Angiopeptin-Eluting Stents: Observations in Human Vessels and Pig Coronary Arteries

Coronary stenting is generally associated with lower rates of restenosis than balloon angioplasty.1,2 However, in-stent restenosis (ISR) remains a significant clinical problem.(3) Pharmacological inhibition of ISR has shown potential in laboratory animals, but has not been associated with clinical benefit in large randomized clinical trials in man.(4,5) Local drug delivery is an attractive proposition, but delivery catheter devices are inconvenient, may cause further injury to the vessel wall, and result in inefficient delivery and regional rather than local distribution.(6–11) Therefore, there has been much recent interest in the use of drug eluting stents.(12–16) Angiopeptin, a synthetic analogue of somatostatin, has been shown to reduce neointima formation after balloon injury when given systemically in some animal models,(17,18) and to reduce adverse events in a clinical trial.(19) However, the drug is rapidly degraded by the liver(20) and therefore local delivery strategies have been attempted.21 Angiopeptin-loaded stents reduced luminal narrowing in a pig model of in-stent stenosis compared to non-loaded stents, although the effect observed was due to inhibition of myointimal proliferation arising as a consequence of the poly(organo)phosphazene stent coating.(22) The BiodivYsio drug delivery stent (Biocompatibles Ltd., Farnham, United Kingdom) is a stainless-steel, balloon-expandable stent coated with a phosphorylcholine (PC) polymer. PC occurs naturally on the external surface of cell membrane lipid bilayers. PC coating confers hemocompatibility,(23–25) and can act as a drug reservoir, capable of controlled release. PC is stable in saline and ethanol (Terry Vick, unpublished observations, 1999), and the durability of the PC coating has been demonstrated using atomic force microscopy following implantation into pig coronary arteries for periods up to 6 months.(26) The aims of the present study were: 1) to determine the release of I125 angiopeptin from PC-coated stents into human saphenous vein ex vivo, and pig coronary arteries in vivo; and 2) to investigate the safety and efficacy of angiopeptin-loaded PC-coated stents in porcine coronary restenosis. Methods Stent preparation BiodivYsio PC-coated stents were used. The thickness of the PC coating for the ex vivo studies was 100 nm on both the luminal and abluminal aspects of the stent. Following initial ex vivo experiments (see below), the thickness of the PC coating on the abluminal aspect of the stents was increased to 1 mm (“drug delivery” PC-coated stent, or DD-PC), allowing for greater loading of angiopeptin. I125 angiopeptin (prepared by Nycomed Amersham Imaging, Princeton, New Jersey) was used for assessment of release characteristics. Stents were placed into I125 angiopeptin solution, incubated for 30 minutes at 37 °C, and dried at 40 ºC for 1 hour. The level of radioactivity per stent was quantified using a gamma counter (Wallac Compugamma, Perkin Elmor, Connecticut) and the amount of angiopeptin loaded, calculated with reference to a 500 µg standard, was 3.9 ± 0.4 µg for ex vivo studies and 8.32 ± 0.8 µg for in vivo delivery studies. Angiopeptin (generic, Lanreotide; kindly provided by Beaufour Ipsen, United Kingdom) was used for safety and efficacy studies. Balloon-mounted DD-PC stents were dipped into a 10 mg/ml solution of angiopeptin in 100% ethanol as described above, and an additional 20 µl of angiopeptin solution was applied directly to the stent surface using a pipette tip following the drying step to increase loading. High-performance liquid chromatography (HPLC) was used to determine angiopeptin loading. Angiopeptin was eluted from a Vydac C18 HPLC column with acetonitrile/sodium perchlorate mobile phase at a flow rate of 1 ml/minute and monitored at 280 nm with an ultraviolet detector. An average loading of 126 µg of angiopeptin was achieved per stent used for the safety and efficacy study. Release and delivery of I125 angiopeptin from PC-coated stents Ex vivo studies in human saphenous vein. The I125 angiopeptin-loaded stents were deployed using a 3.5 x 20 mm PTCA balloon (Bard Corporation, Billerica, Massachusetts) into segments of human saphenous vein obtained from patients undergoing coronary artery bypass grafting (n = 6). The protocol was approved by the Northern General Hospital Ethics Committee and specimens were collected following informed patient consent. The vein and stent were then washed in 10 ml of HEPES-buffered RPMI 1640 culture medium (LIFE, Paisley, United Kingdom) supplemented with penicillin (100 µl/ml), streptomycin (100 units/ml) and glutamine (2 mmol/L) (all from ICN Flow labs) for 10 seconds. The vein was secured in an organ culture chamber and perfused at physiological pressure and flow rates with 100 ml culture medium using a peristaltic pump for either 1 or 24 hours (n = 3 at each time point). At the end of each experiment the vein was cut from the chamber, opened longitudinally to remove the stent, and divided into proximal, stented and distal segments. Tissue samples and the explanted stent were counted for the amount of radioactivity present using a gamma counter. All counts were adjusted for radioactive decay (I125 half life = 60 days). In vivo studies in porcine coronary arteries. Surgical procedures and stent implantation. All animal procedures were conducted according to United Kingdom Home Office Regulations. The investigation conforms with the guide for the care and use of laboratory animals published by the United States National Institute of Health (NIH publication no. 85-23, revised 1996). Yorkshire pigs weighing 16–20 kg were given aspirin (150 mg) pre-operatively and for up to 5 days. General anesthesia was induced and coronary angiography was performed as previously described.27 I125 angiopeptin-loaded PC-coated stents were mounted onto 3.5 mm coronary angioplasty balloons with minimal handling and deployed at 8 atm in 1 segment of left anterior descending coronary artery, 2.8 mm diameter by quantitative angiography, achieving a mid-stent stent:artery ratio of 1.25:1. The animals (n = 8) were killed at 1 hour, 24 hours, 7 days and 28 days (2 animals at each time point). Non-radio-labelled angiopeptin-loaded stents (n = 8) were implanted into coronary arteries of 4 additional animals that were subsequently killed at 1 hour, 7 days and 28 days, prior to analysis of angiopeptin by HPLC. Measurement of surface radioactivity. A Geiger counter (Scintillation meter type 5.40, Mini Instruments, Ltd., Essex, United Kingdom) was used to record over-the-skin radioactivity at 5-minute intervals for the first 25 minutes following stent deployment and immediately before sacrifice. Measurements were taken over the heart, abdomen, kidney, head, leg muscles and bladder. Detection of I125 angiopeptin in blood, urine and tissue samples. Arterial blood samples were obtained immediately before and after each stent deployment, at 5 and 30 minutes following stent deployment and when the animal was killed. One blood sample was also collected from the delivery sheath immediately after stent deployment. Urine and tissue samples were obtained immediately after sacrifice of the animal. Tissue samples were taken from skeletal muscle, cartilage, small intestine, thyroid gland, lung, liver, spleen, kidney, skin, aorta, right and left atria, right and left ventricles, pericardium, left anterior descending (LAD), right coronary (RCA) and circumflex coronary arteries, and myocardium from beneath the stent. The stented coronary arteries were divided into stented, proximal and distal regions. The stented segment was cut longitudinally to remove the stent, with the exception of the 28-day stents, which were left in situ because of encasement in neointima. Tissue samples were weighed and counted with a gamma counter as described above. In addition, formalin-fixed paraffin sections were prepared for microautoradiography by dipping in photographic emulsion (Hypercoat LM-1, Amersham) and exposed at 4 ºC for 11 weeks. For 28 day experiments, stented arteries were formalin-fixed, processed and embedded in T8100 resin (Taab Laboratories, Berkshire, United Kingdom) and transverse sections of vessel with stent in situ were cut and polished28 for autoradiography. HPLC analysis of angiopeptin. Following stent removal, tissue lysates were prepared and HPLC analyses were performed, as described above. Angiopeptin remaining on the explanted stents was eluted with ethanol and also subjected to HPLC analysis. Safety and efficacy of angiopeptin delivery from DD-PC-coated stents Study protocol. Stents were deployed as described, this time using both RCA and LAD in each animal. Five thousand units of sodium heparin were given at the start of catheterization. The animals were killed at 28 days and the arterial segments with stents in situ were excised and immersion-fixed in formalin for 24 hours, processed and embedded in T8100 resin (Taab Laboratories) and transverse sections were cut for histology. There were 4 study groups: angiopeptin-loaded DD-PC coated stents (12 arteries, 6 pigs); DD-PC control stents (12 arteries, 6 pigs); standard (non-DD) PC-coated BiodivYsio stents (10 arteries, 5 pigs); and uncoated DivYsio stents (8 arteries, 4 pigs). Morphometrical analysis. Of 12–20 sections per stent, three were randomly selected (1 from each of the proximal, middle and distal segments of the stent). Sections were discarded if they were incomplete or distorted. These were analyzed by semi-automated quantitative morphometry (Lucia Image Analysis software, Nikon, United Kingdom). The lumen, neointima and total vessel cross-sectional areas were measured and recorded. A cumulative injury score (a modification of the Schwartz Score),29 to reflect mild and moderate arterial injury, was used. Each strut was scored: 0 = no imprint on vessel wall; 1 = internal elastic lamina deformed 45º; 3 = internal elastic lamina broken; and 4 = external elastic lamina broken. The injury score for each section was calculated as the sum of the scores for each strut. To correct for the amount of injury and vessel size, each measurement was divided by the vessel area of that section and then by the injury score. This value was then multiplied by 104 to give an integer of arbitrary units. Statistics All descriptive statistics are expressed as mean ± the standard error of the mean. Morphometrical data in the efficacy study were analyzed using a 1-way ANOVA. The level of significance was taken as p

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