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    • 3X FLAG In the nearly years since the clevidipine

    2018-10-30

    In the nearly 18years since the clevidipine investigational new drug application was filed, our understanding of the structure, tissue distribution and molecular biology of the l-type calcium channels (LTCC) has evolved dramatically (Zuccotti et al., 2011; Abernethy and Soldatov, 2002). The CaV-α-1 family comprises 10 genes, of which 4 genes (CACNA1S, CACNA1C, CACNA1D CACNA1F) encode the LTCC referred to as CaV1.1, CaV1.2, CaV1.3 and CaV1.4 channels. LTCC are distinguished from the other 6 CaV channels by their selective sensitivity to 1,4-dihydropyridines (DHPs), phenylalkylamines (PAAs) and benzothiazepines and by their characteristic slowly inactivating currents. LTCCs are composed of a central pore-forming α-1 subunit and additional α-2/δ, β, and γ subunits. The α-1 subunit confers most of the functional properties to the channel, including voltage sensing, permeability, calcium-dependent inactivation, and sensitivity to organic channel blockers. Although the α-1 subunit defines the basic channel properties, four different β genes (β1–β4; genes CACNB1–4) and extensive splice variants of each gene exist that distinctly modify activation and inactivation kinetics, voltage gating, and drug sensitivity (Hullin et al., 2003). At least 20 of the 56 exons in the human CACNA1C transcript are alternatively spliced (Liao et al., 2007) (Fig. 1B). Splice variants are known to confer different electrophysiological and pharmacological properties on the CaV1.2 channel and to exhibit tissue-specific differences (e.g. 3X FLAG vs. vascular smooth muscle (VSM)) (Liao et al., 2007; Cheng et al., 2009). Smooth muscle is known to be more sensitive to DHPs than cardiac muscle (Moosmang et al., 2003). These tissues express slightly different CaV1.2 splice variants (Cheng et al., 2009; Saada et al., 2003; Liao et al., 2005): exon 8 is expressed in smooth muscle, while exon 8a is expressed in cardiac muscle. Exon 8a in cardiac tissue reduces the affinity of CaV1.2 for DHPs (Welling et al., 1997). Lastly, in addition to the molecular heterogeneity conferred by differing subunit combinations and alternative splice variants, disease-based differences in tissue distribution and expression levels of any given channel complex are common (Hullin et al., 2003; Firth et al., 2011). In this report, we tested the hypothesis that, in lung tissue, there are specific CACNA1C splice variants encoding for CaV1.2 with different molecular pharmacologic profiles for clevidipine compared to CaV1.2 in other peripheral smooth muscles (Fig. 1A). In parallel to our increased understanding of CaV1.2 channels during the last decade, a more detailed understanding of the molecular basis of BP regulation has emerged. For example, pannexin 1 (gene PANX1), which serves as the major ATP-release channel in many cell types (including erythrocytes, endothelial cells, airway epithelial cells and astrocytes (Locovei et al., 2006a; Ransford et al., 2009; Dahl and Keane, 2012)), is involved in two antagonistic ways for blood flow and blood pressure regulation. 1) Erythrocytes sensing low oxygen content and/or subjected to shear stress release ATP through Panx1 channels (Locovei et al., 2006a; Sridharan et al., 2010). The ATP binds to purinergic receptors on endothelial cells, triggering a propagated calcium wave that eventually results in the release of nitric oxide (NO). NO then relaxes vascular smooth muscle cells which increases local perfusion and oxygen supply (Fig. 1A). 2) Activation of α-adrenergic receptors leads to opening of Panx1 channels in VSM cells and blocking of Panx1 channels in these cells attenuates the vasopressor activity of α-agonists (Billaud et al., 2012). Thus, it appears that Panx1 is tied into the α-adrenergic control of blood pressure (Fig. 1A). In this report, we therefore also considered the hypotheses that the clevidipine-induced dyspnea relief is due to clevidipine acting on Panx1 in lung tissue. It is also known that Panx1 co-localizes with voltage-gated calcium channels (CaV1.1) in skeletal muscle (Jorquera et al., 2013). We therefore also considered the possibility that Panx1 associates with CaV1.2 in lung tissue and increases the affinity of CaV1.2 to clevidipine.