Design and evaluation of self-nanoemulsifying drug delivery systems (SNEDDSs) for senicapoc.

Senicapoc (SEN), a potent antisickling agent, shows poor water solubility and poor oral bioavailability. To improve the solubility and cell permeation of SEN, self-nanoemulsifying drug delivery systems (SNEDDSs) were developed. Capryol PGMC®, which showed the highest solubilization capacity, was selected as the oil. The self-emulsification ability of two surfactants, viz., Cremophor-EL® and Tween® 80, was compared. Based on a solubility study and ternary phase diagrams, three optimized nanoemulsions with droplet sizes less than 200 nm were prepared. An in vitro dissolution study demonstrated the superior performance of the SNEDDS over the free drug. During in vitro lipolysis, 80% of SEN loaded in the SNEDDS remained solubilized. An in vitro cytotoxicity study using the Caco-2 cell line indicated the safety of the formulations at 1 mg/mL. The transport of SEN-SNEDDSs across Caco-2 monolayers was enhanced 115-fold (p < 0.01) compared to that of the free drug. According to these results, SNEDDS formulations could be promising tools for the oral delivery of SEN.

KCa3.1 channel inhibition sensitizes malignant gliomas to temozolomide treatment.

Malignant gliomas are among the most frequent and aggressive cerebral tumors, characterized by high proliferative and invasive indexes. Standard therapy for patients, after surgery and radiotherapy, consists of temozolomide (TMZ), a methylating agent that blocks tumor cell proliferation. Currently, there are no therapies aimed at reducing tumor cell invasion. Ion channels are candidate molecular targets involved in glioma cell migration and infiltration into the brain parenchyma. In this paper we demonstrate that: i) blockade of the calcium-activated potassium channel KCa3.1 with TRAM-34 has co-adjuvant effects with TMZ, reducing GL261 glioma cell migration, invasion and colony forming activity, increasing apoptosis, and forcing cells to pass the G2/M cell cycle phase, likely through cdc2 de-phosphorylation; ii) KCa3.1 silencing potentiates the inhibitory effect of TMZ on glioma cell viability; iii) the combination of TMZ/TRAM-34 attenuates the toxic effects of glioma conditioned medium on neuronal cultures, through a microglia dependent mechanism since the effect is abolished by clodronate-induced microglia killing; iv) TMZ/TRAM-34 co-treatment increases the number of apoptotic tumor cells, and the mean survival time in a syngeneic mouse glioma model (C57BL6 mice implanted with GL261 cells); v) TMZ/TRAM-34 co-treatment reduces cell viability of GBM cells and cancer stem cells (CSC) freshly isolated from patients.Taken together, these data suggest a new therapeutic approach for malignant glioma, targeting both glioma cell proliferating and migration, and demonstrate that TMZ/TRAM-34 co-treatment affects both glioma cells and infiltrating microglia, resulting in an overall reduction of tumor cell progression.

Complementary roles of KCa3.1 channels and ß1-integrin during alveolar epithelial repair.

BACKGROUND: Extensive alveolar epithelial injury and remodelling is a common feature of acute lung injury and acute respiratory distress syndrome (ARDS) and it has been established that epithelial regeneration, and secondary lung oedema resorption, is crucial for ARDS resolution. Much evidence indicates that K(+) channels are regulating epithelial repair processes; however, involvement of the KCa3.1 channels in alveolar repair has never been investigated before. RESULTS: Wound-healing assays demonstrated that the repair rates were increased in primary rat alveolar cell monolayers grown on a fibronectin matrix compared to non-coated supports, whereas an anti-ß1-integrin antibody reduced it. KCa3.1 inhibition/silencing impaired the fibronectin-stimulated wound-healing rates, as well as cell migration and proliferation, but had no effect in the absence of coating. We then evaluated a putative relationship between KCa3.1 channel and the migratory machinery protein ß1-integrin, which is activated by fibronectin. Co-immunoprecipitation and immunofluorescence experiments indicated a link between the two proteins and revealed their cellular co-distribution. In addition, we demonstrated that KCa3.1 channel and ß1-integrin membrane expressions were increased on a fibronectin matrix. We also showed increased intracellular calcium concentrations as well as enhanced expression of TRPC4, a voltage-independent calcium channel belonging to the large TRP channel family, on a fibronectin matrix. Finally, wound-healing assays showed additive effects of KCa3.1 and TRPC4 inhibitors on alveolar epithelial repair. CONCLUSION: Taken together, our data demonstrate for the first time complementary roles of KCa3.1 and TRPC4 channels with extracellular matrix and ß1-integrin in the regulation of alveolar repair processes.

Development of a QPatch automated electrophysiology assay for identifying KCa3.1 inhibitors and activators.

The intermediate-conductance Ca(2+)-activated K(+) channel KCa3.1 (also known as KCNN4, IK1, or the Gárdos channel) plays an important role in the activation of T and B cells, mast cells, macrophages, and microglia by regulating membrane potential, cellular volume, and calcium signaling. KCa3.1 is further involved in the proliferation of dedifferentiated vascular smooth muscle cells and fibroblast and endothelium-derived hyperpolarization responses in the vascular endothelium. Accordingly, KCa3.1 inhibitors are therapeutically interesting as immunosuppressants and for the treatment of a wide range of fibroproliferative disorders, whereas KCa3.1 activators constitute a potential new class of endothelial function preserving antihypertensives. Here, we report the development of QPatch assays for both KCa3.1 inhibitors and activators. During assay optimization, the Ca(2+) sensitivity of KCa3.1 was studied using varying intracellular Ca(2+) concentrations. A free Ca(2+) concentration of 1 µM was chosen to optimally test inhibitors. To identify activators, which generally act as positive gating modulators, a lower Ca(2+) concentration (∼200 nM) was used. The QPatch results were benchmarked against manual patch-clamp electrophysiology by determining the potency of several commonly used KCa3.1 inhibitors (TRAM-34, NS6180, ChTX) and activators (EBIO, riluzole, SKA-31). Collectively, our results demonstrate that the QPatch provides a comparable but much faster approach to study compound interactions with KCa3.1 channels in a robust and reliable assay.

Targeting potassium channels Kv1.3 and KC a 3.1: routes to selective immunomodulators in autoimmune disorder treatment?

The Kv1.3 and KC a 3.1 potassium channels are promising targets for the treatment of autoimmune disorders. Many Kv1.3 and KC a 3.1 blockers have a more favorable adverse event profiles than existing immunosuppressants, suggesting the selectivity of Kv1.3 and KC a 3.1 blockade. The Kv1.3 and KC a 3.1 blockers exert differential effects in different autoimmune diseases. The Kv1.3 inhibitors or gene deletion have been shown to have benefits in multiple sclerosis, type 1 diabetes, rheumatoid arthritis, psoriasis, and rapidly progressive glomerulonephritis. The KC a 3.1 blockers have demonstrated efficacy in human primary biliary cirrhosis and showed protective effects in animal models of severe colitis, allergic encephalomyelitis, inflammatory bowel disease, and multiple sclerosis. The KC a 3.1 blockers are not considered candidates for treatment of multiple sclerosis. The selective immunosuppressive effects of the Kv1.3 and KC a 3.1 blockers are due to the differences in their distribution on autoimmune-related immune cells and tissues and ß1 integrin (very late activating antigen)-Kv1.3 channel cross-talk.

Role of the K(Ca)3.1 K+ channel in auricular lymph node CD4+ T-lymphocyte function of the delayed-type hypersensitivity model.

BACKGROUND AND PURPOSE: The intermediate-conductance Ca(2+)-activated K(+) channel (K(Ca)3.1) modulates the Ca(2+) response through the control of the membrane potential in the immune system. We investigated the role of K(Ca)3.1 on the pathogenesis of delayed-type hypersensitivity (DTH) in auricular lymph node (ALN) CD4(+) T-lymphocytes of oxazolone (Ox)-induced DTH model mice. EXPERIMENTAL APPROACH: The expression patterns of K(Ca)3.1 and its possible transcriptional regulators were compared among ALN T-lymphocytes of three groups [non-sensitized (Ox-/-), Ox-sensitized, but non-challenged (Ox+/-) and Ox-sensitized and -challenged (Ox+/+)] using real-time polymerase chain reaction, Western blotting and flow cytometry. KCa 3.1 activity was measured by whole-cell patch clamp and the voltage-sensitive dye imaging. The effects of K(Ca)3.1 blockade were examined by the administration of selective K(Ca)3.1 blockers. KEY RESULTS: Significant up-regulation of K(Ca)3.1a was observed in CD4(+) T-lymphocytes of Ox+/- and Ox+/+, without any evident changes in the expression of the dominant-negative form, K(Ca)3.1b. Negatively correlated with this, the repressor element-1 silencing transcription factor (REST) was significantly down-regulated. Pharmacological blockade of K(Ca)3.1 resulted in an accumulation of the CD4(+) T-lymphocytes of Ox+/+ at the G0/G1 phase of the cell cycle, and also significantly recovered not only the pathogenesis of DTH, but also the changes in the K(Ca)3.1 expression and activity in the CD4(+) T-lymphocytes of Ox+/- and Ox+/+. CONCLUSIONS AND IMPLICATIONS: The up-regulation of K(Ca)3.1a in conjunction with the down-regulation of REST may be involved in CD4(+) T-lymphocyte proliferation in the ALNs of DTH model mice; and K(Ca)3.1 may be an important target for therapeutic intervention in allergy diseases such as DTH.

[Clinical trials of new therapeutic pharmacology for sickle cell disease]. / Essais cliniques de nouvelles thérapeutiques pharmacologiques de la drépanocytose.

Following the generation of transgenic mouse models of sickle cell disease, pre-clinical trials have shown the beneficial effects of various potential therapeutic molecules for the acute or chronic manifestations of the disease. Several molecules are upon evaluation in phase I to phase III clinical trials. These therapeutic approaches target: 1) membrane cation transport systems and channels involved in sickle cell dehydration; 2) adherence of erythrocytes to endothelium; 3) activation of circulating and endothelial cells participating in the vasoocclusive events and local ischemia. The Gardos channel (calcium activated potassium channel KCNN4) is inhibited by the clotrimazole metabolite ICA17043, in phase III trial. The K-Cl co-transport (KCC1/3/4) activated by the depletion of erythrocyte magnesium is inhibited by Magnesium pidolate; dipyridamole inhibits ion transports upon deoxygenation. Sulfasalazyne (inhibitor of the NF-jB pathway) inhibits the abnormal activation of endothelial cells. Nitric oxide (NO) is the most potent vasodilator. It prevents the activation of leucocytes, platelets and endothelial cells in patients with sickle cell disease and vascular remodelling. The L-arginine, the NO precursor, provides could be beneficial in sickle cell patients.