The KCa3.1 blocker TRAM-34 reduces infarction and neurological deficit in a rat model of ischemia/reperfusion stroke.

Microglia and brain infiltrating macrophages significantly contribute to the secondary inflammatory damage in the wake of ischemic stroke. Here, we investigated whether inhibition of KCa3.1 (IKCa1/KCNN4), a calcium-activated K(+) channel that is involved in microglia and macrophage activation and expression of which increases on microglia in the infarcted area, has beneficial effects in a rat model of ischemic stroke. Using an HPLC/MS assay, we first confirmed that our small molecule KCa3.1 blocker TRAM-34 effectively penetrates into the brain and achieves micromolar plasma and brain concentrations after intraperitoneal injection. Then, we subjected male Wistar rats to 90 minutes of middle cerebral artery occlusion (MCAO) and administered either vehicle or TRAM-34 (10 or 40 mg/kg intraperitoneally twice daily) for 7 days starting 12 hours after reperfusion. Both compound doses reduced infarct area by ≈ 50% as determined by hematoxylin & eosin staining on day 7 and the higher dose also significantly improved neurological deficit. We further observed a significant reduction in ED1(+)-activated microglia and TUNEL-positive neurons as well as increases in NeuN(+) neurons in the infarcted hemisphere. Our findings suggest that KCa3.1 blockade constitutes an attractive approach for the treatment of ischemic stroke because it is still effective when initiated 12 hours after the insult.

Environmentally persistent free radicals decrease cardiac function before and after ischemia/reperfusion injury in vivo.

Exposure to airborne particles is associated with increased cardiovascular morbidity and mortality. During the combustion of chlorine-containing hazardous materials and fuels, chlorinated hydrocarbons chemisorb to the surface of transition metal-oxide-containing particles, reduce the metal, and form an organic free radical. These radical-particle systems can survive in the environment for days and are called environmentally persistent free radicals (EPFRs). This study determined whether EPFRs could decrease left ventricular function before and after ischemia and reperfusion (I/R) in vivo. Male Brown-Norway rats were dosed (8 mg/kg, intratracheal) 24 h prior to testing with particles containing the EPFR of 1, 2-dichlorobenzene (DCB230). DCB230 treatment decreased systolic and diastolic function. DCB230 also produced pulmonary and cardiac inflammation. After ischemia, systolic, but not diastolic function was significantly decreased in DCB230-treated rats. Ventricular function was not affected by I/R in control rats. There was greater oxidative stress in the heart and increased 8-isoprostane (biomarker of oxidative stress) in the plasma of treated vs. control rats after I/R. These data demonstrate for the first time that DCB230 can produce inflammation and significantly decrease cardiac function at baseline and after I/R in vivo. Furthermore, these data suggest that EPFRs may be a risk factor for cardiac toxicity in healthy individuals and individuals with ischemic heart disease. Potential mechanisms involving cytokines/chemokines and/or oxidative stress are discussed.

Formulation-based approach to support early drug discovery and development efforts: a case study with enteric microencapsulation dosage form development for a triarylmethane derivative TRAM-34; a novel potential immunosuppressant.

BACKGROUND: Enteric microencapsulation of the potential immunosuppressant TRAM-34 was investigated as a means of enhancing oral drug delivery and minimizing or eliminating hydrolysis of pyrazole-substituted triarylmethane to the respective alcohol. METHOD: TRAM-34 was successfully enteric microencapsulated by a coacervation method using the pH-sensitive Eudragit L 100 polymer. In this study, we utilized water-miscible organic solvents such as acetone and ethanol, which are considered safe class 3 solvents according to the ICH guideline. We deemed such an approach suitable for safe scale up and for enteric coating application to other compounds of a similar lipophilicity. RESULTS: The resulting microparticles were spherical and uniform with an average particle size of 460 microm at 15% theoretical loading. The encapsulation efficiency was 90 +/- 1.9% and the percentage yield was found to be 91.5 +/- 0.3%. The oral administration in rhesus macaques of TRAM-34-loaded enteric-coated microparticles illustrated six times enhancement in its oral bioavailability. However, the TRAM-34 plasma concentration was less than the therapeutic effective level. CONCLUSION: The low oral bioavailability, even after enteric coating, could be attributed to the compound's inherent absorption characteristics and high lipophilicity.

Renal fibrosis is attenuated by targeted disruption of KCa3.1 potassium channels.

Proliferation of interstitial fibroblasts is a hallmark of progressive renal fibrosis commonly resulting in chronic kidney failure. The intermediate-conductance Ca(2+)-activated K(+) channel (K(Ca)3.1) has been proposed to promote mitogenesis in several cell types and contribute to disease states characterized by excessive proliferation. Here, we hypothesized that K(Ca)3.1 activity is pivotal for renal fibroblast proliferation and that deficiency or pharmacological blockade of K(Ca)3.1 suppresses development of renal fibrosis. We found that mitogenic stimulation up-regulated K(Ca)3.1 in murine renal fibroblasts via a MEK-dependent mechanism and that selective blockade of K(Ca)3.1 functions potently inhibited fibroblast proliferation by G(0)/G(1) arrest. Renal fibrosis induced by unilateral ureteral obstruction (UUO) in mice was paralleled by a robust up-regulation of K(Ca)3.1 in affected kidneys. Mice lacking K(Ca)3.1 (K(Ca)3.1(-/-)) showed a significant reduction in fibrotic marker expression, chronic tubulointerstitial damage, collagen deposition and alphaSMA(+) cells in kidneys after UUO, whereas functional renal parenchyma was better preserved. Pharmacological treatment with the selective K(Ca)3.1 blocker TRAM-34 similarly attenuated progression of UUO-induced renal fibrosis in wild-type mice and rats. In conclusion, our data demonstrate that K(Ca)3.1 is involved in renal fibroblast proliferation and fibrogenesis and suggest that K(Ca)3.1 may represent a therapeutic target for the treatment of fibrotic kidney disease.

Naphtho[1,2-d]thiazol-2-ylamine (SKA-31), a new activator of KCa2 and KCa3.1 potassium channels, potentiates the endothelium-derived hyperpolarizing factor response and lowers blood pressure.

Small-conductance (KCa2.1-2.3) and intermediate-conductance (KCa3.1) calcium-activated K(+) channels are critically involved in modulating calcium-signaling cascades and membrane potential in both excitable and nonexcitable cells. Activators of these channels constitute useful pharmacological tools and potential new drugs for the treatment of ataxia, epilepsy, and hypertension. Here, we used the neuroprotectant riluzole as a template for the design of KCa2/3 channel activators that are potent enough for in vivo studies. Of a library of 41 benzothiazoles, we identified 2 compounds, anthra[2,1-d]thiazol-2-ylamine (SKA-20) and naphtho[1,2-d]thiazol-2-ylamine (SKA-31), which are 10 to 20 times more potent than riluzole and activate KCa2.1 with EC(50) values of 430 nM and 2.9 microM, KCa2.2 with an EC(50) value of 1.9 microM, KCa2.3 with EC(50) values of 1.2 and 2.9 microM, and KCa3.1 with EC(50) values of 115 and 260 nM. Likewise, SKA-20 and SKA-31 activated native KCa2.3 and KCa3.1 channels in murine endothelial cells, and the more "drug-like" SKA-31 (half-life of 12 h) potentiated endothelium-derived hyperpolarizing factor-mediated dilations of carotid arteries from KCa3.1(+/+) mice but not from KCa3.1(-/-) mice. Administration of 10 and 30 mg/kg SKA-31 lowered mean arterial blood pressure by 4 and 6 mm Hg in normotensive mice and by 12 mm Hg in angiotensin-II-induced hypertension. These effects were absent in KCa3.1-deficient mice. In conclusion, with SKA-31, we have designed a new pharmacological tool to define the functional role of the KCa2/3 channel activation in vivo. The blood pressure-lowering effect of SKA-31 suggests KCa3.1 channel activation as a new therapeutic principle for the treatment of hypertension.

The intermediate-conductance calcium-activated potassium channel KCa3.1 contributes to atherogenesis in mice and humans.

Atherosclerosis remains a major cause of death in the developed world despite the success of therapies that lower cholesterol and BP. The intermediate-conductance calcium-activated potassium channel KCa3.1 is expressed in multiple cell types implicated in atherogenesis, and pharmacological blockade of this channel inhibits VSMC and lymphocyte activation in rats and mice. We found that coronary vessels from patients with coronary artery disease expressed elevated levels of KCa3.1. In Apoe(-/-) mice, a genetic model of atherosclerosis, KCa3.1 expression was elevated in the VSMCs, macrophages, and T lymphocytes that infiltrated atherosclerotic lesions. Selective pharmacological blockade and gene silencing of KCa3.1 suppressed proliferation, migration, and oxidative stress of human VSMCs. Furthermore, VSMC proliferation and macrophage activation were reduced in KCa3.1(-/-) mice. In vivo therapy with 2 KCa3.1 blockers, TRAM-34 and clotrimazole, significantly reduced the development of atherosclerosis in aortas of Apoe(-/-) mice by suppressing VSMC proliferation and migration into plaques, decreasing infiltration of plaques by macrophages and T lymphocytes, and reducing oxidative stress. Therapeutic concentrations of TRAM-34 in mice caused no discernible toxicity after repeated dosing and did not compromise the immune response to influenza virus. These data suggest that KCa3.1 blockers represent a promising therapeutic strategy for atherosclerosis.

Isolation and purification of closely related citrus limonoid glucosides by flash chromatography.

Several citrus limonoid glycosides have proved to be particularly difficult to purify using conventional techniques. A reversed-phase flash chromatographic technique has been developed for the separation and isolation of the closely related limonoid glucosides, nomilin 17-beta-D-glucopyranoside and nomilinic acid 17-beta-D-glucopyranoside, with confirmation of their identities by electrospray ionisation mass spectrometry. Furthermore, the semi-purification of the mixture of glucosides enriched with flavanone glucosides such as naringin, narirutin and other limonoid glucosides was obtained. The closely eluting glucosides were successfully separated to achieve a good yield and purity of 93%.