Mapping transmembrane residues of proteinase activated receptor 2 (PAR2) that influence ligand-modulated calcium signaling.

Proteinase-activated receptor 2 (PAR2) is a G protein-coupled receptor involved in metabolism, inflammation, and cancers. It is activated by proteolysis, which exposes a nascent N-terminal sequence that becomes a tethered agonist. Short synthetic peptides corresponding to this sequence also activate PAR2, while small organic molecules show promising PAR2 antagonism. Developing PAR2 ligands into pharmaceuticals is hindered by a lack of knowledge of how synthetic ligands interact with and differentially modulate PAR2. Guided by PAR2 homology modeling and ligand docking based on bovine rhodopsin, followed by cross-checking with newer PAR2 models based on ORL-1 and PAR1, site-directed mutagenesis of PAR2 was used to investigate the pharmacology of three agonists (two synthetic agonists and trypsin-exposed tethered ligand) and one antagonist for modulation of PAR2 signaling. Effects of 28 PAR2 mutations were examined for PAR2-mediated calcium mobilization and key mutants were selected for measuring ligand binding. Nineteen of twenty-eight PAR2 mutations reduced the potency of at least one ligand by >10-fold. Key residues mapped predominantly to a cluster in the transmembrane (TM) domains of PAR2, differentially influence intracellular Ca2+ induced by synthetic agonists versus a native agonist, and highlight subtly different TM residues involved in receptor activation. This is the first evidence highlighting the importance of the PAR2 TM regions for receptor activation by synthetic PAR2 agonists and antagonists. The trypsin-cleaved N-terminus that activates PAR2 was unaffected by residues that affected synthetic peptides, challenging the widespread practice of substituting peptides for proteases to characterize PAR2 physiology.

Pathway-selective antagonism of proteinase activated receptor 2.

BACKGROUND AND PURPOSE: Proteinase activated receptor 2 (PAR2) is a GPCR associated with inflammation, metabolism and disease. Clues to understanding how to block PAR2 signalling associated with disease without inhibiting PAR2 activation in normal physiology could be provided by studies of biased signalling. EXPERIMENTAL APPROACH: PAR2 ligand GB88 was profiled for PAR2 agonist and antagonist properties by several functional assays associated with intracellular G-protein-coupled signalling in vitro in three cell types and with PAR2-induced rat paw oedema in vivo. KEY RESULTS: In HT29 cells, GB88 was a PAR2 antagonist in terms of Ca(2+) mobilization and PKC phosphorylation, but a PAR2 agonist in attenuating forskolin-induced cAMP accumulation, increasing ERK1/2 phosphorylation, RhoA activation, myosin phosphatase phosphorylation and actin filament rearrangement. In CHO-hPAR2 cells, GB88 inhibited Ca(2+) release, but activated G(i/o) and increased ERK1/2 phosphorylation. In human kidney tubule cells, GB88 inhibited cytokine secretion (IL6, IL8, GM-CSF, TNF-α) mediated by PAR2. A rat paw oedema induced by PAR2 agonists was also inhibited by orally administered GB88 and compared with effects of locally administered inhibitors of G-protein coupled pathways. CONCLUSIONS AND IMPLICATIONS: GB88 is a biased antagonist of PAR2 that selectively inhibits PAR2/G(q/11)/Ca(2+)/PKC signalling, leading to anti-inflammatory activity in vivo, while being an agonist in activating three other PAR2-activated pathways (cAMP, ERK, Rho) in human cells. These findings highlight opportunities to design drugs to block specific PAR2-linked signalling pathways in disease, without blocking beneficial PAR2 signalling in normal physiology, and to dissect PAR2-associated mechanisms of disease in vivo.

Bidirectional interaction of valproate and lamotrigine in healthy subjects.

OBJECTIVE: To evaluate the steady-state pharmacokinetics of lamotrigine and valproate at three dosing levels of lamotrigine in normal volunteers receiving steady-state therapeutic doses of valproate. METHODS: This was an open-label, randomized, three-way crossover study of 18 normal male volunteers. Subjects received oral valproate (500 mg Depakote twice a day) throughout the study. Each subject subsequently received three oral dosage regimens of lamotrigine (50, 100, or 150 mg/day) for 1 week each, with a 2-week washout period between lamotrigine treatment periods. Valproate and lamotrigine trough plasma samples were determined by a capillary gas chromatography method and immunofluorometric assay, respectively. Urine samples were assayed for 11 valproate metabolites by gas chromatography/mass spectrometry. RESULTS: When compared to other studies in which lamotrigine was administered with no concurrent antiepileptic drug, concomitant valproate markedly increased the half-life of lamotrigine and decreased lamotrigine clearance, without substantial alteration in the linear kinetics of the drug. The addition of lamotrigine was associated with a small but significant 25% decrease in steady-state valproate plasma concentration. Oral clearance of valproate was increased (from 7.2 +/- 1.1 ml/hr/kg before lamotrigine treatment to 9.0 +/- 2.0 ml/hr/kg on day 28; p < 0.05). The formation clearance of the hepatotoxic valproate metabolites, 2-n-propyl-4-pentenoic acid (4-ene-valproate) and 2-propyl-2,4-pentadienoic acid [2(E),4-diene-valproate], was unaffected by lamotrigine administration. CONCLUSIONS: As a consequence of the interaction between lamotrigine and sodium valproate, a dosage reduction of lamotrigine should be considered in patients taking a combination of valproate and lamotrigine.

Winter storms over Canada

Various type of winter storms occur over Canada and produce major impacts on society. Canada is subjected to extra-tropical cyclones with all their embedded structures, as well as blizzards, mountain-induced storms, lake effect storms and polar lows. Many of these storms are accompanied by heavy precipitation in the form of snow or freezing precipitation, bitterly cold conditions, strong winds, and blowing snow. The occurrence and nature of these storms are discussed in this review article.(AU)

Placental transfer of chloroquine in pregnant rabbits.

The pharmacokinetics of chloroquine was determined in pregnant and nonpregnant rabbits following intravenous administration. Blood concentrations of chloroquine and desethylchloroquine in the mother and fetuses were assayed using HPLC. Chloroquine rapidly crossed the placenta, resulting in equivalent fetal and maternal blood concentrations 15-30 min after dosing the mother. The subsequent 1 and 2 hr fetal blood concentrations were about two-fold higher than the corresponding maternal blood concentrations. The desethylchloroquine metabolite blood concentrations were low relative to the drug, although the metabolite was detected in all blood samples including the initial 5 min maternal blood samples and 15 min fetal blood samples. Comparing the pharmacokinetics of chloroquine in the pregnant and nonpregnant animals revealed no difference in total body clearance. However, there was an approximately two-fold decrease in the apparent volumes of distribution and terminal half life in the pregnant rabbits. Because of the relatively large inter-animal variability in these parameters, the differences were not statistically significant (p greater than 0.05), but do indicate a trend toward changes in chloroquine pharmacokinetics during pregnancy.

In vivo-in vitro correlations with a commercial dissolution simulator II: papaverine, phenytoin, and sulfisoxazole.

The dissolution profiles of 11 commercially available papaverine, phenytoin, and sulfisoxazole dosage forms were determined using a dissolution simulator. The products had been the subject of earlier in vivo bioavailability studies with human subjects. The use of an absorption simulator, which is designed to provide an estimate of the optimum sampling scheme for the dissolution simulator, did not provide useful data for this purpose. Good in vivo-in vitro correlations were found for the papaverine dosage forms, which included nine controlled-release products. Less satisfactory correlations were obtained for the phenytoin capsules and the sulfisoxazole tablet products.

Nonlinear ethotoin kinetics.

Five healthy subjects were given single 500-, 1500-, and 2500-mg doses of ethotoin as 250-mg tablets at 7-day intervals. Plasma samples were collected for 49 hr after dosing and were assayed by HPLC for ethotoin. The drug was more slowly absorbed after the two higher doses. There was a disproportionate increase in AUC in all subjects with the escalating doses. Individual subject data were fitted to a first-order model and one incorporating Michaelis-Menten elimination kinetics. Eleven of the 15 data sets were best described by the nonlinear model. All subjects reported visual disturbances with the two higher doses, and four of five experienced dyscoordination of gait with the 2500-mg dose. These effects did not appear to be related to plasma ethotoin concentration.

Hexoprenaline pharmacokinetics in pregnant and nonpregnant sheep.

Hexoprenaline, a beta 2-sympathomimetic agent, is used to suppress uterine contractions in the treatment of premature labor. However, little is known regarding the potential of this drug to undergo placental transfer or whether the pregnant state alters any of the pharmacokinetic parameters. Using sheep as a model, intravenous doses of 14C-hexoprenaline were administered to pregnant and non-pregnant animals. Measurable levels of radioactivity did not appear in fetal blood samples. After intravenous bolus administration, blood concentrations in the ewe could be fitted by a triexponential curve characteristic of a three-compartment pharmacokinetic model. Following intravenous infusion, a biexponential curve described the decline in blood concentrations. Mean terminal half lives for total radioactivity ranged from 2.5 to 4.2 hours. The pregnant animals tended to exhibit smaller apparent volumes of distribution and lower values for total body clearance, normalized to body weight, compared to non-pregnant sheep.

In vivo-in vitro correlations with a commercial dissolution simulator. I: Methenamine, nitrofurantoin, and chlorothiazide.

Dissolution profiles were determined for nine methenamine, 14 nitrofurantoin, and six chlorothiazide dosage forms using a dissolution simulator. Various in vivo-in vitro correlations were examined. The best correlation for methenamine was between the maximum urinary excretion rate and the time for 15% dissolution. A good correlation for the 50-mg nitrofurantoin tablets was also found between cumulative percent of drug excreted in 12 hr and the percent dissolved in 1 hr. There were no significant correlations for the 100-mg nitrofurantoin dosage forms. Good correlations were also observed for the 250- and 500-mg chlorothiazide tablets between the percent of drug dissolved in 1 min or the time for 15% dissolution and the maximum excretion rate.