Investigation of the in vivo activity of CYP3A in Brazilian volunteers: comparison of midazolam and omeprazole as drug markers

Objective This study compares midazolam with omeprazole as marker drugs for the evaluation of CYP3A activity in nine healthy self-reported white Brazilian volunteers. Methods Omeprazole was also used to evaluate the CYP2C19 phenotype. The volunteers received p.o. 20 mg omeprazole, and blood samples were collected 3.5 h after drug administration. After a washout period of 10 days, the volunteers received p.o. 15 mg midazolam maleate, and serial blood samples were collected up to 6 h after administration of the drug. CYP2C19 was genotyped for the allelic variants CYP2C19*1, CYP2C19*2, CYP2C19*3, and CYP2C19*17. Analysis of omeprazole, hydroxyomeprazole, omeprazole sulfone, and midazolam in plasma was carried out by LC-MS/MS. Results The volunteers genotyped as CYP2C19*1*17, CYP2C19*17*17, CYP2C19*1*1 (n=8), or CYP2C19*17*2 (n=1) presented a median hydroxylation index (omeprazole/hydroxyomeprazole) of 1.35, indicating that all of them were extensive metabolizers of CYP2C19. The volunteers (n=9) presented a 0.12 log of the omeprazole/sulfone ratio and a median oral clearance of midazolam of 17.89 ml min(-1) kg(-1), suggesting normal CYP3A activity. Conclusions Orthogonal regression analysis between midazolam clearance and log of the plasma concentrations of the omeprazole/omeprazole sulfone ratio (R=-0.7544, P < 0.05) suggests that both midazolam and omeprazole can be used as markers of CYP3A activity in the population investigated.

Discordant Nadir GH After Oral Glucose and IGF-I Levels on Treated Acromegaly: Refining the Biochemical Markers of Mild Disease Activity

Biochemical markers for remission on acromegaly activity are controversial. We studied a subset of treated acromegalic patients with discordant nadir GH levels after oral glucose tolerance test (oGTT) and IGF-I values to refine the current consensus on acromegaly remission. We also compared GH results by two GH immunoassays. From a cohort of 75 treated acromegalic patients, we studied 13 patients who presented an elevated IGF-I despite post-oGTT nadir GH of <= 1 mu g/l. The 12-h daytime GH profile (GH-12 h), nadir GH after oGTT, and basal IGF-I levels were studied in patients and controls. Bland-Altman method showed high concordance between GH assays. Acromegalic patients showed higher mean GH-12 h values (0.71+/-0.36 vs. 0.31+/-0.28 mu g/l; p<0.05) and nadir GH after oGTT (0.48+/-0.32 vs. 0.097+/-0.002 mu g/l; p<0.05) as compared to controls. Nadir GH correlated with mean GH-12 h (r=0.92, p<0.05). The mean GH-12 h value from upper 95% CI of controls (0.54 mu g/l) would correspond to a theoretical normal nadir GH of <= 0.27 mu g/l. Patients with GH nadir <= 0.3 mu g/l had IGF-I between 100-130% ULNR (percentage of upper limit of normal range) and mean GH-12 h of 0.35+/-0.15, and patients with GH nadir >0.3 and <= 1 mu g/l had IGF-I >130% ULNR and mean GH-12 h of 0.93+/-0.24 mu g/l. Our data integrate daytime GH secretion, nadir GH after oGTT, and plasma IGF-I concentrations showing a continuum of mild residual activity in a subgroup of treated acromegaly with nadir GH values <= 1 mu g/l. The degree of increased IGF-I levels and nadir GH after oGTT are correlated with the subtle abnormalities of daytime GH secretion.

Nitric oxide inhibition in paraventricular nucleus on cardiovascular and autonomic modulation after exercise training in unanesthetized rats

It is well known that regular physical exercise alter cardiac function and autonomic modulation of heart rate variability (HRV). The paraventricular nucleus of hypothalamus (PVN) is an important site of integration for autonomic and cardiovascular responses, where nitric oxide (NO) plays an important role. The aim of our study was to evaluate the cardiovascular parameters and autonomic modulation by means of spectral analysis after nitric oxide synthase (NOS) inhibition in the PVN in conscious sedentary (S) or swimming trained (ST) rats. After swimming training protocol, adult male Wistar rats, instrumented with guide cannulas to PVN and femoral artery and vein catheters were submitted to mean arterial pressure (MAP) and heart rate (HR) recording. At baseline, the physical training induced a resting bradycardia (S: 374 +/- 5, ST: 346 +/- 1 bpm) and promoted adaptations in HRV characterized by an increase in high-frequency oscillations (HF; 26.43 +/- 6.91 to 88.96 +/- 244) and a decrease in low-frequency oscillations (LF; 73.57 +/- 6.91 to 11.04 +/- 2.44) in normalized units. The microinjection of N(omega)-nitro-L-arginine methyl ester (L-NAME) in the PVN of sedentary and trained rats promoted increase in MAP and HR. L-NAME in the PVN did not significantly alter the spectral parameters of HRV of sedentary animals, however in the trained rats increased LF oscillations (11.04 +/- 2.44 to 27.62 +/- 6.97) and decreased HF oscillations (88.96 +/- 2.44 to 72.38 +/- 6.97) in normalized units compared with baseline. Our results suggest that NO in the PVN may collaborate to cardiac autonomic modulation after exercise training. (c) 2010 Elsevier B.V. All rights reserved.

INHIBITION OF GUANYLYL CYCLASE RESTORES NEUTROPHIL MIGRATION AND MAINTAINS BACTERICIDAL ACTIVITY INCREASING SURVIVAL IN SEPSIS

Sepsis results from an overwhelming response to infection and is a major contributor to death in intensive care units worldwide. In recent years, we and others have shown that neutrophil functionality is impaired in sepsis. This correlates with sepsis severity and contributes to aggravation of sepsis by precluding bacterial clearance. Nitric oxide (NO) is a major contributor to the impairment of neutrophil function in sepsis. However, attempts to inhibit NO synthesis in sepsis resulted in increased death despite restoring neutrophil migration. This could be in part attributed to a reduction of the NO-dependent microbicidal activity of neutrophils. In sepsis, the beneficial effects resulting from the inhibition of soluble guanylyl cyclase (sGC), a downstream target of NO, have long been appreciated but poorly understood. However, the effects of sGC inhibition on neutrophil function in sepsis have never been addressed. In the present study, we show that TLR activation in human neutrophils leads to decreased chemotaxis, which correlated with chemotactic receptor internalization and increased G protein-coupled receptor kinase 2 expression, in a process involving the NO-sGC-protein kinase G axis. We also demonstrate that inhibition of sGC activity increased survival in a murine model of sepsis, which was paralleled by restored neutrophil migratory function and increased bacterial clearance. Finally, the beneficial effect of sGC inhibition could also be demonstrated in mice treated after the onset of sepsis. Our results suggest that the beneficial effects of sGC inhibition in sepsis could be at least in part attributed to a recovery of neutrophil functionality.

Paraventricular nucleus modulates autonomic and neuroendocrine responses to acute restraint stress in rats

The paraventricular nucleus of the hypothalamus (PVN) has been implicated in several aspects of neuroendocrine and cardiovascular control The PVN contains parvocellular neurons that release the corticotrophin release ha mone (CRH) under stress situations In addition this brain area is connected to several limbic structures implicated in defensive behavioral control as well to forebrain and brainst m structures involved in cardiovascular control Acute restraint is an unavoidable stress situation that evokes corticosterone release as well as marked autonomic changes the latter characterized by elevated mean arterial pressure (MAP) intense heart rate (HR) Increases and decrease in the tail temperature We report the effect of PVN inhibition on MAP and HR responses corticosterone plasma levels and tail temperature response during acute restraint in rats Bilateral microinjection of the nonspecific synaptic blocker CoCl(2) (1 mM/100 nL) into the PVN reduced the pressor response it inhibited the increase in plasma corticosterone concentration as well as the fall in tail temperature associated with acute restraint stress Moreover bilateral microinjection of CoCl(2) into areas surrounding the PVN did not affect the blood pressure hormonal and tail vasoconstriction responses to restraint stress The present results show that a local PVN neurotransmission is involved in the neural pathway that controls autonomic and neuroendocrine responses which are associated with the exposure to acute restraint stress (C) 2010 Elsevier B V All rights reservi.d

Phosphoinositide-3 Kinase gamma Activity Contributes to Sepsis and Organ Damage by Altering Neutrophil Recruitment

Rationale Sepsis is a leading cause of death in the intensive care unit, characterized by a systemic inflammatory response (SIRS) and bacterial infection, which can often induce multiorgan damage and failure. Leukocyte recruitment, required to limit bacterial spread, depends on phosphoinositide-3 kinase gamma (PI3K gamma) signaling in vitro; however, the role of this enzyme in polymicrobial sepsis has remained unclear. Objectives: This study aimed to determine the specific role of the kinase activity of PI3K gamma in the pathogenesis of sepsis and multiorgan damage. Methods. PI3K gamma wild-type, knockout, and kinase-dead mice were exposed to cecal ligation and perforation induced sepsis and assessed for survival; pulmonary, hepatic, and cardiovascular damage; coagulation derangements; systemic inflammation; bacterial spread; and neutrophil recruitment. Additionally, wild-type mice were treated either before or after the onset of sepsis with a PI3K gamma inhibitor and assessed for survival, neutrophil recruitment, and bacterial spread. Measurements and Main Results: Both genetic and pharmaceutical PI3K gamma kinase inhibition significantly improved survival, reduced multiorgan damage, and limited bacterial decompartmentalization, while modestly affecting SIRS. Protection resulted from both neutrophil-independent mechanisms, involving improved cardiovascular function, and neutrophil-dependent mechanisms, through reduced susceptibility to neutrophil migration failure during severe sepsis by maintaining neutrophil surface expression of the chemokine receptor, CXCR2. Furthermore, PI3K gamma pharmacological inhibition significantly decreased mortality and improved neutrophil migration and bacterial control, even when administered during established septic shock. Conclusions: This study establishes PI3K gamma as a key molecule in the pathogenesis of septic infection and the transition from SIRS to organ damage and identifies it as a novel possible therapeutic target.

Cannabidiol injected into the bed nucleus of the stria terminalis modulates baroreflex activity through 5-HT(1A) receptors

Cannabidiol (CBD) is a non-psychotomimetic constituent of the Cannabis sativa plant that inhibits behavioral and cardiovascular responses to aversive situations. facilitating 5-HT(1A)-mediated neurotransmission. Previous results from our group suggest that the bed nucleus of the stria terminalis (BNST) may be involved in CBD`s anti-aversive effects. To investigate whether the cardiovascular effects of the CBD could involve a direct drug effect on the BNST, we evaluated the effects of CBD microinjection into this structure on baroreflex activity. We also verified whether these effects were mediated by the activation of 5-HT(1A) receptors. Bilateral microinjection of CBD (60 nmol/100 nL) into the BNST increased the bradycardiac response to arterial pressure increases. However, no changes were observed in tachycardiac responses evoked by arterial pressure decreases. Pretreatment of the BNST with the selective 5-HT(1A) receptor antagonist WAY100635 (0.37 nmol/100 nL) prevented CBD effects on the baroreflex activity. Moreover, microinjection of the 5-HT(1A) receptor agonist 8-OH-DPAT (4 nmol/100 nL) caused effects that were similar to those observed after the microinjection of CBD, which were also blocked by pretreatment with WAY100635. In conclusion, the present studies show that the microinjection of CBD into the BNST has a facilitatory influence on the baroreflex response to blood pressure increases, acting through the activation of 5-HT(1A) receptors. (C) 2010 Elsevier Ltd. All rights reserved.

Antileishmanial activity of ruthenium(II)tetraammine nitrosyl complexes

The complexes trans-[Ru(NO)(NH(3))(4)L](X)(3) (X = BF(4)(-), PF(6)(-) or Cl(-) and L = N-heterocyclic ligands, P (OEt)(3), SO(3)(-2)), and [Ru(NO)Hedta)] were shown to exhibit IC(50pro) in the range of 36 (L = imN) to 5000 mu M (L = imC). The inhibitory effects of trans-[Ru(NO)(NH(3))(4)imN](BF(4))(3) and of the Angeli`s salt on the growth of the intramacrophage amastigote form studied were found to be similar while the trans-[Ru(NH(3))(4)imN(H(2)O)](2+) complex was found not to exhibit any substantial antiamastigote effect. The trans-[Ru(NO)(NH(3))(4)imN](BF(4))(3) compound, administered (500 nmol kg(-1) day(-1)) in BALB/c mice infected with Leishmania major, was found to exhibit a 98% inhibition on the parasite growth. Furthermore, this complex proved to be at least 66 times more efficient than glucantime in in vivo experiments. (C) 2010 Elsevier Masson SAS. All rights reserved.

Antinociceptive activity and toxicology of the lectin from Canavalia boliviana seeds in mice

The aim of the present study was to evaluate the potential antinociceptive and toxicity of Canavalia boliviana lectin (CboL) using different methods in mice. The role of carbohydrate-binding sites was also investigated. CboL given to mice daily for 14 days at doses of 5 mg/kg did not cause any observable toxicity. CboL (1, 5, and 10 mg/kg) administered to mice intravenously inhibited abdominal constrictions induced by acetic acid and the two phases of the formalin test. In the hot plate and tail immersion tests, the same treatment of CboL induced significant increase in the latency period. In the hot plate test, the effect of CboL (5 mg/kg) was reversed by naloxone (1 mg/kg), indicating the involvement of the opioid system. In the open-field and rota-rod tests, the CboL treatment did not alter animals` motor function. These results show that CboL presents antinociceptive effects of both central and peripheral origin, involving the participation of the opioid system via lectin domain.

Insular cortex alpha(1)-adrenoceptors modulate the parasympathetic component of the baroreflex in unanesthetized rats

The insular cortex (IC) has been reported to modulate the cardiac parasympathetic activity of the baroreflex in unanesthetized rats. However, which neurotransmitters are involved in this modulation is still unclear. In the present study, we evaluated the possible involvement of local IC-noradrenergic neurotransmission in modulating reflex bradycardiac responses. Bilateral microinjection of the selective alpha(1)-adrenoceptor antagonist WB4101 (15 nmol/100 nL), into the IC of male Wistar rats, increased the gain of reflex bradycardia in response to mean arterial pressure (MAP) increases evoked by intravenous infusion of phenylephrine. However, bilateral microinjection of equimolar doses of either the selective alpha(2)-adrenoceptor antagonist RX821002 or the non-selective beta-adrenoceptor antagonist propranolol into the IC did not affect the baroreflex response. No effects were observed in basal MAP or heart rate values after bilateral microinjection of noradrenergic antagonists into the IC, thus suggesting no tonic influence of IC-noradrenergic neurotransmission on resting cardiovascular parameters. In conclusion, these data provide evidence that local IC-noradrenergic neurotransmission has an inhibitory influence on baroreflex responses to blood pressure increase evoked by phenylephrine infusion through activation of alpha(1)-adrenoceptors. (C) 2009 Elsevier B.V. All rights reserved.

Anti-inflammatory activity and possible mechanism of extract from Mikania laevigata in carrageenan-induced peritonitis

Objectives The aim was to test the potential use of an extract of Mikania laevigata (popularly known in Brazil as guaco), made from leaves harvested in different months of the year, oil neutrophil migration after all inflammatory Stimulus and investigate the underlying molecular mechanisms. Methods We examined the effect of guaco on vascular permeability and leucocyte function in carrageenan-induced peritonitis in mice. Key findings Our results demonstrated that guaco extract administered subcutaneously (3 mg/kg) decreased the vascular permeability and also leucocyte rolling and adhesion to the inflamed tissues by a mechanism dependent on nitric oxide. Specifically, inhibitors of nitric oxide synthase remarkably abrogated the guaco extract-mediated suppression of neutrophil migration to the inflammatory site. In addition, guaco extract-mediated suppression of neutrophil migration appeared to be dependent on the production of the cytokines interleukin-1 beta and tumour necrosis factor-alpha. One of the major constituents of the guaco extract, coumarin, was able to inhibit the neutrophil migration towards the inflammatory focus. Conclusions In conclusion the anti-inflammatory effect induced by guaco extract may be by inhibition of pro-inflammatory cytokine production at the inflammatory site.

Neuromuscular activity of BaTX, a presynaptic basic PLA(2) isolated from Bothrops alternatus snake venom

We have previously isolated a Lys49 phospholipase A(2) homolog (BaTX) from Bothrops alternatus snake venom using a combination of molecular exclusion chromatography and reverse phase HPLC and shown its ability to cause neuromuscular blockade. In this work, we describe a one-step procedure for the purification of this toxin and provide further details of its neuromuscular activity. The toxin was purified by reverse phase HPLC and its purity and molecular mass were confirmed by SIDS-PAGE, MALDI-TOF mass spectrometry, amino acid analysis and N-terminal sequencing. BaTX (0.007-1.4 mu M) produced time-dependent, irreversible neuromuscular blockade in isolated mouse phrenic nerve-diaphragm and chick biventer cervicis preparations (time to 50% blockade with 0.35 mu M toxin: 58 +/- 4 and 24 +/- 1 min, respectively; n = 3-8; mean +/- S.E.) without significantly affecting the response to direct muscle stimulation. In chick preparations, contractures to exogenous acetylcholine (55 and 110 mu M) or KCl (13.4 mM) were unaltered after complete blockade by all toxin concentrations. These results, which strongly suggested a presynaptic mechanism of action for this toxin, were reinforced by (1) the inability of BaTX to interfere with the carbachol-induced depolarization of the resting membrane, (2) a significant decrease in the frequency and amplitude of miniature end-plate potentials, and (3) a significant reduction (59 +/- 4%, n=12) in the quantal content of the end-plate potentials after a 60 min incubation with the toxin (1.4 mu M). In addition, a decrease in the organ bath temperature from 37 degrees C to 24 degrees C and/or the replacement of calcium with strontium prevented the neuromuscular blockade, indicating a temperature-dependent effect possibly mediated by enzymatic activity. (C) 2009 Elsevier Inc. All rights reserved.

Effect of baroreceptor denervation on the autonomic control of arterial pressure in conscious mice

This study evaluated the role of arterial baroreceptors in arterial pressure (AP) and pulse interval (PI) regulation in conscious C57BL mice. Male animals, implanted with catheters in a femoral artery and a jugular vein, were submitted to sino-aortic (SAD), aortic (Ao-X) or carotid sinus denervation (Ca-X), 5 daysprior to the experiments. After basal recording of AP, the lack of reflex bradycardia elicited by administration of phenylephrine was used to confirm the efficacy of SAD, and cardiac autonomic blockade with methylatropine and propranolol was performed. The AP and PI variability were calculated in the time and frequency domains (spectral analysis/fast Fourier transform) with the spectra quantified in low-(LF; 0.25-1Hz) and high-frequency bands (HF; 1-5Hz). Basal AP and AP variability were higher after SAD, Ao-X or Ca-X than in intact mice. Pulse interval was similar among the groups, whereas PI variability was lower after SAD. Atropine elicited a slight tachycardia in control mice but did not change PI after total or partial denervation. The bradycardia caused by propranolol was higher after SAD, Ao-X or Ca-X compared with intact mice. The increase in the variability of AP was accompanied by a marked increase in the LF and HF power of the AP spectra after baroreceptor denervation. The LF and HF power of the PI were reduced by SAD and by Ao-X or Ca-X. Therefore, both sino-aortic and partial baroreceptor denervation in mice elicits hypertension and a remarkable increase in AP variability and cardiac sympathetic tonus. Spectral analysis showed an important contribution of the baroreflex in the power of LF oscillations of the PI spectra. Both sets of baroreceptors seem to be equally important in the autonomic regulation of the cardiovascular system in mice.