QT PRODACT: a multi-site study of in vitro action potential assays on 21 compounds in isolated guinea-pig papillary muscles.

To construct a non-clinical database for drug-induced QT interval prolongation, the electrophysiological effects of 11 positive and 10 negative compounds on action potentials (AP) in guinea-pig papillary muscles were investigated in a multi-site study according to a standard protocol. Compounds with a selective inhibitory effect on the rapidly activated delayed rectifier potassium current (IKr) prolonged action potential duration at 90% repolarization (APD90) in a concentration-dependent manner, those showing Ca2+ current (ICa) inhibition shortened APD30, and those showing Na+ current (INa) inhibition decreased action potential amplitude (APA) and Vmax. Some of the mixed ion-channel blockers showed a bell-shaped concentration-response curve for APD90, probably due to their blockade of INa and/or ICa, sometimes leading to a false-negative result in the assay. In contrast, all positive compounds except for terfenadine and all negative compounds with IKr-blocking activity prolonged APD30-90 regardless of their INa- and/or ICa-blocking activities, suggesting that APD30-90 is a useful parameter for evaluating the IKr-blocking activity of test compounds. Furthermore, the assay is highly informative regarding the modulation of cardiac ion channels by test compounds. Therefore, when APD90 and APD30-90 are both measured, the action potential assay can be considered a useful method for assessing the risk of QT interval prolongation in humans in non-clinical safety pharmacology studies.

QT PRODACT: evaluation of the potential of compounds to cause QT interval prolongation by action potential assays using guinea-pig papillary muscles.

Certain compounds that prolong QT interval in humans have little or no effect on action-potential (AP) duration used traditionally, but they inhibit rapidly-activated-delayed-rectifier potassium currents (IKr) and/or human ether-a-go-go-related gene (hERG) currents. In this study using isolated guinea-pig papillary muscles, we investigated whether new parameters in AP assays can detect the inhibitory effects of various compounds on IKr and/or hERG currents with high sensitivity. The difference in AP duration between 60% and 30% repolarization, 90% and 60% repolarization, and 90% and 30% repolarization (APD30-60, APD60-90, and APD30-90, respectively) were calculated as the new parameters. All the 15 IKr and/or hERG current inhibitors that have been reported (9 compounds) or not reported (6 compounds) to inhibit calcium currents prolonged APD30-60, APD60-90, and/or APD30-90; and 8 of the 15 inhibitors prolonged APD30-60, APD60-90, and/or APD30-90 more potently than APD90. The APD30-60, APD60-90, and APD30-90 measurements revealed no difference in sensitivity when evaluating the effects of the IKr and/or hERG current inhibitors on the three parameters. On the other hand, compounds with little or no effect on hERG currents had no effect on APD30-60, APD60-90, or APD30-90. Therefore, it is concluded that in AP assays using isolated guinea-pig papillary muscles, APD30-60, APD60-90, and APD30-90 are useful indexes for evaluating the inhibitory effects of compounds including mixed ion-channel blockers on IKr and/or hERG currents.

Disassembly of amyloid beta-protein fibril by basement membrane components.

Amyloid beta-protein (A3) fibril in senile plaque may be related to the pathogenesis of Alzheimer's disease (AD). Basement membrane (BM) components are associated with the plaques in AD brain. It suggests that the BM components may play an important role in the deposition of the plaque. We investigated the potential of BM components, such as type IV collagen (collagen IV) and entactin, to induce disassembly of preformed Abeta1-42 (Abeta42) fibrils in direct comparison to laminin. Thioflavin T assays revealed that these BM components disrupted preformed Abeta42 fibrils in a dose-dependent manner. The high concentration of BM components, 100 microg/mL laminin, 50 microg/mL collagen IV and 50 microg/mL entactin, had most effect on disassembly of preformed Abeta42 fibrils (Molar ratio; Abeta42:laminin = 90:1, Abeta42:collagen IV = 34:1, Abeta42:entactin = 20:1). Circular dichroism spectroscopy data indicated that the high concentration of BM components induced structural transition in Abeta42 from beta-sheet to random structures. These results suggest that collagen IV and entactin, as well as laminin, are effective inducers of disassembly of Abeta42 fibrils. The ability of these BM components to induce random structures may be linked to the disassembly of preformed Abeta42 fibrils.

Type IV collagen prevents amyloid beta-protein fibril formation.

The potential of targeting through molecular therapeutics the underlying amyloid beta-protein (A beta) fibrillogenesis causing the initiation and progression of Alzheimer's disease (AD) offers an opportunity to improve the disease. Type IV collagen (collagen IV) is localized in senile plaques in patients with AD. By using thioflavin T fluorescence spectroscopy and electron microscopy, we found that collagen IV inhibited A beta1-40 (A beta40) fibril formation. The critical concentration of collagen IV for this inhibition was 5 microg/mL. Circular dichroism data indicate that collagen IV prevents formation of a beta-structured aggregate of A beta40. These studies demonstrated that collagen IV is apparently a potent inhibitor of A beta fibril formation.