Inhibition of rat platelet aggregation by the diazeniumdiolate nitric oxide donor MAHMA NONOate

1 Inhibition of rat platelet aggregation by the nitric oxide (NO) donor MAHMA NONOate (Z-1-{N-methyl-N-[6-(N-methylammoniohexyl)amino]}diazen-l-ium-1,2-diolate) was investigated. The aims were to compare its anti-aggregatory effect with vasorelaxation, to determine the effects of the soluble guanylate cyclase inhibitor, ODQ (1H-[1,2,4]oxadiazolo[4,3-ajquinoxalin-1-one), and to investigate the possible role of activation of sarco-encloplasmic reticulum calcium-ATPase (SERCA), independent of soluble guanylate cyclase, using thapsigargin. 2 MAHMA NONOate concentration-dependently inhibited sub-maximal aggregation responses to collagen (2 - 10 mug ml(-1)) and adenosine diphosphate (ADP; 2 mum) in platelet rich plasma. It was (i) more effective at inhibiting aggregation induced by collagen than by ADP, and (ii) less potent at inhibiting platelet aggregation than relaxing rat pulmonary artery. 3 ODQ (10 mum) caused only a small shift (approximately half a log unit) in the concentration-response curve to MAHMA NONOate irrespective of the aggregating agent. 4 The NO-independent activator of soluble guanylate cyclase, YC-1 (3-(5'-hydroxymethyl-2'-furyl)-1-benzy] indazole; 1 - 100 mum), did not inhibit aggregation. The cGMP analogue, 8-pCPT-cGMP (8-(4-chlorophenylthio)guanosine 3'5' cyclic monophosphate; 0.1 - 1 mm), caused minimal inhibition. 5 On collagen-aggregated platelets responses to MAHMA NONOate (ODQ 10 PM present) were abolished by thapsigargin (200 nm). On ADP-aggregated platelets thapsigargin caused partial inhibition. 6 Results with S-nitrosoglutathione (GSNO) resembled those with MAHMA NONOate. Glyceryl trinitrate and sodium nitroprusside were poor inhibitors of aggregation. 7 Thus inhibition of rat platelet aggregation by MAHMA NONOate (like GSNO) is largely ODQ-resistant and, by implication, independent of soluble guanylate cyclase. A likely mechanism of inhibition is activation of SERCA.

Autosomal dominant hypocalcemia: A novel activating mutation (E604K) in the cysteine-rich domain of the calcium-sensing receptor

We report a novel activating mutation (E604K) of the calcium-sensing receptor in a family with autosomal dominant hypocalcemia. Whereas all affected individuals exhibited marked hypocalcemia, some cases with untreated hypocalcemia exhibited seizures in infancy, whereas others were largely asymptomatic from birth into adulthood. The missense mutation E604K (G2182A, GenBank accession no. U20759), which affects an amino acid residue in the C terminus of the cysteine-rich domain of the extracellular head, co-segregated with hypocalcemia in all seven individuals for whom DNA was available. Two unaffected, normocalcemic members of the family did not exhibit the mutation. The molecular impact of the mutation on two key components of the signaling response was assessed in HEK-293 cells transiently transfected with cDNA corresponding to either the wild-type calcium-sensing receptor or the E604K mutation derived by site-directed mutagenesis. There was a significant leftward shift in the concentration response curves for the effects of extracellular Ca2+ on both intracellular Ca2+ mobilization (determined by aequorin luminescence) and MAPK activity (determined by luciferase expression). The C terminus of the cysteine-rich domain of the extracellular head may normally act to suppress receptor activity in the presence of low extracellular Ca2+ concentrations.

Acute lead-induced vasoconstriction in the vascular beds of isolated perfused rat tails is endothelium-dependent

Chronic lead exposure induces hypertension in humans and animals, affecting endothelial function. However, studies concerning acute cardiovascular effects are lacking. We investigated the effects of acute administration of a high concentration of lead acetate (100 µΜ) on the pressor response to phenylephrine (PHE) in the tail vascular bed of male Wistar rats. Animals were anesthetized with sodium pentobarbital and heparinized. The tail artery was dissected and cannulated for drug infusion and mean perfusion pressure measurements. Endothelium and vascular smooth muscle relaxation were tested with acetylcholine (5 µg/100 µL) and sodium nitroprusside (0.1 µg/100 µL), respectively, in arteries precontracted with 0.1 µM PHE. Concentration-response curves to PHE (0.001-300 µg/100 µL) were constructed before and after perfusion for 1 h with 100 µΜ lead acetate. In the presence of endothelium (E+), lead acetate increased maximal response (Emax) (control: 364.4 ± 36, Pb2+: 480.0 ± 27 mmHg; P < 0.05) and the sensitivity (pD2; control: 1.98 ± 0.07, 2.38 ± 0.14 log mM) to PHE. In the absence of endothelium (E-) lead had no effect but increased baseline perfusion pressure (E+: 79.5 ± 2.4, E-: 118 ± 2.2 mmHg; P < 0.05). To investigate the underlying mechanisms, this protocol was repeated after treatment with 100 µM L-NAME, 10 µM indomethacin and 1 µM tempol in the presence of lead. Lead actions on Emax and pD2 were abolished in the presence of indomethacin, and partially abolished with L-NAME and tempol. Results suggest that acute lead administration affects the endothelium, releasing cyclooxygenase-derived vasoconstrictors and involving reactive oxygen species.

Médicaments et grossesse : modifications pharmacocinétiques et place du suivi thérapeutique pharmacologique [Pharmacokinetic Alterations in Pregnancy and Use of Therapeutic Drug Monitoring].

Following the thalidomide tragedy, pharmacological research in pregnant women focused primarily on drug safety for the unborn child and remains only limited regarding the efficacy and safety of treatment for the mother. Significant physiological changes during pregnancy may yet affect the pharmacokinetics of drugs and thus compromise its efficacy and/or safety. Therapeutic drug monitoring (TDM) would maximize the potential effectiveness of treatments, while minimizing the potential risk of toxicity for the mother and the fetus. At present, because of the lack of concentration-response relationship studies in pregnant women, TDM can rely only on individual assessment (based on an effective concentration before pregnancy) and remains reserved only to unexpected situations such as signs of toxicity or unexplained inefficiency.

Fipronil Insecticide: Novel Application against Triatomine Insect Vectors of Chagas Disease

We investigated the efficacy and the residual effect of fipronil® against two species of triatomine bugs, Triatoma infestans and Rhodnius neglectus, in laboratory conditions measuring concentration-response and residual activity on different surfaces (dried mud and lime coated mud). Lethal concentrations (LC50,90) were determined on filter paper. The higher insecticide efficacy against R. neglectus when compared to T. infestans may be partially attributed to the differences in their biological cycles and genetic structures. Comparison with lambdacyhalothrin wettable powder showed that fipronil mortality rates (above 50%) were observed on mud blocks and lime-coated mud blocks up to 3 months when fipronil was sprayed at 100 and 200 mg a.i./m². Residual effect deeply decayed after 3 months; and at 6 months post treatment mortality was not observed. In contrast, lambdacyhalothrin showed a long lasting residual effect on both surfaces up to 6 months. Also, it should be mentioned that fipronil had a slow, but lethal activity on the triatomine bugs when wettable formulations were used on porous surfaces.

Evaluation of aggregating brain cell cultures for the detection of acute organ-specific toxicity.

As part of the ACuteTox project aimed at the development of non-animal testing strategies for predicting human acute oral toxicity, aggregating brain cell cultures (AGGR) were examined for their capability to detect organ-specific toxicity. Previous multicenter evaluations of in vitro cytotoxicity showed that some 20% of the tested chemicals exhibited significantly lower in vitro toxicity as expected from in vivo toxicity data. This was supposed to be due to toxicity at supracellular (organ or system) levels. To examine the capability of AGGR to alert for potential organ-specific toxicants, concentration-response studies were carried out in AGGR for 86 chemicals, taking as endpoints the mRNA expression levels of four selected genes. The lowest observed effect concentration (LOEC) determined for each chemical was compared with the IC20 reported for the 3T3/NRU cytotoxicity assay. A LOEC lower than IC20 by at least a factor of 5 was taken to alert for organ-specific toxicity. The results showed that the frequency of alerts increased with the level of toxicity observed in AGGR. Among the chemicals identified as alert were many compounds known for their organ-specific toxicity. These findings suggest that AGGR are suitable for the detection of organ-specific toxicity and that they could, in conjunction with the 3T3/NRU cytotoxicity assay, improve the predictive capacity of in vitro toxicity testing.

Relationship of imatinib-free plasma levels and target genotype with efficacy and tolerability

Imatinib has revolutionised the treatment of chronic myeloid leukaemia (CML) and gastrointestinal stromal tumours (GIST). Using a nonlinear mixed effects population model, individual estimates of pharmacokinetic parameters were derived and used to estimate imatinib exposure (area under the curve, AUC) in 58 patients. Plasma-free concentration was deduced from a model incorporating plasma levels of alpha(1)-acid glycoprotein. Associations between AUC (or clearance) and response or incidence of side effects were explored by logistic regression analysis. Influence of KIT genotype was also assessed in GIST patients. Both total (in GIST) and free drug exposure (in CML and GIST) correlated with the occurrence and number of side effects (e.g. odds ratio 2.7+/-0.6 for a two-fold free AUC increase in GIST; P<0.001). Higher free AUC also predicted a higher probability of therapeutic response in GIST (odds ratio 2.6+/-1.1; P=0.026) when taking into account tumour KIT genotype (strongest association in patients harbouring exon 9 mutation or wild-type KIT, known to decrease tumour sensitivity towards imatinib). In CML, no straightforward concentration-response relationships were obtained. Our findings represent additional arguments to further evaluate the usefulness of individualizing imatinib prescription based on a therapeutic drug monitoring programme, possibly associated with target genotype profiling of patients.

Correlation between vasoconstrictor roles and mRNA expression of alpha1-adrenoceptor subtypes in blood vessels of genetically engineered mice.

We examined the contribution of each alpha(1)-adrenoceptor (AR) subtype in noradrenaline (NAd)-evoked contraction in the thoracic aortas and mesenteric arteries of mice. Compared with the concentration-response curves (CRCs) for NAd in the thoracic aortas of wild-type (WT) mice, the CRCs of mutant mice showed a significantly lower sensitivity. The pD(2) value in rank order is as follows: WT mice (8.21) > alpha(1B)-adrenoceptor knockout (alpha(1B)-KO) (7.77) > alpha(1D)-AR knockout (alpha(1D)-KO) (6.44) > alpha(1B)- and alpha(1D)-AR double knockout (alpha(1BD)-KO) (5.15). In the mesenteric artery, CRCs for NAd did not differ significantly between either WT (6.52) and alpha(1B)-KO mice (7.12) or alpha(1D)-KO (6.19) and alpha(1BD)-KO (6.29) mice. However, the CRC maximum responses to NAd in alpha(1D)- and alpha(1BD)-KO mice were significantly lower than those in WT and alpha(1B)-KO mice. Except in the thoracic aortas of alpha(1BD)-KO mice, the competitive antagonist prazosin inhibited the contraction response to NAd with high affinity. However, prazosin produced shallow Schild slopes in the vessels of mice lacking the alpha(1D)-AR gene. In the thoracic aorta, pA(2) values in WT mice for KMD-3213 and BMY7378 were 8.25 and 8.46, respectively, and in alpha(1B)-KO mice they were 8.49 and 9.13, respectively. In the mesenteric artery, pA(2) values in WT mice for KMD-3213 and BMY7378 were 8.34 and 7.47, respectively, and in alpha(1B)-KO mice they were 8.11 and 7.82, respectively. These pharmacological findings were in fairly good agreement with findings from comparison of CRCs, with the exception of the mesenteric arteries of WT and alpha(1B)-KO mice, which showed low affinities to BMY7378. We performed a quantitative analysis of the mRNA expression of each alpha(1)-AR subtype in these vessels in order to examine the correlation between mRNA expression level and the predominance of each alpha(1)-AR subtype in mediating vascular contraction. The rank order of each alpha(1)-AR subtype in terms of its vasoconstrictor role was in fairly good agreement with the level of expression of mRNA of each subtype, that is, alpha(1D)-AR > alpha(1B)-AR > alpha(1A)-AR in the thoracic aorta and alpha(1D)-AR > alpha(1A)-AR > alpha(1B)-AR in the mesenteric artery. No dramatic compensatory change of alpha(1)-AR subtype in mutant mice was observed in pharmacological or quantitative mRNA expression analysis.

Characterization of a selective antagonist of neuropeptide Y at the Y2 receptor. Synthesis and pharmacological evaluation of a Y2 antagonist.

Neuropeptide Y (NPY) is a potent inhibitor of neurotransmitter release through the Y2 receptor subtype. Specific antagonists for the Y2 receptors have not yet been described. Based on the concept of template-assembled synthetic proteins we have used a cyclic template molecule containing two beta-turn mimetics for covalent attachment of four COOH-terminal fragments RQRYNH2 (NPY 33-36), termed T4-[NPY(33-36)]4. This structurally defined template-assembled synthetic protein has been tested for binding using SK-N-MC and LN319 cell lines that express the Y1 and Y2 receptor, respectively. T4-[NPY(33-36)]4 binds to the Y2 receptor with high affinity (IC50 = 67.2 nM) and has poor binding to the Y1 receptor. This peptidomimetic tested on LN319 cells at concentrations up to 10 microM shows no inhibitory effect on forskolin-stimulated cAMP levels (IC50 for NPY = 2.5 nM). Furthermore, we used confocal microscopy to examine the NPY-induced increase in intracellular calcium in single LN319 cells. Preincubation of the cells with T4-[NPY(33-36)]4 shifted to the right the dose-response curves for intracellular mobilization of calcium induced by NPY at concentrations ranging from 0.1 nM to 10 microM. Finally, we assessed the competitive antagonistic properties of T4-[NPY(33-36)]4 at presynaptic peptidergic Y2 receptors modulating noradrenaline release. the compound T4-[NPY(33-36)]4 caused a marked shift to the right of the concentration-response curve of NPY 13-36, a Y2-selective fragment, yielding a pA2 value of 8.48. Thus, to our best knowledge, T4-[NPY(33-36)]4 represents the first potent and selective Y2 antagonist.

Clinical consequences of imatinib plasma concentrations variability in hemato-oncologic patients

Background: Imatinib has revolutionized the treatment of chronic myeloid leukemia (CML) and gastrointestinal stromal tumors (GIST). Considering the large inter-individual differences in the function of the systems involved in its disposition, exposure to imatinib can be expected to vary widely among patients. This observational study aimed at describing imatinib pharmacokinetic variability and its relationship with various biological covariates, especially plasma alpha1-acid glycoprotein (AGP), and at exploring the concentration-response relationship in patients. Methods: A population pharmacokinetic model (NONMEM) including 321 plasma samples from 59 patients was built up and used to derive individual post-hoc Bayesian estimates of drug exposure (AUC; area under curve). Associations between AUC and therapeutic response or tolerability were explored by ordered logistic regression. Influence of the target genotype (i.e. KIT mutation profile) on response was also assessed in GIST patients. Results: A one-compartment model with first-order absorption appropriately described the data, with an average oral clearance of 14.3 L/h (CL) and volume of distribution of 347 L (Vd). A large inter-individual variability remained unexplained, both on CL (36%) and Vd (63%), but AGP levels proved to have a marked impact on total imatinib disposition. Moreover, both total and free AUC correlated with the occurrence and number of side effects (e.g. OR 2.9±0.6 for a 2-fold free AUC increase; p<0.001). Furthermore, in GIST patients, higher free AUC predicted a higher probability of therapeutic response (OR 1.9±0.5; p<0.05), notably in patients with tumor harboring an exon 9 mutation or wild-type KIT, known to decrease tumor sensitivity towards imatinib. Conclusion: The large pharmacokinetic variability, associated to the pharmacokinetic-pharmacodynamic relationship uncovered are arguments to further investigate the usefulness of individualizing imatinib prescription based on TDM. For this type of drug, it should ideally take into consideration either circulating AGP concentrations or free drug levels, as well as KIT genotype for GIST.

A quantitative analysis of antagonism and inverse agonism at wild-type and constitutively active hamster alpha1B-adrenoceptors.

In order to characterize inverse agonism at alpha1B-adrenoceptors, we have compared the concentration-response relationships of several quinazoline and non-quinazoline alpha1-adrenoceptor antagonists at cloned hamster wild-type (WT) alpha1B-adrenoceptors and a constitutively active mutant (CAM) thereof upon stable expression in Rat-1 fibroblasts. Receptor activation or inhibition thereof was assessed as [3H]inositol phosphate (IP) accumulation. Quinazoline (alfuzosin, doxazosin, prazosin, terazosin) and non-quinazoline alpha1-adrenoceptor antagonists (BE 2254, SB 216,469, tamsulosin) concentration-dependently inhibited phenylephrine-stimulated IP formation at both WT and CAM with Ki values similar to those previously found in radioligand binding studies. At CAM in the absence of phenylephrine, the quinazolines produced concentration-dependent inhibition of basal IP formation; the maximum inhibition was approximately 55%, and the corresponding EC50 values were slightly smaller than the Ki values. In contrast, BE 2254 produced much less inhibition of basal IP formation, SB 216,469 was close to being a neutral antagonist, and tamsulosin even weakly stimulated IP formation. The inhibitory effects of the quinazolines and BE 2254 as well as the stimulatory effect of tamsulosin were equally blocked by SB 216,469 at CAM. At WT in the absence of phenylephrine, tamsulosin did not cause significant stimulation and none of the other compounds caused significant inhibition of basal IP formation. We conclude that alpha1-adrenoceptor antagonsits with a quinazoline structure exhibit greater efficacy as inverse agonists than those without.

Systematic review of population pharmacokinetic analyses of imatinib and relationships with treatment outcomes

Several population pharmacokinetic (PPK) analyses of the anticancer drug imatinib have been performed to investigate different patient populations and covariate effects. The present analysis offers a systematic qualitative and quantitative summary and comparison of those. Its primary objective was to provide useful information for evaluating the expectedness of imatinib plasma concentration measurements in the frame of therapeutic drug monitoring. The secondary objective was to review clinically important concentration-effect relationships to provide help in evaluating the potential suitability of plasma concentration values. Nine PPK models describing total imatinib plasma concentration were identified. Parameter estimates were standardized to common covariate values whenever possible. Predicted median exposure (Cmin) was derived by simulations and ranged between models from 555 to 1388 ng/mL (grand median: 870 ng/mL and interquartile "reference" range: 520-1390 ng/mL). Covariates of potential clinical importance (up to 30% change in pharmacokinetic predicted by at least 1 model) included body weight, albumin, α1 acid glycoprotein, and white blood cell count. Various other covariates were included but were statistically not significant or seemed clinically less important or physiologically controversial. Concentration-response relationships had more importance below the average reference range and concentration-toxicity relationships above. Therapeutic drug monitoring-guided dosage adjustment seems justified for imatinib, but a formal predictive therapeutic range remains difficult to propose in the absence of prospective target concentration intervention trials. To evaluate the expectedness of a drug concentration measurement in practice, this review allows comparison of the measurement either to the average reference range or to a specific range accounting for individual patient characteristics. For future research, external PPK model validation or meta-model development should be considered.

Computerized analysis of isometric tension studies provides important additional information about vasomotor activity

Concentration-response curves of isometric tension studies on isolated blood vessels are obtained traditionally. Although parameters such as Imax, EC50 and pA2 may be readily calculated, this method does not provide information on the temporal profile of the responses or the actual nature of the reaction curves. Computerized data acquisition systems can be used to obtain average data that represent a new source of otherwise inaccessible information, since early and late responses may be observed separately in detail

Vasorelaxant effects of the potassium channel opener SR 47063 on the isolated human saphenous vein and rat aorta

The vasorelaxant effects of SR 47063 (4-(2-cyanimino-1,2-dihydropyrid-1-yl)-2,2-dimethyl-6-nitrochromene), a new K+-channel opener structurally related to levcromakalim, were examined in isolated human saphenous vein (HSV) and rat aorta (RA). HSV or RA rings were precontracted with either KCl or noradrenaline and cumulative relaxant concentration-response curves were obtained for SR 47063 (0.1 nM to 1 µM) in the presence or absence of 3 µM glibenclamide. SR 47063 potently relaxed HSV and RA precontracted with 20 mM (but not 60 mM) KCl or 10 µM noradrenaline in a concentration-dependent manner, showing slightly greater activity in the aorta. The potency of the effect of SR 47063 on HSV and RA was 12- and 58-fold greater, respectively, than that reported for the structurally related K+-channel opener levcromakalim. The vasorelaxant action of SR 47063 in both blood vessels was strongly inhibited by 3 µM glibenclamide, consistent with a mechanism of action involving ATP-dependent K+-channels.

Protective effect of N-acetylcysteine against oxygen radical-mediated coronary artery injury

The present study investigated the protective effect of N-acetylcysteine (NAC) against oxygen radical-mediated coronary artery injury. Vascular contraction and relaxation were determined in canine coronary arteries immersed in Kreb's solution (95% O2-5% CO2), incubated or not with NAC (10 mM), and exposed to free radicals (FR) generated by xanthine oxidase (100 mU/ml) plus xanthine (0.1 mM). Rings not exposed to FR or NAC were used as controls. The arteries were contracted with 2.5 µM prostaglandin F2alpha. Subsequently, concentration-response curves for acetylcholine, calcium ionophore and sodium fluoride were obtained in the presence of 20 µM indomethacin. Concentration-response curves for bradykinin, calcium ionophore, sodium nitroprusside, and pinacidil were obtained in the presence of indomethacin plus Nomega-nitro-L-arginine (0.2 mM). The oxidative stress reduced the vascular contraction of arteries not exposed to NAC (3.93 ± 3.42 g), compared to control (8.56 ± 3.16 g) and to NAC group (9.07 ± 4.0 g). Additionally, in arteries not exposed to NAC the endothelium-dependent nitric oxide (NO)-dependent relaxation promoted by acetylcholine (1 nM to 10 µM) was also reduced (maximal relaxation of 52.1 ± 43.2%), compared to control (100%) and NAC group (97.0 ± 4.3%), as well as the NO/cyclooxygenase-independent receptor-dependent relaxation provoked by bradykinin (1 nM to 10 µM; maximal relaxation of 20.0 ± 21.2%), compared to control (100%) and NAC group (70.8 ± 20.0%). The endothelium-independent relaxation elicited by sodium nitroprusside (1 nM to 1 µM) and pinacidil (1 nM to 10 µM) was not affected. In conclusion, the vascular dysfunction caused by the oxidative stress, expressed as reduction of the endothelium-dependent relaxation and of the vascular smooth muscle contraction, was prevented by NAC.