Novel approaches for cardiovascular drug eluting devices based on cellular pharmacokinetic determinants of coronary artery cells

Cardiovascular diseases represent the main cause of mortality in industrialized countries; clinical manifestations include angina pectoris, myocardial infarction, and chronic coronary heart disease. Changes in fluid dynamics disturb the physiological functions of the vascular endothelium (Li et al., 2005) and subsequent vascular remodeling accompanied by proliferation of cells and infiltration of inflammatory cells results in atherosclerosis within the vessel. The formation of atherosclerotic plaques causes a flow-limiting stenosis, thus restraining the coronary blood flow (Libby et al., 2011).
The most frequently performed invasive procedure to reopen a stenotic vessel in clinics is percutaneous transluminal coronary angioplasty. To prevent a spontaneous occlusion and reduce restenosis rates, a coronary stent is deployed. However, the use of bare-metal stents (BMS) and drug-eluting stents (DES) is associated with two severe complications. A mechanical-induced injury provokes a remodeling of the arterial wall resulting in a neointima formation within the stented segment, namely in-stent restenosis (ISR). This has been defined as one major drawback of BMS. Restenosis evolves by increased vascular smooth muscle cells (SMC) migration and proliferation from the intimal layer of the vessel wall that ultimately obstruct the vessel lumen. Even if the use of DES reduces the incidence of ISR, the unspecific cytotoxicity of the loaded substances is believed to promote the development of the rare but more severe complication, known as late stent-thrombosis (LST) (Holmes et al., 2010). Based on the current understanding, a permanent inhibition of endothelial cell (EC) proliferation and migration hampers the re-endothelialization of the stent struts. Additionally, a hypersensitive reaction to the stent material and polymer supports the development of thrombosis.
Considering the pathophysiological basis for development of ISR and LST it seems evident that stent material and especially the drug coating are key features that should be modulated to inhibit the progressive proliferation of SMCs and to promote the re-endothelialization.
Within the context of dual-drug technology that combines the different cellular effects of two compounds (Kukreja N et al., 2008) , we developed a DES with a luminal located atorvastatin and an abluminal applied sirolimus. This approach inhibits ISR without provoking a long-term impact on re-endothelialization. Novel concepts predict the use of an abluminal located antiproliferative drug to ensure a targeted tissue release while reducing the systemic exposure (Petersen et al., 2013). Based on this concept, we analyzed the effects of atorvastatin and sirolimus on cellular proliferation (section 3.1). Atorvastatin was found not to impede the antiproliferative effect of sirolimus on SMCs. Furthermore, atorvastatin revealed a less pronounced effect on ECs proliferation. Given these results, re-endothelialization may be less impaired by using dual DES with an abluminal/luminal coating strategy.
From a pharmacological point of view there are two different strategies to improve cell-specific effects of chemotherapeutics which includes identifying of cellular targets and modulating the pharmacokinetics of candidate drugs. One mechanism that contributes to the drug`s pharmacokinetic profile is the expression of drug transporters that mediate the uptake or efflux of compounds (Giacomini et al., 2010). Specifically, the expression of efflux transporters, including P-glycoprotein, modulates the biological cellular activity of chemotherapeutics thus hampering the therapeutic effect (Silverman, 1999). The uptake transporter OCT1 has been shown to transport a variety of substances including the established DES-compound paclitaxel (Gupta et al., 2012). Therefore, we tested whether an adenoviral-induced overexpression of OCT1 using the SMC-specific promotor of transgelin (SM22α) would enhance the antiproliferative effects of paclitaxel in vascular SMCs (section 3.2). First, the activity of SM22α was assessed in various cell types; a muscle cell-specific expression was demonstrated. The activity of OCT1 was then compared in adenoviral infected ECs and SMCs, with a higher accumulation of OCT1 substrates found in SMCs. To test the findings from the concept study relating to cell-specific drug effects, we studied the impact of paclitaxel treatment of ECs and SMCs, finding a significantly increased effect in SMCs. These results suggest that cell-specific expression of transport proteins serves as a mechanism for producing a selective effect on target cells.
Another approach to improve the outcome of DES is the use of drugs that show benefits in the treatment of atherosclerosis. Since pleiotropic activities of statins were associated with a high potential in restenosis reduction after systemic therapy (Pasceri et al., 2004; Serruys et al., 2002), statins were suggested as suitable drug candidates for local application. Following data that shows a high impact of cerivastatin in inhibiting SMCs proliferation and neointima formation without impairing ECs cellular behavior (Corpataux et al., 2005; Jaschke et al., 2005), we studied which cellular mechanism may account for the cell-specific activity (section 3.3). Endothelial cells and SMCs were treated with different statins, with atorvastatin especially presenting an SMC-specific inhibition of proliferation. Quantifying the expression of the primary drug target revealed comparable levels of HMG-CoA reductase mRNA and protein expression, leading to the assumption that pharmacokinetics may account for different cellular activity. We detected a higher accumulation of atorvastatin in SMCs; this has been associated with a higher endogenous expression of OATP2B1, a high affinity transporter for atorvastatin (Grube et al., 2006). Adenoviral-induced overexpression of OATP2B1 supported our previous finding. Assuming that the expression of OATP2B1 is a determinant of drug effects in SMCs, we used a cell line overexpressing OATP2B1 to identify cytotoxic drugs suitable for SMC specific inhibition. The screening provided evidence that teniposide may be an OATP2B1 substrate. This was supported by subsequent proliferation assays demonstrating a higher efficacy of teniposide on SMC proliferation in the presence of heterogeneously expressed OATP2B1.
A variety of limus agents have entered clinics for local application on DES. While several drugs including sirolimus, zotarolimus, and everolimus demonstrated high efficiency and safety, some failed to do so (Ota et al., 2015). The anti-inflammatory and immunomodulatory compound pimecrolimus especially showed excessive neointimal growth in humans despite promising data from a preclinical assessment (Berg et al., 2007; Ormiston et al., 2009). Nevertheless, the underlying mechanisms contributing to the failure of pimecrolimus eluting stents are unknown. We therefore studied the impact of pimecrolimus on SMCs and ECs proliferation and viability (section 3.4). According to our study, pimecrolimus had a cytostatic effect in both cells. Since preliminary data from an mRNA microarray suggested that pimecrolimus induced the expression of genes involved in the interferon signaling pathway, we analyzed their expression by real-time quantitative PCR. Importantly, pimecrolimus but not sirolimus led to an upregulation of these genes. This could in part be associated with inhibition of the phosphatase calcineurin, a downstream target of the pimecrolimus/FK506-binding protein 12-complex and known to modulate the interferon pathway (Wang et al., 2012). Specifically, the interaction of calcineurin with toll-like receptor 4 may modulate the expression of interferon-inducible genes upon pimecrolimus treatment. In accordance were our findings showing that silencing of the toll-like receptor 4 reduced the activation of gene expression. This crosstalk between the interferon and toll-like receptor 4 signaling may be a molecular mechanisms explaining the failure of pimecrolimus-eluting stents in clinical trials.