Using 99mTc-(V)-DMSA to follow the vascular calcification process in vascular smooth muscle cells based on pit-1 expression.

BACKGROUND: Vascular calcification is an established feature of atherosclerosis process. The sodium/phosphate transporter PiT-1 acts as a biosensor in vascular calcification of VSMCs. [99mTc]-Pentavalent dimercaptosuccinic acid (99mTc-(V)-DMSA) was mediated by PiT-1 transporter in tumoral cells and we propose its evaluation in a vascular calcification in vitro model. The aim of this study was to determine if 99mTc-(V)-DMSA can follow the vascular calcification process in vascular smooth muscle cells (VSMCs) based on PiT-1 expression. METHODS: From a rat aortic VSMC cell line (A7r5), we set up a model of calcification within 7 days using a calcifying medium containing a high inorganic phosphate concentration. Phosphocalcic deposits were monitored with Alizarin red and Von Kossa staining and with phase contrast microscopy. PiT-1 expression was evaluated with an immunofluorescence assay and osteopontin expression, with whole cell ELISA assay. 99mTc-(V)-DMSA uptake was measured in control and calcifying conditions and compared with optical microscopy evaluation. RESULTS: Under hyperphosphatemia conditions, the VSMC cells progressively overexpressed osteopontin protein, PiT-1 transporter, and synthetized mineralized matrix with phosphocalcic deposition. 99mTc-(V)-DMSA uptake was to 2.8±2.08%DA/mg-protein in control cells and 42±24%DA/mg-protein in calcified cells (P<0.001). PiT-1 inhibition with phosphonoformic acid completely reverse the calcium deposition as well as the 99mTc-(V)-DMSA uptake. These results demonstrated that 99mTc-(V)-DMSA in-vitro uptake is mediated by PiT-1 transporter and follow the VSMC calcification process. CONCLUSIONS: These preliminary in-vitro results showed 99mTc-(V)-DMSA uptake follow the phospho-calcic deposition mediated by PiT-1 transporter. This radiotracer may have some potential to detect changes of VSMC metabolism occurring in the atherosclerosis process.

In vitro assessment of P-gp and BCRP transporter-mediated drug-drug interactions of riociguat with direct oral anticoagulants.

As an alternative to vitamin K antagonists (VKAs), direct oral anticoagulants (DOACs) are increasingly prescribed in combination with riociguat in the treatment of chronic thromboembolic pulmonary hypertension (CTEPH). Pharmacokinetics of riociguat and DOACs are influenced by efflux transporters, such as P-glycoprotein (P-gp) and Breast Cancer Resistance Protein (BCRP). This work aimed to assess P-gp and BCRP-mediated drug-drug interactions of riociguat with DOACs using in vitro models. Bidirectional permeabilities of apixaban and rivaroxaban were investigated across MDCK-MDR1 and MDCK-BCRP models, in the absence and in the presence of increasing concentrations of riociguat (0.5-100 µm). Calculated efflux ratios were subsequently used to determine riociguat inhibition percentages and half maximal inhibitory concentration (IC50). P-gp-mediated efflux of apixaban and rivaroxaban was inhibited by 8% and 21%, respectively, in the presence of 100 µm riociguat. BCRP-mediated transport of apixaban and rivaroxaban was inhibited by 36% and 77%, respectively. IC50s of riociguat on MDCK-MDR1 and MDCK-BCRP models were higher than 100 µm for apixaban and higher than 100 µm and 46.5 µm for rivaroxaban, respectively. This work showed an in vitro inhibition of BCRP-mediated DOACs transport by riociguat. In vivo studies may be required to determine the clinical relevance of these transporter-mediated interactions.

Pharmacological characterization of the 3D MucilAir™ nasal model.

The preclinical evaluation of nasally administered drug candidates requires screening studies based on in vitro models of the nasal mucosa. The aim of this study was to evaluate the morpho-functional characteristics of the 3D MucilAir™ nasal model with a pharmacological focus on [ATP]-binding cassette (ABC) efflux transporters. We initially performed a phenotypic characterization of the MucilAir™ model and assessed its barrier properties by immunofluorescence (IF), protein mass spectrometry and examination of histological sections. We then focused on the functional expression of the ABC transporters P-glycoprotein (P-gp), multidrug resistance associated protein (MRP)1, MRP2 and breast cancer resistance protein (BCRP) in bidirectional transport experiments. The MucilAir™ model comprises a tight, polarized, pseudo-stratified nasal epithelium composed of fully differentiated ciliated, goblet and basal cells. These ABC transporters were all expressed by the cell membranes. P-gp and BCRP were both functional and capable of actively effluxing substrates. The MucilAir™ model could consequently represent a potent tool for evaluating the interaction of nasally administered drugs with ABC transporters.

In Vitro Assessment of Pharmacokinetic Drug-Drug Interactions of Direct Oral Anticoagulants: Type 5-Phosphodiesterase Inhibitors Are Inhibitors of Rivaroxaban and Apixaban Efflux by P-Glycoprotein.

Because of their lower bleeding risk and simplicity of use, direct oral anticoagulants (DOACs) could represent an interesting alternative to conventional anticoagulant treatment with vitamin K antagonists for patients with pulmonary arterial hypertension (PAH). P-glycoprotein (P-gp) plays a key role in DOAC pharmacokinetics. Type 5-phosphodiesterase inhibitors (PDE5is), a drug class commonly used in the treatment of PAH, have been shown to strongly inhibit P-gp. This work aimed to assess potential P-gp-mediated drug-drug interactions between PDE5is and DOACs using in vitro methods. A cellular model of drug transport assay, using P-gp-overexpressing Madin-Darby canine kidney cells (transfected with the human P-gp gene), was used to determine the bidirectional permeabilities of two DOACs (rivaroxaban and apixaban) in the absence and presence of increasing concentrations (0.5-100 µM) of three PDE5is (sildenafil, tadalafil, and vardenafil). Permeabilities and efflux ratios were calculated from DOAC concentrations, were measured with liquid chromatography coupled with mass spectrometry, and were subsequently used to determine the PDE5i percentage of inhibition and half maximal inhibitory concentration (IC50 ). Rivaroxaban efflux was inhibited by 99%, 66%, and 100% with 100 µM sildenafil, tadalafil, and vardenafil, respectively. Similarly, apixaban efflux was inhibited by 97%, 74%, and 100%, respectively. The IC50 values of the three PDE5is were 8, 28, and 5 µM for rivaroxaban and 23, 15, and 3 µM for apixaban, respectively. This study showed strong in vitro inhibition of DOAC efflux by PDE5is. In vivo studies are required to determine the clinical relevance of these interactions.

In Vitro Comparison of the Role of P-Glycoprotein and Breast Cancer Resistance Protein on Direct Oral Anticoagulants Disposition.

BACKGROUND: Pharmacokinetics of direct oral anticoagulants (DOACs) are influenced by ATP-binding cassette (ABC) transporters such as P-glycoprotein (P-gp) and Breast Cancer Resistance Protein (BCRP). OBJECTIVES: To better understand the role of transporters in DOAC disposition, we evaluated and compared the permeabilities and transport properties of these drugs. METHODS: Bidirectional permeabilities of DOACs were investigated across Caco-2 cells monolayer. Transport assays were performed using different concentrations of DOAC and specific inhibitors of ABC transporters. Cell model functionality was evaluated by transport assay of two positive control substrates. RESULTS: The results of transport assays suggest a concentration-dependent efflux of apixaban, dabigatran etexilate and edoxaban, whereas the efflux transport of rivaroxaban did not seem to depend on concentration. Verapamil, a strong inhibitor of P-gp, decreased DOAC efflux in the Caco-2 cell model by 12-87%, depending on the drug tested. Ko143 reduced BCRP-mediated DOAC efflux in Caco-2 cells by 46-76%. CONCLUSION: This study allowed identification of three different profiles of ABC carrier-mediated transport: predominantly P-gp-dependent transport (dabigatran), preferential BCRP-dependent transport (apixaban) and approximately equivalent P-gp and BCRP-mediated transport (edoxaban and rivaroxaban).