Search

Organic anion transporting polypeptide (OATP) 1B3–1B7 (LST-3TM12) is a member of the OATP1B [solute carrier organic anion transporter ( SLCO) 1B] family. This transporter is not only functional but also expressed in the membrane of the smooth endoplasmic reticulum of hepatocytes and enterocytes. OATP1B3–1B7 is a splice variant of SLCO1B3 in which the initial part is encoded by SLCO1B3, whereas the rest of the mRNA originates from the gene locus of SLCO1B7. In this study, we not only showed that SLCO1B3 and the mRNA encoding for OATP1B3–1B7 share the 5′ untranslated region but also that silencing of an initial SLCO1B3 exon lowered the amount of SLCO1B3 and of SLCO1B7 mRNA in Huh-7 cells. To validate the assumption that both transcripts are regulated by the same promoter we tested the influence of the bile acid sensor farnesoid X receptor (FXR) on their transcription. Treatment of Huh-7 and HepaRG cells with activators of this known regulator of OATP1B3 not only increased SLCO1B3 but also OATP1B3–1B7 mRNA transcription. Applying a heterologous expression system, we showed that several bile acids interact with OATP1B3–1B7 and that taurocholic acid and lithocholic acid are OATP1B3–1B7 substrates. As OATP1B3–1B7 is located in the smooth endoplasmic reticulum, it may grant access to metabolizing enzymes. In accordance are our findings showing that the OATP1B3–1B7 inhibitor bromsulphthalein significantly reduced uptake of bile acids into human liver microsomes. Taken together, we report that OATP1B3–1B7 transcription can be modulated with FXR agonists and antagonists and that OATP1B3–1B7 transports bile acids. NEW & NOTEWORTHY Our study on the transcriptional regulation of the novel organic anion transporting polypeptide (OATP) 1B3–1B7 concludes that the promoter of solute carrier organic anion transporter ( SLCO) 1B3 governs SLCO1B3–1B7 transcription. Moreover, the transcription of OATP1B3–1B7 can be modulated by farnesoid X receptor (FXR) agonists and antagonists. FXR is a major regulator in bile acid homeostasis that links OATP1B3–1B7 to this physiological function. Findings in transport studies with OATP1B3–1B7 suggest that this transporter interacts with the herein tested bile acids.

Recent studies have focused on coproporphyrin (CP)-I and CP-III (CPs) as endogenous biomarkers for organic anion transporting polypeptides (OATPs). Previous data showed that CPs are also substrates of multidrug resistance-associated protein (MRP/Mrp) 2 and 3. This study was designed to examine the impact of loss of Mrp2 function on the routes of excretion of endogenous CPs in wild-type (WT) Wistar compared to Mrp2-deficient TR(−) rats. To exclude possible confounding effects of rat Oatps, the transport of CPs was investigated in Oatp-overexpressing HeLa cells. Results indicated that CPs are substrates of rodent Oatp1b2, and that CP-III is a substrate of Oatp2b1. Quantitative targeted absolute proteomic (QTAP) analysis revealed no differences in Oatps, but an expected significant increase in Mrp3 protein levels in TR(−) compared to WT rat livers. CP-I and CP-III concentrations measured by LC-MS/MS were elevated in TR(−) compared to WT rat liver, while CP-I and CP-III estimated biliary clearance was decreased 75- and 840-fold in TR(−) compared to WT rats, respectively. CP-III concentrations were decreased 14-fold in the feces of TR(−) compared to WT rats, but differences in CP-I were not significant. In summary, the disposition of CPs was markedly altered by loss of Mrp2 and increased Mrp3 function as measured in TR(−) rats.

The family of Organic Anion Transporting Polypeptides are known to facilitate the transmembrane transport. OATP1B3-1B7 is a novel member of the OATP1B-subfamily, and is encoded by SLCO1B3-SLCO1B7 readthrough deriving from the genes SLCO1B3 and SLCO1B7 on chromosome 12. The resulting protein is expressed in the smooth endoplasmatic reticulum of hepatocytes, is functional, and transports dehydroepiandrosterone-sulfate (DHEAS). In the gene area encoding for the 1B7-part of the protein, there are coding polymorphisms. It was the aim of this study to test the frequency and the impact of these genetic variants on transport activity. The minor allele frequency (MAF) of the coding polymorphisms was determined in a cohort of 192 individuals. DHEAS transport function was determined by applying the vTF-7 based heterologous expression system using plasmids encoding for OATP1B3-1B7 or the respective variants. The genetic variants 641 T (MAF 0.021), 1073 G (MAF 0.169) and 1775 A (MAF 0.013) significantly reduced DHEAS accumulation in cells transfected with OATP1B3-1B7, albeit without significantly influencing expression of the transporter as determined by Western blot analysis and immunofluorescence after heterologous expression. Genotyping revealed complete linkage of the variants 884A, 1073 G and 1501C. Presence of the haplotype abolished the DHEAS-transport function of OATP1B3-1B7. Naturally and frequently occurring genetic variants located within the gene region of SLCO1B7 encoding for the 1B7-part of OATP1B3-1B7 influence the in vitro function of this member of the OATP1B-family. With their functional characterisation, we provide the basis for pharmacogenetic studies, which may help to understand the in vivo relevance of this transporter.

AIMS: The anthelminthic ivermectin is receiving new attention as it is being repurposed for new indications such as mass drug administrations for the treatment of scabies or in malaria vector control. As its pharmacokinetics are still poorly understood, we aimed to characterize the population pharmacokinetics of ivermectin in plasma and dried blood spots (DBS), a sampling method better suited to field trials, with special focus on the influence of body composition and enterohepatic circulation. METHODS: We performed a clinical trial in 12 healthy volunteers who each received a single oral dose of 12 mg ivermectin, and collected peripheral venous and capillary DBS samples. We determined ivermectin concentrations in plasma and DBS by liquid chromatography tandem mass spectrometry using a fully automated and scalable extraction system for DBS sample processing. Pharmacokinetic data were analysed using non‐linear mixed effects modelling. RESULTS: A two‐compartment model with a transit absorption model, first‐order elimination, and weight as an influential covariate on central volume of distribution and clearance best described the data. The model estimates (inter‐individual variability) for a 70 kg subject were: apparent population clearance 7.7 (25%) l h(−1), and central and peripheral volumes of distribution 89 (10%) l and 234 (20%) l, respectively. Concentrations obtained from DBS samples were strongly linearly correlated (R (2) = 0.97) with plasma concentrations, and on average 30% lower. CONCLUSION: The model accurately depicts population pharmacokinetics of plasma and DBS concentrations over time for oral ivermectin. The proposed analytical workflow is scalable and applicable to the requirements of mass drug administrations.

The protein family of Organic Anion Transporting Polypeptides (OATPs) summarizes various transporters known to facilitate cellular uptake of xenobiotics. One member of this family is OATP2B1. This transporter is ubiquitously expressed and possesses a PDZ-binding motif at the C-terminus. PDZK1 (PDZ domain-containing 1) is a scaffold protein that influences function of different membrane proteins by sorting/stabilization of their membrane localization. It was aim of the herein reported study to investigate whether there is an interaction between OATP2B1 and PDZK1, and to further characterize its impact on transport function. At first expression of both OATP2B1 and PDZK1 was evaluated in liver, kidney and intestine. Based on the existence of a C-terminal PDZ-class I binding motif in OATP2B1 and the co-expression in all tested tissues an interaction was likely. Testing the influence of PDZK1 on OATP2B1 transport function revealed enhanced transport capacity for estrone 3-sulfate, thereby suggesting a change in OATP2B1 amount in the membrane. This assumption was validated by Western blot analysis. Finally, deletion of the C-terminal PDZ-binding motif in OATP2B1 lowered the impact of PDZK1 on transport function. Taken together, we report an interaction of PDZK1 with OATP2B1, which influences localization and function of the transporter. Changes in PDZK1 expression may therefore be one factor contributing to interindividual differences in OATP2B1 mediated pharmacokinetic processes.

Organic anion transporting polypeptides (OATPs) and particularly the two members of the OATP1B family are known for their role in pharmacokinetics. Both SLCO1B3 and SLCO1B1 are located on chromosome 12 encompassing the gene locus SLCO1B7. Hitherto, this particular gene has been assumed to be a pseudogene, even though there are published mRNA sequences linked to this chromosomal area. It was aim of this study to further investigate SLCO1B7 and the associated mRNA LST-3TM12. In a first step, we aligned all mRNAs linked to the chromosomal region of SLCO1B-transporters. This in silico analysis revealed that LST-3TM12 is a product of splicing of SLCO1B3 and SLCO1B7, and encodes for a protein with twelve transmembrane domains. The existence of LST-3TM12 mRNA was verified by polymerase chain reaction showing liver enriched expression. In addition, immunohistological staining showed that LST-3TM12 protein was expressed in the endoplasmic reticulum (ER) of hepatocytes. Localization in the ER was further verified by immunoblot analysis showing high amounts of LST-3TM12 in liver microsomes. Function of LST-3TM12 was assessed by transport studies after heterologous expression in HeLa cells, where the transporter was shown to be expressed not only in the ER but also in the plasma membrane. Overexpression of LST-3TM12 was associated with enhanced cellular accumulation of dehydroepiandrosterone sulfate (Vmax 300.2 pmol mg−1 min−1; Km 34.2 µm) and estradiol 17β-glucuronide (Vmax 29.9 mol mg−1 min−1 and Km 32.8 µM). In conclusion, LST-3TM12 is a functional splice variant of SLCO1B3 and SLCO1B7 expressed in the ER of human liver.

The human drug transporter Organic Anion Transporting Polypeptide (hOATP)2B1 facilitates cellular uptake of its substrates. Various studies suggest that hOATP2B1 is involved in intestinal absorption, but preclinical evaluations performed in rodents do not support this. Thus, our study aimed to compare the expression and function of hOATP2B1 with its orthologue in rats (rOatp2b1). Even if the general expression pattern was comparable, the transporters exhibited substantial differences on functional level. While bromosulfophthalein and atorvastatin were substrates of both transporters, the steroid sulfate conjugates estrone 3-sulfate (E1S), progesterone sulfate and dehydroepiandrosterone sulfate were only transported by hOATP2B1. To further elucidate these functional differences, experiments searching for the E1S substrate recognition site were conducted generating human-rat chimera as well as partly humanized variants of rOatp2b1. The rOatp2b1-329-hOATP2B1 chimera led to a significant increase in E1S uptake suggesting the C-terminal part of the human transporter is involved. However, humanization of various regions within this part, namely of the transmembrane domain (TMD)-9, TMD-10 or the extracellular loop-5 did not significantly change E1S transport function. Replacement of the intracellular loop-3, slightly enhanced cellular accumulation of sulfated steroids. Taken together, we report that OATP2B1 exhibited differences in recognition of steroid sulfate conjugates comparing the rat and human orthologues.

In the renal proximal tubule the secretion and reabsorption of glomerularly filtrated compounds is realized by a functional network of uptake and efflux transporters. The activity and localization of several transporters expressed at the apical tubular membrane are regulated by the membrane-associated protein PDZ domain-containing 1 (PDZK1). We aimed to characterize the transcriptional regulation of this modulator of renal transport. Coexpression analyses of PDZK1 and putative regulators were performed using human kidney samples. Protein and mRNA expression of PDZK1 in renal proximal tubule epithelial cells after adenoviral transfer and siRNA knockdown of transcription factor hepatocyte nuclear factor-1α (HNF1α) was assessed by quantitative real-time PCR and Western blot analysis. Transactivation of the PDZK1 promoter was quantified in cell-based reporter gene assays. Subsequently, the binding of HNF1α to the PDZK1 promoter was verified by in silico analyses and chromatin immunoprecipitation assay. HNF1α positively regulated the promoter activity of PDZK1. Adenoviral overexpression of HNF1α in renal proximal tubule epithelial cells (RPTEC) increased PDZK1 mRNA and protein expression, whereas siRNA knockdown of HNF1α resulted in decreased expression of PDZK1. Our results show that HNF1α, which has previously been described as a modulator of several transporters of the renal transportosome, is also a key determinant of PDZK1 transcription.

Active targeting and specific drug delivery to parenchymal liver cells is a promising strategy to treat various liver disorders. Here, we modified synthetic lipid-based nanoparticles with targeting peptides derived from the hepatitis B virus large envelope protein (HBVpreS) to specifically target the sodium-taurocholate cotransporting polypeptide (NTCP; SLC10A1) on the sinusoidal membrane of hepatocytes. Physicochemical properties of targeted nanoparticles were optimized and NTCP-specific, ligand-dependent binding and internalization was confirmed in vitro. The pharmacokinetics and targeting capacity of selected lead formulations was investigated in vivo using the emerging zebrafish screening model. Liposomal nanoparticles modified with 0.25 mol% of a short myristoylated HBV derived peptide, that is Myr-HBVpreS2-31, showed an optimal balance between systemic circulation, avoidance of blood clearance, and targeting capacity. Pronounced liver enrichment, active NTCP-mediated targeting of hepatocytes and efficient cellular internalization were confirmed in mice by 111In gamma scintigraphy and fluorescence microscopy demonstrating the potential use of our hepatotropic, ligand-modified nanoparticles.

Background: It was our intention to develop cathepsin B-sensitive nanoparticles for tumor-site-directed release. These nanoparticles should be able to release their payload as close to the tumor site with a decrease of off-target effects in mind. Cathepsin B, a lysosomal cysteine protease, is associated with premalignant lesions and invasive stages of cancer. Previous studies have shown cathepsin B in lysosomes and in the extracellular matrix. Therefore, this enzyme qualifies as a trigger for such an approach. Methods: Poly(dimethylsiloxane)-b-poly(methyloxazoline) (PDMS-PMOXA) nanoparticles loaded with paclitaxel were formed by a thin-film technique and standard coupling reactions were used for surface modifications. Despite the controlled release mechanism, the physical properties of the herein created nanoparticles were described. To characterize potential in vitro model systems, quantitative polymerase chain reaction and common bioanalytical methods were employed. Conclusions: Stable paclitaxel-loaded nanoparticles with cathepsin B digestible peptide were formed and tested on the ovarian cancer cell line OVCAR-3. These nanoparticles exerted a pharmacological effect on the tumor cells suggesting a release of the payload.

Katharina Prestin,1,* Maria Olbert,2,* Janine Hussner,1 Tamara L Isenegger,1 Daniel G Gliesche,1 Kerstin Böttcher,2 Uwe Zimmermann,3 Henriette E Meyer zu Schwabedissen1 1Department of Pharmaceutical Sciences, Biopharmacy, University of Basel, Basel, Switzerland; 2Center of Drug Absorption and Transport, Institute of Pharmacology, 3Department of Urology, University Medicine Greifswald, Greifswald, Germany *These authors contributed equally to this work Abstract: Mammalian nuclear receptors (NRs) are transcription factors regulating the expression of target genes that play an important role in drug metabolism, transport, and cellular signaling pathways. The orphan and structurally unique receptor small heterodimer partner 1 (syn NR0B2) is not only known for its modulation of drug response, but has also been reported to be involved in hepatocellular carcinogenesis. Indeed, previous studies show that NR0B2 is downregulated in human hepatocellular carcinoma, suggesting that NR0B2 acts as a tumor suppressor via inhibition of cellular growth and activation of apoptosis in this tumor entity. The aim of our study was to elucidate whether NR0B2 may also play a role in other tumor entities. Comparing NR0B2 expression in renal cell carcinoma and adjacent nonmalignant transformed tissue revealed significant downregulation in vivo. Additionally, the impact of heterologous expression of NR0B2 on cell cycle progression and proliferation in cells of renal origin was characterized. Monitoring fluorescence intensity of resazurin turnover in RCC-EW cells revealed no significant differences in metabolic activity in the presence of NR0B2. However, there was a significant decrease of cellular proliferation in cells overexpressing this NR, and NR0B2 was more efficient than currently used antiproliferative agents. Furthermore, flow cytometry analysis showed that heterologous overexpression of NR0B2 significantly reduced the amount of cells passing the G1 phase, while on the other hand, more cells in S/G2 phase were detected. Taken together, our data suggest that downregulation of NR0B2 may also play a role in renal cell carcinoma development and progression. Keywords: small heterodimer partner, SHP1, NR0B2, nuclear receptor, kidney cancer, proliferation

Clinical success of coronary drug-eluting stents (DES) is hampered by simultaneous reduction of smooth muscle cell (HCASMC) and endothelial cell proliferation due to unspecific cytotoxicity of currently used compounds. Previous in vitro data showing SMC-specific inhibition of proliferation suggested that statins may be suitable candidates for DES. It was aim of this study to further investigate statins as DES drug candidates to identify mechanisms contributing to their cell-selectivity. In vitro proliferation assays comparing the influence of various statins on HCASMC and endothelial cells confirmed that atorvastatin exhibits HCASMC-specificity. Due to similar expression levels of the drug target HMG-CoA reductase in both cell types, cellular accumulation of atorvastatin was assessed, revealing enhanced uptake in HCASMC most likely driven by significant expression of OATP2B1, a known uptake transporter for atorvastatin. In accordance with the finding that endogenous OATP2B1 influenced cellular accumulation in HCASMC we used this transporter as a tool to identify teniposide as new DES candidate drug with HCASMC-specific effects. We describe OATP2B1 as a determinant of pharmacokinetics in the coronary artery. Indeed, endogenously expressed OATP2B1 significantly influences the uptake of substrate drugs, thereby governing cell specificity. Screening of candidate drugs for interaction with OATP2B1 may be used to promote SMC-specificity.

Small heterodimer partner 1 (SHP1, NR0B2) is a member of the superfamily of nuclear receptors (NRs). Even if this orphan receptor, unlike other NRs, lacks the DNA-binding domain, it is capable of regulating transcription by repressing the activity of other NRs by direct protein-protein interaction. Accordingly, SHP1 is part of negative feedback loops of the transcriptional regulation of genes involved in drug metabolism and various metabolic pathways including bile acid and glucose homeostasis. Although it is known that several interacting partners of SHP1 also modulate its expression, there is little information about genetic variability of this regulatory mechanism. Our study aimed to identify genetic variants in the NR0B2 promoter region and to determine their impact on NR0B2 transcription. For this, DNA samples originating from 119 participants of the population-based cohort Study of Health in Pomerania were analyzed by Sanger sequencing revealing four genetic variants: NR0B2:c.-594T>C (rs71636795), NR0B2:c.-414G>C (newly identified), NR0B2:c.-423C>T (rs78182695), and NR0B2:c.-224delCTGA (rs145613139) localized in the 5' untranslated region of NR0B2. The impact of these variants on transactivation of the NR0B2 promoter by NRs known to be regulators of SHP1 expression (hepatocyte nuclear factor 4α, liver receptor homolog-1, and farnesoid X receptor) was assessed in a cell-based reporter gene assay, showing that transactivation by hepatocyte nuclear factor 4α and liver receptor homolog-1 was significantly decreased in the presence of the genetic variant NR0B2:c.-594T>C, even though this effect was cell specific. However, SHP1 mRNA expression in a small collection of human kidney samples was not affected by these genetic variants.

Genome wide association studies revealed single nucleotide polymorphisms (SNP) located within the promoter of PDZ domain containing protein 1 (PDZK1) to be associated with serum uric acid levels. Since modulation of transporters and particularly of membrane proteins involved in uric acid handling by PDZK1 has previously been reported, the aim of this study was to analyze the impact of the polymorphisms rs1967017, rs1471633, and rs12129861 on promoter activity and thereby transcription of PDZK1. Cell-based reporter gene assays showed transactivation of the PDZK1-promoter by triiodothyronine mediated by thyroid hormone receptors (THR) α and β. In silico analysis verified localization of the polymorphism rs1967017 within the most likely THR binding site whose deletion reduced THR-mediated transactivation. Furthermore, our study shows regulation of PDZK1 by thyroid hormones, thereby providing a mechanistic basis for the previously reported associations between thyroid hormone status and uric acid homeostasis.

Renal tubular handling of urate is realized by a network of uptake and efflux transporters, including members of drug transporter families such as solute carrier proteins and ATP-binding cassette transporters. Solute carrier family 2, member 9 (SLC2A9), is one key factor of this so called “urate transportosome.” The aim of the present study was to understand the transcriptional regulation of SLC2A9 and to test whether identified factors might contribute to a coordinated transcriptional regulation of the transporters involved in urate handling. In silico analysis and cell-based reporter gene assays identified a hepatocyte nuclear factor (HNF)4α-binding site in the promoter of SLC2A9 isoform 1, whose activity was enhanced by transient HNF4α overexpression, whereas mutation of the binding site diminished activation. HNF4α overexpression induced endogenous SLC2A9 expression in vitro. The in vivo role of HNF4α in the modulation of renal SLC2A9 gene expression was supported by findings of quantitative real-time RT-PCR analyses and chromatin immunoprecipitation assays. Indeed, mRNA expression of SLC2A9 and HNF4α in human kidney samples was significantly correlated. We also showed that in renal clear cell carcinoma, downregulation of HNF4α mRNA and protein expression was associated with a significant decline in expression of the transporter. Taken together, our data suggest that nuclear receptor family member HNF4α contributes to the transcriptional regulation of SLC2A9 isoform 1. Since HNF4α has previously been assumed to be a modulator of several urate transporters, our findings support the notion that there could be a transcriptional network providing synchronized regulation of the functional network of the urate transportosome.

Cardiovascular diseases are the leading causes of death in industrialized countries. Atherosclerotic coronary arteries are commonly treated with percutaneous transluminal coronary intervention followed by stent deployment. This treatment has significantly improved the clinical outcome. However, triggered vascular smooth muscle cell (SMC) proliferation leads to in-stent restenosis in bare metal stents. In addition, stent thrombosis is a severe side effect of drug eluting stents due to inhibition of endothelialization. The aim of this study was to develop and test a stent surface polymer, where cytotoxic drugs are covalently conjugated to the surface and released by proteases selectively secreted by proliferating smooth muscle cells. Resting and proliferating human coronary artery smooth muscle cells (HCASMC) and endothelial cells (HCAEC) were screened to identify an enzyme exclusively released by proliferating HCASMC. Expression analyses and enzyme activity assays verified selective and exclusive activity of the matrix metalloproteinase-9 (MMP-9) in proliferating HCASMC. The principle of drug release exclusively triggered by proliferating HCASMC was tested using the biodegradable stent surface polymer poly-l-lactic acid (PLLA) and the MMP-9 cleavable peptide linkers named SRL and AVR. The specific peptide cleavage by MMP-9 was verified by attachment of the model compound fluorescein. Fluorescein release was observed in the presence of MMP-9 secreting HCASMC but not of proliferating HCAEC. Our findings suggest that cytotoxic drug conjugated polymers can be designed to selectively release the attached compound triggered by MMP-9 secreting smooth muscle cells. This novel concept may be beneficial for stent endothelialization thereby reducing the risk of restenosis and thrombosis.

Activation of the immune system is a way for host tissue to defend itself against tumor growth. Hence, treatment strategies that are based on immunomodulation are on the rise. Conventional cytostatic drugs such as the anthracycline doxorubicin can also activate immune cell functions of macrophages and natural killer cells. In addition, cytotoxicity of doxorubicin can be enhanced by combining this drug with the cytokine interferon-γ (IFNγ). Although doxorubicin is one of the most applied cytostatics, the molecular mechanisms of its immunomodulation ability have not been investigated thoroughly. In microarray analyses of HeLa cells, a set of 19 genes related to interferon signaling was significantly over-represented among genes regulated by doxorubicin exposure, including signal transducer and activator of transcription (STAT) 1 and 2, interferon regulatory factor 9, N-myc and STAT interactor, and caspase 1. Regulation of these genes by doxorubicin was verified with real-time polymerase chain reaction and immunoblotting. An enhanced secretion of IFNγ was observed when HeLa cells were exposed to doxorubicin compared with untreated cells. IFNγ-neutralizing antibodies and inhibition of Janus tyrosine kinase (JAK)-STAT signaling [aurintricarboxylic acid (ATA), (E)-2-cyano-3-(3,4-dihydrophenyl)-N-(phenylmethyl)-2-propenamide (AG490), STAT1 small interfering RNA] significantly abolished doxorubicin-stimulated expression of interferon signaling-related genes. Furthermore, inhibition of JAK-STAT signaling significantly reduced doxorubicin-induced caspase 3 activation and desensitized HeLa cells to doxorubicin cytotoxicity. In conclusion, we demonstrate that doxorubicin induces interferon-responsive genes via IFNγ-JAK-STAT1 signaling and that this pathway is relevant for doxorubicin's cytotoxicity in HeLa cells. Immunomodulation is a promising strategy in anticancer treatment, so this novel mode of action of doxorubicin may help to further improve the use of this drug among different types of anticancer treatment strategies.

The pharmacodynamics of the loaded compounds defines clinical failure or success of a drug-eluting device. Various limus derivatives have entered clinics due to the observed positive outcome after stent implantation, which is explained by their antiproliferative activity resulting from inhibition of the cytosolic immunophilin FK506-binding protein 12. Although pimecrolimus also binds to this protein, pimecrolimus-eluting stents failed in clinics. However, despite its impact on T lymphocytes little is known about the pharmacodynamics of pimecrolimus in cultured human coronary artery cells. We were able to show that pimecrolimus exerts antiproliferative activity in human smooth muscle and endothelial cells. Furthermore in those cells pimecrolimus induced transcription of interferon-inducible genes which in part are known to modulate cell proliferation. Modulation of gene expression may be part of an interaction between calcineurin, the downstream target of the pimecrolimus/FK506-binding protein 12-complex, and the toll-like receptor 4. In accordance are our findings showing that silencing of toll-like receptor 4 by siRNA in A549 a lung carcinoma cell line reduced the activation of interferon-inducible genes upon pimecrolimus treatment in those cells. Based on our findings we hypothesize that calcineurin inhibition may induce the toll-like receptor 4 mediated activation of type I interferon signaling finally inducing the observed effect in endothelial and smooth muscle cells. The crosstalk of interferon and toll-like receptor signaling may be a molecular mechanism that contributed to the failure of pimecrolimus-eluting stents in humans.

The implantation of drug-eluting stents (DES) is a common therapy for atherosclerotic occlusions whereby the loaded compounds often exhibit unspecific cytotoxicity. In detail, clinical success is hampered by simultaneous reduction of smooth muscle (SMC) and endothelial cell proliferation, the latter being associated with reduced endothelialization resulting in prothrombotic surfaces. It was aim of this study to investigate statins as DES candidates to identify mechanisms contributing to a previously reported cell-selectivity. In vitro proliferation assays confirmed the SMC-specific activity of atorvastatin. Due to similar expression levels of the drug target HMG-CoA reductase in both cell types, cellular accumulation of atorvastatin was assessed, revealing enhanced uptake in SMCs most likely driven by significant expression of OATP2B1, a transporter known to be involved in pharmacokinetics. The higher accumulation of atorvastatin and its associated specific effect in SMCs was further confirmed by data dem...

Enhanced proliferation of human coronary artery smooth muscle cells (HCASMCs) and thereby formation of neointima is one of the factors contributing to failure of coronary stents. Even if the use of drug eluting stents (DES) and thereby the local delivery of cytotoxic compounds has significantly improved the clinical outcome, unselective cytotoxic effects are assumed to hamper clinical success. Novel pharmacological approaches are required to enhance cellular selectivity of locally delivered drugs. Cell specific overexpression of a drug transporter could be used to enhance cellular accumulation and therefore cell specificity. In the herein reported study we tested the possibility of cell specific transporter expression to enhance drug effects in HCASMCs. We generated adenoviral constructs to overexpress the organic cation transporter 1 (OCT1) under control of the promoter of SM22α, which had been previously reported as muscle cell specific gene. First the activity of the SM22α-promoter was assessed in various cell types supporting the notion of muscle cell specificity. Subsequently, the activity of the transporter was compared in infected HCAECs and HCASMCs revealing enhanced accumulation of substrate drugs in HCASMCs in presence of the SM22α-promoter. Testing the hypothesis that this kind of targeting might serve as a mechanism for cell-specific drug effects, we investigated the impact on paclitaxel treatment in HCASMC and HCAECs, showing significantly increased antiproliferative activity of this substrate drug on muscle cells. Taken together, our findings suggest that cell-specific expression of transport proteins serves as mechanism governing the uptake of cytotoxic compounds for a selective impact on targeted cells.

Within the context of novel stent designs we developed a dual drug-eluting stent (DDES) with an abluminally focussed release of the potent anti-proliferative drug sirolimus and a luminally focussed release of atorvastatin with stabilizing effect on atherosclerotic deposits and stimulating impact on endothelial function, both from biodegradable poly(L-lactide)-based stent coatings. With this concept we aim at simultaneous inhibition of in-stent restenosis as a result of disproportionally increased smooth muscle cell proliferation and migration as well as thrombosis due to failed or incomplete endothelialisation. The especially adapted spray-coating processes allowed the formation of smooth form-fit polymer coatings at the abluminal and luminal side with 70% respectively 90% of the drug/polymer solution being deposited at the intended stent surface. The impacts of tempering, sterilization, and layer composition on drug release are thoroughly discussed making use of a semi-empirical model. While tempering at 80 °C seems to be necessary for the achievement of adequate and sustained drug release, the coating sequence for DDES should be rather abluminal-luminal than luminal-abluminal, as reduction of the amount of sirolimus eluted luminally could then potentially minimize the provocation of endothelial dysfunction. In vitro proliferation and viability assays with smooth muscle and endothelial cells underline the high potential of the developed DDES.