UNIVERSITY OF CINCINNATI

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1 UNIVERSITY OF CINCINNATI Date: 11/16/2004 I, Murad Rasem Melhem, hereby submit this work as part of the requirements for the degree of: in: Doctorate of Philosophy (Ph.D.) Pharmaceutical Sciences It is entitled: NOVEL PERI-VASCULAR DRUG DELIVERY FOR THE TREATMENT OF NEOINTIMAL HYPERPLASIA ASSOCIATED WITH PTFE DIALYSIS GRAFT FAILURE This work and its defense approved by: Chair: Pankaj Desai, Ph.D. William Cacini, Ph.D. Gerald Kasting, Ph.D. Sue Heffelfinger, M.D., Ph.D. Prabir Roy-Chaudhury, M.D., Ph.D.

2 NOVEL PERI-VASCULAR DRUG DELIVERY FOR THE TREATMENT OF NEOINTIMAL HYPERPLASIA ASSOCIATED WITH PTFE DIALYSIS GRAFT FAILURE A dissertation submitted to the Division of Research and Advanced Studies of the University of Cincinnati in partial fulfillment of the requirements for the degree of DOCTORATE OF PHILOSOPHY (Ph.D.) in the Department of Pharmaceutical Sciences of the College of Pharmacy 2004 by Murad Rasem Melhem B. Pharm., University of Jordan, Jordan 1998 Committee Chair: Pankaj Desai, B.S., M.S., Ph.D.

3 ABSTRACT Intimal hyperplasia (IH), that leads to hemodialysis access dysfunction, is a major problem affecting patients undergoing renal dialysis. This work aimed at developing a novel approach that entails peri-vascular delivery of cytotoxic agents such as paclitaxel for the inhibition of IH. We formulated a polymeric delivery system for use as a peri-vascular wrap around the graft-vein junction. The formulation was rigorously optimized with respect to its mechanical strength and in vitro drug release kinetics. The optimized formulation used ethylene-vinyl acetate polymer and PEG4000 as the channeling agent (15%) with paclitaxel loading of 5%. This formulation exhibited a biphasic release profile (initial burst followed by sustained release for > 28 days), which is coincident with the time course of intimal thickening. The formulation was then tested for paclitaxel cytotoxicity against a panel of cells that are known to be involved in IH such as smooth muscle and endothelial cells MTT cell viability assay. The formulation retained paclitaxel cytotoxicity against all cell types investigated. Pharmacodynamic assessment entailed a comparative evaluation of IH employing a validated pig model (Yorkshire pigs). Dialysis grafts were implanted between the femoral artery and vein. One side received drug-loaded perivascular wrap, while the contra-lateral side received drug-free wraps (controls) for days. Histomorphometric analysis of sections from control versus drug-treated tissues indicated complete inhibition of IH on the treatment side, while the control side exhibited ~ 56 % neointimal blockage. Pharmacokinetic studies indicated that paclitaxel was not detectable in plasma samples (limit of quantitation, 50 nm). Taken together, our studies propose an exciting novel drug delivery approach for the treatment of vascular stenosis. They provide strong scientific bases for further pre-clinical and clinical evaluation for prevention of dialysis grafts failure and other vascular abnormalities where IH is the underlying pathology.

4 To the all knowing GOD

5 ACKNOWLEDGEMENTS The work presented here ahs been a truly rewarding experience to me. I would like to begin by glorifying and expressing my heart-filled gratitude to the almighty and all-knowing God for his blessings and for giving me the intellectual power and perseverance to go through all the trials and tribulations in my life. This scientific work was based on the research ideas of my graduate advisor Dr. Pankaj Desai. To him I say that I am very thankful for giving me the opportunity to work on a demanding but rewarding area of research that really fits my interests coming into graduate school. I am very thankful for his generosity in providing the resources that made this work possible. Dr. Desai was more than an academic advisor. I am grateful to him for all the guidance and the friendship he offered that greatly helped me to focus on my research and my life objectives. This project was supported by a generous grant from the U.S. Department of health and Human Services / National Institutes of Health (Bethesda, MD). I am also equally thankful to our collaborator Dr. Prabir Roy-Chaudhury, research faculty member and nephrologists at the College of Medicine, University of Cincinnati. I thank him for all the help we received from his research resources in working with the animal model as well as guidance in the assessment of the clinically-relevant end point. I am very thankful for the insight that he provided for me to plan the next few steps in my life. I would like to express my gratitude for Dr. Gerald Kasting for serving on my comprehensive exam and dissertation committees. His enthusiastic suggestions and critique at the different

6 stages of this work always provided me with curiosity to explore the means to make the best out of this research. I am very thankful to Dr. Burnett Kelly, surgeon at the College of Medicine, University of Cincinnati, for his friendship and all the help he provided with the in vivo experiments. His expertise in surgery and vascular pathology were a great asset to the project. I would like to thank Dr. Sue Heffelfinger, pathologist at the College of Medicine, University of Cincinnati, for the advice and the training our group had in carrying out both the in vitro and in vivo experiments. I am also grateful to Dr. William Cacini for serving on my comprehensive exam and dissertation committees. I am very thankful To Drs. Patrick Marroum, Brian Booth and Jogarao Gobburu, senior reviewers the Center for Drug Evaluation and Research at the Food and Drug Administration (Rockville, MD), for providing me with the one of a kind opportunity to intern under their supervision. In retrospect, the experience I got from the few projects I worked on has positively changed my outlook on research. I would like to thank the Department of Pharmaceutical Sciences and the College of Pharmacy for all the academic, financial and administrative support. I thank the faculty at the Department of Pharmaceutical Sciences for their academic guidance. In specific, I thank Drs. Shenouda, Sakr, Al-Khalidi, Caperelli, Pauletti and Gudelsky for offering me curricular and academic guidance. I am grateful for the kindness, affection and patience of Marcie, Donna, Julie,

7 Paulette, Alfreda, Paula and Marilyn. I thank Sue Ryan for her friendship and unique loving attitude. I thank Dr. Acosta and Dr. Lee for extending the opportunity to join the graduate program and making my stay here a comfortable one. I would like to thank my parents for all the hardships they went through bringing me up and helping me attain higher education. I truly and deeply believe that their love and prayers keep me going wherever I am. I hope that I can always live up to their expectations. I would like to thank my parents in-law and their family for their blessings, love and wishes. I would like to thank my colleagues Srikanth, Abhijeet, Pattie, Sandhya, Rucha, Niresh, Purvi and Fang for their friendship and good times. I also thank Santosh, Marisa, John, Hatim, Sunila, Altaf, Saja, Namrata, Varsha and many other graduate students for their company and the assistance they provided over the years. Last but not least, in fact most, I would like to express my deepest gratitude to my wife Lubna for sharing life with me. She has been there for the better or worse. She has been the comforting hands and affectionate heart through all the good and hard times. I couldn t have asked for a better gift from God for the years and years to come. I have had a challenging but wonderful academic experience at the University of Cincinnati over the past five years. I thank the University of Cincinnati for providing me with this opportunity.

8 By dedicating this work to the all-knowing God, I share with the world what is really God s work and blessing in the first place.

9 TABLE OF CONTENTS ABSTRACT DEDICATION ACKNOWLEDGEMENTS TABLE OF CONTENTS LIST OF FIGURES LIST OF TABLES i v ix 1 INTRODUCTION HEMODIALYSIS VASCULAR ACCESS DYSFUNCTION (HVAD): A MAJOR CLINICAL PROBLEM AFFECTING RENAL DIALYSIS PATIENTS NEOINTIMAL HYPERPLASIA Pathogenesis of Venous Neointimal Hyperplasia Cell types, Growth Factors and Molecular Mechanisms Time Course of Neointimal Formation Arterial versus Venous Stenosis The Role of Adventitia and Adventitial Remodeling in Vascular Stenosis Overview of Available Interventions Drug eluting stents (DES) Animal Studies with DES Clinical Trials Antisense Technologies Brachytherapy TAXANES Chemistry and Mechanism of Cytotoxic Action i

10 1.3.2 Pharmacology and Administration Pharmacokinetics of Paclitaxel Paclitaxel Metabolism Toxic and Adverse Effects Paclitaxel as an Anti-stenotic Agent: In Vitro and In Vivo Experience LOCALIZED VERSUS SYSTEMIC APPROACHES Rationale for Formulating Paclitaxel-Based Localized Treatments Rationale for Enhanced Target Tissue/Plasma Ratio with Localized Delivery Polymeric Formulations: Successful Formulation Attributes and Applications Endo-vascular versus Peri-vascular Delivery of Paclitaxel Peri-Vascular Delivery in Failing PTFE Dialysis Access Grafts: Rationale and Novelty HYPOTHESIS AND SPECIFIC AIMS MATERIALS Chemicals Cell lines and Biological Reagents Assay methodology Animal Experiments METHODS ii

11 4.1 PREPARATION AND TESTING OF PACLITAXEL-LOADED POLYMERIC SYSTEMS Preparation of Paclitaxel-Loaded Polymeric Matrices Mechanical Properties of Loaded Polymeric Matrices Drug Content Uniformity. / Chemical Stability of Paclitaxel Embedded into Polymeric Matrices Fluid Uptake and Weight Loss Experiments Morphological Analysis Using Environmental Electron Microscopy (ESEM) In Vitro Paclitaxel Release from Polymeric Formulations Statistical Analysis IN VITRO CYTOTOXICITY OF PACLITAXEL RELEASED FROM POLYMERIC FORMULATIONS Inhibition of Cell Proliferation by Polymeric Paclitaxel Statistical Analysis DETERMINATION OF IN VIVO OF EFFICACY OF THE OPTIMIZED PACLITAXEL LOADED SLABS Preparation of Paclitaxel-Loaded Polymeric Matrices Surgical procedures Treatment Duration and Protocols Digitization and Hisomorphometric Analysis Blood Sampling Protocols, Complete Blood Counts (CBC) Analysis and Gross Assessment of Toxicity Statistical Analysis iii

12 5 RESULTS PREPARATION AND TESTING OF PACLITAXEL-LOADED POLYMERIC SYSTEMS Drug Content Uniformity Chemical Stability of Paclitaxel Embedded into Polymeric Matrices Mechanical Properties of Loaded Polymeric Sheets Water Sorption and Weight Loss Experiments Morphological Analysis Using Environmental Electron Microscopy (ESEM) In Vitro Paclitaxel Release from Polymeric Formulations IN VITRO CYTOTOXICITY OF PACLITAXEL RELEASED FROM POLYMERIC FORMULATIONS Inhibition of Cell Proliferation by Polymeric Paclitaxel DETERMINATION OF IN VIVO OF EFFICACY OF THE OPTIMIZED PACLITAXEL LOADED SLABS Histomorphometric Measurements Systemic Exposure, Complete Blood Counts (CBC) Analysis and Gross Assessment Of Toxicity DISCUSSION REFERENCES iv

13 LIST OF FIGURES Fig Cell types and cytokines involved in neointimal formation Fig Matrix and structural proteins...7 Figure Diagrammatic presentation of the net effect of vascular remodeling in the presence of equal volumes of neointimal hyperplasia. (a) unfavorable remodeling due to vascular constriction resulting in a net decrease in luminal cross sectional area, (d) favorable remodeling due to vascular dilatation resulting in a patent vessel even in the presence of prominent neointimal thickening Figure Chemical structures of taxanes: (1) paclitaxel and (2) docetaxel Fig Drug release kinetics from polymers can be modulated by the addition of a channeling agent: a diagrammatic presentation illustrating the physical interaction between the polymeric matrix, the loaded therapeutic agent and the polymeric channeling agent Fig Diagrammatic representation of the venous anastomosis to demonstrate how the harvested samples were sectioned. Maximal neointimal hyperplasia occurs at the site of graftvein junction (Area B) v

14 Fig Percent of paclitaxel retained in matrices at the end of different storage periods under dry (diamonds) and in-solution (triangles) conditions Fig Stress-strain analysis for two formulations. Young s modulus was calculated from the slope of the initial portion of the plots. Note the steeper slope of the initial portion for the formulation containing 15% PEG4000 indicating higher elasticity Fig Effect of the channeling agent (PEG4000) on the porosity of polymeric matrices: increase in total weight of the polymer with increasing concentrations of PEG4000 and eventual weight loss upon drying Fig Scanning electron photomicrographs of EVA matrices loaded with 5% paclitaxel; a) EVA surface image (2000X), b) cross-sectional image of EVA matrix showing closely adherent stacks of polymeric layers (2000X) Fig Scanning electron phtomicrographs of EVA matrices loaded with 5% paclitaxel/15% PEG4000; a) polymer surface image (250X) showing channels running along the orientation of imagining, b) EVA matrices at higher maginification showing a marked increase in surface porosity due to partial leaching of PEG4000 (2000X) Fig In vitro release kinetics of paclitaxel from EVA matrices loaded with 5% paclitaxel by weight and increasing loading of PEG4000 (Mean ± S.D.).. 76 vi

15 Fig Relationship between the mean increase in weight of polymeric matrices and the fraction of drug load released from EVA matrices loaded with 5% paclitaxel / 15% PEG4000 by weight Fig In vitro effects of the paclitaxel-loaded polymers on HUVECs: addition of the paclitaxel-loaded polymers to cell cultures resulted in a significant inhibition of in vitro proliferation of these cell types Fig In vitro effects of the paclitaxel-loaded polymers on NHDFs: addition of the paclitaxel-loaded polymers to cell cultures resulted in a significant inhibition of in vitro proliferation of these cell types Fig In vitro effects of the paclitaxel-loaded polymers on HASMCs: addition of the paclitaxel-loaded polymers to cell cultures resulted in a significant inhibition of in vitro proliferation of these cell types Fig Peri-vascular paclitaxel wraps block venous stenosis. Note the complete absence of luminal stenosis at the graft-vein anastomoses of animals treated with the paclitaxel loaded polymers vii

16 Fig Peri-vascular paclitaxel wraps block venous stenosis: Note the marked decrease in neointimal hyperplasia at the graft-vein anastomosis in panel A (treated with a paclitaxel loaded peri-vascular wraps) as compared to panel B (treated with a control wraps) in animals 1 and Fig Peri-vascular paclitaxel wraps block venous stenosis: Note the marked decrease in neointimal hyperplasia at the graft-vein anastomosis in panel A (treated with a paclitaxel loaded peri-vascular wraps) as compared to panel B (treated with a control wraps) in animals 3 and Fig Explant specimen of the graft, artery and vein as a composite block. Note the perivascular polymer in place around the explanted vessel viii

17 LIST OF TABLES Table Summary of clinical studies with paclitaxel- and sirolimus-drug eluting stents Table Composition of different polymeric formulations tested.. 53 Table Paclitaxel content in EVA loaded slabs Table Effect of PEG4000 loading on the mechanical strength of paclitaxel-loaded EVA matrices Table The cumulative % of paclitaxel content released from matrices with different loadings of PEG4000. The initial 60% of the total amount released over the sampling period was fitted to a square root of time relationship and the regression coefficient is reported as a measure of goodness of fit Table Statistical comparisons in HUVEC survival following exposure to different polymeric formulations Table Statistical comparisons in HASMC survival following exposure to different polymeric formulations ix

18 Table Statistical comparisons in NHDF survival following exposure to different polymeric formulations Table Peri-vascular paclitaxel wraps block neointimal hyperplasia. Summary of the results from histomorphomeric analysis x

19 1 INTRODUCTION 1.1 HEMODIALYSIS VASCULAR ACCESS DYSFUNCTION (HVAD): A MAJOR CLINICAL PROBLEM AFFECTING RENAL DIALYSIS PATIENTS. End-stage kidney disease is a complete or near complete failure of the kidneys to function to excrete wastes, concentrate urine, and regulate electrolytes. This condition is also called Endstage renal disease (ESRD). End-stage kidney disease occurs when the kidneys are no longer able to function at a level that is necessary for day to day life. It usually occurs as chronic renal failure progresses to the point where kidney function is less than 10% of baseline. At this point, the kidney function is so low that without dialysis or kidney transplantation, complications are multiple and severe, and death is eminent to occur from accumulation of fluids and waste products in the body. In the United States, nearly 300,000 people are on long-term dialysis and more than 20,000 have a functioning transplanted kidney. The most common cause of ESRD in the US is diabetes. ESRD almost always follows chronic kidney failure, which may exist for 10 to 20 years or more before progression to ESRD. Other risk factors include hypertension, atherosclerosis, rheumatic conditions and genetic predisposition. Terminal renal conditions are associated with life-threatening complications such as congestive heart failure, encephalopathy, nervous system damage, dementia, peripheral neuropathy, platelet dysfunction, gastrointestinal loss of blood; duodenal or peptic ulcers and seizures. As for 1

20 prognosis, ESRD is fatal unless treated with dialysis or transplantation. Both of these treatments can have serious risks and consequences. The outcome varies and is unique to each individual. The most common type of dialysis is hemodialysis. Hemodialysis is a medical procedure that uses a special machine (a dialyzer) to filter waste products and excess fluid from the blood and to restore normal constituents to it. This shuffling of multiple substances is accomplished by virtue of the differences in the rates of their diffusion through a semi-permeable membrane (a dialysis membrane). The dialysate is then pumped out to a disposal tank and new dialysate is pumped in. The process of removing excess fluid is known as ultrafiltration. The blood is circulated and diffused numerous times during a dialysis session; each circulation through the machine removes more waste and excess fluid. Hemodialysis is usually performed three or more times a week for 4-5 hours. For long-term hemodialysis, usually a patient will need vascular access to be connected to the dialysis machine. The access insertion is a surgical procedure that connects an artery to a vein, so two needles have to be inserted each time the patient comes in for dialysis. The connection between an artery and vein is often achieved using tubes known as dialysis access arterio-venous grafts (AVG). In the United States, the most common surgical procedure to connect an artery and a vein is via placing a synthetic graft (artificial vessel) mainly made of polytetrafluoroethylene (PTFE). These synthetic grafts tend to have problems with clotting, blockage or infection that lead to dysfunctional access. 2

21 Hemodialysis vascular access dysfunction is currently a major clinical problem amongst patients with ESRD in the United States. It is the single most important cause of hospitalization and morbidity in the hemodialysis patient population. As such, loss of functional access is considered one of the contributing factors to the overall mortality in the hemodialysis patient population. Medicare data suggest that the annual cost of vascular access failure-related morbidity currently exceeds one billion dollars per year [Feldman., 1996]. As mentioned earlier, the most common type of vascular access procedure performed in the United States is the PTFE arterio-venous graft insertion. This accounts for more than 70% of all hemodialysis access procedures. Therefore, the subsequent discussion will be focused on this form of dialysis access. The current patency rates associated with PTFE gtafts and the proximal veins for years 1 and 2 are 50% and 25%, respectively [Schwab, 1999]. Graft blockage, known as in-graft stenosis, is responsible for more than 80% of all vascular access dysfunction. In more than 90% of these cases the underlying pathology for graft stenosis is venous neointimal hyperplasia (VNH) [Beathard, 1994]. VNH is characterized by smooth muscle cell (SMC) / myofibroblasts proliferation and migration, angiogenesis and extra-cellular matrix deposition. Surprisingly, despite the magnitude of the problem, it remains an under-explored area of research. Consequently, there are currently no effective interventions to treat this condition [Himmelfarb, 1996]. This is unfortunate since the VNH in the setting of PTFE grafts appears to be more aggressive than the arterial neointimal hyperplasia that occurs following angioplasties and in peripheral bypass grafts. This is obvious when one compares the 88% patency rates of aorto-iliac grafts at five years post insertion to the 50% patency at one year post insertion in the PTFE access setting 3

22 [Abbott, 1995]. Moreover, venous stenoses in the PTFE access grafts also have poorer response to the mechanical angioplasty (40% three-month survival) compared to arterial stenoses having over 80% six-month survival rate [Beathard, 1992]. 1.2 NEOINTIMAL HYPERPLASIA: Pathogenesis of Venous Neointimal Hyperplasia: Cell types, Growth Factors and Molecular Mechanisms. As mentioned earlier, there are to date very few studies that have attempted to analyze the pathology of venous stenosis in PTFE dialysis grafts [Swedberg, 1989; Roy-Chaudhury, 2001]. These studies, however, shared the conclusion that the predominant lesion leading to access dysfunction is VNH characterized by SMC/myofibroblasts proliferation and migration, matrix deposition and angiogenesis (Fig and ). Based on events extracted from other model systems, such as arterial angioplasty and bypass models, the initiating event for neointimal formation is hemodynamic stress and/or direct vessel wall injury. These initiating events result in the activation, proliferation and migration of smooth muscle cells, fibroblasts, macrophages and endothelial cells (EC) as a result of growth factor and cytokine release. Such growth factors include tissue transforming growth factor- β (TGF-β), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bfgf) [Olson, 1992; Schwartz 1995; Jawien, 1992, Braddock, 1994]. The central event in this paradigm appears to be SMC migration and proliferation into the media and intima. 4

23 1a 1b 1c 1d 1e 1f 1g 1h Fig Cell types and cytokines involved in neointimal formation (adventitia to left and lumen to right). (a) PTFE graft (hematoxylin and eosin 200). Note the significant venous neointimal hyperplasia (extent of arrow) between the graft (G) and the lumen (L). (b) Downstream vein (hematoxylin and eosin 200). Note the presence of microvessels (thin arrows) within the adventitia (A). Also note the thickened (arterialized) media (M, double-headed arrow) and the significant amount of neointimal hyperplasia (N, bar). (c) Downstream vein; neointima (vwf 400). Note the prominent angiogenesis within the neointima (arrows), as assessed by this endothelial cell marker (d) downstream vein; neointima (vwf + Ki67 800). High-power view of a microvessel within the neointima of downstream vein. Note the distinct colocalization of blue (endothelial) and brown (proliferating) cells indicating active endothelial cell proliferation (angiogenesis). (e) Upstream graft; neointima (PG-M1 2000). High-power view of a macrophage giant cell adjacent to the neointimal surface of PTFE graft (G). Also note the large 5

24 number of macrophages in this area (thin arrows). (f) Downstream vein; neointima (SMA + Ki ). High-power view of a portion of the neointima stained for smooth muscle cells (brown) and proliferating cells (blue). Note that almost all the active cellular proliferation in this specimen (arrows) is occurring within the neointimal microvessels (angiogenesis). At the time that this specimen was harvested, there was no ongoing smooth muscle cell proliferation (g) PTFE graft; adventitia (bfgf 500). Note the strong expression of bfgf in adventitial vessels (thick arrow) and by the macrophage giant cell layer (thin arrow) lining the graft. (f) Downstream vein; media and neointima (PDGF 400). There is strong expression of this cytokine in the venous media (M) and by smooth muscle cells/myofibroblasts within the neointima (N, bar) [adapted from Roy-Chaudhury, 2001]. 6

25 2a 2b 2c 2d Fig Matrix and structural proteins. (a) PTFE graft (tenascin 200). There is strong expression of tenascin in the region of the macrophage giant cell layer (thin arrow) surrounding PTFE graft (G) and on the abluminal side of the neointima (thick arrow). (b) PTFE graft (collagen IV 160). Collagen is present as expected within the walls of prominent adventitial blood vessels (thin arrows) and in this particular sample within the luminal portion of the neointima (thick arrow). (c) Downstream vein; neointima (fibronectin 500). There is strong diffuse expression of fibronectin by ECM components. (d) Downstream vein; neointima (laminin 500). Laminin is a prominent component of microvessels (arrows) within the neointima [adapted from Roy-Chaudhury, 2001]. 7

26 Time Course of Neointimal Formation. We are unaware of any reports to date that describe the time course of neointimal formation in venous models. However, from what is known about the arterial injury models and the process of wound healing, the initial trigger for stenosis appears to be vascular injury. The sequence of events that follow the surgical intervention can be divided into three stages: StageI (0-3 days), where radial injury leads to endothelial denudation resulting in a storm of growth factors release followed by SMCs proliferation which experimentally peaks within hours [Chesebro, 1993]; Stage II (3-14 days), SMCs start attaining the so-called secretory phenotype (myofibroblasts) and begin secreting large amounts of extracellular matrix elements [Chen, 1997]; StageIII (beyond 14 days), re-endothelialization occurs and ushers in SMCs re-attaining their contractile type but the extracellular matrix deposition continues for a few weeks to several months [Clowes, 1975] Arterial versus Venous Stenosis. Although there are some similarities between venous and arterial response to injuries, there are several important differences between the two models, especially in the context of arterio-venous PTFE dialysis grafts. VNH appears to be more aggressive than arterial neointimal hyperplasia. Also, VNH does not respond to surgical interventions as well as the arterial lesions [Beathard, 2002]. Other differences include: (i) at a hemodynamic (flow mechanics) level, the PTFE dialysis graft is currently the only clinical setting in which a non-compliant material is anastomosed to a relatively compliant vein. This creates changes in the pattern of blood flow at the graft-vein junction. Rather than flowing in a laminar course, blood flow tends to be turbulent. This is likely to result in significant differences in the degree of shear stress and 8

27 compliance mismatch at the graft-vein junction. (ii) At an anatomic level, veins have a poorly defined internal elastic lamina which could easily allow the migration of SMCs and myofibroblasts from the media and adventitia into the intima in response to endothelial denudation caused by increased shear stress. (iii) At a cellular and physiologic level, there are multiple differences between veins and arteries, which may contribute to an increased propensity for venous stenosis. Briefly, veins have relatively low nitric oxide and prostacyclin levels, an increase in vasoconstrictor sensitivity and relatively high numbers of basic fibroblast growth factor receptors as compared to arteries [Motwani, 1998]. (iv) Finally, there are factors that are specific to the dialysis patient which include (a) the presence of uremia as a modifying factor and (b) insertion of needles into PTFE dialysis grafts which may result in platelet aggregation and unorganized thrombi formation, aggravation of hemodynamic stress and the consequent downstream release of mediators of SMCs proliferation such as platelet-derived growth factor (PDGF) [Albers, 1994]. We believe that it is critical to identify the reasons for the clinical differences between venous and arterial neointimal hyperplasia at a cellular and molecular level to be able to identify the macro- as well as the micro-targets for interventions. In other words, this could mean that therapies which are based on studies done in the context of arterial neointimal hyperplasia, may have limited efficacy in the setting of venous neointimal hyperplasia The Role of Adventitia and Adventitial Remodeling in Vascular Stenosis. Recent studies have focused on assessing the role of adventitial remodeling in vascular stenosis [Libby, 1997; Nakamura, 1998]. It is speculated that in the presence of equivalent volumes of neointimal hyperplasia, vascular constriction will result in a smaller luminal area, 9

28 while vascular dilatation will result in a larger luminal area [Schwartz, 1998] as illustrated in Figure In the setting of experimental coronary angioplasty models, it is the general notion that vascular or adventitial remodeling is responsible for approximately 50% of overall luminal stenosis [Libby, 1997]. Blood flow through the arterio-venous graft is one of the main determinants of the functionality of the access. Blood flow is dependent more on the overall luminal cross sectional area than on just the neointimal volume. Therefore, repeated reference will be made throughout this overview to mechanisms and interventions that have the potential to positively impact vascular remodeling. The role of vascular remodeling in venous stenosis has been underscored by several studies. First, Shi et al. demonstrated that angioplasty results in a transformation of α-actin-negative adventitial fibroblasts to α-actin-positive adventitial myofibroblasts. Twenty eight days post angioplasty, this event is followed, by the development of a collagen rich scar in the adventitia which could contribute to adverse vascular remodeling [Shi, 1996]. Moreover, findings by the same group provide evidence for the migration of adventitial fibroblasts from the adventitia through the media and into the intima, where they contribute to neointimal hyperplasia following angioplasty. During the course of this migration, these adventitial fibroblasts transform into α- actin-positive myofibroblasts [Shi, 1996; Kalra, 2000]. In the PTFE access setting, studies performed by Roy-Chaudhury et al. on venous specimens from PTFE dialysis access subjects suggest that up to 40% of neointimal cells are myofibroblasts [Roy-Chaudhury, 2001]. Thus, it can be reasonably hypothesized that the peri-vascular modulation of adventitial contents may be more effective in positively influencing adventitial remodeling than the systemic administration or endovascular delivery of the same anti-stenotic agent. 10

29 (a) (b) Figure Diagrammatic presentation of the net effect of vascular remodeling in the presence of equal volumes of neointimal hyperplasia. (a) unfavorable remodeling due to vascular constriction resulting in a net decrease in luminal cross sectional area, (d) favorable remodeling due to vascular dilatation resulting in a patent vessel even in the presence of prominent neointimal thickening. 11

30 1.2.2 Overview of Available Interventions. A number of attempts for the treatment /prevention of stenotic vascular proliferative diseases have been reported. These include: localized drug delivery utilizing porous balloon catheters, endovascular radiation and drug-eluting stents. Rgardless of whether these approaches entailed pre-clinical or clinical studies, they have been limited to the arterial setting. Several approaches for localized delivery of anti-stenotic drugs utilizing such as rapamycin [Morice, 2002 and Moses, 2003], mithramycin [Fishbein, 2001], tyrphostin [Golomb, 1996] and heparin [Teomim, 1999] have been implemented Drug eluting stents (DES): In conjunction to angioplasty, the use of endoluminal stents gained widespread acceptance in percutaneous coronoray artery interventions. This is aided by the fact that clinical trials showed evidence of decreased stenosis rate when compared to angioplasty alone. This success can be attributed to the elimination of recoil and chronic negative remodeling. Despite these advances, stenosis rates in patients receiving stents are unacceptably high. This was explained by the observation of exaggerated neointimal hyperplastic growth with the same pathogenetic basis discussed earlier. The concurrent systemic administration of a number of therapeutic agents has not shown any significant impact on the rates of in-stent re-stenosis. A primary reason for the low overall rates of success of systemically administered agents may be related to inadequate drug levels reaching the site of lesion. Therefore, the idea of localized drug delivery emerged in the early nineties. 12

31 By means of specifically designed drug delivery balloons, various drugs were administered at the injury site to maximize target tissue concentrations. However, the balloon-driven technique was observed to provide only marginal advantages. This is mainly because this approach was associated with low efficiency in delivering the drug and high rates of systemic washout. This led to the belief that high sustained regional concentrations in a reservoir-type device provides a significant advantage. A logical technique for this purpose appeared to be stents. Although there is some degree of systemic exposure, this could only be viewed as a downside only if dose dumping occurs due to the engineering of drug coated stents. Drug-eluting stents contain agents that target thrombus formation (heparin), inflammation (dexamethasone) and/or cellular proliferation (sirolimus and paclitaxel). We will be discussing the efficacy of drug-eluting stents here in two parts. First, we will review the design of stents and some of the animal studies performed with different agents. In the second part, we will summarize the available data from clinical trials. There are several approaches for the loading the different therapeutic agents onto stents. Some drugs can be loaded onto the metallic stent surfaces by dipping the stents into a drug-containing solution. Other drugs can be applied to stent surfaces utilizing more complicated methods such as spray coating. However, due to the lack of a reservoir mechanism that would keep the drug at the desired site, only highly hydrophobic agents can be delivered to the vessel wall utilizing this approach. Thus, further efforts have been focused on the use of polymers that could serve to control the rate and extent of drug release. 13

32 The drug-containing polymeric matrices can be loaded onto the stent utilizing dipping or spray coating techniques. It is obviously desirable to use biocompatible polymers in stent coating. Biocompatible polymers are of two main types: biostable and biodegradable. Drugs can be liberated by diffusion from biostable matrices or by both diffusion and non-enzymatic polymeric erosions in biodegradable polymers. The polymers that were used had to be tested for biocompatibility to minimize tissue reactions against foreign substances. The selection of an inert coating matrix is the first challenge that one might face during development since many of these synthetic polymers induce a profound inflammatory response and neointimal hyperplasia on their own. Examples of biocompatible polymers that mimic or are compatible with biological matrices include poly-lactic acid (PLA), cellulose, phosphoryl choline and ethylene-vinyl acetate Animal Studies with DES: Antithrombotic Agents Because of the catastrophic consequences of stent thrombosis (clot formation within the stent), efforts have been directed towards localized anti-thrombotic approaches. These approaches included drug-coated stents. Since thrombus formation is one of the initiating events in neointimal formation, the strategy for using drug-coated stents have the potential to be used in inhibiting neointimal hyperplasia. Some of the drugs and approaches are discussed below. Heparin: Initial animal experiments assessed the effects of heparin on subacute thrombus formation and whether these effects led to reduction of intimal formation. These studies reported potential beneficial effects on thrombus formation but variable reductions of neointimal 14

33 thickness at 4 and 12 weeks following surgery [Hardhammar, 1996]. A recent study by Matsumoto et al has investigated the effects of stents coated with multiple layers of releasable heparin on neointimal formation in coronary arteries of pigs. Intra-vascular ultrasound showed that the neointimal area (mm 2 ) was significantly suppressed at the heparin-coated stent site (2.12 +/- 0.58) compared with the control stent site (3.92 +/- 0.33) (P < 0.01). Histologic analysis also demonstrated that neointimal area (mm 2 ) was significantly less at the heparin-coated stent (2.94 +/- 0.43) than at the control stent site (4.41 +/- 0.38) (P < 0.01). These results suggest that the stent coated with releasable heparin is beneficial in reducing neointimal formation and subsequent in-stent restenosis [Matsumoto, 2002]. Hirudin/iloprost: Hirduin is a direct inhibitor of thrombin and iloprost is a prostaglandin-i analogue which inhibits platelet aggregation. Both agents have been combined into a poly-llactic acid (PLLA) coated stents [Alt, 1998] and used as a stent coating. In a pig model, Alt et al examined the vascular response to implantation of these coronary stents. Twenty-eight days after implantation, morphometric analysis demonstrated that the coating was associated with a greater lumen diameter through a reduction in the mean restenosis area by 24.8% (P<0.02) in the overstretch pig model compared with uncoated control stents. The use of these stents did not induce a local inflammatory response [Alt, 2000]. Anti-proliferative Agents: Corticosteroids: The inflammatory component in the pathophysiology of neointimal hyperplasia has led many researchers to the test the hypothesis that anti-inflammatory agents may be useful in the treatment of neointimal hyperplasia. Corticoids have long been shown to inhibit SMC 15

34 proliferation and monocyte and macrophage influx which is a major event in vascular inflammation (Furutama, 1998). Although dexamethasone-coated stents reduced the inflammatory reactions induced by synthetic polymers, the effect on neointimal hyperplasia was minimal as observed in porcine studies [Muller, 1994 and Lincoff, 1997]. In contrast, De Schreeder and colleagues reported three studies in which methylprednisolone coated stents had significant inhibitory effects on both inflammation and neointimal blockage at 4-6 weeks compared to controls [De Scheerder 1996]. Sirolimus: or rapamycin is a naturally occurring macrolide antibiotic produced by the fungus Streptomyces hygroscopicus. It is highly effective in inhibiting the onset and severity of autoimmune diseases in various animal models. It is also an FDA approved drug for the prophylaxis of renal transplant rejection since In addition, it possesses potent antiproliferative effects against SMCs since it causes cell cycle arrest at the G1 and S phases. Suzuki et al investigated the efficacy of this agent in inhibiting neointimal hyperplasia in the procine model. Stents coated with thin layers of methacrylate or polyethylene vinyl acetate and containing 185 µg of sirolimus were inserted into major coronary arteries of pigs following vascular overstretch injuries. Angiographic analysis and histological assessment 28 days following implantation revealed 35-50% difference in inflammatory response, neointimal area and the magnitude of stenosis between the control and the treatment groups [Suzuki, 2001]. However, more fibrin deposition was found in the neointima at the sirolimus-coated stent sites. This may reflect a delay in healing and impaired fibrin clearance or degradation secondary to the effects of the drug. 16

35 In another related study, Klugherz et al observed that sirolimus-coated stents when loaded with various amounts of sirolimus ( µg) efficaciously reduced in-stent re-stenosis at 30 days in porcine coronary arteries. The group demonstrated sufficient tissue concentrations, low wholeblood concentrations and no detrimental effects on vascular tissue [Klugherz, 2002]. In another study, Aggarwal et al also showed beneficial effects of sirlomus-eluting stents at 30 days in porcine coronary model. However, these beneficial effects were lost at 90 and 180 days post treatment [Aggarwal, 2002]. Collectively, these studies show that the use sirolimus-coated stents is an effective approach to prevent re-stenosis. Paclitaxel: is a well-known anti-proliferative agent used in the treatment of solid tumors. It exerts its pharmacological effects through formation of numerous decentralized and unorganized microtubules, thus arresting cell cycle progression at the G2/M phase. With regards to its use as an anti-stenotic agent, encouraging observations have been made in several animal studies investigating different approaches for paclitaxel coating. These approaches included: (i) coats of co-polymers impregnated with paclitaxel, (ii) self expanding paclitaxel-eluting stents and (iii) stents with honey-combed metallic struts. Studies with these systems were performed in both pigs and rabbits [Heldman, 2001; Farb, 2001; Drachman, 2000; Hong, 2001 and Finkelstein, 2003]. All these studies consistently revealed a significant, dosedependent reduction of neointimal hyperplasia (up to 60% when compared to controls). Furthermore, the use of these stents showed signs of positive remodeling at the stented segments. 17

36 However, these studies also revealed signs of incomplete healing and persistence of large number of inflammatory macrophages and dense fibrin with very little collagen. Other unresolved issues pertaining to paclitaxel-eluting stents is the duration of their effectiveness. Farb et al reported that inhibition of neointimal formation was no longer evident at 3 months using polymer-coated stents contaning 20.2 or 42 µg of paclitaxel in a rabbit iliac artery model [Farb, 2001]. Drachman and colleagues used a much higher dose of paclitaxel (200 µg) in the same animal model. The group reported that at 6 months after stenting, neointimal area was still two-fold lower in the animals which received paclitaxel-eluting stents [Drachman, 2000]. Taken together, these studies clearly demonstrate that paclitaxel may be effective in preventing of in-stent stenosis. Furthermore, they suggest that the efficacy of the paclitaxel-coated stents is dependent on the rate and extent of drug release. Again, as observed with sirolimus, there are contradicting reports on the duration of effectiveness. Furthermore, in some cases only incomplete healing was observed. Tacrolimus: is a highly hydrophobic immunosuppressant drug that primarily prevents release of various cytokines. In vitro testing showed that tacrolimus is 100-fold less potent than sirolimus in inhibiting SMC proliferation [Gregory, 1993 and Mohacsi, 1997]. Notwithstanding its lower potency, tacrolimus has been shown to be effective in preventing in-stent stenosis in porcine coronary arteries. Tacrolimus-eluting stents (40 µg) showed significant (64%) reduction in intimal hyperplasia as compared to bare stents at 4 weeks follow-up. Complete healing and re- 18

37 endothelialization were observed without any signs of profound inflammation [Alt, 2002 and Rihm,2002]. 17-β-estradiol: Estrogens can inhibit SMC proliferation and accelerate re-endothelialization and healing following angioplasty. New and colleagues [New, 2002] investigated the use of low (67µg) and high (240 µg) doses of 17-β-estradiol-coated stents in the treatment of coronary arteries in a pig model. After 4 weeks, a 40% reduction in the intimal area was observed in the high dose group as compared to polymer-coated stents. Complete endothelial regeneration was seen in all stent groups by that time. Miscellaneous Agents: A number of drugs failed to inhibit neointimal hyperplasia when investigated for their potential as anti-stenotic agents in DES. Accordingly, Quaniprilat (an ACE inhibitor), simvastatin (a lipid lowering drug) and a combination of cis-platinum and mitoxantrone (anti-cancer agents) were tested as candidates for drugs in stent coatings. These agents were tested in phsophorylcholine-coated stents and implanted in porcine coronary arteries [Van Beusekom, 2002 and Stefanadis, 2002]. None of these agents showed significant effects on neointimal formation at 4-5 weeks follow-up. Also, the loading of vascular endothelial growth factor (VEGF) on polymer-coated stents did not produce and significant effects on re-stenosis in rabbit iliac artery stent model [Swanson, 2003] Clinical Trials: While heparin was considered as the gold standard in anti-stenotic therapy based on in vitro and animal studies, findings from clinical studies were disappointing [Haude, 2003]. 19

38 However, based on promising results from some of the above indicated studies, several followup clinical studies were conducted. Trials with only two agents, sirolimus and paclitaxel, had favorable outcomes. For these agents, initial studies were conducted at a pilot stage. Well controlled randomized studies followed these initial assessments. Therefore, only results from randomized studies are described below. Clinical Trials with Sirolimus-Eluting Stents: Morice et al reported the results from the RAVEL (Randomized study with sirolimuseluting Velocity balloon-expandable stents in the treatment of patients with de novo native coronary artery Lesions). The trial was conducted in 238 patients with single primary lesions in native coronary arteries. Patients were randomized to receive a Bx Velocity sirolimus-eluting stent (140 µg/mm 2 of sirolimus per unit of stent surface area, n = 120) or bare metal stent (control group, n = 118). Only patients who had a single primary lesion with a diameter between 2.5 and 3.5 mm that could be covered by an 18 mm stent were recruited. Patients with history of narrowing stenosis > 50% or had a thrombus within the target lesion were excluded. The groups were comparable in demographics and lesion characteristics except that the sirolimus-treated group was comprised of significantly higher number of males compared to the control group. At 6 months the degree of intimal proliferation, measured as the degree of late luminal loss, was significantly lower in the sirloimus-treated group (0.01 mm) compared to the control group (0.8 mm, P < 0.001). None of the patients in the treated group had in-stent re-stenosis compared to 26.6% of patients in the bare metal stent group (P < 0.001) at 6 months. There were no episodes of stent thrombosis in either group. At 1-year follow-up, the overall rate of Major Adverse 20

39 Clinical Events (MACE) was 5.8% in the sirolimus-treated group and 28.8% in the bare metal stent group (P < 0.001) [Morice, 2002]. Another study which evaluated the performance of the SIRIUS Bx Velocity sirolimus-coated stents at 9 months was reported by Moses et al [Moses, 2003]. Patients with re-stenotic or de novo lesions of mm length and mm in diameter were randomized to receive a Bx Velocity sirolimus-eluting stents (n = 545) or bare metal stents (n= 556). Angiographic restenotic rates at 8 months were 3.2% in the sirolimus-eluting stent group versus 35.4% in the control group (P < 0.001). The MACE rates at 9 months were 7.1% (38 events) in the sirolimustreated group and 18.9% (99 events) in the control group (P < 0.001). There were only 4 events of stent thrombosis in the control group compared to 2 events in the sirolimus-eluting stent group. Clinical Studies with Paclitaxel-Eluting Stents: Grube et al published the findings from the TAXUS I clinical study that investigated the outcomes of using the TAXUS paclitaxel-coated stents (1 µg/mm 2 of paclitaxel per unit of stent surface area) for one year. Volunteer patients with de novo or restenotic lesions (12 mm or less) and vessel diameter of mm were enrolled. The subjects were randomized to receive a TAXUS (slow release (TAXUS-SR, n =31) stent or bare metal stent (control group, n = 30). The groups were comparable with regards to demographics and lesion characteristics. There were no MACE events at 30 days and no signs of stent thrombosis at 1, 6, 9, 12 months post stenting in either group. At 12-months follow-up, the MACE rates were 3% in the TAXUS-SR group (1 event) and 10% in the control group (4 events). There was no evidence of wrapper effect 21

40 (stenosis at the 5 mm on either sides of the stent) in either group. Angiographic in-stent restenosis rates at 6 months were 0% for TAXUS-treated patients versus 10% in the control group. Intra-vascular ultrasound (IVUS) showed significant reduction in neointimal hyperplasia in the TAXUS-SR group (14.8 mm 2 ) as compared to the control group (21.6 mm 2, P < 0.05) [Grube, 2003]. Colombo et al reported the findings from the TAXUS II clinical study that investigated the outcomes of using the TAXUS paclitaxel-coated stents (1 µg/mm 2 of paclitaxel per unit of stent surface area) for one year. The study enrolled volunteer patients with de novo or restenotic lesions (12 mm or less) and vessel diameter of mm. The subjects were randomized to receive a TAXUS slow release (TAXUS-SR, n =131) stent, moderate release (TAXUS-MR, n = 135) stent or bare metal stent (control group, n = 270). All stents were 15 mm in length and 3.0 or 3.5 mm in diameter. The major adverse cardiac events (MACE) explored included: Q-wave myocardial infarction (MI), target vessel revascularization (TVR), stent stenosis and death. The cumulative rates of such events at 12 months follow-up were reported to be 21.7% in the control group (57 events), 10.9% in the TAXUS SR group (14 events, P= versus controls) and 9.9 % in the TAXUS MR group (13 events, P = versus controls). The rates of TVR alone at 12 months were reported to be 17.5% in the control group (46 events), 10.1% in the TAXUS SR group (13 events, P= versus controls) and 6.9 % in the TAXUS MR group (9 events, P = versus controls). At 12 months follow-up there was no evidence of stent thrombosis in the control group. One incident was reported in each of the treatment groups. Angiographic instent re-stenosis rate at 6 months were reported to be 19.0% in the control group, 9.3% in the 22

41 TAXUS SR group (P= versus controls) and 4.7 % in the TAXUS MR group (P = versus controls) [Colombo, 2003]. Park et al published the results of the ASPECT trial (Asian Paclitaxel-Eluting stent Clinical Trial) that evaluated the efficacy of paclitaxel DES in preventing coronary re-stenosis over a 6 month period. They conducted a multi-center, randomized, triple blind controlled trial on the Supra-G paclitaxel DES (Cook Group Inc.). This study enrolled 177 patients with discrete coronary lesions (< 15 mm) and vessel diameters of mm. The patients were then randomized to receive a low-dose paclitaxel-eluting stent (1.3 mg/mm 2, n = 58), a high-dose paclitaxel-eluting stent (6.2 µg/mm 2, n = 60) and a bare metal stent (n = 59). Different groups of these patients received one of the following anti-platelet agents; include: clopidogrel, ticlopidine or cilostazol. At an average follow-up of 174 days, the high-dose group had significantly better results compared to the control group. These measured outcomes included: degree of stenosis, 14% versus 39% of luminal diameter, P = 0.001), delayed loss of luminal diameter (0.3 mm versus 1.0 mm, P = 0.001) and re-stenosis rates (4% versus 27%, P = 0.001). Again, the researchers employed intra-vascular ultrasound and observed a dose-dependent reduction in the volume of intimal hyperplasia (31 mm 3, 18 mm 3 and 13 mm 3 in the high-dose, low-dose and control groups, respectively). The effects of co-administered anti-platelet therapies were also investigated. Overall, there was a higher rate of MACE in patients receiving cilostazol compared to those receiving ticlopidine or clopidrogel (96% versus 81%, respectively, P = 0.007) at 6 months [Park, 2003]. The major relevant points of all the studies for both Paclitaxel and sirolimus are summarized in Table

42 Table Summary of clinical studies with paclitaxel- and sirolimus-drug eluting stents. Study Drug Stent Characteristics Colombo et al Paclitaxel Slow Release Moderate Release Bare Metal No. of Patients Follow Up 1 year 4.7% 2.3% 19.0% Angiographic ISR MACE (%) days days Grube et al Paclitaxel Slow Release Bare Metal year 0% 10% Park et al Paclitaxel Low Dose High Dose Bare Metal days 14% 39% Morice et al Sirolimus Coated Stent Bare Metal months 0% 26.6% Moses et al Sirolimus Coated Stent Bare Metal months 3.2% 35.4% ISR: in-stent restenosis, MACE: major adverse clinical events

43 The VElocity (rapamycin-coated) stents and the TAXUS EXPRESS 2 (paclitaxel-coated) stents, highlighted in Table , have been approved by the Food and Drug Administration and are currently in use for the treatment of de novo stenotic lesions following coronary angioplasty. The clinical use of DESs loaded with paclitaxel or sirolimus underscores the following points: a) the need for such interventions, b) while DES are currently in clinical use, concurrent efforts are underway to further optimize DESs in terms of reducing localized toxicities, increasing efficacy and prolonging beneficial effects Antisense Technologies: As described earlier, in the hyperplastic vascular proliferative diseases, the normally quiescent SMCs can be transformed into the migratory and proliferating phenotype that expand and modify the surrounding matrix. This suggests that the molecular mechanisms underlying SMC transformation are potential targets against which anti-proliferative therapies may be effective [Dilley, 1987; Schwartz, 1987]. However, given the large variety of mitogens known to initiate SMC proliferation, it seems unlikely that therapies directed against any single growth factor or transduction pathway would be as effective as these targeting downstream elements of mitogenesis [Schwartz, 1986; Ross, ]. A review of the myriad of cyclins, cyclin-dependent kinases (cdk), phosphatases (ckp) known to participate in cell cycle progression is outside the scope of this overview [Ross, 1993 and Grana, 1995]. However, the role of key cellular factors is highlighted here. Following surgery (in vivo) or disruption of cellular matrix of growth arrested (G 0 ) SMCs in vitro, an early increase in the expression of the proto-oncogene c-myc over the course of 2-6 hours is elicited [Kindy, 1986; Gadeau, 1991; Bennett, 1994 and Edelman, 1995]. Following this, cells become competent to 25

44 undergo further G 1 to S phase cell cycle progression. In mid to late G1 phase, which occurs 8-12 hours after injury, marked increases in the expression of the c-myb transcription factor have been observed [Thompson, 1986 and Brown, 1992]. Then beginning in the late G1 phase and persisting through the S-phase (16-24 hours), expression of the proliferating cell nuclear antigen (PCNA) is also increased. As early (c-myc), mid (c-myb) and late (PCNA) G1/S-associated transcription factors, these three proteins play distinct and critical roles in the regulation of SMC proliferation [Bravo, ]. Consequently, a number of investigators have investigated the use of antisense oligodeoxynucleotides (AS-ODN) to alter the proliferation of cells in the vascular tissues. Antisense technology refers to the use of synthetic oligonucleotides (short RNA transcripts) designed to interrupt the synthesis of specific proteins. In the course of protein synthesis, genetic information coded on a double-stranded DNA molecules are transcribed into a single-stranded (sense) messenger RNA chain (mrna) that is then translated into proteins. Therefore, numerous steps during this process can be targeted to affect the final outcome. One approach is to target specific RNA molecules (antisense) by introducing oligonucleotides that can specifically bind to normal coding RNA. Such oligonucleotides can cause hybrid arrest of translation by introducing non-readable double-stranded areas in the molecule or can mark the mrna for cleavage by creating double-stranded regions that can be cleaved by RNAases. Oligonucleotides employed in antisense studies are usually nucleotide long molecules and can be readily synthesized in any desired sequence. In the specific setting of stenotic lesions, AS-ODNs have been employed against c-myc, c-myb and PCNA. 26

45 AS-c-myc-ODNs: Animal Studies: Bennett and co-workers investigated the effects of a 15-mer AS-c-myc-ODN applied in a gel formulation to the adventitial aspect of the injured segments of rat carotid arteries. It was observed that peak c-myc mrna was reduced by 75%. Histological analysis 14 days later showed significantly reduced neointimal formation in the injured arteries exposed to the AS-cmyc-ODN. This suggested that intial mitogenic stimulation is critical to the development of the neointima and that AS-c-myc-ODNs have the potential to be effective in preventing re-stenosis [Bennett, 1994]. Shi et al employed trans-catheter delivery of an AS-c-myc-ODN in a pig model of coronary artery balloon injury. Following balloon injury for 30 seconds, the balloon was replaced with a porous catheter and treatment was delivered intra-murally. The animals were randomized to a control group (saline) and a treatment group (AS-c-myc-ODN). This study demonstrated significant reduction in the maximal neointimal area by 30% in the treatment group. The neointimal thickness was also reduced to 40% of controls [Shi, 1994]. Human Studies: Given that SMCs derived from human lesions produce mitogens that are known to augment c-myc expression [Miano, 1993], it seems logical to have carefully-designed clinical trials to assess the efficacy of AS-c-myc-ODN in the treatment of post-injury stenosis. The results from a single center randomized placebo controlled trial of a 15-mer AS-c-myc-ODN for the treatment of re-stenosis were reported by Kutryk and colleagues [Kutryk, 2002]. Eighty-five 27

46 patients were randomly assigned to receive either 10 mg of phosphorothioate-modified 15-mer antisense ODN or saline vehicle by intracoronary local delivery after coronary stent implantation. The primary end point was percent neointimal volume obstruction measured by computerized intra-vascular ultrasound at six-month follow-up. Secondary end points included clinical outcome and quantitative coronary angiography analysis. The researchers utilized intravascular ultrasound to measure the extent of stenosis in 77 patients. In-stent volume obstruction was similar between the two groups (44 % placebo versus 46 % ODN, P = 0.57). There were no differences in angiographic re-stenosis rates (38.5% placebo versus 34.2% ODN, P = Despite the promising in vivo results, the findings from clinical trials suggest that AS-c-myc- ODNs are not effective in humans at least at the doses investigated. The reasons for this are unclear but might be related to low rates of uptake into the target cells, low transfection efficiency or mechanistic differences between humans and animals. AS-c-myb-ODNs: In vivo studies conducted by Simons et al employed gel-delivered 18-mer targeting the highly conserved nucleotides 4-22 of the c-myb sequence to the injured segments of rat carotid arteries. The investigators revealed that at two weeks following the vascular injury, message levels of c-myb were reduced by 65 folds in arteries treated with ODNs compared to controls [Simons, 1992]. Neointimal formation in AS-c-myb-ODN treated animals was markedly diminished (16% of controls). Edelman and colleagues also compared the efficacy of two AS-c-myb-ODN sequences (targeting nucleotides 4-21 vs of rat c-myb). Two different polymers (Pluronic gel and EVA matrices) were used for the peri-vascular delivery of the oligonucleotides. Irrespective of the 28

47 mode of delivery both AS-c-myb-ODNs, sequences showed distinct inhibition of neointimal thickening at 7 and 14 days post injury (intima:media ratios were >1.0 in control arterial segments while the ratio was <0.2 in treated segments) [Edelman, 1995]. AS-PCNA-ODNs: Simons et al tested the delivery of two AS-PCNA-ODNs in a peri-adventitial manner. The first sequence targeted the nucleotide 4-21 and the second ODN was against nucleotides of rat PCNA mrna in the rat carotid artery model of balloon injury. This study showed that AS-PCNA-ODNs, but not the sense controls, produce a 77% reduction in mrna at 24 hours and a 52% reduction in the number of medial PCNA expressing SMCs at 3 days. This reduction in PCNA expression was accompanied by 59% decrease in the number of proliferating SMCs and an 80% reduction in neointimal proliferation at 2 weeks post injury [Simons, 1994]. Current Status of Antisense Technologies: Although antisense treatment of accelerated vascular diseases shows a lot of promise, a number of major problems remain. Foremost among them are frequency of non-specific effects, toxicity associated with the use of oligonucleoitides and ineffective delivery of these molecules to the target site. A number of approaches are being developed to reduce binding of unintended sequences via partial matches. Reducing the length of the ODNs to nucleotides is one such approach. The other major issue facing antisense therapies is the lack of effective delivery of the ODNs to target tissues. A few delivery systems provide an effective means of localized targeting [Henke, 2000 and Miyano-Kurosaki, 2004]. 29

48 Moreover, the use of AS-ODNs is associated with incomplete effectiveness against key targeted cellular elements. Criticism is directed towards this approach since it targets upstream events in the transduction cellular machinery. Multiple pathways resulting from multiple cytokinereceptor interactions might allow the cell to escape the blockade effect of antisense oligonucleotides delivered locally. This was underscored by the cellular uptake of an overload of antisense oligonucleotide sequences against cdk2 and PCNA was found to be relatively low [Morishita, ]. However, efforts for gene expression profiling of human stenotic lesions by cdna array analysis are rendering the antisense approaches more appealing and feasible [Hilker, 2003]. On the other hand, anti-proliferative and immunosuppressive agents target a single major event (paclitaxel) or multiple central events (rapamycin) in the cell cycle, resulting in a caging situation for stimulated SMCs and myofibroblasts Brachytherapy: The general notion is that stenosis presents as a chronic proliferative response to injury and inflammation caused by the balloon/stent or other devices in the arterial settings. Therefore, the hypothesis that radiation can limit the proliferative response was formulated by several groups. The effect of vascular brachytherapy (radiation) was tested in a variety of experimental models. These studies collectively show that re-stenosis could be reduced to roughly 50% of its control value using beta or, less frequently, gamma radiation [Teirstein, 1997; Waksman, 2000 and Verin, 2001]. Brachytherapy is typically employed in the laboratory by an intra-vascular radiation source (usually a beta emitter) for 5-30 min and produces a long-term response of reduction of restenosis. However, by limiting the tissue growth and regeneration, brachytherapy produces an adverse response related to delayed and impaired healing [Costa, 1999]. Delayed 30

49 healing due to brachytherapy may cause the mechanical device such as a stent, which otherwise are fully embedded in the wall within a month, to remain uncovered for longer periods. Therefore, brachytherapy increases the chance of late vessel thrombosis. Furthermore, the effect on delayed healing of the vessel wall after the balloon trauma may lead to the formation of aneurysm around the stent. This clinical effect is similar to that caused by thrombosis leading to adverse endpoints such as myocardial infarction and sometimes death. Overall, studies show that brachytherapy is an effective tool to reduce re-stenosis, but it comes at the price of a higher likelihood of long-term complications related to delayed healing of the vessel wall. 1.3 TAXANES: Taxanes are a novel family of structurally related compounds that share a core ring structure called baccatin III. Among a number of compounds tested, paclitaxel and docetaxel (the active ingredients in Taxol and Taxotere ) exhibited the most potent activities against a variety of tumors. Extensive research in the late 80s and early 90s led to the approval of paclitaxel (1993) and docetaxel (1995) by regulatory bodies. Currently, these compounds gained great clinical significance through the past two decades in the treatment of ovarian, breast and colon cancers as well melanomas. Paclitaxel is originally a natural product from the north american Yew tree Taxus Brevifolia [Wani, 1971]. French researchers produced semisynthetic derivatives of baccatin III, and modified the ring with synthesized side chains. Docetaxel emerged as a result of these efforts and entered clinical trials in the 1990s [Ringel,1991]. 31

50 1.3.1 Chemistry and Mechanism of Cytotoxic Action. The taxane ring comprises a 4-member oxetan ring attached at positions C-4 and C-5, and a bulky ester side chain at C-13 [Wani, 1971; Ringel, 1991 and Kearns, 1997] (Figure ). The configuration of this ester chain is essential for the antitumor activity. Docetaxel configuration differs structurally from paclitaxel in two ways: in the structure of the attachment to the C59 carbonyl in the C-13 side chain; in the loss of the acetyl group esterified to the C-10 hydroxyl of the baccatin ring [Rowinsky, 1997 and Trudeau, 1996]. The mechanism of action of taxoids entails microtubule stabilization, which makes them resistant to disintegration and, hence, interferes with the normal mitotic process. Both taxoids bind to the β-subunit of tubulin, but the microtubules produced in the presence of docetaxel (average 13.4 tubulin subunits) are larger than those produced in the presence of paclitaxel (average 12 tubulin subunits). The binding site for paclitaxel is distinct from that of colchicine, podophyllotoxins, or vinca alkaloids [Rowinsky, 1997]. Docetaxel binds to tubulin more avidly than paclitaxel and is retained inside intracellular compartments for longer periods than paclitaxel. This may explain why docetaxel may be two to four times more potent than paclitaxel as an antitumor agent [Gelmon, 1994]. In addition to disruption of cellular mitosis, there is evidence that the cytotoxicity of taxanes entails apoptosis. Studies in human myeloid leukemia cell lines corroborate this observation of paclitaxel-induced apoptosis involving phosphorylation pathways. The induction and modulation of p53 have been demonstrated, but are not essential for the initiation of apoptosis. Bcl-2 over-expression was also found to delay paclitaxel-induced apoptosis [Wahl, 1996]. Paclitaxel results in the direct phosphorylation of bcl-2, induced expression of bcl-x and Raf- 1(which causes a decrease of bcl-2). All of these mechanisms have been shown to increase 32

51 apoptosis [Sackett, 1997]. Other studies also attributed anti-angiogenic effects to taxanes [Lennernas, 2003] Pharmacology and Administration. Taxoid compounds are insoluble in aqueous solution. Therefore, paclitaxel is formulated in 49.7% alcohol and 50.3% polyoxyethylated castor oil derivative, known as Cremophor EL [Dorr, 1994]. After dilution into sodium chloride for injection, or 5% dextrose solution in glass containers, it is usually administered through specific in-line filters and tubing sets. The observation of hypersensitivity reactions to paclitaxel in early phase I trials led to changing the infusion rate. It was observed that infusion of paclitaxel ( mg/m 2 ) over a 24-hour period resulted in similar anti-cancer activities but much lower hypersensitivity compared to its delivery over 6 hours. Presumably, this was because paclitaxel activity was related to the overall AUC, while hypersensitivity was more a consequence of peak Cremophor levels. However, a 24-hour infusion requires overnight hospitalization. Alternatively, the dosage regimen currently employed is a 3-hour infusion with the use of corticosteroids such as dexamethasone [Kearns, 1995; Gelmon, 1994 and Trudeau, 1996]. In current practices, the recommended dose of paclitaxel as a single agent or in combination now ranges from 135 to 250 mg/m 2, generally as a 3-hour infusion repeated every 3 weeks. These adult dosages and treatment schedules cannot be directly extrapolated into the pediatric setting. On a given schedule, children tolerate higher doses better than adults do on a body surface area basis. However, the average clearance rate in children at 290 mg/m 2 lies within the range reported for adults at mg/m 2 (135 ml/min/m 2 in children versus 102 to 359 ml/min/m 2 33

52 in adults) [Sonnichsen, 1994]. In addition, similar C max values of approximately 1 mmol/l were achieved in pediatric and adult patients by the end of 24-hour infusions. These data do not identify the underlying reason for the pediatric difference, but do indicate that it cannot be a difference in the rate of drug elimination Pharmacokinetics of Paclitaxel. The taxanes are highly bound to plasma protein (paclitaxel 95% bound, docetaxel 90% bound) [Trudeau, 1996]. Tissue distribution and binding influence the rate of plasma clearance; paclitaxel shows saturable distribution and nonlinear pharmacokinetics [Trudeau, 1996 and Eisenhauer, 1998], which results in a disproportionate increase in area under the curve (plasma AUC) or C max with dose. The mean clearance of paclitaxel appears to decrease as the dose is increased if the schedule remains constant. For a dose of 135 mg/m 2, the clearance rate is 14.7 L/hr/m 2 ; for a dose of 250 mg/m 2, the clearance rate is 8 L/hr/m 2. Hence, the severity and duration of toxicity increase disproportionately with dose escalation [Kearns, 1997]. This phenomenon has multiple clinical ramifications in the observed efficacy and adverse effects of the drug. The ramifications of saturable elimination have greater influence at higher doses where the plasma concentrations exceed the affinity constant for elimination (K m ). For 3-hour infusions, a 30% increase in dose ( from 135 mg/m 2 to 175 mg/m 2 ) results in an 80% increase in AUC (from 10.9 mmol/l. hr to 18.5 mmol/l. hr). Similarly, if the dose of paclitaxel is reduced because of excess toxicity from one cycle to another, then it decreases the AUC significantly and may compromise efficacy. Consequently, the comparison of response rates to dose intensity within or across clinical trials is very difficult if different dosing schedules of paclitaxel are used. 34

53 The nonlinear pharmacokinetics of paclitaxel could be attributed to its vehicle of formulation. Sparreboom et al demonstrated in a mouse model that when paclitaxel was formulated in 50% polyoxyethylated castor oil derivative, it had a nonlinear disposition; when formulated in Tween 80, the kinetics changed to a linear pattern [Sparreboom, 1996]. This issue requires further investigation since there are significant inter-species differences in paclitaxel disposition Paclitaxel Metabolism. The systemic clearance of paclitaxel is through hepatic metabolism. Renal clearance is minimal and accounts for less than 5% [Monsarrat, 1997]. Both paclitaxel and docetaxel are metabolized by hepatic cytochrome P450 enzyme systems and eliminated by biliary excretion [Rahman, 1994; Cresteil, 1994 and Harris, 1994]. Paclitaxel undergoes stereospecific CYP2C8 hydroxylation at the C6 position of the taxane nucleus to form 6-α-hydroxypaclitaxel, the major metabolite in humans. Paclitaxel is also hydroxylated by CYP3A4 in the para position on the phenyl group attached to C3 of the C-13 side chain to generate 3 -p-hydroxyphenyl-paclitaxel. In vitro, the levels of each mono-hydroxylated species can be influenced by the inductive effects of co-medications. Each monohydroxylated species can be further metabolized, probably by the other pathway, to converge to the dihydroxy taxol metabolite, 6-α-hydroxyl- 3 -phydroxyphenyl-paclitaxel. The half-life of total metabolites (5.6 ± 0.4 hours) greatly exceeds that of unchanged taxol (2.9 ± 0.3 hours) [Harris, 1994]. The hydroxylation significantly reduces potency in cytotoxicity assays, but not in microtubule binding assays. Thus, this suggests that.even though the hydroxylated products may retain the potential anti-microtubule activity; they may not be able to penetrate cellular membranes. 35

54 O R 2 O O OH R1 Ph NH OH O O OH H OBz OAc O Paclitaxel (1): R = Ph, R1 = Ac Docetaxel (2): R = t-buo, R1 = H Figure Chemical structures of taxanes: (1) paclitaxel and (2) docetaxel 36

55 1.3.5 Toxic and Adverse Effects. Myelosuppression is the dose limiting toxicity of clinically used taxanes. Pharmacology studies during clinical trials of paclitaxel have found that the severity of neutropenia is most closely associated with the duration that plasma levels remain above nmol/l [Huizing, 1993]. Thus, the severity of the neutropenia is related to the infusion duration of paclitaxel; it increases with longer infusions. Mucositis follows the same pattern [Rowinsky, 1997 and Trudeau, 1996]. Docetaxel causes neutropenia equivalent to that with a 24-hour infusion of paclitaxel. Alopecia, mild gastrointestinal toxicity, and hypersensitivity reactions are observed with equal frequency during therapy with either taxane. Cardiac arrhythmias, especially asymptomatic bradycardias, are seen with paclitaxel [Kearns, 1997; Rowinsky, 1996; Trudeau, 1996 and Eisenhauer, 1998]. The presence of a pacemaker or a prior history of cardiac conduction defects are relative contraindications to paclitaxel. Combinations with doxorubicin increase the incidence of congestive heart disease [Gianni, 1995]. No adverse cardiac effects have been reported with docetaxel. Dose-related myalgia and neuropathy are seen with paclitaxel; particularly increases in neurosensory symptoms are observed with cisplatin combinations [Huizing, 1993] Paclitaxel as an Anti-stenotic Agent: In Vitro and In Vivo Experience. As described in the above sections, the pathophysiology of stenosis consists of complex interaction of cytokines and several growth factors with cellular and acelullar elements. The conclusion that can be logically derived is that blockade of any one factor is often not enough to inhibit the stenotic cascade. Therefore, attention has been focused on disrupting essential central cellular processes that would consequently affect major downstream events that ultimately result 37

56 in intimal thickening. Microtubules are major contributors to many cell functions including cell division, motility, transport and extracellular secretory processes. The unique mechanism of action of paclitaxel, acting on polymerizing microtubules, makes it a good candidate for the treatment/prevention of stenotic lesions. Previous studies by several groups demonstrated that paclitaxel can inhibit the growth and migration of SMCs in vitro both after single high dose application or continuous drug exposure. Continuous incubation with paclitaxel concentrations between 0.01 and 10 µm showed highly significant inhibition even of growth factor-stimulated SMCs in culture [Axel, 1997]. As angiogenesis is a major event contributing to intimal growth following vascular injury, the ability of paclitaxel to inhibit proliferation of ECs was examined by several groups. Single dose exposure to 0.01 µm paclitaxel did not compromise EC proliferation significantly. However, continuous exposure to paclitaxel (concentrations range µm for 8 days) in vitro significantly inhibits of EC proliferation. These promising results have been substantiated with in vivo studies that used locally applied paclitaxel to prevent neointimal formation after balloon dilatation of plaques that were induced by electrical stimulation of rabbit carotid arteries. Histopathological examination of the vessel lumens 28 days after balloon angioplasty revealed that the animal group treated with locally delivered paclitaxel had a mean degree of stenosis around 26% while controls had a mean of 34% stenotic extent [Axel, 1997]. 38

57 1.4 LOCALIZED VERSUS SYSTEMIC APPROACHES: Rationale for Formulating Paclitaxel-Based Localized Treatments. Most anti-proliferative drugs in clinical use are non-specific and they affect all rapidly dividing cells including normal tissues. It is well-known that this non-specific activity results in extensive toxicities to normal tissues. Hence, a preferred approach would be to use pharmacokinetic principles to optimize drug administration. In this regard, targeted drug delivery has the potential to reduce the adverse effects while enhancing the desired therapeutic activity. One main principle is targeted drug delivery. Furthermore, localized drug delivery systems is a method of first order targeting by enhancing drug delivery to tissues harboring the lesions involved in pathogenesis. A number of studies have investigated such approaches. These include: paclitaxel [Winternitz, 1996; Fung, 1998, Zhang, 1997] and BCNU [Fung, 1998] among others. Advantages of localized anti-proliferative therapies include: (a) reduction in bone marrow suppression and drug-specific toxicities such as cardiac rhythm disturbances [Boye, 1995; Perez, 1998], (b) requirements for reduced drug doses [Sonnichsen, 1994], (c) maximal localized drug exposure measured by exposure time and tissue concentration. This is also closely related to drug loss resulting from first pass effects, since the drug is directly available to the target tissue [Markman, 1995], (d) reduction / elimination of the need to employ pre-medications. 39

58 1.4.2 Rationale for Enhanced Target Tissue/Plasma Ratio with Localized Delivery Most of our understanding of the regional exposure advantage of anti-proliferative agents is derived from the investigation of localized delivery for purposes of treating solid tumors. The concept of regional drug exposure advantage (R de ) for anti-neoplastic agents is related to drug clearance and the blood perfusion rate into the target region by the formula [Eckman, 1974 and Dedrick, 1988]: R de = 1 + CL T /(Q R (1-E R ) where CL T is the total body clearance, Q R is the blood flow rate and E R is the extraction ratio of the drug at the target region. The implications of this equation are that: (i) a clinically viable candidate for localized delivery would be a drug that has high extraction ratio and high body clearance or both, (ii) an effective drug delivery system would lead to high drug levels sustained at the site of injury by releasing ample drug amounts while decreasing the blood perfusion at the target site. Application of this equation to calculate R de in the theoretical situation of paclitaxel can be viewed in the following manner: a) paclitaxel disposition systemically is biexponential. Alpha and beta half lives of paclitaxel are 2.9 and 5.0, respectively [Harris, 1994 and Rowinsky, 1990]. b) the molecule is highly lipophilic (log P O:W = 3.5) [Heimans, 1994]. The above values make R de of localized paclitaxel delivery particularly interesting; that is, the drug is extracted by the target tissue in its first pass resulting in increased target tissue burden. Hence, the amount of drug available to the systemic circulation and potentially partioning into peripheral compartments (non-target tissues) for toxic drug reactions is considerably reduced. Thus, localized paclitaxel delivery may result in alleviating the body burden both pharmacokinetically and pharmacodynamically. 40

59 1.4.3 Polymeric Formulations: Successful Formulation Attributes and Applications. Polymeric systems, that release the drug by diffusion, erosion or a combination of both mechanisms, have been utilized for several applications for controlled and localized drug delivery. Parenteral use, such as that investigated here, requires the use of biocompatible polymers. Such polymers are further characterized as being biodegradable or biostable. In the case of biodegradable polymers, matrix erosion is usually controlled via hydrolytic reactions or enzymatic processes. Involvement of hydrolytic processes is more favorable since they are less prone to inter-subject variability. Sterilization of such polymers can be achieved through gas, gamma irradiation or UV surface methods. From a clinical perspective, drug release rates from polymeric matrices must be controlled so that drug levels in the target compartment are neither below the therapeutic window nor they are on the asymptotic portion of the dose-response curve. At the asymptotic portion of relationship profile, a large increase in the drug levels results in minute increase in the inhibitory effects. The excess drug will have the chance to be washed into the systemic circulation causing adverse drug reactions. Also, drug release patterns are tailored to coincide with the series of major events leading to the development of the lesion. To summarize, in order to develop a clinically useful polymeric delivery system for a drug, a good formulation would possess flexibility in terms of its physical properties and drug release characteristics. The rate and extent of drug release are governed by the drug solubility in the polymer and in the release medium, the surface area of the device and the ability of the leaching medium to imbibe into the loaded matrix. As experience has been with many research groups, engineering such dosage forms to the desired release pattern is a challenge. In many instances, the drug release 41

60 profiles follow a square root of time relationship. A plot the percentage of the drug released versus the square root of time yields a straight line. The slope of such a plot is termed (D eff ), which is effective diffusivity of the drug through the releasing matrix. Such a relationship indicates a diffusion-controlled mechanism [Desai, and Siepmann, 2001]. In case of bio-degradable polymers erosion takes over as the primary mechanism by which the drug load is gradually released once the initial burst effect is complete. Release of hydrophobic agents from bio-stable polymeric matrices, particularly those that are hydrophobic, is virtually incomplete. The extent of drug liberation from such matrices can be increased in the presence of additives possessing some swelling nature [Dordunno, 1997 and Zhang, 1996]. The presence of such additives with appropriate hydrophilic/lipophilic balance in the delivery system aids in achieving the desired drug release patterns. The adjuvants with high swelling abilities create microchannels upon partial sorption. This facilitates the escape of drug molecules from the matrix (Fig ). Materials that can aid in drug liberation through the aforementioned mechanism include polyethylene glycols (PEG4600 [Fishbein, 2001], PEG400, PEG4600), sodium chloride, gelatin [Di Colo, 1982] and low molecular weight PLA amongst others Endo-vascular versus Peri-vascular Delivery of Paclitaxel. As described in previous sections, local drug delivery to the vascular wall can be achieved either through the endo-vascular route, utilizing porous catheters and drug-loaded stents, or through surgically implanted peri-vascular release devices. Regardless of the approach, the transfer of drug from the point of release to the target tissue relies on mechanisms of drug transport, such as diffusion and convection as well as the partitioning of the drug in and around target tissues. These mechanisms in turn depend on the physicochemical properties of 42

61 the drug and the surrounding environment. For example, hydrophobicity or absolute charge can regulate transport and distribution of drug in and around arterial tissues. Water-soluble drugs readily permeate into tissues. However, while this facilitates the rapid equilibration and distribution of the drug in tissue, this also leads to rapid clearance from the site of delivery. As a result, there is increasing interest in less soluble, more hydrophobic compounds. Hydrophobic compounds are relatively insoluble in the aqueous phase, and tissue partitioning and retention are achieved by binding to available hydrophobic elements on fixed tissue sites. Unlike hydrophilic drugs, hydrophobic compounds can possibly remain in and around target vascular tissues for a long time after application. The complexity of the forces that govern the coupled transport and binding of hydrophobic drugs requires in-depth characterization in order to achieve clinical goals. Paclitaxel is a good example of a hydrophobic compound with high therapeutic potential in proliferative vascular diseases. Its ultimate clinical use may depend on thorough characterization of the binding and diffusional mechanisms of this drug in vascular tissues. Creel et al performed ex vivo experiments to assess the disposition of paclitaxel across the layers of arterial wall following peri-vascular and endovascular application [Creel, 2000]. Paclitaxel deposition at equilibrium within the arterial wall varied across the arterial wall and but was found to be significantly higher at the layer in direct contact with the drug pool. Permeation into the wall increased with exposure time and varied with the origin of delivery. In contrast to hydrophilic compounds, the concentration in tissue exceeded the applied concentration and the rate of transport was markedly slower (lower effective diffusivity). Furthermore, endo-vascular and peri-vascular paclitaxel application lead to markedly differential deposition across the blood vessel wall. These data suggest that 43

62 paclitaxel interacts with non-specific arterial tissue elements as it moves under the forces of diffusion and convection and can establish substantial partitioning and spatial gradients across the tissue. Total arterial deposition with endovascular application was at least twice that of the peri-vascular one with the exposure times beyond 15 minutes. Different structures in the vessel wall have different affinities to paclitaxel and this is apparent with the differential partitioning of the drug for both the endovascular and peri-vascular applications. Accordingly, application of paclitaxel perfusate via the endo-vascular route results in highest drug partitioning within the intima followed by markedly lower levels within the media and adventitia. With the perivascular application, however, maximal tissue concentrations are found within the adventitia and declining levels were observed towards the intima [Creel, 2000]. These findings underscore the fact that paclitaxel concentrations following endovascular application are expected to be high within the intima which may result in delayed intimal healing. Moreover, while endo-vascular application using DES are quite effective in the clinical setting, it has much higher chance of resulting in systemic exposure to cumulatively high amounts of paclitaxel, at least theoretically. This is less likely to occur with peri-vascular delivery. As indicated earlier, endo-vascular delivery of paclitaxel via coated-stents is a successful strategy for the treatment of vascular stenosis. Peri-vascular delivery is an emerging approach and has not been as intensively investigated. Only a few efforts have been made to utilize the peri-vascular techniques to deliver anti-proliferative agents such as tyrphostin, mithramycin and heparin. We came across only one study that describes peri-vascular paclitaxel delivery 44

63 [Signore, 2001]. All of these studies focused on the balloon-injured carotid or coronary arteries. Moreover, none of the aforementioned research applications attempted to test or modify the drug release patterns or to correlate the applied doses with the pharmacodynamic end points. In the study by Signore et al, paclitaxel was applied in polymeric wraps to the peri-vascular aspect of balloon-expanded carotids in rats. Injured non-treated arteries exhibited a pronounced intimal hyperplasia ( / mm 2 at 14 days and / mm 2 at 28 days). A marked reduction in luminal area was also observed in non-treated groups (44% at 14 days and 43% at 28 days). Injured arteries treated with peri-vascular paclitaxel did not show any intimal hyperplasia, and luminal area was increased in five of six treated groups (unchanged in one group). A group of animals received drug-free polymeric matrices to test whether the polymer itself possesses anti-stenotic properties. Injured arteries treated with sham polymeric implants exhibited intimal hyperplasia and luminal narrowing Peri-Vascular Delivery in Failing PTFE Dialysis Access Grafts: Rationale and Novelty. In the special context of PTFE dialysis access grafts, we believe that the better choice for localized delivery systems for paclitaxel would be the peri-vascular application. The factors supporting such a choice include: (i) Ease of delivery in the clinical setting: it would be relatively easy to wrap the mechanically-coherent polymer around the graft-vein anastomosis at the time of surgery, (ii) Potentially higher efficacy: As indicated earlier, this technique has been successfully employed to block hyperplastic vascular growth in pre-clinical animal models for a variety of agents in the arterial setting, (iii) Potential effects on adventitial remodeling: it is likely perivascular delivery approaches will positively impact all three layers of the vessel wall (adventitia, media and intima) as a result of continuous diffusion and differential partitioning through these 45

64 layers. Given that adventitial remodeling and fibroblast migration play a major role in stenosis, this pattern of partitioning may be advantageous to achieve favorable remodeling. In addition, such a delivery system can also result in significant anti-angiogenic effects, thus further contributing to the inhibition on VNH, (iv) Intimal healing: as discussed earlier, complete reendothelialization is a major event in alleviating the continuous injurious process to vessel walls. Moreover, complete intimal restoration is a desired clinical end point. The amounts of pacltaxel reaching the intimal layer following peri-vascular delivery are expected to be relatively low. As such, this approach is more likely to facilitate complete repair of the intima. 46

65 Fig Drug release kinetics from polymers can be modulated by the addition of a channeling agent: a diagrammatic presentation illustrating the physical interaction between the polymeric matrix, the loaded therapeutic agent and the polymeric channeling agent. 47

66 2 HYPOTHESIS AND SPECIFIC AIMS As described earlier, the main cause of dialysis access failure is venous neointimal hyperplasia (VNH). There are three major events leading to the development of VNH: (i) proliferation and migration of SMCs in response to de-endothelialization and cytokine release (ii) vascular remodeling and contribution from adventitial fibroblasts and (iii) angiogenesis. Paclitaxel is a potent anti-proliferative / anti-angiogenic agent and is an excellent candidate for localized drug delivery based on the properties described earlier. VNH leading to PTFE dialysis failure is pathologically aggressive and responds poorly to surgical interventions. We believe that PTFE dialysis grafts are ideally suited for peri-vascular local therapeutic approaches in view of their superficial location, their distance from vital organs and the relative ease of implantation, either at time of graft placement or during subsequent dialyses. Therefore, the main thrust of this proposal was to develop a novel polymeric drug system for effective delivery of antiproliferative therapies to treat VNH. In this context, the central hypothesis of our research was: Localized, controlled delivery of paclitaxel at the graft-vein junction effectively inhibits neointimal growth while minimizing the systemic exposure to the cytotoxic agents. The hypothesis was tested through the following specific aims: Specific aim 1: To develop peri-vascular polymeric wraps for phased delivery paclitaxel, for effective control of neointimal formation. Employing a biocompatible polymer, ethylene vinyl acetate (EVA), and a channeling agent, polyethylene glycol (PEG), efforts were made to prepare an optimized formulation that releases biologically active paclitaxel. The bases for product optimization included: 1) drug loading 48

67 uniformity, 2) physico-chemical properties, 3) paclitaxel stability and 4) rate and extent of in vitro drug release (initial burst and sustained release phases). Specific aim 2: To examine the cytotoxic activity of the polymeric drug devices employing clinically relevant in vitro primary cell cultures and cell lines. This entailed an evaluation of the cytotoxic activity and cellular disposition of the active agents embedded in the polymeric matrices. The cytotoxic activity was tested against different cell types known to participate in VNH, namely smooth muscle cells, endothelial cells and fibroblasts. Specific aim 3: This aim focused on the evalutaion of the pharmacokinetics / pharmacodynamics of the polymeric drug delivery system employing a clinically relevant animal model. For this purpose, at the time of graft insertion into each animal, the optimized polymeric wraps were implanted at the graft-vein anastomosis on one side with appropriate controls implanted on the contra-lateral side. At different time intervals, animals were sacrificed and grafts with attached tissues were harvested. In-graft stenosis was assessed utilizing auscultations and histomorphometric analysis. To assess for systemic exposure following peri-vascular implantation, blood samples were drawn at different time points from a number of treated animals and assayed for paclitaxel content in plasma. 49

68 3 MATERIALS 3.1 Chemicals. Paclitaxel and bovine serum albumin (BSA) were obtained from Sigma Chemical Co., (St Louis, MO). EVA (Elvax 40) was obtained from Du Pont Chemical Co., (Wilmington, DE). Granular polyethylene glycol4000 (PEG4000) was obtained from Dow Chemical Co. (Danbury, CT). Methylene chloride, ethyl acetate, dimethylsulfoxide (DMSO), isopropyl alcohol, methanol and all other chemicals (HPLC grade) were obtained from Fisher Scientific (Santa Clara, CA). 3.2 Cell lines and Biological Reagents. Human umbilical vein endothelial cells (HUVEC), neonatal human dermal fibroblasts (NHDF) and human aortic smooth muscle cells (HASMC) and respective culture media (SmGM- 2 and EBM-2 with supplements) were obtained from Cambrex Bio Science Walkersville, Inc. (East Rutherford, New Jersey). Growth supplements in smooth muscle cell culture media included human endothelial growth factor (hegf), insulin, human fibroblast growth factor-b (hfgf-b), fetal bovine serum (FBS), glutamine. Supplements for endothelial and fibroblast growth media included hydrocortisone, hfgf-b, vascular endotheial growth factor (VEGF), ascorbic Acid, heparin, FBS, hegf and GA. According to information supplied by the company, routine characterization of HUVEC includes morphological observation throughout serial passages as well as adhesion molecules and specific markers. Cryopreserved cells were thawed on receipt, seeded in T-25 flasks for 5-9 days at 37 C under 5% CO 2. Following the first passage, cells were sub-cultured every 5-9 days and used in experimentation up to passage 15 as recommended by the company. 50

69 3.3 Assay methodology. We adopted and validated a combination of two previously published methods [Jamis- Dow, 1993 and Waters Corp.; in-house files) for the analysis of paclitaxel in different biological media. The method utilized high performance liquid chromatography with ultraviolet detection (HPLC/UV). Sample Preparation: a) Plasma and interstitial fluid: Samples were extracted on a C 18 solid-phase column (6 ml Bond Elut, Varian Sample Preparation Products, Harbor City, CA). Each column was preconditioned with 3 ml water followed by 3 ml acetonitrile and then a final wash with 3 ml water. The sample (200µl) was applied to the column and washed with 3 ml of water to remove any traces of water-soluble impurities. The extract was collected by eluting the column with 4 ml acetonitrile twice. The eluent was dried on heating blocks at temperatures 60 C under a constant stream of nitrogen using the Reactivap / Reactitherm III drying system (Pierce Inc., Rockford, IL). The extract was reconstituted in 200µl methanol: acetonitrile: water (9:34:57). b) In vitro drug release medium: A sample of 2 ml of the medium used for drug release kinetics experiments was drawn and paclitaxel was extracted twice with 3 ml ethyl acetate. The supernatant organic phase was withdrawn out and evaporated under a continuous stream of nitrogen as described above. The extract was reconstituted in 200 µl methanol:acetonitrile:water (9:34:57). 51

70 HPLC Conditions: A volume of 100 µl of reconstituted extracts were isocratically chromatographed with acetonitrile: methanol:water 34:9:57 at a flow rate of 1 m/min. Separation was achieved on a Symmetry C 18, 5µm, mm column (Waters Corporation, Milford, MA). Pre-column C 18 inserts were replaced approximately every 40 samples to maintain optimum resolution. Docetaxel (Aventis Pharmaceuticals, Bridgewater, NJ) was used as the internal standard. Paclitaxel and docetaxel peaks were detected at 229 nm using Waters 486 ultraviolet tunable absorbance detector. A standard curve of paclitaxel and docetaxel concentrations dissolved in mobile phase was obtained in the µm concentration range and used directly to quantitate the amount of paclitaxel released employing linear regression. 3.4 Animal Experiments. Species and Gender: Domestic swine Yorkshire-cross male pigs (mean body weight = 47 kg) were purchased from Yeazel and Co. (Wolverton, OH). Animal care and housing and surgical procedures were performed at the Department of Laboratory Animal Medicine Services (LAMS), an Assessment and Accreditation of Laboratory Animal Care (AAALAC)-approved animal facility. Programs for preventive medicine, husbandry, pre- and post-surgical care were developed to assure adequate veterinary care and were provided at all times. Protocols for the animal studies were approved by the Institutional Animal Care and Use Committees (IACUC). 52

71 4 METHODS 4.1 PREPARATION AND TESTING OF PACLITAXEL-LOADED POLYMERIC SYSTEMS Preparation of Paclitaxel-Loaded Polymeric Matrices. Polymeric films were prepared using solvent casting technique. Paclitaxel and EVA were dissolved in methylene chloride to yield a clear polymeric solution. Paclitaxel content (expressed as % w/w of EVA) was kept at 5%. In our initial experiments, we observed that paclitaxel embedded in polymeric matrices exhibited extremely slow and incomplete drug release patterns. Therefore, we tested the effects of incorporating PEG4000 as a channeling agent on the rate and extent of drug release. EVA films with different % w/w loadings of paclitaxel and PEG4000 in EVA were prepared as described in Table The bubble-free solution was poured into PTFE evaporation dishes, air-dried and the cast polymeric matrices were cut into rectangular pieces ( cm, 0.3 mm thickness) for the assessment of in vitro drug release, swelling (fluid uptake), drug stability and in vitro cytotoxicity. Table Composition of different polymeric formulations tested. Formulation I II III IV Composition (% of film weight) 5% paclitaxel + 95% EVA 5% paclitaxel+ 8% PEG % EVA 5%paclitaxel+15% PEG % EVA 5%paclitaxel+20% PEG % EVA 53

72 4.1.2 Mechanical Properties of Loaded Polymeric Matrices. Freshly prepared polymeric matrices described above were cut into rectangular pieces of cm with a thickness of 0.3 mm thick for stress-strain measurements at room temperature. The apparatus consisted of a cell in which the sample was mounted between two clamps, one attached to the bottom and the other connected to a movable force transducer (Statham Model G ) operated at a constant voltage and a dc power supply (Hewlett-Packared Model 6234A). The output from the transducer was displayed on a recorder (Goerz, Servogor 124) that was calibrated using a set of standard balance weight. Test sections were demarcated on the center of each slab. An isothermal stress-strain test was carried out as follows: the sample slabs were mounted between the clamps and the unstressed length of the test section (L i ) was measured to an accuracy of ±0.002 cm by means of a cathetometer (Mitutoyo model ). The sample was then elongated to a pre-determined length and the force employed was monitored until it remained constant for at least 5 minutes. This was considered to be the equilibrium value of the force. This force was recorded along with the initial length (L o ) and the stretched lengthlength (L) of the stretched sample. A series of measurements at five or more elongations consecutively with increasing L/L o of at least 1.2 were carried out in this manner. Hooke s Law states that stress is directly proportional to the strain according to the following equation: F/A = E (L-L o /L o ) Where F is the applied force (N), A is the cross-sectional area of the slab (mm 2 ). Young s modulus (the modulus of elasticity) is a measure of the hardness, stiffness or rigidity of the slab the was then calculated from the slope of the initial linear portion of the stress-strain plot as shown in Figure

73 4.1.3 Drug Content Uniformity. Polymeric slabs ( cm, 0.3 mm thick) were cut and accurately weighed. The loaded matrices were then dissolved in 300 µl of methylene chloride and sonicated for 10 minutes. Methanol (3 ml) was added to the methylene chloride solution in increments of 1 ml until a polymeric precipitate was formed or turbidity was evident. The turbid solution was sonicated for 10 minutes and then centrifuged at 4500 rpm for 10 minutes. The supernatant was drawn and evaporated under a continuous nitrogen stream at temperature 40 C until dryness. The residue was reconstituted in 250 µl of methanol: acetonitrile: water mixture (9: 34: 57) and assayed for paclitaxel utilizing a validated HPLC/UV method. The paclitaxel content was then calculated and compared to the theoretical content in the weighed matrices employing one-way analysis of variance (ANOVA) Chemical Stability of Paclitaxel Embedded into Polymeric Matrices. The stability of paclitaxel embedded in EVA matrices and released over time, in PBS (ph 7.4) containing 0.4 g/l albumin as well as upon dry storage in the dark, was tested over a period of 21 days. The amount of unchanged paclitaxel retained in matrices and/or released into the medium was determined by analyzing both the amount of intact drug released and the amount remaining in the matrix. Paclitaxel released into the release solution was extracted using two volumes of 3 ml ethyl acetate extraction. The organic phase was separated, dried under a continuous stream of nitrogen. The residue was reconstituted in mobile phase and analyzed utilizing the HPLC method described earlier. The amount of intact drug retained in the matrices (dry stored and partially desorbed) was determined by sonicating the tested matrices in 1 ml methylene chloride for 5 minutes. Four volumes of methanol were slowly added (1 ml at a time) 55

74 to the methylene chloride to precipitate EVA while keeping paclitaxel in solution. Extracts were then dried under a stream of nitrogen and analyzed utilizing the HPLC method described earlier Fluid Uptake and Weight Loss Experiments. EVA matrices with different amounts of the channeling agent, PEG4000, were cast and cut as mentioned earlier and the initial dry weight of each slab recorded. These matrices were subsequently incubated in 10 ml of PBS (ph 7.4) containing 0.4 g/l bovine serum albumin and 3% isopropyl alcohol under gentle agitation at room temperature. At different time points (up to 10 days), the matrices were removed, their surfaces were blotted dry and each sample was weighed to determine the extent of swelling (weight gain). At each sampling time point, the bathing medium was replaced to provide sink conditions. At the end of the experiment, the polymeric slabs were air dried for 72 hours to remove any trapped moisture. Matrices were then weighed to determine eventual mass loss. Percent swelling was defined as: 100 [(the wet weight at time point (t x )- the initial dry weight(t 0 ))/ the initial dry weight(t 0 )]. For purposes of stating results, dry designates the final slab weight following the 13 day period Morphological Analysis Using Environmental Electron Microscopy (ESEM). The structural features of EVA films containing 5% paclitaxel or a combination of 5% paclitaxel/15% PEG 4000 were assessed using environmental scanning electron microscopy (Philips XL30 ESEM-FEG) operating at 1-20 kev to minimize irradiation damage to the matrices. Rectangular polymeric slabs cm, 0.3 mm thickness containing 5% paclitaxel by weight were cut and accurately weighed. Each film sample was visualized at different 56

75 magnifications under two different perspectives (surface, cross sections) at different beam angles. Post-desorption samples were incubated at room temperature for 24 hours in 10 ml of 70% (v/v) ethanol solution with mild agitation. The incubation with ethanol was performed to accelerate the dissolution for PEG4000. The films were then transferred to and incubated in 10 ml of PBS (ph 7.4) containing 0.4 g/l bovine serum albumin for an additional 48 hours with mild agitation. The polymeric samples were then removed from desorption medium, washed with de-ionized water, air-dried for 72 hours and then imaged on ESEM In Vitro Paclitaxel Release from Polymeric Formulations. Rectangular slabs cm were cut from the above described formulated films and accurately weighed. The cast matrices were placed in 20 ml scintillation vials with 10 ml of PBS (ph 7.4) containing 0.4 g/l bovine serum albumin. The vials were kept at 37 C in the dark with rotational mixing at 50 rpm. At pre-selected time points, 2 ml samples were withdrawn for paclitaxel analysis. The bathing medium was replaced with fresh medium to maintain sink conditions. Sample medium was placed in glass test tubes and paclitaxel was extracted using 3 ml of ethyl acetate. The tubes were capped and vortex-mixed for 30 seconds to allow paclitaxel to partition into the supernatant organic phase. The tubes were then centrifuged at 2000 g for two minutes. The top organic ethyl acetate layer was withdrawn. The extraction procedure was repeated one more time. The two organic fractions were combined and evaporated under nitrogen stream. The dried residue was then reconstituted in 250 µl of acetonitrile:water 43:57 mixture. Samples were then analyzed utilizing a modified HPLC method to the one described earlier. 57

76 4.1.8 Statistical Analysis. Data are presented as mean ± S.D. Comparisons of release profiles (extent at several time points) of different formulations were assessed using the analysis of variance (ANOVA) with Tukey s analysis for pai-wise comparisons when applicable. A value of p <0.05 was considered to be significant. 4.2 IN VITRO CYTOTOXICITY OF PACLITAXEL RELEASED FROM POLYMERIC FORMULATIONS Inhibition of Cell Proliferation by Polymeric Paclitaxel. HASMC, NHDF and HUVEC were used as representatives of cells involved in neointimal formation. For each experiment, cells were plated into each of 12-wells and allowed to attach and grow for 24 hours. Cells were then exposed to the following formulations in triplicates (three wells per formulation): drug-free EVA, 5% paclitaxel/eva, 5% paclitaxel/15% PEG4000/80%EVA and 15% PEG4000/85%EVA. Triplicates of wells with no added drug or polymer (only DMSO) served as universal controls. Cells were incubated with these formulations for 6 days under gentle mixing. The medium was changed every other day to maintain sink conditions. At the end of 6 days, cells were washed with sterile PBS and cell viability was assessed utilizing a validated (3-(4, 5- dimethylthiazolyl-2)-2, 5- diphenyltetrazolium bromide) (MTT) assay. This method is based on the ability of mitochondrial dehydrogenases of viable cells to reduce the yellow MTT into purple formazan which absorbs at 540 nm. At the end of 4-hour incubation with MTT, culture medium was removed and 500 µl of DMSO were added to the cells. The absorbance of treated cells was compared to that of 58

77 controls as a measure of cell viability. Cell survival was then expressed as % of untreated controls Statistical Analysis. The comparison between the cytotoxic effects of paclitaxel released from different formulations was carried out employing analysis of variance (ANOVA) with Tukey s analysis for paiwise comparisons when applicable. Data are presented as mean ± S.D. Differences were considered significant at p DETERMINATION OF IN VIVO OF EFFICACY OF THE OPTIMIZED PACLITAXEL- LOADED SLABS Preparation of Paclitaxel-Loaded Polymeric Matrices. Paclitaxel-loaded matrices were prepared according to the experimental protocols described under section Matrices prepared were cut into rectangular pieces mm, 0.3 mm thick and were sterilized under UV light for 30 minutes before implantation into animals. To ensure unidirectional flux of the drug, a backing EVA sheet (thickness = 0.6 mm) was attached to the polymeric slab using few drops of methylene chloride as a glue before sterilization Surgical procedures. Anesthesia: Telazol (4-5 mg/kg) and atropine (0.05 mg/kg) were combined in a 10 ml syringe. The cocktail was then injected intramuscularly into the pig gluteal muscle. In our observations, approximately 10 minutes were required for full anesthesia to occur. The pig was 59

78 then transferred to the clinical laboratory and a mask was placed around the nose and mouth. Isoflorane and oxygen were then administered via the mask. The physiological parameters (temperature, heart rate, respiration and mucus membrane color) were closely monitored and recorded. The abdomen was shaved, eyes were lubricated and catheters were placed in the animals ears. The surgical areas were scrubbed with alcohol thrice. Intubation: The endotracheal tube was coated with a thin layer of KY jelly. The oxygen flow was stopped and the mask was removed. The animal was held down and the epiglottis was located and pulled down utilizing a laryngoscope. The endotracheal tube was inserted carefully to avoid injury and fixed in place. Animal Preparation: The pig was then moved to the surgery room and placed on the lift table. Animals were then anesthetized as described above and fluid drip was started. The surgical sites are scrubbed three times with betadine and alcohol. The cleaned site was then sprayed with betadine. Heparin (200 unit / kg) was administered through the ear catheter at the time of the first incision. Additional heparin doses (5000 unit) were administered if the surgical procedure lasted more than 4 hours. The duration of anesthesia was 4 hours for of the dialysis grafts insertion and polymeric devices implantation. Surgical Procedure: The skin overlying the femoral artery and femoral vein was shaved and draped in a sterile fashion. A single skin incision was made on each side and the femoral artery and vein were exposed and mobilized. A venotomy was then made in the femoral vein approximately 2 cm below the inguinal ligament. One end of a 4 mm diameter dialysis graft (6.5 mm long) was sutured to the venotomy while the other end was sutured to an arteriotomy in the 60

79 femoral artery. The PTFE graft was then positioned in a subcutaneous pocket. Surgical hemostasis was confirmed prior to suturing and stapling skin edges at the incision site. Animals were given low doses of aspirin (325 mg/day) as an anti-coagulant from the day before surgery till the time of sacrifice Treatment Duration and Protocols. At the time of surgery, drug-loaded matrices were placed (wrapped) around the graft-vein junction. EVA matrices loaded with 15% PEG4000 were placed around the junction on one side which served as control side. EVA matrices containing 5% paclitaxel / 15% PEG4000 were wrapped around the on the junction on the contra-lateral side of each animal (treatment side). The decision whether the left or the right side received control or drug-loaded devices was made in an unbiased (random) fashion. Grafts were auscultated (checked for flow using a stethoscope) every 72 hours and at the end of the experiment, just prior to sacrificing the animal. Each animal served as its own control. At different time intervals (ranging from 4 to 42 days post surgery), pigs were euthanized with large intravenous dose of pentobarbital (3900 mg). The entire graft along with the attached artery and vein was perfusion fixed in situ using 10% formalin. The samples were stored in formalin for 24 hours and then embedded in paraffin using standard techniques. The arterial and venous specimens were then sectioned into 2 mm blocks and used for histomorphometric analysis. 61

80 4.3.4 Digitization and Histomorphometric Analysis. Video images of hemotoxylin and eosin (H & E) stained venous sections were projected at a final magnification of 200X using an Olympus BH-2 microscope, a Sony camera and a Unix workstation. Image-J software obtained from the National Institutes of Health (Bethesda, MD) was used for the morphometric analyses. For sections that contained both graft and vein (Area B in Fig ), the total neointimal area in each 2 mm block was measured using a digitizing pen. The total luminal areas enclosed by the graft or vein in all 3 mm blocks were also measured. A mean value for luminal stenosis was then calculated by dividing the total neointimal area by the total area of the graft and vein. Neointimal area was calculated as follows: Neointimal area = Internal elastic lamina area (IEL) Luminal area. For the determination of mean values of neointimal blockage, data for 2-3 sections of the 2-mm blocks were combined. The morphometric analysis as described above allowed us to identify whether locally delivered anti-proliferative therapy can in fact reduce venous neointimal hyperplasia and luminal stenosis Blood Sampling Protocols, Complete Blood Counts (CBC) Analysis and Gross Assessment of Toxicity. To determine the extent of systemic exposure following device implantation, blood samples (2 ml) were drawn from a group of treated animals (n = 4) at different time points (0, 30 mins, 1 hr, 1, 7,10, 14, 28 days). The time schedule of sampling coincided with the different times of animal sacrifice. Plasma was separated from the collected blood and assayed for paclitaxel content utilizing the HPLC/UV analysis method described earlier. Seromas 62

81 (interstitial fluid-filled capsules) were formed in some cases around the grafts. Seromal fluid were also collected and assayed for paclitaxel utilizing the HPLC/UV method. Additionally, blood samples (2 ml) were drawn from a group of 4 pigs at the times of receipt of animals on site, time of surgery and 7 days post-surgery. Standard whole blood counts (full CBC analysis) were performed to assess myelosuppression (reduced while blood cell counts). All animals were examined daily for evidence of breakdown of the vascular anastomosis or any other symptoms/signs of discomfort due to the paclitaxel-loaded or control polymers Statistical Analysis. For sample size calculation, the variability observed in earlier experiments was considered as the base for our calculations. In previous experiments designed to assess the efficacy of radiation to reduce neointimal hyperplasia (Roy-Chaudhury, 2002), a 25% standard deviation was observed in the treatment group. Accordingly, a group of at least 12 pigs was required to facilitate the detection of differences as large as 40% in luminal stenosis at a significance level of 0.05 (α = 0.05) and a power of 80% (β = 0.2) when using ANOVA. A number greater than 12 animals (15 in our case) was suggested to allow for animal drop-outs. Due to logistical and practical limitations, we were able to assess pharmacodynamic end points only in 4 out of the 15 pigs. Statistical Comparisons: Differences in the mean morphometric data between controls and treatment sides (including historical controls) were analyzed utilizing a one-way ANOVA. A p- value of or less than 0.05 was considered significant. Results from an additional group were included in this analysis. This group includes the 15 pigs from the radiation study carried out by 63

82 Roy-Chaudhury and colleagues (Roy-Chadhury, 2002) that did not receive any treatment. This comparison is important to ascertain that the drug-free polymers do not have significant inhibitory effects on the formation of neointimal lesions. GRAFT VEIN B A C Fig Diagrammatic representation of the venous anastomosis to demonstrate how the harvested samples were sectioned. Maximal neointimal hyperplasia occurs at the site of graftvein junction (Area B). 64

83 5 RESULTS 5.1 PREPARATION AND TESTING OF PACLITAXEL-LOADED POLYMERIC MATRICES Drug Content Uniformity. To determine the uniformity of paclitaxel content in cast matrices from different batches, weighed slabs from each batch were dissolved in methylene chloride. EVA was precipitated using methanol with paclitaxel remaining in the supernatant solution. The supernatant was dried and the residue was re-constituted and analyzed utilizing HPLC/UV. Table lists the paclitaxel content in matrices prepared with 5% w/w paclitaxel loading. Paclitaxel content in samples from the three batches was 5.02% ± 0.07%. There was marginal or no statistically significant difference in the amount of paclitaxel recovered from the matrices between different batches (P = 0.150) Chemical Stability of Paclitaxel within the Polymeric Matrices. Paclitaxel-loaded polymeric matrices were incubated in albumin-containing PBS solution or stored under dry storage in the dark. The chemically intact paclitaxel retained in matrices and/or released into the medium over a period of 21 days was determined utilizing the HPLC/UV procedure outlined earlier. When calculating the amount of paclitaxel, we took into consideration both, the amount of unchanged drug released and that retained in the matrix. The effects of storage conditions and exposure to the medium on paclitaxel stability are shown in Figure At the end of 21 days, the amount of paclitaxel that remained chemically intact 65

84 under the dry and aqueous environment was 91.2% ± 6.8% and 82.8% ± 4.6%, respectively. The loss of these amounts of paclitaxel were motly due to chemical degradation, mainly epimerization in the case of exposure to the aqeous environment. A statistical comparison of the paclitaxel stability profiles under dry and aqueous conditions indicated that at day 21, there was a significant difference in the amount of paclitaxel recovered (P = 0.047). As indicated earlier, paclitaxel was lost due to chemical instability by day Mechanical Properties of Loaded Polymeric Sheets. Freshly prepared polymeric matrices containing different amounts of PEG4000 (0, 13%, 15%, 17% and 20%), were cut into rectangular pieces and subjected to an isothermal stress-strain test. Young s modulus, a measure of the the rigidity or stiffness of the slabs, was then calculated from the slope of the initial linear part of the stress-strain plot, an example of which is shown in Figure Table lists the Young s moduli of matrices representing different formulations. The calculated values indicate that up to 15% (w/w) of PEG4000 loading resulted in a decrease in the elasticity of the slab. However, at the loading of 17% and 20% of the channeling agent, detrimental effects on the mechanical strength of the polymeric matrices were observed. It is apparent that an optimal ratio of the channeling agent to the polymer is required to achieve acceptable mechanical strength and an increase in the percentage of the channeling agent beyond a certain level may decrease the elasticity. 66

85 Table Paclitaxel content in EVA loaded slabs Batch Number Paclitaxel Content (% average of n=3) CV% Grand Average Table Effect of PEG4000 loading on the the modulud of elasticity of paclitaxel-loaded EVA matrices. Formulation Young s Modulus (N/mm 2 ) 5%paclitaxel, 0% PEG %paclitaxel, 13% PEG %paclitaxel, 15% PEG %paclitaxel, 17% PEG %paclitaxel, 20% PEG

86 % Paclitaxel Retained Dry Storage Aqueous Medium Time (days) Fig Percent of paclitaxel retained in matrices at the end of different storage periods under dry (diamonds) and in-solution (triangles) conditions. 68

87 Stress (N/mm 2 ) E= 0.77 Paclitaxel 5% / PE4000 0% Strain (L-L o /L o ) Fig Stress-strain analysis for two formulations. Young s modulus was calculated from the slope of the initial portion of the plots. Note the steeper slope of the initial portion for the formulation containing 15% PEG4000 indicating higher elasticity. 69

88 5.1.4 Water Sorption and Weight Loss Experiments. To evaluate the efficiency of PEG4000 as a channeling agent, slabs of different formulations were incubated in albumin-containing PBS solution for 10 days. At each sampling time point, the leaching solution was replaced with fresh release medium to maintain sink conditions. Slabs were removed and weighed periodically to assess weight gain. The gain in weight is reflective of the effectiveness of PEG4000 in facilitating water sorption into the EVA matrix. At the end of 10 days of incubation, the slabs were air dried for three days and weighed to assess for eventual weight loss corresponding to the dissolution of the channeling agent. As shown in Figure , the weight gain in the case of matrices not containing PEG4000 was negligible. At the end of 10 days of incubation, the 8% PEG4000-loaded matrices gained more than 17% of initial weight and eventually lost around 8% of its initial weight after drying. Also, matrices containing15% PEG4000 experienced a weight gain of approximately 42% and lost approximately 23% of its initial weight upon drying Morphological Analysis Using Environmental Electron Microscopy (ESEM). Pre- and post-sorption slabs were imaged on ESEM from different aspects following brittle fracture to detect any morphological changes caused by the presence of PEG4000. Figure shows ESEM scanning electron photomicrographs of partially desorbed matrices consisting of 5% paclitaxel in EVA. These images suggest that the hydrophobic EVA polymer had a dense homogenous morphology with no appreciable porosity (Figure a). Figure b shows a cross section following brittle fracture of the same matrices. The figure represents a cross-section image following brittle fracture and reveals plate-like domains closely stacked. Figure shows ESEM images of partially desorbed sample matrices containing 70

89 % Weight Gain Time (days) 0% PEG4000 8% PEG % PEG4000 Fig Effect of the channeling agent (PEG4000) on the porosity of polymeric matrices: increase in total weight of the polymer with increasing concentrations of PEG4000 and eventual weight loss upon drying. 71

90 A B Fig Scanning electron photomicrographs of EVA matrices loaded with 5% paclitaxel; a) EVA surface image (2000X), b) cross-sectional image of EVA matrix showing closely adherent stacks of polymeric layers (2000X). A B Pores Fig Scanning electron phtomicrographs of EVA matrices loaded with 5% paclitaxel/15% PEG4000; a) polymer surface image (250X) showing channels running along the orientation of imagining, b) EVA matrices at higher maginification showing a marked increase in surface porosity due to partial leaching of PEG4000 (2000X). 72

91 15% PEG4000 by weight. At a magnification of 250X, a two-phase system with typical channel morphology measuring few microns in length is clearly observed on the surface. Figures a and b exhibit a surface view of EVA matrices without and with PEG4000, respectively, at a magnification of 2000X. A comparison of the two clearly indicated the presence of large pores in PEG4000-containing matrices In Vitro Paclitaxel Release from Polymeric Formulations. Formulations with 5% w/w loading of paclitaxel and varying amounts of the channeling agent (0, 10, 13, 15, 17 and 20% by weight) in EVA were tested for paclitaxel release kinetics. The cast matrices were placed in 10 ml of albumin-containing PBS at 37 C under rotational mixing. At pre-selected time points, samples were withdrawn for paclitaxel analysis and the release medium was replaced with fresh medium to maintain sink conditions. Paclitaxel was then extracted and the extract was dried and reconstituted in mobile phase. Samples were then analyzed using the HPLC/UV method described earlier. Figure shows the cumulative fraction of paclitaxel load released from different formulations as a function of time. As shown, except for the matrices containing 20% PEG4000, all other formulations showed comparative biphasic release characteristics, with an initial burst occurring in the first few hours followed by a sustained release phase over 10 days. Several mathematical models were employed to correlate the initial percent drug release (< 60% of the cumulative amount eventually released) with time. Based on the goodness of fit criteria (R 2 and visual inspection), the square root of time model was fitted to the obtained data. For the early 73

92 Table The cumulative % of paclitaxel content released from matrices with different loadings of PEG4000. The initial 60% of the total amount released over the sampling period was fitted to a square root of time relationship and the regression coefficient is reported as a measure of goodness of fit. Formulation Paclitaxel 5% EVA 95% Paclitaxel 5% PEG % EVA 80% Paclitaxel 5% PEG % EVA 75% Paclitaxel 5% PEG % EVA 80% Paclitaxel 5% PEG % EVA 80% Paclitaxel 5% PEG % EVA 80% Released over 3 days (%) Released over entire period (%) M t /M = 7.68 t 1/ M t /M = 6.90 t 1/ M t /M = t 1/ M t /M = t 1/ M t /M = t 1/ M t /M = t 1/ t 1/2 Equation R 2 Sampling period (days)

93 portion of the release profiles, R 2 was always larger than 0.9. The results from release from the analysis of data evaluating the square root of time relationship are reported in Table Comparisons of the total AUC under the cumulative drug release-time curve and the fraction of drug load released at day 1, day 3 and day 8 from different formulations were performed utilizing ANOVA with Tukey s testing for pair-wise comparisons. A pair-wise comparison of the 0%, 10% and 13% PEG4000 formulations indicated no statistically significant difference in the AUC of the paclitaxel release versus time profiles. However, there was a highly significant difference in the AUC between 15% PEG4000-containing EVA matrices versus these containing 0% PEG4000. Also, pair-wise comparisons of the 15%, 17% and 20% PEG4000-containing formulations exhibited statistically significant differences (P < 0.001). Similar observations were evident for the pair-wise comparisons for the fraction of drug released at different time points between the six formulations. To further understand the effect of the channeling agent and the increase in water sorption with higher loads of PEG4000, we plotted the extent of weight gain against the cumulative percentage of paclitaxel released for the formulation consisting of 5% paclitaxel /15% PEG4000 / 80% EVA (Figure ) at different time points. This plot shows that the cumulative amount of paclitaxel released from the polymers is directly proportional to extent of enhancement of fluid uptake (R 2 = 0.92). Based on the findings derived from mechanical testing, weight gain and release kinetics, we selected the formulation containing 5% paclitaxel /15% PEG4000 / 80% EVA for further 75

94 120 Cumulative % Paclitaxel Relesaed Time (days) Paclitaxel 5% / PEG4000 0% Paclitaxel 5% / PEG % Paclitaxel 5% / PEG % Paclitaxel 5% / PEG % Paclitaxel 5%/ PEG % Paclitaxel 5% / PEG % Fig In vitro release kinetics of paclitaxel from EVA matrices loaded with 5% paclitaxel by weight and increasing loading of PEG4000 (Mean ± S.D.) 76

95 Paclitaxel Relesaed (% of initial loading) Weight Gain (mean %) Fig Relationship between the mean increase in weight of polymeric matrices and the fraction of drug load released from EVA matrices loaded with 5% paclitaxel / 15% PEG4000 by weight at different time points. 77

96 investigation. This formulation exhibited increased ability to withstand peeling, handling and implantation. Also, this formulation exhibited favorable release profiles with an initial burst corresponding to the release of >35% of the drug load followed by a sustained release phase. This pattern of drug release (a significant burst effect followed by a continuous controlled phase) from the formulation containing both paclitaxel and 15% PEG40000 fits well with what is suggested as the temporal profile of neointimal hyperplasia. Both the release kinetics and the mechanical properties of this formulation were the d=criteris upon which we selected this formulation for further in vitro and in vivo testing. 5.2 IN VITRO CYTOTOXICITY OF PACLITAXEL RELEASED FROM POLYMERIC FORMULATIONS Inhibition of Cell Proliferation by Polymeric Paclitaxel Primary cultures of physiologically-relevant cells known to be involved in the formation of neointimal hyperplasic lesions were used to assess the cytotoxicity of polymeric paclitaxel. Accordingly, cultures of HUVECs, NHDFs and HASMCs were plated onto 12-well plates and allowed to seed for 24 hours. Cells were then exposed to the following formulations: (a) 100% EVA (b) 15% PEG4000 / 85%EVA (c) 5% paclitaxel / 95%EVA (d) 5% paclitaxel / 15%PEG4000 / 80%EVA and (e) controls (media only). Cells were incubated with the above formulations for 6 days. At the end of 6 days, cell proliferation was assessed utilizing a MTT assay. The differences in effects of the different formulations on cell survival were assessed utilizing ANOVA with Tukey s testing for pair-wise comparisons. 78

97 Figure shows survival of HUVECs exposed to the different formulations. Compared to untreated controls, all the formulations tested, except for EVA alone, caused a significant decrease in cell survival. Exposure to slabs loaded with PEG4000 alone resulted in cell kill levels comparable to those observed with EVA alone (P = 0.878). Moreover, exposure to the formulation containing 5% paclitaxel /15% PEG4000 resulted in the most prominent cell kill. There was no statistically significant difference in cell kill between the two paclitaxel-loaded formulations. However, exposure to both the paclitaxel-loaded formulations resulted in a significant decrease in cell survival compared to their respective controls (P <0.001 for both formulations). For the NHDFs, only the formulations containing polymeric paclitaxel caused a significant inhibition of cell proliferation compared to untreated controls (P < 0.001; Fig ). Comparing the two paclitaxel-loaded formulations to their respective controls, highly significant differences in cell survival were observed (P < in both cases). There was no statistically significant difference in cell survival between cells exposed to the paclitaxel-loaded formulation containing 15% PEG4000 and that containing paclitaxel alone (P = 0.433). As shown in Fig , the survival of HASMCs relative to untreated controls was significantly affected only by the formulations containing paclitaxel (P <0.001 and P = for the formulation containing 5% paclitaxel / 15% PEG4000 / 80% EVA and the one containing 5% paclitaxel / 95% EVA respectively). A summary of the results from statistical comparisons for the three cell types is presented in Tables , ,

98 Table Statistical comparisons in HUVEC survival following exposure to different polymeric formulations. Comparison P- value No added polymer vs. Pac5%, PEG %, EVA 80% <0.001 EVA 100% vs. Pac5%, PEG %, EVA 80% <0.001 EVA 100% vs. Pac5%, EVA95% <0.001 EVA 100% vs. PEG %/EVA 85% Pac5%, PEG %, EVA 80% vs. PEG %, EVA 85% No added polymer vs. EVA 100% No added polymer vs. PEG %, EVA 85% Table Statistical comparisons in HASMC survival following exposure to different polymeric formulations. Comparison p-value No added polymer vs. Pac5%, PEG %, EVA 80% EVA 100% vs. Pac5%, PEG %, EVA 80% EVA 100% vs. Pac5%, EVA95% EVA 100% vs. PEG %/EVA 85% Pac5%, PEG %, EVA 80% vs. PEG %, EVA % No added polymer vs. EVA 100% No added polymer vs. PEG %, EVA 85% Table Statistical comparisons in NHDF survival following exposure to different polymeric formulations. Comparison p-value No added polymer vs. Pac5%, PEG %, EVA 80% <0.001 EVA 100% vs. Pac5%, PEG %, EVA 80% <0.001 EVA 100% vs. Pac5%, EVA95% <0.001 EVA 100% vs. PEG %, EVA 85% Pac5%, PEG %, EVA 80% vs. PEG %, EVA < % No added polymer vs. EVA 100% No added polymer vs. PEG %, EVA 85%

99 % Cell Survival (relative to controls) P < P = Ctrls PEG 15%, Pac 5%, EVA 80% Pac 5%, EVA 95% EVA 100% Formulation PEG 15% Fig In vitro effects of the paclitaxel-loaded polymers on HUVECs: addition of the paclitaxel-loaded polymers to cell cultures resulted in a significant inhibition of in vitro proliferation of these cell types (refer to Table for the detailed statistical comparisons). 81

100 P = % Cel Survival (relative to controls) P = Ctrls PEG 15%, Pac 5%, EVA 80% Pac 5%. EVA 95% EVA 100% Formulation PEG 15% Fig In vitro effects of the paclitaxel-loaded polymers on NHDFs: addition of the paclitaxel-loaded polymers to cell cultures resulted in a significant inhibition of in vitro proliferation of these cell types (refer to Table for the detailed statistical comparisons). 82

101 P < % Cell Survival (relative to controls) P < Ctrls PEG 15%, Pac 5%, EVA 80% Pac 5%, EVA 95% EVA 100% Formulation PEG 15% Fig In vitro effects of the paclitaxel-loaded polymers on HASMCs: addition of the paclitaxel-loaded polymers to cell cultures resulted in a significant inhibition of in vitro proliferation of these cell types (refer to Table for the detailed statistical comparisons). 83

102 5.3 DETERMINATION OF THE IN VIVO EFFICACY OF THE OPTIMIZED PACLITAXEL- LOADED SLABS Histomorphometric Measurements As described in the Materials and Methods section, in vivo efficacy was determined in Yorkshire cross swine pigs. Following insertion of the arterio-venous grafts, a paclitaxel loaded polymer (5% paclitaxel + 15% PEG % EVA) was carefully placed around the graft-vein anastomosis on one side. A control polymer (15% PEG % EVA) was placed around the contra-lateral anastomosis. Each pig served as its own control. Special care was taken to ensure that the perivascular polymers fitted snugly around the graft-vein anastomoses. Animals were sacrificed on days 23, 24, 32 and 37 post graft placement. These time points were selected based on the results from the study by Kelly et al on swine pigs (Kelly, 2002). The findings from this study indicated that neointimal blockage at the anastomoses was prominent around 28 days. At the time of sacrifice, the grafts together with the attached artery and vein were carefully dissected and fixed in formalin. The harvested sample was then cut into 3 mm blocks which were embedded in paraffin and cut into 4 µm thick sections. The sections were then stained with H &E and imaged. The images were digitized for histomorphometric analysis. Briefly, measurements were taken of the area of neointimal cellular formation and of the entire luminal area subtended by PTFE graft. Percentage luminal stenosis was evaluated employing measurements of luminal area blocked by the neointimal growth for each section obtained. Results from histomorphometric analysis of luminal stenosis for control and paclitaxel treated sides were analyzed and compared for statistical significance. A comparison of luminal stenosis on the paclitaxel-treated side with that in historical controls was also made. Historical controls represent a group of 12 pigs that did not receive any treatment around the anastomoses and were 84

103 scarficed around 28 days post-surgery. All comparisons were performed utilizing one-way ANOVA with Tukey test for pair-wise comparisons. The results are shown in Figure Fig illustrates the percentage luminal stenosis for the paclitaxel- treated anastomoses compared to the contra-lateral controls as well historical control. Note that there was no discernable luminal stenosis (0%) at any of the graft-vein anastomoses treated with the paclitaxel loaded wraps compared to a mean luminal stenosis of 56.5% ± 35.0% at the anastomoses treated with the control wraps (P = 0.002). With regards to historical controls (% luminal stenosis = 52.7% ± 7.4%), no significant difference in the degree of luminal stenosis between the two control groups was observed (P = 0.91). This indicates that it is appropriate to include the historical control group in our analyses. This also indicates that polymers loaded with PEG4000 did not cause significant inhibition of neointimal formation. Figure and Figure represent digital images showing inhibition of neointimal hyperplasia at the graft-vein anastomosis following treatment with peri-vascular paclitaxel wraps compared to controls. Panels A shows the absence of any cellular formations within the graft at the anastomosis of the treated animals. The colored segments within the grafts are mostly acellular fibrin deposition that takes place as part of the wound healing process. Spots within the treated anastomoses that show intense colors are blood clots that most probably occurred postmortum. A summary of the results from histomorphometric analysis is reported in Table

104 5.3.2 Systemic Exposure, Complete Blood Counts (CBC) Analysis and Gross Assessment of Toxicity. There was no evidence of significant local toxicity from the peri-vascular paclitaxel wraps. Of the 17 pigs that we have included in our studies, we had only one anastomotic breakdown. This indicated that paclitaxel released from the polymeric matrices did not hamper or delay wound healing at the treated anastomoses. All the specimens were successfully harvested intact as a composite block (Fig ). Harvesting a composite tissue sample indicated the absence of noticeable necrosis. This suggested that paclitaxel was released in a unidirectional manner and that neighboring tissues were exposed to negligible amounts of paclitaxel. Upon macroscopic examination, there was no evidence of significant necrosis at the adventitial aspect of the attached veins.. This further demonstrates the lack of local toxicity of the paclitaxel-loaded formulation. However, it should be emphasized that these findings were made through a subset of the animals. Moreover, inflammation was evident on both sides and more prominent on the treatmemt side which was quite expected. Due to logistical procedures and the nature of these experiments, we were unable to measure paclitaxel concentration in the target tissue in order not to lose a lot of tissues before we can do extensive histomorphometry. Attempts were also made to measure paclitaxel levels in local tissue fluid (seroma fluid) as well as the systemic circulation. Paclitaxel was not detected in the plasma separated from peripheral blood at any of the time points tested. Moreover, paclitaxel was not detected in seroma fluids around the treated anastomoses at the time of sacrifice. This further supports the previous observations that paclitaxel diffusion was occurring in a unidirectional manner into the target tissue. Further, our contention that only minimal systemic 86

105 exposure may result from paclitaxel-loaded formulations is determined by the white blood cell count. 87

106 Table Peri-vascular paclitaxel wraps block neointimal hyperplasia. Summary of the results from histomorphomeric analysis. Animal Animal Total Luminal Area Neointimal Area % Luminal Stenosis Number ID (pixels 2 ) (pixels 2 ) 1 8 treated control treated control treated control treated control

107 % ± 35.0% 52.0% ± 7.42% % Luminal Stenosis % Treated Control Historical n = 4 n = 4 Controls n = 12 Fig Peri-vascular paclitaxel wraps block venous stenosis. Note the complete absence of luminal stenosis at the graft-vein anastomoses of animals treated with the paclitaxel loaded polymers. 89

108 1 A 1 B 2 A 2 B Fig Peri-vascular paclitaxel wraps block venous stenosis: Note the marked decrease in neointimal hyperplasia at the graft-vein anastomosis in panel A (treated with a paclitaxel loaded peri-vascular wraps) as compared to panel B (treated with a control wraps) in animals 1 and 2. The colored segments within the treated grafts represent acellular collagen arrangements. 90

109 3 A 3 B 4 A 4 B Fig Peri-vascular paclitaxel wraps block venous stenosis: Note the marked decrease in neointimal hyperplasia at the graft-vein anastomosis in panel A (treated with a paclitaxel loaded peri-vascular wraps) as compared to panel B (treated with a control wraps) in animals 3 and 4. The colored segments within the treated grafts represent acellular collagen arrangements. 91

110 Polymer Femoral artery Femoral vein Fig Explant specimen of the graft, artery and vein as a composite block. Note the perivascular polymer in place around the explanted vessel. 92