Stent-based delivery of adeno-associated viral vectors with sustained vascular transduction and iNOS-mediated inhibition of in-stent restenosis.

In-stent restenosis remains an important clinical problem in the era of drug eluting stents. Development of clinical gene therapy protocols for the prevention and treatment of in-stent restenosis is hampered by the lack of adequate local delivery systems. Herein we describe a novel stent-based gene delivery platform capable of providing local arterial gene transfer with adeno-associated viral (AAV) vectors. This system exploits the natural affinity of protein G (PrG) to bind to the Fc region of mammalian IgG, making PrG a universal adaptor for surface immobilization of vector-capturing antibodies (Ab). Our results: 1) demonstrate the feasibility of reversible immobilization of AAV2 vectors using vector tethering by AAV2-specific Ab appended to the stent surface through covalently attached PrG, 2) show sustained release kinetics of PrG/Ab-immobilized AAV2 vector particles into simulated physiological medium in vitro and site-specific transduction of cultured cells, 3) provide evidence of long-term (12 weeks) arterial expression of luciferase with PrG/Ab-tethered AAV2Luc, and 4) show anti-proliferative activity and anti-restenotic efficacy of stent-immobilized AAV2iNOS in the rat carotid artery model of stent angioplasty.

Local delivery of mithramycin restores vascular reactivity and inhibits neointimal formation in injured arteries and vascular grafts.

Arterial restenosis is responsible for the high failure rates of vascular reconstruction procedures. Local sustained drug delivery has shown promise in the prevention of restenosis. The drug release rate from mithramycin-loaded EVA matrices (0.1%) was evaluated, and their antirestenotic effect was studied in the rat carotid model and rabbit model of vascular grafts. The modulation of c-myc expression by mithramycin treatment was examined by immunohistochemistry in the rat carotid model. The proliferative response of injured rat arteries was studied by bromdeoxyuridine (BrdU) immunostaining. The impact of mithramycin treatment on vasomotor responses of the venous segments grafted into arterial circulation was studied ex vivo using vasoreactive compounds. Mithramycin was released exponentially from EVA matrices in PBS. Matrices co-formulated with PEG-4600 revealed enhanced release kinetics. The perivascular implantation of drug-loaded EVA-PEG matrices led to 50% reduction of neointimal formation, and reduced the c-myc expression and BrdU labeling in comparison to control implants. Decreased sensitivity of mithramycin-treated grafts to serotonin-induced vasoconstriction was observed. Local perivascular mithramycin treatment limits the functional alteration caused by the grafting of venous segments in high-pressure arterial environment, and potently inhibits stenosis secondary to grafting and angioplasty injury. The antirestenotic effect is associated with reduced c-myc expression and with subsequent decrease in SMC proliferation.

Formulation and delivery mode affect disposition and activity of tyrphostin-loaded nanoparticles in the rat carotid model.

Poor drug residence in the arterial wall hinders clinical implementation of local drug delivery strategies for the treatment of restenosis. A rat carotid model of vascular injury and intraluminal delivery of tyrphostin-containing polylactic acid (PLA) nanoparticles (NPs) were used to determine the relationship between residence properties and biological activity of different formulations and administration modes. The effects of delivery modes (denudation and delivery time) and formulation variables (adsorbed vs encapsulated drug, and NP size) on arterial drug/NP retention were examined. Antirestenotic effects of large (160 nm) and small (90 nm) tyrphostin-containing NPs, surface-absorbed tyrphostin, and systemic treatment were compared. Fluorescent NPs were used to study the spatial distribution of the carrier in the arterial wall. The decrease in arterial tyrphostin level over time fitted a biexponential model. Delivery time and pressure, endothelium integrity, particle size, and drug-polymer association affected local pharmacokinetics and the antirestenotic results after 14 days. The PLA-based tyrphostin NP formulation ensured a prolonged drug residence at the angioplasty site after single intraluminal application. Several readily adjustable formulation and procedural factors considerably modified arterial ingress of the drug-loaded NPs and governed their subsequent redistribution, tissue binding, elimination, and ensuing antirestenotic effect.

Drug delivery systems for the treatment of restenosis.

Attempts to achieve revascularization of coronary arteries blocked by atherosclerotic plaques are hampered by restenotic hyperproliferative response of the treated vessels. The uniform failure of clinical trials using systemic therapies to prevent restenosis has prompted development of methods for arterial drug delivery systems. This review describes technologies of polymeric-based, perivascular, and intraluminal drug and gene delivery systems. The critical assessment of controversies including drug and vehicle type, dose and release rate, and preclinical validation is reviewed.

Nanoparticulate delivery system of a tyrphostin for the treatment of restenosis.

Restenosis, the principal complication of percutaneous transluminal coronary angioplasty is responsible for the 35-40% long-term failure rate following coronary revascularization. The neointimal formation, a morphological substrate of restenosis, is dependent on smooth muscle cells (SMC) proliferation and migration. Signal transduction through the platelet-derived growth factor (PDGF)/PDGF receptors system is involved in the process of post-angioplasty restenosis. The unsuccessful attempts to control restenosis by systemic pharmacological interventions have prompted many researchers to look for more promising therapeutic approaches such as local drug delivery. Tyrphostins are low molecular weight inhibitors of protein tyrosine kinases. We assessed the release kinetics and in vivo effects of nanoparticles containing PDGF-Receptor beta (PDGFRbeta) tyrphostin inhibitor, AG-1295. AG-1295-loaded poly(DL-lactide) (PLA) nanoparticles were prepared by spontaneous emulsification/solvent displacement technique. In vitro release rate and the impact of drug/polymer ratio on the nanoparticle size were determined. The degree of tyrosine phosphorylation was assessed by Western blot with phosphotyrosine-specific antibody in rat SMC extracts. Several bands characteristic of PDGF BB-stimulated SMC disappeared or weakened following tyrphostin treatment. Local intraluminal delivery of AG-1295-loaded PLA nanoparticles to the injured rat carotid artery had no effect on proliferative activity in medial and neointimal compartments of angioplastisized arteries, indicating a primary antimigration effect of AG-1295 on medial SMC.

Local delivery of platelet-derived growth factor receptor-specific tyrphostin inhibits neointimal formation in rats.

Signal transduction through the platelet-derived growth factor (PDGF)/PDGF receptor (PDGFR) system is involved in the process of postangioplasty restenosis. Tyrphostins are low molecular weight inhibitors of protein tyrosine kinases. We assessed the antiproliferative effects of PDGFRbeta-specific tyrphostin AG-1295 in vitro and in vivo. AG-1295 significantly inhibited rat smooth muscle cell growth stimulated by PDGF-BB or FCS. This antiproliferative effect was paralleled by reversible reduction of the total phosphotyrosine level and the degree of PDGFRbeta phosphorylation by the drug in vitro. Local sustained delivery of the drug from perivascularly implanted polymeric matrices resulted in focal AG-1295 levels of 711 and 29.1 ng/mg of dry arterial tissue 1 and 14 days after implantation in rats. AG-1295 delivered from polymeric matrices resulted in a 35% reduction of neointimal formation on day 14 after balloon injury in the rat carotid model. Tyrosine phosphorylation of certain transduction proteins in arterial tissue extracts was significantly upregulated by balloon injury on day 3 but was essentially returned to or below basal levels 14 days after injury. Tyrphostin treatment decreased tyrosine phosphorylation at both time points below the basal levels. Moreover, the enhancement of PDGFRbeta expression 3 and 14 days after arterial injury was strongly inhibited by AG-1295 treatment. It can be concluded that AG-1295 reduces neointimal formation by inhibiting PDGFbeta-triggered tyrosine phosphorylation.

Sustained delivery and expression of DNA encapsulated in polymeric nanoparticles.

Sustained release polymeric gene delivery systems offer increased resistance to nuclease degradation, increased amounts of plasmid DNA (pDNA) uptake, and the possibility of control in dosing and sustained duration of pDNA administration. Furthermore, such a system lacks the inherent problems associated with viral vectors. Biodegradable and biocompatible poly(DL-lactide-co-glycolide) polymer was used to enacapsulate pDNA (alkaline phosphatase, AP, a reporter gene) in submicron size particles. Gene expression mediated by the nanoparticles (NP) was evaluated in vitro and in vivo in comparison to cationic-liposome delivery. Nano size range (600 nm) pDNA-loaded in poly(DL-lactide-co-glycolide) polymer particles with high encapsulation efficiency (70%) were formulated, exhibiting sustained release of pDNA of over a month. The entrapped plasmid maintained its structural and functional integrity. In vitro transfection by pDNA-NP resulted in significantly higher expression levels in comparison to naked pDNA. Furthermore, AP levels increased when the transfection time was extended, indicating sustained activity of pDNA. However, gene expression was significantly lower in comparison with standard liposomal transfection. Seven days after i.m. injections in rats, naked pDNA and pDNA-NP were found to be significantly more potent (1-2 orders of magnitude) than liposomal pDNA. Plasmid DNA-NP treatment exhibited increased AP expression after 7 and 28 days indicating sustained activity of the NP.

PDGF-receptor tyrosine kinase blocker AG1295 selectively attenuates smooth muscle cell growth in vitro and reduces neointimal formation after balloon angioplasty in swine.

BACKGROUND: Signaling through protein tyrosine kinases (PTKs) is a major contributor to the transmission of mitogenic stimuli to the interior of the cell and nucleus. The present study was designed to determine the effect of the tyrphostin AG1295, a selective blocker of PDGF-receptor PTK, on the growth of porcine and human smooth muscle cells (SMCs) in culture, on the outgrowth kinetics of SMCs from porcine and human arterial explants, and on neointimal formation after balloon injury in pigs. METHODS AND RESULTS: SMCs for culture were obtained from porcine abdominal aortas, human internal mammary arteries, and endarterectomy tissue from a single human carotid artery. Addition of AG1295 to SMCs before PDGF stimulation completely inhibited PDGF-beta-receptor tyrosine phosphorylation without affecting the level of PDGF-beta-receptor. AG1295 resulted in a selective, reversible inhibition of SMC proliferation in culture (76%) with only mild (13.5%) inhibition of endothelial cell proliferation. The number of SMCs accumulating around explants of porcine carotid arteries and human endarterectomy specimens 12, 15, 19, 22, and 24 days after plating was reduced by 82% to 92% in AG1295-treated compared with nontreated specimens, and initiation of SMC outgrowth was markedly delayed. The numbers of cells accumulated 10 days after initiation of outgrowth were significantly lower in treated versus control explants. Local intravascular delivery of AG1295-impregnated polylactic acid-based nanoparticles (130+/-25 nm) to the site of balloon injury to porcine femoral arteries resulted in significant reductions in intima/media area ratio and luminal cross-sectional area narrowing by neointima compared with contralateral control arteries to which empty nanoparticles were applied (0.15+/-0.07 versus 0.09+/-0.03, P=.046 and 20+/-4% versus 10+/-4%, P=.0009, n=6 for both). CONCLUSIONS: The tyrphostin AG1295, a selective blocker of PDGF-receptor kinase, exerts a marked inhibitory effect on the activation, migration, and proliferation of porcine and human SMCs in vitro and an approximately 50% inhibitory effect on neointimal formation after balloon injury in porcine femoral arteries when delivered via biodegradable nanoparticles. Further studies appear to be warranted to evaluate the applicability of this novel approach to the interventional setting.

Controlled periadventitial administration of verapamil inhibits neointimal smooth muscle cell proliferation and ameliorates vasomotor abnormalities in experimental vein bypass grafts.

OBJECTIVE: Inhibition of early myointimal proliferation may improve longterm patency of vein grafts, but the clinical use of many experimental drugs is limited by systemic toxicity. To determine whether this goal can be achieved by low-dose targeted drug administration, we constructed a polymeric system delivering verapamil and evaluated the effects on local and downstream vein graft morphology, neointimal smooth muscle cell proliferation, and vasomotor function. METHODS: Ethylene-vinyl acetate polymeric delivery systems were constructed, containing 2% verapamil by weight. These are flexible, biocompatible, and nonbiodegradable matrices, delivering the drug at a rate of 10 micrograms/day. The autologous external jugular vein was used to create a carotid artery bypass graft in hypercholesterolemic (n = 22) rabbits. Verapamil-containing matrices (n = 12) or plain polymers (control, n = 10) were wrapped around the proximal third of the veins after reperfusion. Graft vasomotor function was evaluated and was also compared with function of an additional group of normocholesterolemic vein grafts (n = 8). RESULTS: Twenty-eight days after grafting, intimal index (intima/media thickness ratio) was 31% lower, neointima/original lumen surface ratio was 26% lower, and residual luminal area was 71% greater (4.00 +/- 1.2 mm2 versus 2.34 +/- 0.9 mm2, all p < 0.01) under verapamil matrices compared with control grafts. Neointimal smooth muscle cell content was reduced from 45.4% to 28.2%, and net neointimal smooth muscle cell thickness was reduced by 47% (30 microns vs 15.8 microns, both p < 0.01). Verapamil-treated segments distal to the matrices also showed significantly lower neointimal smooth muscle cell density and increased lumen size. Sensitivity to serotoin and vasomotor responses to serotonin, norepinephrine, and sodium nitroprusside in distal segments were significantly lower in verapamil-treated grafts than in controls. CONCLUSIONS: Periadventitial controlled administration of verapamil below 1% of the systemic dose effectively inhibits myointimal hyperplasia in vein grafts. Local polymeric drug delivery may be readily applicable to coronary revascularization operations.

Controlled delivery of a tyrphostin inhibits intimal hyperplasia in a rat carotid artery injury model.

We examined the inhibitory effect of AG-17, a potent inhibitor of protein tyrosine kinase activity on injury-induced vascular SMC proliferation by polymeric-based, periadventitial controlled release implant in the balloon catheter carotid injury model in rats. The AG-17 delivery system was formulated from ethylenevinyl acetate copolymer and the release kinetics as well as drug stability were determined. Polymeric matrices containing 2 or 10% AG-17 were implanted perivascularly in rats following balloon catheter injury. Western blot analysis of explanted arterial segments revealed enhanced tyrosine phosphorylation in injured arteries that was essentially reduced to normal levels in treated arteries. The mean neointima to media ratios were significantly reduced in both 2% (0.79 +/- 0.17, n = 9, P < 0.02) and 10% AG-17 (0.59 +/- 0.09, n = 12, P < 0.001) groups in comparison to the control group (1.38 +/- 0.18, n = 16). The mean areas of the media in the control and the 2% AG-17 group did not differ significantly but a significant reduction of the mean area of the media was observed in 10% AG-17 group. Embedding of the unstable tyrphostin compound, AG-17, in a hydrophobic matrix stabilizes the drug both in vitro and in vivo, and allows delivery-rate modulation as well as protracted site-specific therapy. Perivascular controlled release delivery of the tyrphostin AG-17 inhibits neointimal formation in the rat carotid injury model.