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Angiopeptin-Eluting Stents: Observations in Human Vessels and Pig Coronary Arteries
May 2002
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 PC is stable in saline and ethanol (Terry Vick, unpublished observations, 1999), and the durability of the PC coating has been demonstrated using atomic force microscopy following implantation into pig coronary arteries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
1. Serruys PW, de Jaegere P, Kiemeneij F, et al., for the BENESTENT Study Group. A comparison of balloon-expandable stent implantation with balloon angioplasty in patients with coronary disease. N Engl J Med 1994;331:489–495.
2. Fischman DL, Leon M, Bain DS, et al., for the Stent Restenosis Study Investigators. A randomized comparison of coronary stent placement and balloon angioplasty in the treatment of coronary artery disease. N Engl J Med 1994;331:496–501.
3. Dussaillant GR, Mintz GS, Pichard AD, et al. Small stent size and intimal hyperplasia contribute to restenosis: A volumetric intravascular ultrasound analysis. J Am Coll Cardiol 1995;26:720–724.
4. The Multicenter European Research Trial with Cilazapril After Angioplasty to Prevent Transluminal Coronary Obstruction and Restenosis (MERCATOR) Study Group. Does the new angiotensin converting enzyme inhibitor cilazapril prevent restenosis after percutaneous transluminal coronary angioplasty? Results of the MERCATOR Study. Circulation 1992;86:100–110.
5. Faxon D, Spiro T, Minor S, et al. Low molecular weight heparin in prevention of restenosis after angioplasty. Results of enoxoparin restenosis (ERA) trial. Circulation 1994;90:908–914.
6. Wolinsky H, Thung SN. Use of a perforated balloon catheter to deliver concentrated heparin into the wall of the normal canine artery. J Am Coll Cardiol 1990;15:475–481.
7. Hong MK, Wong SC, Popma JJ, et al. A dual purpose angioplasty-drug infusion catheter for the treatment of intragraft thrombus. Cathet Cardiovasc Diagn 1994;32:193–195.
8. Stadius ML, Collins C, Kernoff R. Local infusion balloon angioplasty to obviate restenosis compared with conventional balloon angioplasty in an experimental model of atherosclerosis. Am Heart J 1993;126:47–56.
9. Azrin MA, Mitchel JF, Fram DB, et al. Decreased platelet deposition and smooth muscle cell proliferation after intramural heparin delivery with hydrogel-coated balloons. Circulation 1994;90:433–441.
10. Gunn J, Francis SE, Holt CM, et al. Is local drug delivery truly a low efficiency procedure? (Abstr). Am J Cardiol 1997;80:67S.
11. Mitchel JF, Azrin MA, Fram DB, et al. Inhibition of platelet deposition and lysis of intracoronary thrombus during balloon angioplasty using urokinase-coated hydrogel balloons. Circulation 1994;90:1979–1988.
12. Aggarwal R, Ireland D, Azrin MA, et al. Antithrombotic potential of polymer coated stents eluting platelet glycoprotein IIb/IIIa receptor antibody. Circulation 1996;94:3311–3317.
13. Baron JH, Gershlick AH, Hogrefe K, et al. In vitro evaluation of c7E3-Fab (ReoPro®) eluting polymer-coated coronary stents. Cardiovasc Res 2000;46:585–594.
14. Heldman AW, Cheng L, Jenkins M, et al. Paclitaxel stent coating inhibits neointimal hyperplasia at 4 weeks in a porcine model of coronary restenosis. Circulation 2001;103:2289–2295.
15. Serruys PW, Morice MC, Sousa JE, et al. The RAVEL study: A randomized study with the sirolimus coated Bx velocity balloon-expandable stent in the treatment of patients with de novo native coronary artery lesions (Abstr). Eur Heart J 2001;22:484.
16. Honda Y, Grube E, de la Fuente LM, et al. Novel drug-delivery stent. Intravascular ultrasound observations from the first human experience with the QP2-eluting polymer stent system. Circulation 2001;104:380–383.
17. Foegh ML, Asotra S, Conte JV, et al. Early inhibition of myointimal proliferation by angiopeptin after balloon injury in the rabbit. J Vasc Surg 1994;19:1084–1091.
18. Santoian ED, Schneider JE, Gravanis MB, et al. Angiopeptin inhibits intimal hyperplasia after angioplasty in porcine coronary arteries. Circulation 1993;88:11–14.
19. Eriksen UH, Amtorp O, Bagger JP, et al. Randomized double-blind Scandinavian trial of angiopeptin versus placebo for the prevention of clinical events and restenosis after coronary balloon angioplasty. Am Heart J 1995;130:1–8.
20. Cathapermal S, Foegh ML, Rau C, et al. Disposition and tissue distribution of angiopeptin in the rat. J Drug Metabol Dispos 1991;19:735–739.
21. Hong MK, Bhaytti T, Matthews BJ, et al. The effect of porous balloon-delivered angiopeptin on myointimal hyperplasia after balloon injury in the rabbit. Circulation 1993;88:638–648.
22. De Scheerder I, Wilkzek K, Van Dorpe J, et al. Local angiopeptin delivery using coated stents reduces neointimal proliferation in overstretched porcine coronary arteries. J Invas Cardiol 1996;8:215–222.
23. Campbell EJ, O’Byrne V, Stratford PW, et al. Biocompatible surfaces using methacryloylphosphorylcholine laurylmethacrylate copolymer. ASAIO J 1994;40:M853–M857.
24. Veil KR, Chronos NAF, Palmer SJ, et al. Phosphorylcholine-a biocompatible coating for coronary angioplasty devices (Abstr). Circulation 1995;92:(Suppl I):685.
25. Kuiper KKJ, Robinson KA, Chronos NAF, et al. Phosphorylcholine coated metallic stents in rabbit iliac and porcine coronary arteries. Scand Cardiovasc J 1998;32:261–268.
26. Tolhurst L. The analysis of post implant phosphorylcholine coatings on cardiovascular stents by AFM. Proceedings of the Medical Devices Conference, 1999:427(Abstr).
27. Post MJ, de Smet BJ, van der Helm Y, et al. Arterial remodeling after balloon angioplasty or stenting in an atherosclerotic experimental model. Circulation 1997;96:996–1003.
28. Malik N, Gunn J, Holt CM, et al. Intravascular stents: A new technique for tissue processing for histology, immunohistochemistry and transmission electron microscopy. Heart 1998;80:509–516.
29. Schwartz RS, Huber KC, Murphy JG, et al. Restenosis and the proportional neointimal response to coronary artery injury: Results in a porcine model. J Am Coll Cardiol 1992;19:267–274.
30. Light JT, Bellan JA, Chen I-L, et al. Angiopeptin enhances acetylcholine-induced relaxation and inhibits intimal hyperplasia after vascular injury. Am J Physiol 1993;265:H1265–H1274.
31. Sousa JE, Costa MA, Abizaid AC, et al. Sustained suppression of neointimal proliferation by sirolimus-eluting stents. Circulation 2001;104:2007–2011.
32. Lincoff AM, Furst JG, Ellis SG, et al. Sustained local delivery of dexamethasone by a novel intravascular eluting stent to prevent restenosis in the porcine coronary injury model. J Am Coll Cardiol 1997;29:808–816.
33. Strecker E-P, Gabelmann A, Boos I, et al. Effect on intimal hyperplasia of dexamethasone from coated metal stents compared with non-coated stents in canine femoral arteries. Cardiovasc Intervent Radiol 1998;21:487–496.
34. Dev V, Eigler N, Sheth S, et al. Kinetics of drug delivery to the arterial wall via polyurethane-coated removable nitinol stent: Comparative study of two drugs. Cathet Cardiovasc Diagn 1995;34:272–278.
35. Lambert T, Dev V, Rechavia E, et al. Localised arterial wall drug delivery from a polymer-coated removable metallic stent. Kinetics, distribution and bioactivity of forskolin. Circulation 1994;90:1003–1011.
36. Malik N, Gunn J, Shepherd S, et al. Phosphorylcholine-coated stents in porcine coronary arteries: In vivo assessment of biocompatibility. J Invas Cardiol 2001;13:193–201.
37. Van der Giessen WJ, Lincoff M, Schwartz R, et al. Marked inflammatory sequelae to implantation of biodegradable and non-biodegradable polymers in porcine coronary arteries. Circulation 1996;94:1690–1697.
38. Moreau JP, Defeudis FU. Pharmacologic studies of somatostatin and somatostatin analogues: Therapeutic advances and perspectives. Life Sci 1987;40:419–437.
39. Howell M, Orskov H, Frystyk F, et al. Lanreotide, a somatostatin analogue, reduces insulin-like growth factor I accumulation in proliferating aortic tissue in rabbits in vivo. Eur J Endocrinol 1994;130:422–425.
40. Lundergan C, Foegh ML, Vargas R, et al. Inhibition of myointimal proliferation of the rat carotid artery by the peptides angiopeptin and BIM 23034. Atherosclerosis 1989;80:49–55.
41. Bauters C, van Belle E, Wernert N, et al. Angiopeptin inhibits oncogene induction in rabbit aorta after balloon denudation. Circulation 1994;89:2327–2331.
42. Colas B, Cambillau A, Buscail L, et al. Stimulation of a membrane tyrosine phosphatase activity by somatostatin analogues in rat pancreatic acinar cells. Eur J Biochem 1992;207:1017–1024.
43. Foegh ML. Angiopeptin: A treatment for accelerated myointimal hyperplasia. J Heart Lung Transplant 1992;11:528–531.
