Ilia Fishbein

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.

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.

Biodegradable implant strategies for inhibition of restenosis

Restenosis of coronary arteries within 6 months of angioplasty has severely limited the long-term success of this procedure. As with any procedure that involves vascular manipulation, thrombosis and stenosis due to intimal proliferation and blood vessel remodeling are the processes that interfere with prolonged patency. This review explores the latest strategies in the form of biodegradable implants designed to inhibit arterial restenosis. The devices discussed herein have potential usefulness not only in coronary artery disease but also in a broad variety of vascular procedures and settings.

Study of the drug release mechanism from tyrphostin AG-1295-loaded nanospheres by in situ and external sink methods.

The present study focused on in vitro release of polylactide-nanoencapsulated tyrphostin AG-1295, a potential agent for local therapy of restenosis. The drug was formulated in matrix-type nanoparticles, termed nanospheres (NS) using the nanoprecipitation method. AG-1295 is a model for low-molecular weight lipophilic compounds, the release behavior of which cannot be adequately characterized by existing methods. An in vitro release technique suitable for optimizing the nanoparticulate formulation release behavior was developed through a novel external sink method and an in situ release method utilizing the environmental sensitivity of the AG-1295 fluorescence spectrum. Similar tendencies were demonstrated by both methods in drug release studied as a function of selected NS preparation variables. The release properties of the drug fractions varying in their binding mode to the carrier particles were studied by the external sink method. The NS surface-adsorbed drug exhibited a significantly higher release rate compared to the drug entrapped in the polymeric matrix. The in situ release of the encapsulated drug was analyzed using the diffusion models of release from a matrix-type sphere. The release was shown to be a composite process, with a burst phase attributed largely to the rapid dissociation of the surface-bound AG-1295. The diffusion-controlled phase exhibited an alteration in kinetic pattern obviously due to the drug distribution between polymeric matrix compartments differing in their permeability. Drug in vitro release investigation may be effectively used to characterize the drug-carrier interaction and internal carrier structure in nanoparticulate formulations, as well as optimize the release behavior in respect to their therapeutic application.

Metalloproteinase inhibitor attenuates neointima formation and constrictive remodeling after angioplasty in rats: augmentative effect of αvβ3 receptor blockade

Abstract Release of matrix metalloproteinases (MMP) from smooth muscle and foam cells following arterial injury facilitates cell migration, neointimal hyperplasia, and vessel wall remodeling. Inhibition of MMP activity using the hydroxamate, zinc-chelating mimicers of collagen, Batimastat and Marimastat, has shown efficacy in reducing constrictive vascular remodeling 6 weeks after experimental angioplasty but not intimal hyperplasia. Vitronectin receptor (α v β 3 ) blockade interferes with binding of this integrin to MMP-2 and proteolyzed collagen, thereby reducing cell invasion. This study tests the effect of MMP inhibition, with and without vitronectin receptor (α v β 3 ) blockade, on neointima formation and arterial remodeling in a long-term model (up to 212 months) of balloon injury in vivo. Male Sabra rats were treated with Batimastat (BB-94, British Biotech Pharmaceuticals Ltd., 30 mg/kg, intraperitoneally) and/or the α v β 3 receptor inhibiting RGD peptide, G-Pen-GRGDSPCA (GIBCO BRL, 0.1 μmol), administered as a perivascular gel to the common carotid artery after balloon injury. Animals were sacrificed 3, 14, 25, and 75 days ( n =21, 23, 22, and 21) after injury. Animals treated with BB-94, peptide, or both had markedly increased absolute luminal area with markedly reduced luminal cross-sectional-area narrowing by neointima and intima-to-media area ratio at all time points except for 3 days after balloon injury versus non-treated, ballooned animals. Combined treatment was significantly more effective than either one alone. Constrictive remodeling, most marked 212 months after balloon injury, was prevented at this time point in all treated animals. The pattern of reduction in luminal narrowing, neointimal formation, and constrictive remodeling across treatment groups correlated very significantly with the reduction in tissue MMP activity as determined by zymography at 3 days. Confirmation of the efficacy of this strategy in larger animals should be the next step toward testing the applicability of this novel approach to the interventional setting.

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.

Site-specific delivery of colchicine in rat carotid artery model of restenosis

Abstract A periadventitial polymeric drug delivery system is one strategy for obtaining and maintaining high tissue levels of drugs at the site of vascular injury. The inhibitory effect of colchicine, an antiproliferative agent, on neointimal proliferation after vascular injury was examined in the balloon catheter carotid artery injury model in rats. Controlled-release colchicine delivery was achieved by formulating drug-containing ethylene vinylacetate copolymer matrices, and the release kinetics were determined in vitro and in vivo. Polymeric matrices containing 0.1% and 0.01% colchicine, and plain polymers were implanted perivascularly in rats following balloon catheter injury. The arterial specimens were harvested after 21 days, processed histologically and evaluated morphometrically. Site-specific delivery of colchicine from 0.1% loaded perivascular matrices, but not 0.01%, significantly inhibited neointimal proliferation after balloon injury. However, local tissue toxicity was observed in some animals. The results of this study should be reconfirmed in a larger animal model such as the porcine catheter injured artery model.

Tyrphostins, inhibitors of protein tyrosine kinase, in restenosis

Abstract Accelerated proliferative response of smooth muscle cells (SMC) to vessel wall injury is the principal cause of premature coronary occlusion in patients undergoing heart transplantation, coronary artery bypass grafting, and PTCA. Protein tyrosine kinases (PTK) activity is involved in multiple steps of signal transduction of SMC growth factors. It is essential for normal cell proliferation, and greatly amplified in proliferative disorders. Thus, blocking the activity of tyrosine kinases may provide a unique and useful strategy for the treatment of syndromes involving accelerated proliferation of vascular SMC. It was shown that a series of low molecular weight PTK inhibitors, termed tyrphostins inhibit PDGF-dependent DNA synthesis and cell growth in vitro and in vivo, and that this occurs in large part by inhibition of PDGF receptor autophosphorilation. This paper reviews the data on tyrphostins activity in cell cultures of vascular SMC, in in vivo models of restenosis, and describes the potential therapeutic approach of tyrphostin delivery in restenosis.