Perivascular delivery of anti-proliferative agents is an attractive approach to inhibit hyperplasia that causes stenosis of synthetic hemodialysis grafts and other vascular grafts. Perivascular drug delivery systems typically release drugs to both the vascular wall and non-target extravascular tissue. The objective of this study was to develop a biodegradable, perivascular delivery system for localized, sustained and unidirectional drug release in the context of synthetic arteriovenous (AV) grafts used for chronic hemodialysis. To this end, a dense non-porous polymer barrier layer was laminated to either i) a drug-loaded non-porous polymer layer, or ii) a porous polymer layer. To provide tuneability, the porous layer could be loaded with drug during casting or later infused with a drug-loaded hydrogel. The polymer bilayer wraps were prepared by a solvent casting, thermal-phase inversion technique using either polylactide-co-glycolide (PLGA) or polycaprolactone (PCL). Sunitinib, a multi-target receptor tyrosine kinase inhibitor, was used as a model drug. In a modified transwell chamber system, the barrier function of the non-porous PLGA backing was superior to the non-porous PCL backing although both markedly inhibited drug diffusion. As assessed by in vitro release assays, drug release duration from the drug-loaded non-porous PCL construct was almost 4-fold greater than release from the porous PCL construct infused with drug-laden hydrogel (22 days vs. 5 days); release duration from the drug-loaded non-porous PLGA construct was prolonged approximately 3-fold over release from the porous PLGA construct infused with drug-laden hydrogel (9 days vs. 3 days). Complete in vitro degradation of the PLGA porous and non-porous constructs occurred by approximately 35 days whereas the PCL constructs remained intact even after most drug was released (49 days). The PLGA non-porous bilayer wrap containing 143±5.5 mg sunitinib in the inner layer was chosen for further pharmacokinetic assessment in vivo where the construct was placed around the external jugular vein in a porcine model. At one week, no drug was detected by HPLC/MS/MS in any examined extravascular tissue whereas high levels of drug were detected in the wrapped vein segment (1048 ng/g tissue). At four weeks, drug was detected in adjacent muscle (52 ng/g tissue) but 13-fold greater amounts were detected in the wrapped vein segment (1742 ng/g tissue). These results indicate that the barrier layer effectively impedes extravascular drug loss. Tensile testing showed that the initially flexible PLGA construct stiffened with hydration, a phenomenon also observed after in vivo placement. This characteristic may be useful to resist undue circumferential venous tensile stress produced in AV grafting. The PLGA wrap bilayer formulation is a promising perivascular drug delivery design for local treatment of hemodialysis AV graft hyperplasia and possibly other hyperplastic vascular disorders.

Sustained delivery of anti-proliferative drugs to the perivascular area using an injectable polymeric platform is a strategy to inhibit vascular hyperplasia and stenosis. In this study, the concentrations of sirolimus in vascular tissues were evaluated after delivery using an injectable platform made of poly(lactic-co-glycolic acid)-polyethylene glycol-poly(lactic-co-glycolic acid) (PLGA-PEG-PLGA). In order to optimize the drug release profile, the effect of two solvents or solid loading of the sirolimus into the polymer gel was first examined in vitro. The early release was slower with loading of dry drug into the polymer, compared to drug dissolution in solvents. Dry sirolimus was therefore used to load the polymer and applied to the perivascular surface of the graft-venous anastomosis at the time of surgical placement of a carotid-jugular synthetic hemodialysis graft in a porcine model. This was replenished by ultrasound-guided injection of additional drug-laden polymer at one, two and three weeks post-operatively. Magnetic resonance imaging (MRI) using pulse sequences specifically designed for optimal detection of the polymeric gel showed that the polymer injected post-operatively remained at the juxta-anastomotic perivascular site at two weeks. Sirolimus was extracted from various segments of the juxta-anastomotic tissues and the drug concentrations were determined using HPLC-MS/MS. Tissue sirolimus concentrations at one and two weeks were highest near the venous anastomosis, which were approximately 100- to 500-fold greater than the concentrations necessary to inhibit vascular smooth muscle cell proliferation placement of a carotid-jugular synthetic hemodialysis graft in a porcine model in vitro. Drug concentrations remained above the inhibitory concentrations for at least six weeks postoperatively. Thus, serial injections of sustained-delivery polymer gel loaded with sirolimus can provide high localized concentrations at target vascular tissues and thus may be useful for the prevention and treatment of vascular proliferative disorders such as hemodialysis graft stenosis. In addition, MRI is useful for the monitoring of the location of the drug depot.

Synthetic arteriovenous (AV) grafts, placed between an artery and vein, are used for hemodialysis but often fail due to stenosis, typically at the vein-graft anastomosis. This study recorded T lymphocyte and macrophage accumulation at the vein-graft anastomosis, suggesting a role for inflammation in stenosis development. Epoxyeicosatrienoic acids (EETs), products of cytochrome P-450 epoxidation of arachidonic acid, have vasculoprotective and anti-inflammatory effects including inhibition of platelet activation, cell migration, and adhesion. EETs are hydrolyzed by soluble epoxide hydrolase (sEH) to less active diols. The effects of a specific inhibitor of sEH (sEHI) on cytokine release from human monocytes and mouse bone marrow-derived macrophages (BMMΦ) from wild-type (WT) and sEH knockout (KO) animals were investigated. Expression of sEH protein increased over time at the anastomosis as evaluated by immunohistochemistry. Pre-exposure of adherent human monocytes to sEHI (5 μM) significantly inhibited lipopolysaccharide-induced release of monocyte chemotactic protein-1 (MCP-1) and tumor necrosis factor-α and enhanced the EET-to-diol ratio. Release of MCP-1 from WT BMMΦ was significantly inhibited but release from sEH KO BMMΦ was not attenuated indicating the specificity of the sEHI. In contrast, sEHI did not inhibit the release of macrophage inflammatory protein-1 or interleukin-6. Nuclear translocation of NF-κB, as assessed by immunocytochemical staining, was not decreased with sEHI in monocytes, but the phosphorylation of JNK was completely abrogated, suggesting this pathway is the target of sEHI effects in monocytes. These results suggest that sEHI may be useful for inhibition of inflammation and subsequently stenosis in AV grafts.

The insulin sensitizing glitazone drugs, rosiglitazone (ROS) and pioglitazone (PGZ) both have anti-proliferative and anti-inflammatory effects and induce adipose tissue (fat) to produce the vaso-protective protein adiponectin. Stenosis due to intimal hyperplasia development often occurs after placement of arteriovenous synthetic grafts used for hemodialysis. This work was performed to characterize the in vitro and in vivo effects of ROS or PGZ incorporation in fat and to determine if fat/PGZ depots could decrease vascular hyperplasia development in a porcine model of hemodialysis arteriovenous graft stenosis. Powdered ROS or PGZ (6–6000 μM) was mixed with fat explants and cultured. Drug release from fat was quantified by HPLC/MS/MS, and adiponectin and monocyte chemotactic protein-1 (MCP-1) levels in culture media were measured by ELISA. The effect of conditioned media from the culture of fat with ROS or PGZ on i) platelet-derived growth factor-BB (PDGG-BB)-stimulated proliferation of human venous smooth muscle cells (SMC) was measured by a DNA-binding assay, and ii) lipopolysaccharide (LPS)-induced human monocyte release of tumor necrosis factor-alpha (TNFα) was assessed by ELISA. In a porcine model, pharmacokinetics of PGZ from fat depots transplanted perivascular to jugular vein were assessed by HPLC/MS/MS, and retention of the fat depot was monitored by MRI. A porcine model of synthetic graft placed between carotid artery and ipsilateral jugular vein was used to assess effects of PGZ/fat depots on vascular hyperplasia development. Both ROS and PGZ significantly induced the release of adiponectin and inhibited release of MCP-1 from the fat. TNF production from monocytes stimulated with LPS was inhibited 50–70% in the presence of media conditioned by fat alone or fat and either drug. The proliferation of SMC was inhibited in the presence of media conditioned by fat/ROS cultures. Fat explants placed perivascular to the external jugular vein were retained, as confirmed by MRI at one week after placement. PGZ was detected in the fat depot, in the external jugular vein wall and in adjacent tissue at clinically relevant levels, whereas levels in plasma were below detection. External jugular vein exposed to fat incorporated with PGZ had increased adiponectin expression compared to vein exposed to fat alone. However, the development of hyperplasia within the arteriovenous synthetic grafts was unchanged by treatment with fat/PGZ depots compared to no treatment.

Vascular grafts often fail due to neointimal hyperplasia development. Vascular smooth muscle cells (SMC) and macrophages are likely key contributors to hyperplasia growth. Anti‐inflammatory epoxyeicosatrienoic acids (EETs) are derived from arachidonate by cytochrome (CYP) P450 enzymes but are catabolized by sEH. Thus, enhanced sEH activity that decreases EETs may contribute to graft hyperplasia.In a pig arteriovenous graft model, we detected a 6‐fold decrease in CYP450 activity and a 4‐fold increase in sEH activity in the venous‐graft anastomatic tissue after graft placement. Consequently, EET levels decreased by 7‐fold. Further, there was intense tissue immunostaining for TNFα and phospho‐IκbEffects of inhibitors of sEH (sEHI) on human aortic SMC and macrophages were investigated. Polytetrafluoroethylene graft material stimulated the release of TNFα from mouse macrophage (RAW) cells. This release was inhibited by sEHI alone or in combination with EETs. sEHI also inhibited LPS‐induced chemotaxis of RAW cells. In SMC, sEHI prevented TNFα‐induced phosphorylation of Iκb, and PDGF‐induced SMC migration to the same extent as imatinib, a PDGF‐receptor antagonist.These results suggest that sEH participates in vascular graft hyperplasia. Thus sEH inhibitors may be useful in preventing graft hyperplasia.