6-Chloro-3-alkylamino-4H-thieno[3,2-e]-1,2,4-thiadiazine 1,1-dioxide derivatives were synthesized and characterized as activators of adenosine 5'-triphosphate (ATP) sensitive potassium (K(ATP)) channels in the beta-cells by measuring effects on membrane potential and insulin release in vitro. The effects on vascular tissue in vitro were measured on rat aorta and small mesenteric vessels. Selected compounds were characterized as competitive inhibitors of [(3)H]glibenclamide binding to membranes of HEK293 cells expressing human SUR1/Kir6.2 and as potent inhibitors of insulin release in isolated rat islets. 6-Chloro-3-(1-methylcyclobutyl)amino-4H-thieno[3,2-e]-1,2,4-thiadiazine 1,1-dioxide (54) was found to bind and activate the SUR1/Kir6.2 K(ATP) channels in the low nanomolar range and to be at least 1000 times more potent than the reference compound diazoxide with respect to inhibition of insulin release from rat islets. Several compounds, e.g., 3-propylamino- (30), 3-isopropylamino- (34), 3-(S)-sec-butylamino- (37), and 3-(1-methylcyclopropyl)amino-4H-thieno[3,2-e]-1,2,4-thiadiazine 1,1-dioxide (53), which were found to be potent and beta-cell selective activators of K(ATP) channels in vitro, were found to inhibit insulin secretion in rats with minimal effects on blood pressure and to exhibit good oral pharmacokinetic properties.

In activated rat mesenteric small arteries, the effect of pH1 on force, membrane potential, and free cytosolic calcium ([Ca2"]

Compound 1a (NN414) is a potent opener of Kir6.2/SUR1 K(ATP) channels. Compound 1a inhibits insulin release in vitro and in vivo and preserves beta cell function in preclinical animal models suggesting that such a compound could find use in treatment or prevention of type 1 and type 2 diabetes. The crystal structure and a convergent synthesis of 1a are presented together with a range of new analogues of 1a. Several compounds, e.g., 6-chloro-3-(1-methyl-1-phenylethyl)amino-4H-thieno[3,2-e]-1,2,4-thiadiazine 1,1-dioxide (1h), were found to be potent openers of Kir6.2/SUR1 K(ATP) channels and were able to suppress glucose-stimulated insulin release from rat islets in vitro (EC(50) = 0.04 +/- 0.01 muM) and in vivo after intravenous or peroral administration to hyperinsulinemic obese Zucker rats (ED(50) = 4.0 mg/kg). Structural modifications of this series of K(ATP) channel openers have provided compounds with promising pharmacokinetic properties indicating that brief periods of beta cell rest can be achieved.

To evaluate long-term effects of contractile and mitogenic stimuli on the contractile reactivity of arterial smooth muscle, we measured the incorporation of the thymidine analogue 5-bromo-2'-deoxyuridine (BrdUrd) and mechanical responses in arterial segments that had been maintained in tissue culture. The experiments were performed on renal arteries that had been isolated from adult rats, chemically sympathectomized, mechanically denuded from endothelium and mounted under distension. Exposure of arterial segments for up to 3 weeks to culture medium supplemented with fetal calf serum resulted in the following consecutive changes: a strong acute contraction, selective pharmacological changes that included decreased contractile responses to phenylephrine and vasopressin and increased relaxing responses to isoproterenol,

For decades, preclinical toxicology was essentially a descriptive discipline in which treatment-related effects were carefully reported and used as a basis to calculate safety margins for drug candidates. In recent years, however, technological advances have increasingly enabled researchers to gain insights into toxicity mechanisms, supporting greater understanding of species relevance and translatability to humans, prediction of safety events, mitigation of side effects and development of safety biomarkers. Consequently, investigative (or mechanistic) toxicology has been gaining momentum and is now a key capability in the pharmaceutical industry. Here, we provide an overview of the current status of the field using case studies and discuss the potential impact of ongoing technological developments, based on a survey of investigative toxicologists from 14 European-based medium-sized to large pharmaceutical companies.

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We evaluated whether GTP-binding regulatory proteins (G-proteins) are involved in responses of resistance arterial smooth muscle to contractile agonists. We therefore pretreated isolated sympathectomized mesenteric resistance arteries of the rat with pertussis toxin (PTX) and recorded their contractile responses to aluminium fluoride, endothelin, high potassium, phenylephrine, phorbol myristate acetate, serotonin and vasopressin. PTX reduced contractile responses to agonists with the following order of potency: phenylephrine = serotonin greater than vasopressin = endothelin. The toxin reduced responses to phenylephrine in both the presence and absence of extracellular Ca2+. In Ca2(+)-depleted vessels that were exposed to phenylephrine, PTX virtually abolished responses to Ca2+ while hardly affecting responses to Ca2+ in the presence of endothelin. Also aluminium fluoride and phorbol myristate acetate induced contractions. These were dependent on extracellular Ca2+ and inhibited by felodipine. PTX reduced responses to aluminium fluoride but not those to phorbol myristate acetate. These data indicate that PTX sensitive G-proteins are involved in both influx of Ca2+ and release of intracellular Ca2+ following alpha 1-adrenergic and serotonergic stimulation of resistance arteries. The role of G-proteins in stimulated Ca2+ influx could involve a direct effect on calcium channels although an indirect effect through protein kinase-C can not be entirely excluded. The persistance of contractile responses to vasopressin and endothelin following PTX suggests that these agonists engage different pathways to induce contraction or have a higher efficacy in activating similar G-proteins.

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