit carotid artery rapidly removes lipid from fatty streaks. Circ Res. 2002;90:974-80. Kaul S, Rukshin V, Santos R, et al. Intramural delivery of recombinant apolipoprotein A-IMilano/phospholipid complex (ETC-216) inhibits in-stent

105 stenosis in porcine coronary arteries. Circulation. 2003;107:2551-4. Herdeg C, Gohring-Frischholz K, Haase K, Geisler T, Zurn C, Hartmann U, Wohrle J, Nusser T, Dippon J, May AE, Gawaz M. Catheter-Based Delivery of Fluid Paclitaxel for Prevention of Restenosis in Native Coronary Artery Lesions After Stent Implantation. Circulation: Cardiovascular Interventions. 2009; 2: 294-30. LincoffAM, Topol EJ, Ellis SG. Local drug delivery for the prevention of restenosis. Fact, fancy, and

110 future. Circulation. 1994; 90: 2070-208. Herdeg C, Oberhoff M, Baumbach A, Blattner A, Kuttner A, Schroder S, Haase KK, Karsch KR. Visualization and comparison of drug effects after local paclitaxel delivery with different catheter types. Basic Res Cardiol. 1999; 94: 454-463. Dommke C, Haase KK, Suselbeck T, Streitner I, Haghi D, Metz J, Borggrefe M, Herdeg C. Local paclitaxel delivery after coronary stenting in an experimental animal model. Thromb Haemost. 2007; 98:

115 674-680. Tepe G, Zeller T, Albrecht T, Heller S, Schwarzwalder U, Beregi JP, Claussen CD, Oldenburg A, Scheller B, Speck U. Local delivery of paclitaxel to inhibit restenosis during angioplasty of the leg. N Engl J Med. 2008; 358: 689-699. Bultmann A, Herdeg C, Li Z, Munch G, Baumgartner C, Langer H, Kremmer E, Geisler T, May A, Ungerer M, Gawaz M. Local delivery of soluble platelet collagen receptor glycoprotein VI inhibits thrombus formation in vivo. Thromb

120 Haemost. 2006; 95: 763-766. Ikeno F, Lyons J, Kaneda H, Baluom M, Benet LZ, Rezaee M. Novel percutaneous adventitial drug delivery system for regional vascular treatment. Catheter Cardiovasc Interv. 2004 Oct;63(2):222-30. Karanian JW, Peregoy JA, Chiesa OA, Murray TL, Ahn C, Pritchard WF. Efficiency of Drug Delivery to the Coronary Arteries in Swine Is Dependent on the Route of Administration: Assessment of Luminal, Intimal, and Adventitial Coronary Artery and

125 Venous Delivery Methods. Journal of Vascular and Interventional Radiology. Volume 21, Issue 10, October 2010, Pages 1555-1564. Sanders WG, Hogrebe PC, Grainger DW, Cheung AK, Terry CM. A biodegradable perivascular wrap for controlled, local and directed drug delivery. J Control Release. 2012 Jul 10;161(l):81-9. Owens CD, Gasper W, Alley HF, Chong KC, Rapp JH, Conte MS, Seward K, Grenon MS. Adjunctive Dexamethasone Infusion into the Adventitial of the Femoro-

130 popliteal Artery to Enhance Clinical Efficacy Following Endovascular Revascularization: A Proof of Concept First in Man Study. Journal of Vascular Surgery. 2013 Aug; Volume 58, Issue 2 , Page 565. [0012] The following references are pertinent to the use of therapeutics for the treatment of some forms of vascular disease:

135

Getz GS, eardon CA. Apolipoprotein A-l and A-l mimetic peptides: a role in atherosclerosis. J Inflamm Res. 2011;4:83-92. Shah PK, Yano J, Reyes O, Chyu KY, Kaul S, Bisgaier CL, Drake S, Cercek B. High-dose recombinant apolipoprotein A-I(milano) mobilizes tissue cholesterol and rapidly reduces plaque lipid and macrophage content in apolipoprotein e-deficient mice. Potential

140 implications for acute plaque stabilization. Circulation. 2001;103:3047-3050. Nicholls SJ, Cutri B, Worthley SG, Kee P, Rye KA, Bao S, Barter PJ. Impact of short-term administration of high-density lipoproteins and atorvastatin on atherosclerosis in rabbits. Arterioscler Thromb Vase Biol. 2005;25:2416 -2421. Ibanez B, Vilahur G, Cimmino G, Speidl WS, Pinero A, Choi BG, Zafar MU, Santos-Gallego CG, Krause B, Badimon L, Fuster V, Badimon JJ. Rapid change in plaque size,

145 composition, and molecular footprint after recombinant apolipoprotein A-l Milano (ETC-216) administration: magnetic resonance imaging study in an experimental model of atherosclerosis. J Am Coll Cardiol. 2008;51:1104 -1109. Nissen SE, Tsunoda T, Tuzcu EM, Schoenhagen P, Cooper CJ, Yasin M, Eaton GM, Lauer MA, Sheldon WS, Grines CL, Halpern S, Crowe T, Blankenship JC, Kerensky R. Effect of recombinant ApoA-l Milano on coronary atherosclerosis in patients with

150 acute coronary syndromes: a randomized controlled trial. JAMA. 2003;290:2292-2300. Tardif JC, Gregoire J, L'Allier PL, Ibrahim R, Lesperance J, Heinonen TM, Kouz S, Berry C, Basser R, Lavoie MA, Guertin MC, Rodes-Cabau J. Effects of reconstituted high-density lipoprotein infusions on coronary atherosclerosis: a randomized controlled trial. JAMA. 2007;297: 1675-1682. Nicholls SJ, Dusting GJ, Cutri B, Bao S, Drummond GR, Rye KA, Barter PJ. Reconstituted high-density

155 lipoproteins inhibit the acute pro-oxidant and proinflammatory vascular changes induced by a periarterial collar in normocholesterolemic rabbits. Circulation. 2005;111:1543-1550. Rong JX, Li J, Reis ED, Choudhury RP, Dansky HM, Elmalem VI, Fallon JT, Breslow JL, Fisher EA. Elevating high- density lipoprotein cholesterol in apolipoprotein E-deficient mice remodels advanced atherosclerotic lesions by decreasing macrophage and increasing smooth muscle cell content.

160 Circulation. 2001;104:2447-2452. Shah PK, Yano J, Reyes O, Chyu KY, Kaul S, Bisgaier CL, Drake S, Cercek B. High-dose recombinant apolipoprotein A-I(milano) mobilizes tissue cholesterol and rapidly reduces plaque lipid and macrophage content in apolipoprotein e-deficient mice. Potential implications for acute plaque stabilization. Circulation. 2001 Jun 26;103(25):3047-50. Diditchenko S, Gille A, Pragst I, Stadler D, Waelchli M, Hamilton R, Leis A, Wright SD. Novel Formulation of a

165 Reconstituted High-Density Lipoprotein (CSL112) Dramatically Enhances ABCAl-Dependent Cholesterol Efflux. Arteriosclerosis, Thrombosis, and Vascular Biology. 2013; 33: 2202-2211. Shaw JA, Bobik A, Murphy A, Kanellakis P, Blombery P, Mukhamedova N, Woollard K, Lyon S, Sviridov D, Dart AM. Infusion of Reconstituted High-Density Lipoprotein Leads to Acute Changes in Human Atherosclerotic Plaque. Circulation Research. 2008; 103: 1084-109. Bielicki JK, Zhang H, Cortez Y,

170 Zheng Y, Narayanaswami V, Patel A, Johansson J, Azhar S. A new HDL mimetic peptide that stimulates cellular cholesterol efflux with high efficiency greatly reduces atherosclerosis in mice. J Lipid Res. 2010 Jun;51(6):1496-503. Navab M, Anantharamaiah GM, Reddy ST, Van Lenten BJ, Buga GM, Fogelman AM. Peptide Mimetics of Apolipoproteins Improve HDL Function. J Clin Lipidol. 2007 May;l(2):142-7. Ibanez B, Vilahur G, Cimmino G, Speidl WS, Pinero A, Choi BG, Zafar

175 M L⁾, Santos-Gallego CG, Krause B, Badimon L, Fuster V, Badimon JJ. Rapid change in plaque size, composition, and molecular footprint after recombinant apolipoprotein A-l Milano (ETC-216) administration: magnetic resonance imaging study in an experimental model of atherosclerosis. J Am Coll Cardiol. 2008 Mar 18;51(ll):1104-9.

180 [0013] The following references are pertinent to the use of ultrasound guided needle placement:

Raza K, Lee CY, Pilling D, Heaton S, Situnayake RD, Carruthers DM, Buckley CD, Gordon C, Salmon M. Ultrasound guidance allows accurate needle placement and aspiration from small joints in patients with early inflammatory arthritis. Rheumatology (Oxford). 2003 Aug;42(8):976-9. Zhu Y, 185 Magee D, Ratnalingam R, Kessel D. A training system for ultrasound-guided needle insertion procedures. Med Image Comput Comput Assist Interv. 2007;10(Pt l):566-74. Roessel T, Wiessner D, Heller AR, Zimmermann T, Koch T, Litz RJ. High-resolution ultrasound-guided high interscalene plexus block for carotid endarterectomy. Reg Anesth Pain Med. 2007 May-Jun;32(3):247-53.

190 BRIEF SUMMARY OF THE INVENTION

[0014] Methods according to the present invention are able to achieve enhanced concentrations of lipid binding agents in lipid rich necrotic cores contained within a targeted blood vessel. More importantly these methods are safer than currently used delivery techniques in that they significantly avoid the potential to disrupt plaque from the endothelial side when accessed

195 transluminal^. The methods rely on percutaneous, direct injection to the adventitial layer of a lipid binding agent containing at least one class A amphipathic alpha helix using an externally introduced needle. More specifically the needle has been modified to have echogenic properties. The echogenic needle is advanced through the skin adjacent to the targeted lipid rich area under the guidance of an externally placed ultrasound probe. The methods are applicable to deliver a 200 therapeutic to two distinct blood vessels: any of the carotid arteries and the common and superficial femoral artery. For the targeting of the carotid artery, the needle is ideally introduced at an oblique angle to the longitudinal axis of the targeted blood vessel and the targeted injection site is preferably at or proximal to the lipid rich plaque. In a preferred embodiment at least 50% of the injectate is to be delivered within the area outside of the external elastic lamina and the outer

205 extent of the adventitial and perivascular connective tissues that surround the targeted carotid artery. The targeting of high density vasa vasorum within the carotid artery can also be used to further enhance the concentration within the lipid rich necrotic core due to its extension in atherosclerotic patients from the adventitial into the media and intima layer. For the targeting of the femoral artery, at least one injection site is ideally within 5 centimeters distal or proximal to

210 the bifurcation of the common femoral artery into the superficial femoral artery. The targeted injection site can be along either the common femoral artery or the superficial femoral artery. Multiple injection sites along the superficial femoral artery extending distal to the popliteal artery can be used. Ideally at least one injection site should be within 5 em's of the common femoral bifurcation. The patient must have less than a 60% stenosis of the superficial femoral artery

215 adjacent to the bifurcation. The significance of the stenosis cutoff is due to the higher prevalence of fibro-calcific and calcific plaques often seen in stenosis equal to or greater than 60% in the superficial femoral artery. The lipid binding agents described herein have no known effect on these types of plaque. The direct injection to either the carotid or femoral artery can be performed multiple times allowing for sustained therapeutic tissue concentrations.

220

[0015] The methods have been found to have a number of advantages over the prior art.

[0016] First, direct injection into the adventitial region has been found to provide immediate high concentrations to the tissue immediately surrounding the targeted lipid rich necrotic core. These 225 concentrations (a) are well in excess of those seen when administered systemically, (b) create elevated levels seen within ten minutes after the injection and (c) utilize substantially smaller injection volumes than systemically administered drugs.

[0017] Second, it has been found by us that injecting lipid binding agents as described herein into 230 the adventitial region surrounding a lipid rich necrotic core allows the agent to diffuse in a circumferentially and longitudinally concentration dependent manner. This allows for the whole of the lipid rich necrotic core to be blanketed and to reach smaller and less developed plaques. [0018] Third, by using a percutaneous, direct injection approach, repeat drug dosings can be 235 administered in daily intervals. This is a key advantage over catheter based injection protocols.

[0019] Fourth, by using an external ultrasound probe coupled to an echogenic needle, the tip of the needle can be precisely positioned close to the external elastic lamina allowing for maximum transmural diffusion across the external elastic lamina into the media and intima. This is 240 particularly important when using steep insertion angles as non echogenic needles have poor visibility under ultrasound at steep angles. The use of ultrasound coupled to an echogenic needle can also be used to help target the area of high density vasa vasorum within the carotid artery allowing for further enhanced concentrations of drug within the lipid rich necrotic core.

245 [0020] Fifth, by using a percutaneous, direct injection, the risk of plaque disruption from the endothelial side is significantly diminished versus catheter based methods. This is an extremely critical risk factor especially when targeting the carotid artery that is avoided with the methods described herein.

250 [0021] The use of a lipid binding agent is limited to pharmaceutical formulations with an active drug containing at least one class A amphipathic alpha helix as the secondary structure. A more specific embodiment of the current invention uses at least one ApoA-1 protein of SEQ ID NO: 1. The preferred embodiment uses a peptide mimetic between 10 and 40 amino acids in length, such peptide containing at least one class A amphipathic alpha helix mimicking the lipid binding

255 properties of the ApoA-1 protein. While a lipid free or lipid bound mixture can be used, the preferred embodiment is the lipid binding agent complexed in a lipid bound state to a neutral or anionic phospholipid. This is due to the tendency of amphipathic molecules to aggregate in a concentration dependent manner in the lipid free state. The desired tissue concentration is in the range of 10 - 30 mgs per dosing but can be higher or lower depending on the size of the lipid rich

260 necrotic core. In the lipid free state, this would require significantly higher volumes to effectively prevent pre-dosing aggregation such higher volumes being less advantageous. Additionally as the lipid binding agent is delivered precisely where it needs to go based on a specific lipid rich necrotic core, the ideal tissue concentration is based on the size of the core and not on body weight or patient surface area. This is distinct from the dosing regimen for systemic

265 administration.

DETAILED DESCRIPTION OF THE INVENTION [0022] The present invention is designed to address the need for safer and more effective 270 methods for the rapid stabilization of a lipid rich necrotic core contained within a targeted blood vessel for the treatment and prevention of ischemic stroke and peripheral artery disease. As used herein, the stabilization of a lipid rich necrotic core can be performed by the at least partial delipidation of macrophages or foam cells contained within the lipid rich necrotic core or the binding to free cholesterol contained within and adjacent to the lipid rich necrotic core and the at 275 least partial removal of it from the arterial wall. While both ischemic stroke and peripheral artery disease have their own unique pathology, the underlying cause of the elevated risk is often the vulnerability to rupture of the lipid rich necrotic core contained within the artery.

[0023] In order that the present invention may be more readily understood, certain terms are first 280 defined. Additional definitions are set forth throughout the detailed description.

[0024] As used herein, "ApoA-1" refers to the human, wild type protein of apolipoprotein A-l encoded by the APOA1 gene.

285 [0025] As used herein, "peptide mimetic" refers to any peptide containing between 10 and 40 amino acids with at least one Class A amphipathic alpha helix as a secondary structure.

[0026] As used herein, "lipid binding agent" refers to any pharmaceutical formulation with an active ingredient containing at least one class A amphipathic alpha helix as its secondary 290 structure.

[0027] As used herein, "transluminal" refers to the use of an artery for access to a target site.

[0028] As used herein, "percutaneous" refers to access to a target site by way of a needle 295 puncture through the skin.

[0029] As used herein, "complexed", "complexing", "complex" refers to the combining or combination of a lipid binding agent as used and defined in this document with a phospholipid.

300 [0030] As used herein, "lipid free state" refers to an ApoA-1 protein or peptide mimetic that exists substantially in the absence of any phospholipids. [0031] As used herein, "lipid bound state" refers to an ApoA-1 protein or peptide mimetic that exists substantially complexed to a phospholipid.

305

[0032] As used herein, "injectate" refers to the lipid binding agent that gets delivered during an injection procedure as described herein.

[0033] As used herein, "echogenic needle" refers to any needle attached or attachable to a 310 syringe that can be at least partially visualized under ultrasound.

[0034] Methods according to the present invention are able to achieve enhanced concentrations of lipid binding agents in lipid rich necrotic cores contained within a targeted blood vessel capable of reaching a therapeutically effective dose sufficient to at least partially delipidate a lipid rich

315 necrotic core. The targeted blood vessels are any of the common, internal or external carotid arteries or any of the common femoral or superficial femoral arteries. The methods rely on percutaneous, direct injection to the adventitial layer of a lipid binding agent using a percutaneously introduced echogenic needle. In a preferred embodiment, the outer boundary of the external elastic lamina is targeted with a needle trajectory that is oblique to the longitudinal

320 axis of the external elastic lamina. These methods have been found to have a number of advantages over the prior art.

[0035] First, direct injection into the adventitial region has been found to provide immediate high concentrations to the tissue immediately surrounding the targeted lipid rich necrotic core of lipid

325 binding agents such as those described herein. One particular lipid binding agent is the use of high density lipoprotein (HDL) containing at least one ApoA-1 protein. While there are numerous examples in the prior art using systemic infusions of HDL based therapeutics for the treatment of acute coronary syndromes, the immediate and high concentrations are not seen with systemic infusions of such HDL therapeutics due to the delivery method. With these systemic infusions, the

330 HDL therapeutic is subjected to metabolic degradation and modification and is distributed widely across off target tissue beds. When treating ischemic stroke, the time to reach peak concentration directly in the relevant tissue bed is extremely important. The patient is at high risk for a recurrent event, and an immediate and high concentration is needed to reduce the risk. The direct injection into the carotid artery adventitial layer provides a very rapid high concentration as the typical

335 injection cycle is less than 2 minutes. This is in direct contrast to systemic administration where an intravenous infusion is administered over a two to four hour window with peak concentrations being reached several hours later. Even with high systemic doses, the peak concentration in a targeted tissue bed does not reach the levels seen with less than 2% of the dose administered according to the methods herein. For catheter based interventions, there are two common types

340 of administrations: (a) intramural infusion and (b) transluminal delivery to the adventitia using needle based catheters. For either route of administration, a patient needs to have a catheter inserted into the arterial circulation system via a cut down procedure. This in itself takes time and carries risk. The local tissue uptake for intramural infusion is typically 1-2% into the intima layer when the patient has a heavy plaque burden. The rest of the injectate simply washes downstream

345 with the blood flow. The use of transluminal delivery to the adventitia is the most effective catheter based delivery technique with drug concentration reaching up to 60% post delivery. The limitations of this delivery route however, are numerous and include among others the time to get the catheter inserted, the difficulty in knowing where in the adventitia the injection is occurring as the delivery is not performed under ultrasound, the inability to target the area of

350 vasa vasorum, the risk of bleeding from the cut down and the needle perforation across the endothelium and the risk of causing plaque rupture into the blood flow lumen. This last limitation is severe particularly when targeting the carotid artery of the ischemic stroke patient. The transluminal catheter based delivery to the adventitia procedures typically use a stainless steel needle based catheter where the needle penetrates through the endothelial layer, past the media

355 and into the adventitia. This creates several issues. The first issue is the elevated risk of plaque rupture from simply placing the catheter at the site of plaque. The catheter placement is done under the use of fluoroscopy, however, fluoroscopy imaging is not capable if visualizing the lipid rich necrotic core. So the practitioner has no effective way of knowing where the catheter or needle placement is relative to the targeted lipid rich necrotic core. The second issue is that the

360 needle has to perforate the endothelial layer causing a bleeding risk to the patient. This bleeding risk is independent of that seen at the site of catheter insertion into the body. Once the needle passes through the endothelial layer, it has to pass through the plaque filled intima and media layer and into the adventitia. For heavily calcified lesions, this creates a significant potential for plaque disruption and simply is not safe for use in the carotid artery. The needle advancement

365 into the adventitia is an estimate. There is no way to know if the adventitia has been reached as the layers of the blood vessel are not visible under fluoroscopy. One option that has been studied is the use of a small amount of contrast agent injected out of the needle to confirm its position. This confirmation is done by looking at the diffusion pattern of the drug. The problem with this option is that the catheter and needle are now prepped with contrast which can react with lipid 370 binding agents, and this contrast is injected into various parts of the arterial wall. Using this option, if the catheter and needle placement are incorrect, the catheter has to be repositioned and the needle is then advanced through the endothelium again creating a new perforation. This creates a new potential for plaque disruption and greater bleeding risk. Because of these factors, this technique would never be applied in the carotid and has limitations even in the superficial

375 femoral artery. The methods described herein overcome these limitations.

[0036] Second, it has been found by us that when injecting lipid binding agents into the adventitial region surrounding a lipid based plaque, the agent diffuses in a circumferentially and longitudinally concentration dependent manner. This allows for the whole of the lipid rich core to

380 be blanketed and to reach smaller and less developed plaques. This widespread diffusion and targeting of the lipid rich necrotic core are not possible with systemic drug infusions or with catheter based drug delivery. The lipid binding agents as described herein have many functions including anti-inflammatory and anti-oxidant properties, however, the function employed for the methods described herein is the ability to reversibly bind to lipid filled macrophages and to at

385 least partially delipidate them. In order for this reversible binding to occur, it is important that a sufficient amount of the lipid binding agent is delivered directly to the area containing the lipid rich necrotic core in a sufficient concentration.

[0037] Third, by using a percutaneous, direct injection approach, repeat drug dosings can be 390 administered in daily intervals due to the ease of administration and limited patient recovery time from the injection procedure. This is a key advantage over catheter based injection protocols as the patient has a bleeding risk associated with the catheter insertion procedure and often requires some recovery time. Systemic drug infusions are not subject to the limitations of patient recovery as seen with catheter based delivery routes, however, when drugs are administered 395 systemically, the dosing can only be re-administered at half life intervals of the drug. This is not a limiting factor for the methods described herein as the only limiting factor is the time for local drug clearance from the targeted injection site. This is approximately 1 day for an HDL based therapeutic delivered according to the methods contained herein versus approximately 7 days for a systemic infusion of an HDL based therapeutic.

400

[0038] Fourth, by using an external ultrasound probe capable of visualizing an echogenic needle, the tip of the needle can be precisely positioned close to the outer boundary of the external elastic lamina allowing for maximum transmural diffusion across the external elastic lamina into the media and intima. The ultrasound probe can also be used to help target the area of high

405 density vasa vasorum within the carotid artery allowing for further enhanced concentrations of drug within the lipid rich necrotic core. There is also more flexibility for precise needle placement if repeat dosings are used. For example if the size or composition of the lipid rich necrotic core changes between injection times, the placement of the needle can be tailored to the new information. The use of an echogenic needle is important particularly when steep needle

410 insertion angles are used. Steeper angles of standard needles make it much harder if not impossible to visualize the tip of the needle.

[0039] Fifth, by using a percutaneous, direct injection, the risk of plaque disruption from the endothelial side is significantly diminished versus catheter based methods. This is an extremely

415 critical risk factor especially when targeting the carotid artery that is greatly reduced with the methods described herein. The risks of causing a plaque disruption are so great when treating the carotid artery that the standard of care for placing a carotid artery stent by means of a catheter is to use an embolic filtration device. The filtration device is typically placed just distal to the area where the stent will be placed so that any debris dislodged from the artery wall while the stent is

420 being positioned and deployed will be captured and pulled out with the system. This embolic positioning device is so critical that the approval of carotid artery stents requires the use of an embolic filtration device. The placement of a distal embolic filtration device itself is a bit controversial in that the device crosses the treatment area before being positioned and deployed, thus creating a risk of dislodging plaque before the temporary filter is in place to capture any

425 debris. When targeting any artery with a plaque that is potentially vulnerable to rupture (including either the carotid or the superficial femoral artery), the ability to deliver an effective amount of a therapeutic without the potential to disrupt or dislodge plaque is a critical advantage. The methods described herein provide a significant advantage over the current methods for drug delivery.

430

[0040] The present invention consists of the percutaneous, direct echogenic needle based injection of a lipid binding agent containing at least one class A amphipathic alpha helix to a superficial blood vessel that can be accessed percutaneously under the guidance of an external ultrasound probe. In one embodiment the lipid binding agent contains a pharmaceutical 435 formulation with an active ingredient that contains at least one ApoA-1 protein of SEQ ID NO: 1.

In a more preferred embodiment, a peptide mimetic with lipid binding properties similar to ApoA- 1 consisting of SEQ ID NO: 4 is used. It is important to note, however, that any lipid binding agent contained within a pharmaceutical formulation with an active ingredient containing at least one class A amphipathic alpha helix as its secondary structure can be used as the lipid binding agent. The superficial arteries to be targeted consist of any of the common, internal or external carotid arteries for the treatment of ischemic stroke as well as any of the common and superficial femoral arteries for the treatment of peripheral artery disease. For additional clarification, the treatment of ischemic stroke includes a transient ischemic attack. [0041] The methods described herein use an echogenic needle attached to a syringe containing a lipid binding agent. The use of an echogenic needle visualized under the guidance of ultrasound is critical for precise tip visualization particularly at steep insertion angles. While there are various techniques for surface modifications that can be done to the outer surface of a needle to improve its visibility under ultrasound, nothing contained herein is intended to limit the methods to a particular type of modification except to say that the methods contained herein are intended to be performed by way of a stainless steel or polymer needle that was intentionally modified to be more visible under ultrasound than a conventional stainless steel or polymer needle. The preferred embodiment uses a stainless steel needle with a surface that has been textured for enhanced visualization under ultrasound. There are additional methods for visualizing conventional stainless steel and or polymer needles that can also allow for precise tip localization. These include CT and MRI among others. The methods described herein are limited to the use of ultrasound for all needle visualization.

[0042] Such echogenic needle is advanced through the skin adjacent to the lipid rich plaque of a superficial artery and is advanced towards the adventitial layer ideally at or proximal to the lipid rich plaque. The echogenic needle tip is advanced and positioned under the guidance of an externally located ultrasound probe. The basic procedure consists of the use of a handheld or otherwise mounted ultrasound probe (typically a 7.5 Mhz linear array probe but can be any frequency and mode capable of imaging the needle tip) and a small gage needle ideally 22 gage or smaller and 1.5 inches or longer, but can also be of any diameter and length as may be necessary to optimally reach and treat the intended artery. The placement of the ultrasound probe is ideally at or close to perpendicular to the planned needle trajectory. The desired approach angle of the needle towards the adventitia is oblique to the longitudinal axis of the targeted blood vessel and more specifically to the outer boundary of the external elastic lamina. 470 In one aspect of the invention, the echogenic needle can be a hybrid needle capable of electrical stimulation in addition to ultrasound visualization.

[0043] The use of ultrasound guidance for needle placement has been used for the administration of nerve blocks agents as the ultrasound allows for the visualization of both the nerve as well as 475 the needle tip. For the current invention, the ultrasound imaging visualizes both the needle tip and the external elastic lamina. The external elastic lamina serves as a physical barrier between the more densely arranged media layer and the looser adventitia. It is this barrier that provides guidance for needle placement and the optimal delivery zone for the lipid binding agent.

480 [0044] The region of interest to be targeted is the adventitia of the lipid rich containing blood vessel. A typical arterial wall consists of the endothelium as the basement membrane which in turn is surrounded by the intima. The intima is surrounded by the internal elastic lamina over which is located the tunica media. The media is covered by the external elastic lamina which acts as the outer barrier separating the arterial wall from the adventitial layer. It is common to refer to

485 anything lying beyond the external elastic lamina as the perivascular space, including regions within the adventitia and beyond.

[0045] The adventitial layer is the outermost layer of a blood vessel consisting of collagen and elastic fibers with fibroblasts and smooth muscle cells. The layer is loosely organized to permit

490 vasoconstriction and dilatation. This looser arrangement is different than the tightly packed smooth muscle cells in the tunica media layer. Because of this looser arrangement, delivery to a single location within the adventitia allows for a circumferential and longitudinal volume dependent diffusion of injectate. Once the tip of the needle has passed into the adventitial space as determined by ultrasound, the lipid binding agent is delivered into the adventitial layer. The

495 needle can be advanced into the media past the outer boundary of the external elastic lamina but should be pulled back such that most or all of the delivery occurs within the adventitial layer.

[0046] For the treatment of ischemic stroke, a preferred embodiment delivers at least 50% of the lipid binding agent within the adventitia. This ensures that the majority of the injection can 500 diffuse into the media and intima (location of the majority of the lipid rich plaques) while avoiding excessive venous and lymphatic drainage. This is less of an issue when targeting the superficial femoral artery and is not required. As most of the sites of carotid plaque buildup occur at the carotid sinus, an injection site that is proximal to the lipid rich necrotic core area and that targets the adventitia of the common carotid just proximal to the bifurcation is preferred and allows for 505 the diffusion of drug to both the internal and external carotid arteries. The targeting of the common carotid bifurcation also provides for excellent anatomical localization which can be useful for repeat dosing. This procedure can be repeated with as many doses as necessary to at least partially stabilize the lipid rich necrotic core. Additionally the right or left carotid arteries can be targeted individually or together as may be required for each or subsequent dosings.

510

[0047] In a normal vessel there is a shallow infiltration of adventitial vasa vasorum. Vasa vasorum are arterioles that exist to ensure cells farther from larger blood vessels are properly nourished. In an atherosclerotic vessel containing a lipid rich necrotic core, there can be significantly increased vasa vasorum density extending from the adventitia to the inner media and sometimes directly to

515 the intima layer due to the effects of positive vessel remodeling and changes in arterial pressure.

Because the external vasa vasorum originates in the adventitia, targeted delivery to the adventitia in patients with increased external vasa vasorum density allows for an additional conduit for drug diffusion into the media and intima enabling a more direct targeting of the lipid rich necrotic core. For the treatment and prevention of ischemic stroke, the targeting of high density vasa vasorum

520 within a carotid artery allows for an enhanced concentration within the media layer as well as within the lipid rich necrotic core. This targeting is not possible with either systemic infusion or catheter based delivery methods. In one aspect of the invention described herein, an area of high density vasa vasroum can be first located prior to inserting the echogenic needle. In another aspect, the use of ultrasound contrast can be used once the needle is in place within the

525 adventitia to locate the site of highest density vasa vasorum.

[0048] Because of the targeted, local delivery, a significantly smaller amount of lipid binding agent is used than for either systemic or catheter based delivery. The present invention uses doses for the carotid artery that are ideally between 0.5 and more than 5 mi's with a concentration ideally

530 between 0.5 and 20 mg/ml. The dosing for the femoral artery is ideally between 0.5 and 10 mi's with a concentration ideally between 0.1 and 20 mg/ml. It is important to note, however, that the purpose of the delivery is for the rapid stabilization of the lipid rich necrotic core. As such any combination of volume and concentration can be used to achieve this rapid stabilization. For the treatment of peripheral artery disease, the ideal injection site should be within 5 em's of the

535 bifurcation of the common femoral artery in a superficial artery containing less than 60% stenosis.

The significance of the stenosis cutoff is due to the higher prevalence of fibro-calcific and calcific plaques often seen in stenosis equal to or greater than 60% in the superficial femoral artery. Lipid binding agents as described herein have no known effect on these types of plaques. The target tissue concentration is approximately 10 - 30 mg but the dosing is based on the size of the lipid 540 rich necrotic core to be treated and not based on body mass or patient surface area. This is distinct from intravenous (systemic) administration where the doses are typically between 20 mg/ kg and 80 mg/kg of patient weight.

[0049] The injection time is greater than 30 seconds for up to 2 mi's of fluid. In a preferred 545 embodiment, the injection time is at least 60 seconds. Additionally, for larger volumes, the ideal site of injection is more proximal to the lipid rich necrotic core. A general rule is an additional cm for each additional ml of volume.

[0050] The lipid rich necrotic core of a blood vessel consists of highly lipidated macrophage cells, 550 free cholesterol and other lipids and typically originate within the intima layer of an artery. These scavenger cells continue to accumulate modified lipids including oxidized and acetylated low density lipoproteins (LDL) from the surrounding arterial area and progress to foam cells. Eventually these cells die and spill their contents into the surrounding lipid rich pool thereby increasing the size of the lipid rich necrotic core and further destabilizing the local arterial area. 555 Rapidly delipidating these macrophages as well as binding to free cholesterol can reduce the size of the lipid rich necrotic core and reduce the expression of pro-inflammatory cytokines within the microenvironment. As such lipid binding agents that are designed to directly bind to macrophages and remove cholesterol are used to address this need.

560 [0051] The lipid binding agents as described herein have many functions, however, the function employed for the methods described herein is the ability to reversibly bind to lipid filled macrophages to at least partially delipidate them as well as to bind to free cholesterol. These lipid binding agents are further limited to those pharmaceutical formulations with an active ingredient containing at least one class A amphipathic alpha helix as its secondary structure. One aspect of

565 the methods described herein is the use of a pharmaceutical formulation containing at least one ApoA-1 protein. A more preferred embodiment uses a peptide mimetic between 10 and 40 amino acids in length that contains at least one Class A amphipathic alpha helix that mimics the lipid binding properties of ApoA-1. ApoA-1 and peptide mimetics that mimic the Class A amphipathic alpha helix of ApoA-1 are known to bind directly to macrophages and remove cholesterol from 570 the core of the macrophage cells via the S -B1 and ABC-A1 cell surface receptors. SR-B1 and ABC- Al receptors are highly expressed in lipid rich macrophages contained within lipid rich necrotic cores of both carotid and superficial femoral arteries. It is the receptor mediated offloading of cholesterol and the ability to bind free cholesterol that makes the ApoA-1 and peptide mimetics the lipid binding agent used herein for the rapid stabilization of a lipid rich necrotic core. While

575 much focus has been placed on the use of systemic HDL infusions to treat acute coronary syndromes, the main goal of the present methods is the rapid stabilization of the lipid rich necrotic core for the treatment of ischemic stroke and peripheral artery disease. Rapid stabilization can occur by partially delipidating the lipid rich macrophages in a manner that may not statistically reduce the overall size of the plaque burden. However successful treatment can

580 be determined by looking at the changes in the echogenicity of the lipid rich necrotic core, with such successful treatment reducing the incidence of an initial or recurrent ischemic event.

[0052] Any ApoA-1 protein containing at least 90% sequence homology to the human wild type (SEQ ID NO:l) can be used as the lipid binding agent. ApoA-1 is the major protein component of

585 HDL in plasma. The protein promotes cholesterol efflux from tissues to the liver for excretion and constitutes approximately 70% of the total protein content of HDL particles. While any ApoA-1 based compound consisting of at least 90% sequence homology to the human wild type can be used, one particular embodiment consists of the ApoA-1 Milano (SEQ ID No: 2) mutation. The Milano mutation consists of a cysteine substitution for arginine at position 173. This naturally

590 occurring mutation was discovered in 1979 in a small village in the North of Italy. The substitution of the cysteine mutation allows for the formation of dimers via a disulfide bridge. These ApoA-1 Milano dimers can have enhanced stability in both the lipid free and lipid bound form as well as better particle size homogeneity in the lipid bound form when complexed to phospholipids.

595 [0053] Any peptide mimetic containing between 10 and 40 amino acids with at least one class A amphipathic alpha helix as its secondary structure can be used including the following: SEQ ID NO's: 3,4, 5 or 6. The preferred embodiment uses SEQ ID NO: 4. It is important to note, however, that any lipid binding agent contained within a pharmaceutical formulation with an active ingredient containing at least one class A amphipathic alpha helix as its secondary structure can

600 be used as the lipid binding agent.

[0054] There are many known techniques for producing the ApoA-1 proteins and peptide mimetics including various cell based expression systems for the proteins as well as solid phase synthesis for the peptides. Nothing contained herein is intended to limit the methods described 605 herein to a particular method of manufacturing the proteins or peptides.

[0055] While the ApoA-1 or peptide mimetic drug can be administered in the lipid free state, it can also be administered in a lipid bound state alone or in combination with lipid free drug. There are several phospholipids that can be used. Examples include but are not limited to POPC (1-

610 palmitoyl-2-oleoyl-s/i-glycero-3-phosphocholine), PAPC ( l-palmitoyl-2-arachidonoyl-s/i-glycero-3- phosphocholine), DMPC (l,2-dimyristoyl(d54)-s/i-glycero-3-phosphocholine), and DPPC (1,2- dipalmitoyl-s/i-glycero-3-phosphocholine). The complex can consist of a single phospholipid or one or more distinct phospholipids. The phospholipids can be neutral or anionic. The molar ratio of phospholipid to protein is ideally approximately 100:1 but can be anything from 25:1 to 500:1

615 (phospholipid to protein). The molar ratio of phospholipid to peptide mimetic is ideally approximately 10:1 but can be anything from 1:1 to 50:1 (phospholipid to peptide). The preferred complex uses the POPC phospholipid.

[0056] When ApoA-1 or peptide mimetic is complexed to a phospholipid, it forms a nanoparticle 620 sized micelle structure with at least one ApoA-1 protein or peptide mimetic embedded at least partially on the surface of the complex. The hydrophobic core of these particles is largely empty allowing for the accumulation of lipids from lipidated macrophage cells. The use of small amounts of cholesterol can also be added to the formulation to improve the stability of the formulation, however the preferred embodiment does not use any cholesterol added to the formulation. 625 Depending on the number of ApoA-1 proteins or peptides, the particle size can vary. With the ApoA-1 Milano protein in dimeric form, the number of ApoA-1 proteins occurs in even numbers. While any number of proteins can be used, one embodiment consists of a formulation with two to four ApoA-1 proteins per particle with a particle size distribution between 7 and 13 nm in diameter with a pre beta electrophoretic mobility. A formulation containing any number of 630 peptide mimetics per particle can be used with the preferred formulation consisting of a particle size distribution between 7 and 13 nm in diameter also with a pre beta electrophoretic mobility.

[0057] There are many known techniques for complexing ApoA-1 and peptide mimetics with a phospholipid known in the art. These include but are not limited to sonication, extrusion, 635 adsorbent resins and detergent based dialysis. Briefly the creation of these complexes take advantage of the amphipathic nature of ApoA-1 or peptide mimetics including the Class A alpha helical repeating motifs. The lipids are first dissolved in a suitable organic solvent which is then evaporated. The dry lipid film is rehydrated in a suitable buffer. The appropriate ApoA-1 or peptide mimetic is added to a non ionic detergent which is then incubated and added to the 640 rehydrated lipid film. The detergent is removed to allow for membrane incorporation of the ApoA-1 protein or peptide mimetic and self assembly. However, there are numerous variations that can occur when creating these particles and any method suitable for commercial scale up under cGMP conditions can be used.

645 [0058] The lipid binding agent can be supplied as a pharmaceutical formulation in various forms none of which are intended to be limiting. The preferred form is as a lyophilized powder to be re- suspended in suitable physiological buffer at the time of administration. The agent can be supplied in a prefilled syringe with a pre-defined concentration. It can also be supplied in a frozen state and thawed prior to use. In this manner, it can contain small amounts of suitable

650 cryopreservatives customary for storing and shipping material at subfreezing temperatures.

[0059] The following examples are for illustrative purposes only and are not meant to be limiting in any way to only that which is included within the examples.

655 [0060] Example 1 A patient can be identified as moderate or high risk for ischemic stroke from a number of sources, however, the most likely candidate will be one who has at least a 70% carotid vessel stenosis and is symptomatic for a recent ischemic event where the carotid artery is the culprit lesion. Once the patient has been identified, the patient would normally be scheduled for endarterectomy. Using the present invention, the patient would be subjected to the disclosed

660 methods. The patient would be brought into a treatment room. The treatment room can be a cath lab or outpatient clinic equipped with ultrasound. The culprit carotid artery is identified along with the lipid rich necrotic core at or near the culprit artery. Based on the size of the lipid rich plaque, a drug dosing to be administered is determined. A linear array ultrasound probe is placed at the site of the culprit lesion. An echogenic needle is advanced though the skin at or

665 proximal to the lipid rich plaque at an angle less than 90 degrees to the longitudinal axis of the targeted blood vessel. The needle and linear array probe are approximately perpendicular to each other. The needle tip is advanced adjacent to the outer boundary of the external elastic lamina. The lipid binding agent is injected over a period of 60 seconds. The needle is then withdrawn. Additional dosings can be administered as needed including once a day or once a week until the

670 lipid rich necrotic core is adequately stabilized. Stabilization can be assessed in a number of ways but the easiest way is to look at changes in the echogenicity of the lipid core. [0061] Example 2 A patient complains of intermittant claudication and is diagnosed with peripheral artery disease. An angiography is performed on the superficial femoral artery and

675 there is a non flow limiting stenosis less than 60% present. An angioplasty is not performed. The presence of soft, lipid rich plaque is located within the superficial artery using ultrasound or other non invasive imaging, and the patient is subjected to the methods described herein. An echogenic needle attached to a syringe filled with a lipid binding agent is advanced through the skin targeting the superficial femoral artery at a site ideally within 5 em's of the bifurcation. The

680 adventitial is reached, and the lipid binding agent is injected over 60 seconds. A second injection site is targeted 5 em's distal to the first injection site, and the injection procedure is repeated. The needle is withdrawn and pressure is applied against both injection sites to ensure no excessive bleeding.

685 EXPERIMENTAL

[0062] The following Experiments are offered by way of illustration and are not intended to limit the methods to those used in the experiments.

690 [0063] Studies were performed to show visual and quantitative evidence of deposition of agents in the adventitia and the biodistribution of the delivered agents from the various sites of injection. The study was designed to assess the distribution and delivery mechanics of various agents including lipid binding agents. These agents were injected into the adventitia of the superficial femoral arteries of 10 micro pigs. Injections were made with volumes between 1 and 3

695 mi's into various regions within the adventitia and perivascular region surrounding the blood vessels. The use of contrast was used for some injections to visually verify the extent and timing of both circumferential and longitudinal diffusion as well as the volume dependent diffusion effects. Various agents were injected including lipid based agents, lipid based agents in polymeric encapsulation, and genes. Genes encoding for two different ApoA-1 proteins (wild type and

700 Milano) were also delivered to verify the expression patterns within the targeted arterial wall.

Animals were sacrificed at 24 hours and 7 days for assessment. ELISA against the ApoA-1 was used to correlate the visual diffusion pattern with actual cellular uptake and expression.

[0064] In vitro studies were conducted to assess the macrophage binding properties of lipid 705 binding agents such as those described herein. Briefly THP-1 cells were differentiated into macrophages which were then incubated with fluorescently labeled oxidized LDL. These cells were then treated with various lipid binding agents such as those described herein. Studies were conducted against sham and positive controls and demonstrated the ability of lipid binding agents such as those described herein to at least partially delipidate lipidated macrophage and foam 710 cells.

[0065] While the above is a complete description of the preferred embodiments of the invention, various alternatives, modifications, and equivalents may be used. Therefore, the above description should not be taken as limiting the scope of the invention which is defined by the 715 appended claims.

[0066] SEQ ID NO: 1

Met Lys Ala Ala Val Leu Thr Leu Ala Val Leu Phe Leu Thr Gly Ser

Gin Ala Arg His Phe Trp Gin Gin Asp Glu Pro Pro Gin Ser Pro Trp

720 Asp Arg Val Lys Asp Leu Ala Thr Val Tyr Val Asp Val Leu Lys Asp

Ser Gly Arg Asp Tyr Val Ser Gin Phe Glu Gly Ser Ala Leu Gly Lys

Gin Leu Asn Leu Lys Leu Leu Asp Asn Trp Asp Ser Val Thr Ser Thr

Phe Ser Lys Leu Arg Glu Gin Leu Gly Pro Val Thr Gin Glu Phe Trp

Asp Asn Leu Glu Lys Glu Thr Glu Gly Leu Arg Gin Glu Met Ser Lys

Asp Leu Glu Glu Val Lys Ala Lys Val Gin Pro Tyr Leu Asp Asp Phe

Gin Lys Lys rp Gin Glu Glu Met Glu Leu Tyr Arg Gin Lys Val Glu

Pro Leu Arg Ala Glu Leu Gin Glu Gly Ala Arg Gin Lys Leu His Glu

Leu Gin Glu Lys Leu Ser Pro Leu Gly Glu Glu Met Arg Asp Arg Ala

Arg Ala His Val Asp Ala Leu Arg Thr His Leu Ala Pro Tyr Ser Asp

730 Glu Leu Arg Gin Arg Leu Ala Ala Arg Leu Glu Ala Leu Lys Glu Asn

Gly Gly Ala Arg Leu Ala Glu Tyr His Ala Lys Ala Thr Glu His Leu

Ser Thr Leu Ser Glu Lys Ala Lys Pro Ala Leu Glu Asp Leu Arg Gin

Gly Leu Leu Pro Val Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala

Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr Gin

735

[0067] SEQ ID NO: 2

Met Lys Ala Ala Val Leu Thr Leu Ala Val Leu Phe Leu Thr Gly Ser

Gin Ala Arg His Phe Trp Gin Gin Asp Glu Pro Pro Gin Ser Pro Trp

Asp Arg Val Lys Asp Leu Ala Thr Val Tyr Val Asp Val Leu Lys Asp

740 Ser Gly Arg Asp Tyr Val Ser Gin Phe Glu Gly Ser Ala Leu Gly Lys

Gin Leu Asn Leu Lys Leu Leu Asp Asn Trp Asp Ser Val Thr Ser Thr

Phe Ser Lys Leu Arg Glu Gin Leu Gly Pro Val Thr Gin Glu Phe Trp Asp Asn Leu Glu Lys Glu Thr Glu Gly Leu Arg Gin Glu Met Ser Lys

Asp Leu Glu Glu Val Lys Ala Lys Val Gin Pro Tyr Leu Asp Asp Phe

Gin Lys Lys Trp Gin Glu Glu Met Glu Leu Tyr Arg Gin Lys Val Glu

Pro Leu Arg Ala Glu Leu Gin Glu Gly Ala Arg Gin Lys Leu His Glu

Leu Gin Glu Lys Leu Ser Pro Leu Gly Glu Glu Met Cys Asp Arg Ala

Arg Ala His Val Asp Ala Leu Arg Thr His Leu Ala Pro Tyr Ser Asp

Glu Leu Arg Gin Arg Leu Ala Ala Arg Leu Glu Ala Leu Lys Glu Asn

Gly Gly Ala Arg Leu Ala Glu Tyr His Ala Lys Ala Thr Glu His Leu

Ser Thr Leu Ser Glu Lys Ala Lys Pro Ala Leu Glu Asp Leu Arg Gin

Gly Leu Leu Pro Val Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala

Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr Gin

755 [0068] SEQ ID NO: 3

Asp Trp Phe Lys Ala Phe Tyr Asp Lys Val Ala Glu Lys Phe Lys Glu Ala Phe

[0069] SEQ ID NO: 4

760 Asp Trp Phe Lys Ala Phe Tyr Asp Lys Val Ala Glu Lys Phe Lys Glu Ala Phe Pro Asp Trp Phe Lys Ala Phe Tyr Asp Lys Val Ala Glu Lys Phe Lys Glu Ala Phe

[0070] SEQ ID NO: 5

765 Asp Trp Leu Lys Ala Phe Tyr Asp Lys Val Phe Glu Lys Phe Lys Glu Phe Phe

[0071] SEQ ID NO: 6

Trp Leu Lys Ala Phe Tyr Asp Lys Val Ala Glu Lys Leu Lys Glu Al 770 Phe Pro Asp Trp Ala Lys Ala Ala Tyr Asp Lys Ala Ala Glu Lys Al Lys Glu Ala Ala

Claims

What is claimed is:

1 A method for the treatment of ischemic stroke, said method comprising the steps of: identifying a human patient with an accumulation of one or more lipid rich necrotic cores contained within one or more carotid arteries; introducing in a percutaneous manner an echogenic needle towards the outer boundary of the external elastic lamina of the identified one or more carotid arteries whereby such advancement is done under the guidance of external ultrasound; and delivering a lipid binding agent into the adventitial layer of the identified one or more carotid arteries in an amount sufficient to at least partially delipidate the lipid rich necrotic core contained within the one or more identified carotid arteries.

2 A method as in claim 1, wherein at least 50% of the lipid binding agent is delivered between the external elastic lamina and the outer extent of the adventitial and perivascular connective tissues that surround said one or more identified carotid arteries.

3 A method as in claim 1, wherein the primary goal of the delivery is the stabilization of the targeted artery such that endarterectomy is not required.

4 A method as in claim 1, wherein the patient is identified following an ischemic stroke or transient ischemic attack.

5 A method as in claim 1, wherein the delivery volume is sufficient to allow for substantially circumferential distribution around the adventitial tissue at or adjacent to the lipid rich necrotic core.

6 A method as in claim 1, wherein the echogenic needle penetration angle is oblique to the longitudinal axis of the external elastic lamina.

7 A method as in claim 1, further comprising the step of identifying the area of the highest density of adventitial vasa vasorum contained within or near the lipid rich necrotic core followed by delivering a lipid binding agent to the adventitial space nearest that area.

8 A method as in claim 7, wherein the targeting of the adventitial vaso vasorum is intended to serve as an additional conduit for the delivery of the lipid binding agent from the adventitial layer into the area surrounding the lipid rich necrotic core of the targeted artery.

9 A method as in claim 7, wherein the adventitial vasa vasorum is also accompanied by intra plaque neovascularization. 10 A method as in claim 1, wherein the patient is identified following a carotid duplex ultrasound with at least a 50% stenosis.

11 A method as in claim 4, wherein the method is carried out prior to endarterectomy or carotid artery stenting.

12 A method as in claim 4, wherein the method is performed adjunctively to a carotid artery stenting procedure.

13 A method as in claim 1, wherein the lipid binding agent is a pharmaceutical formulation containing an active ingredient with one or more class A amphipathic alpha helices as its secondary structure.

14 A method as in claim 13, wherein the lipid binding agent contains one or more ApoA-1 proteins.

15 A method as in claim 14, wherein the ApoA-1 protein has at least 90% sequence homology to the full length human wild type ApoA-1 protein (SEQ ID No: 1).

16 A method as in claim 15, wherein the ApoA-1 protein consists of ApoA-1 Milano (SEQ ID No: 2) with at least 50% dimers.

17 A method as in claim 1, wherein the lipid binding agent contains a pharmaceutical formulation with an active ingredient that contains at least one peptide between 10 and 40 amino acids in length such peptide with at least one class A amphipathic alpha helix as its secondary structure.

18 A method as in claim 17, wherein the peptide consists of any of SEQ I D No's: 3, 4, 5 or 6.

19 A method as in any of claims 13-18, wherein the lipid binding agent is complexed to at least one neutral or anionic phospholipid.

20 A method as in claim 19, wherein the phospholipid consists of POPC or PLPC.

21 A method as in claim 14 or 17, wherein the one or more ApoA-1 proteins or peptides is located at least partially on the outside surface of a phospholipid based micelle structure between 7 and 20 nanometers in diameter.

22 A method as in claim 1, wherein the dosing per patient is based on the amount of lipid based plaque in the targeted carotid artery and not based on patient weight or surface area.

23 A method as in claim 1, wherein the volume of injection for each administration is prefera bly between 0.5 and 5 mi's.

24 A method as in claim 23, wherein the concentration is between .5 and 20 mg/ml. 25 A method as in claim 1, wherein the agent is delivered at any of 24 hour spacing, once weekly or once a month until a clinically beneficial stabilization of the lipid rich necrotic core is achieved.

26 A method as in claim 1, wherein the method is performed adjunctively to or immediately following the intravenous administration of a thrombolytic agent.

27 A method as in claim 1, wherein the injection site is just proximal to the common carotid bifurcation allowing for the delivery of the lipid binding agent to both the internal and external branches of the targeted carotid artery by way of the adventitia.

28 A method for the treatment of peripheral artery disease, said method comprising: identifying a human patient with an accumulation of one or more lipid rich necrotic cores adjacent to the bifurcation of the common femoral artery into the superficial femoral artery; a further step of confirming that the patient has an arterial lumen stenosis of the superficial femoral artery of less than 60%; followed by positioning an echogenic needle under the guidance of ultrasound to the outer boundary of the external elastic lamina at or adjacent to the lipid rich necrotic core followed by delivering a lipid binding agent into the perivascular space an amount sufficient to at least partially delipidate the lipid rich necrotic core with such delivery being performed through direct percutaneous injection using an echogenic needle under the guidance of an external ultrasound probe.

29 A method as in claim 28, wherein the targeted artery does not meet the requirements for atherectomy or balloon angioplasty.

30 A method as in claim 28, wherein the administration is given at any of 24 hour spacing, once weekly or once a month until a therapeutically beneficial stabilization of the lipid rich necrotic core is achieved.

31 A method as in claim 28, wherein the lipid binding agent contains one or more ApoA-1 proteins.

32 A method as in claim 31, wherein the ApoA-1 protein has at least 90% sequence homology to the full length human wild type ApoA-1 protein (SEQ ID NO:l).

33 A method as in claim 32, wherein the ApoA-1 protein consists of ApoA-1 Milano

(SEQ ID NO: 2) with at least 50% dimers.

34 A method as in claim 28, wherein the lipid binding agent contains at least one peptide between 10 and 40 amino acids such peptide with at least one class A amphipathic alpha helix as its secondary structure. 35 A method as in claim 34, wherein the peptide consists of any of SEQ ID No's: 3, 4,

5 or 6.

36 A method as in any of claims 31-35, wherein the lipid binding agent is complexed to at least one neutral or anionic phospholipid.

37 A method as in claim 36, wherein the phospholipid consists of POPC or PLPC. 38 A method as in any of claims 31-35, wherein the one or more ApoA-1 protein or peptide is located at least partially on the outside surface of a phospholipid based micelle structure between 7 and 20 nanometers.

39 A method as in claim 28, wherein the dosing per patient is based on the amount of lipid based plaque in the targeted artery and not based on patient weight or surface area.

40 A method as in claim 28, wherein the volume of injection is between 0.5 and more than 10 mi's.

41 A method as in claim 40, wherein the concentration is between .1 and 20 mg/ml.

42 A method as in claim 28, wherein the method is performed adjunctively to a diagnostic angiography.