Toward Global Standards for Comparator Pharmaceutical Products: Case Studies of Amoxicillin, Metronidazole, and Zidovudine in the Americas

This study compared in vitro dissolution characteristics and other quality measures of different amoxicillin, metronidazole, and zidovudine products purchased in the Americas to a comparator pharmaceutical product (CPP). These three drugs are classified as Biopharmaceutics Classification System Class I drugs with the possibility that dissolution findings might be used to document bioequivalence. All investigated zidovudine products were found to be in vitro equivalent to the CPP. Only 3 of 12 tested amoxicillin products were found to be in vitro equivalent to the CPP. None of the tested metronidazole products were in vitro equivalent to the CPP. These findings suggest but do not confirm bioinequivalence where in vitro comparisons failed, given that an in vivo blood level study might have confirmed bioequivalence. At times, identifying a CPP in one of the selected markets proved difficult. The study demonstrates that products sold across national markets may not be bioequivalent. When coupled with the challenge of identifying a CPP in different countries, the results of this study suggest the value of an international CPP as well as increased use of BCS approaches as means of either documenting bioequivalence or signaling the need for further in vivo studies. Because of increased movement of medicines across national borders, practitioners and patients would benefit from these approaches.

Is income area level associated with blood pressure in adults regardless of individual-level characteristics? A multilevel approach

This population-based health survey was carried out in Florianopolis, Brazil, to assess the association between adult systolic blood pressure (SBP) and contextual income level, after controlling for potential individual-level confounders. A statistically significant negative association between SBP levels and contextual income was identified after adjusting for individual-level characteristics. SBP levels in the highest and in the intermediate tertiles of contextual income were 5.78 and 2.82 mmHg lower, respectively, than that observed in the bottom tertile. The findings suggest an association between income area level and blood pressure, regardless of well-known individual-level hypertension risk factors. (C) 2012 Elsevier Ltd. All rights reserved.

Angiotensin-(1-7) decreases LPS-induced inflammatory response in macrophages

It has been previously shown that besides its classical role in blood pressure control the reninangiotensin system, mainly by action of angiotensin II on the AT1 receptor, exerts pro-inflammatory effects such as by inducing the production of cytokines. More recently, alternative pathways to this system were described, such as binding of angiotensin-(17) to receptor Mas, which was shown to counteract some of the effects evoked by activation of the angiotensin IIAT1 receptor axis. Here, by means of different molecular approaches we investigated the role of angiotensin-(17) in modulating inflammatory responses triggered in mouse peritoneal macrophages. Our results show that receptor Mas transcripts were up-regulated by eightfold in LPS-induced macrophages. Interestingly, macrophage stimulation with angiotensin-(17), following to LPS exposure, evoked an attenuation in expression of TNF-a and IL-6 pro-inflammatory cytokines; where this event was abolished when the receptor Mas selective antagonist A779 was also included. We then used heterologous expression of the receptor Mas in HEK293T cells to search for the molecular mechanisms underlying the angiotensin-(17)-mediated anti-inflammatory responses by a kinase array; what suggested the involvement of the Src kinase family. In LPS-induced macrophages, this finding was corroborated using the PP2 compound, a specific Src kinase inhibitor; and also by Western blotting when we observed that Ang-(17) attenuated the phosphorylation levels of Lyn, a member of the Src kinase family. Our findings bring evidence for an anti-inflammatory role for angiotensin-(17) at the cellular level, as well as show that its probable mechanism of action includes the modulation of Src kinases activities. J. Cell. Physiol. 227: 21172122, 2012. (C) 2011 Wiley Periodicals, Inc.

Gene expression profiles displayed by peripheral blood mononuclear cells from patients with type 2 diabetes mellitus focusing on biological processes implicated on the pathogenesis of the disease

Patients with type 2 diabetes mellitus (T2DM) exhibit insulin resistance associated with obesity and inflammatory response, besides an increased level of oxidative DNA damage as a consequence of the hyperglycemic condition and the generation of reactive oxygen species (ROS). In order to provide information on the mechanisms involved in the pathophysiology of T2DM, we analyzed the transcriptional expression patterns exhibited by peripheral blood mononuclear cells (PBMCs) from patients with T2DM compared to non-diabetic subjects, by investigating several biological processes: inflammatory and immune responses, responses to oxidative stress and hypoxia, fatty acid processing, and DNA repair. PBMCs were obtained from 20 T2DM patients and eight non-diabetic subjects. Total RNA was hybridized to Agilent whole human genome 4x44K one-color oligo-microarray. Microarray data were analyzed using the GeneSpring GX 11.0 software (Agilent). We used BRB-ArrayTools software (gene set analysis - GSA) to investigate significant gene sets and the Genomica tool to study a possible influence of clinical features on gene expression profiles. We showed that PBMCs from T2DM patients presented significant changes in gene expression, exhibiting 1320 differentially expressed genes compared to the control group. A great number of genes were involved in biological processes implicated in the pathogenesis of T2DM. Among the genes with high fold-change values, the up-regulated ones were associated with fatty acid metabolism and protection against lipid-induced oxidative stress, while the down-regulated ones were implicated in the suppression of pro-inflammatory cytokines production and DNA repair. Moreover, we identified two significant signaling pathways: adipocytokine, related to insulin resistance; and ceramide, related to oxidative stress and induction of apoptosis. In addition, expression profiles were not influenced by patient features, such as age, gender, obesity, pre/post-menopause age, neuropathy, glycemia, and HbA(1c) percentage. Hence, by studying expression profiles of PBMCs, we provided quantitative and qualitative differences and similarities between T2DM patients and non-diabetic individuals, contributing with new perspectives for a better understanding of the disease. (C) 2012 Elsevier B.V. All rights reserved.

Angiotensin-(3-4) counteracts the Angiotensin II inhibitory action on renal Ca2+-ATPase through a cAMP/PKA pathway

We recently demonstrated that Angiotensin-(3-4) [Ang-(3-4)], an Ang II-derived dipeptide, overcomes inhibition of plasma membrane Ca2+-ATPase promoted by nanomolar concentrations of Ang II in basolateral membranes of renal proximal tubule cells, with involvement of a so far unknown AT(2)R-dependent and NO-independent mechanism. The present study investigates the signaling pathway triggered by Ang-(3-4) that is responsible for counteracting the inhibitory effect of Ang II, and attempts to elucidate the functional interaction of the dipeptide with Ang II at the level of AT(2)R. Stimulation by cholera toxin of G(s)alpha protein structurally linked to AT(2)R as revealed by their co-immunoprecipitation mimicked the effect of Ang-(3-4) on Ca2+-ATPase activity. Furthermore, addition of dibutyril-cAMP (db-cAMP) mimicked Ang-(3-4), whereas the specific PKA inhibitor, PKAi((5-24)) peptide, suppressed the counter-regulatory effect of Ang-(3-4) and the AT(2)R agonist, CGP42112A. Membrane-associated PKA activity was stimulated by Ang-(3-4) or CGP42112A to comparable levels as db-cAMP, and the Ang-(3-4) effect was abrogated by the AT(2)R antagonist PD123319, whereas the AT(1)R antagonist Losartan had no effect. Ang-(3-4) stimulated PKA-mediated phosphorylation of Ca2+-ATPase and activated PKA to comparable levels. Binding assays demonstrated that Ang-(3-4) could not displace H-3-Ang II from HEK 293T cells expressing AT(2)R, but 10(-10) mol/L Ang-(3-4) resulted in the appearance of a probable higher-affinity site (picomolar range) for Ang II. The results presented herein demonstrate that Ang-(3-4), acting as an allosteric enhancer, suppresses Ang II-mediated inhibition of Ca2+-ATPase through an AT(2)R/cAMP/PKA pathway, after inducing conformational changes in AT(2)R that results in generation of higher-affinity sites for Ang II. (C) 2012 Elsevier B.V. All rights reserved.

Maternal high-sodium intake alters the responsiveness of the renin-angiotensin system in adult offspring

Aims: The goal of the current study was to evaluate the impact of maternal sodium intake during gestation on the systemic and renal renin-angiotensin-aldosterone-system (RAAS) of the adult offspring. Main methods: Female Wistar rats were fed high- (HSD-8.0% NaCl) or normal-sodium diets (NSD-1.3% NaCl) from 8 weeks of age until the delivery of their first litter. After birth, the offspring received NSD. Tail-cuff blood pressure (TcBP) was measured in the offspring between 6 and 12 weeks of age. At 12 weeks of age, the offspring were subjected to either one week of HSD or low sodium diet (LSD-0.1 6%NaCl) feeding to evaluate RAAS responsiveness or to acute saline overload to examine sodium excretory function. Plasma (PRA) and renal renin content (RRC), serum aldosterone (ALDO) levels, and renal cortical and medullary renin mRNA expression levels were evaluated at the end of the study. Key findings: TcBP was higher among dams fed HSD, but no TcBP differences were observed among the offspring. Male offspring, however, exhibited increased TcBP after one week of HSD feeding, and this effect was independent of maternal diet. Increased RAAS responsiveness to the HSD and LSD was also observed in male offspring. The baseline levels of PRA. ALDO, and cortical and medullary renin gene expression were lower but the RRC levels were higher among HSD-fed male offspring (HSDoff). Conversely, female HSDoff showed reduced sodium excretion 4 h after saline overload compared with female NSDoff. Significance: High maternal sodium intake is associated with gender-specific changes in RAAS responsiveness among adult offspring. (C) 2012 Elsevier Inc. All rights reserved.

Chronic Conventional resistance exercise reduces blood pressure in stage 1 Hypertensive men

Moraes, MR, Bacurau, RFP, Casarini, DE, Jara, ZP, Ronchi, FA, Almeida, SS, Higa, EMS, Pudo, MA, Rosa, TS, Haro, AS, Barros, CC, Pesquero, JB, Wurtele, M, and Araujo, RC. Chronic conventional resistance exercise reduces blood pressure in stage 1 hypertensive men. J Strength Cond Res 26(4): 1122-1129, 2012-To investigate the antihypertensive effects of conventional resistance exercise (RE) on the blood pressure (BP) of hypertensive subjects, 15 middle-aged (46 +/- 3 years) hypertensive volunteers, deprived of antihypertensive medication (reaching 153 +/- 6/93 +/- 2 mmHg systolic/diastolic BP after a 6-week medication washout period) were submitted to a 12-week conventional RE training program (3 sets of 12 repetitions at 60% 1 repetition maximum, 3 times a week on nonconsecutive days). Blood pressure was measured in all phases of the study (washout, training, detraining). Additionally, the plasma levels of several vasodilators or vasoconstrictors that potentially could be involved with the effects of RE on BP were evaluated pre- and posttraining. Conventional RE significantly reduced systolic, diastolic, and mean BP, respectively, by an average of 16 (p < 0.001), 12 (p < 0.01), and 13 mm Hg (p < 0.01) to prehypertensive values. There were no significant changes of vasoactive factors from the kallikrein-kinin or renin-angiotensin systems. After the RE training program, the BP values remained stable during a 4-week detraining period. Taken together, this study shows for the first time that conventional moderate-intensity RE alone is able to reduce the BP of stage 1 hypertensive subjects free of antihypertensive medication. Moreover, the benefits of BP reduction achieved with RE training remained unchanged for up to 4 weeks without exercise.

Acute ethanol intake induces superoxide anion generation and mitogen-activated protein kinase phosphorylation in rat aorta: A role for angiotensin type 1 receptor

Ethanol intake is associated with increase in blood pressure, through unknown mechanisms. We hypothesized that acute ethanol intake enhances vascular oxidative stress and induces vascular dysfunction through renin-angiotensin system (RAS) activation. Ethanol (1 g/kg; p.o. gavage) effects were assessed within 30 min in male Wistar rats. The transient decrease in blood pressure induced by ethanol was not affected by the previous administration of losartan (10 mg/kg; p.o. gavage), a selective ATI receptor antagonist. Acute ethanol intake increased plasma renin activity (PRA), angiotensin converting enzyme (ACE) activity, plasma angiotensin I (ANG I) and angiotensin II (ANG II) levels. Ethanol induced systemic and vascular oxidative stress, evidenced by increased plasma thiobarbituric acid-reacting substances (TBARS) levels, NAD(P) H oxidase-mediated vascular generation of superoxide anion and p47phox translocation (cytosol to membrane). These effects were prevented by losartan. Isolated aortas from ethanol-treated rats displayed increased p38MAPK and SAPK/JNK phosphorylation. Losartan inhibited ethanol-induced increase in the phosphorylation of these kinases. Ethanol intake decreased acetylcholine-induced relaxation and increased phenylephrine-induced contraction in endothelium-intact aortas. Ethanol significantly decreased plasma and aortic nitrate levels. These changes in vascular reactivity and in the end product of endogenous nitric oxide metabolism were not affected by losartan. Our study provides novel evidence that acute ethanol intake stimulates RAS activity and induces vascular oxidative stress and redox-signaling activation through AT(1)-dependent mechanisms. These findings highlight the importance of RAS in acute ethanol-induced oxidative damage. (c) 2012 Elsevier Inc. All rights reserved.

Angiotensin II Facilitates Breast Cancer Cell Migration and Metastasis

Breast cancer metastasis is a leading cause of death by malignancy in women worldwide. Efforts are being made to further characterize the rate-limiting steps of cancer metastasis, i.e. extravasation of circulating tumor cells and colonization of secondary organs. In this study, we investigated whether angiotensin II, a major vasoactive peptide both produced locally and released in the bloodstream, may trigger activating signals that contribute to cancer cell extravasation and metastasis. We used an experimental in vivo model of cancer metastasis in which bioluminescent breast tumor cells (D3H2LN) were injected intra-cardiacally into nude mice in order to recapitulate the late and essential steps of metastatic dissemination. Real-time intravital imaging studies revealed that angiotensin II accelerates the formation of metastatic foci at secondary sites. Pre-treatment of cancer cells with the peptide increases the number of mice with metastases, as well as the number and size of metastases per mouse. In vitro, angiotensin II contributes to each sequential step of cancer metastasis by promoting cancer cell adhesion to endothelial cells, trans-endothelial migration and tumor cell migration across extracellular matrix. At the molecular level, a total of 102 genes differentially expressed following angiotensin II pretreatment were identified by comparative DNA microarray. Angiotensin II regulates two groups of connected genes related to its precursor angiotensinogen. Among those, up-regulated MMP2/MMP9 and ICAM1 stand at the crossroad of a network of genes involved in cell adhesion, migration and invasion. Our data suggest that targeting angiotensin II production or action may represent a valuable therapeutic option to prevent metastatic progression of invasive breast tumors.

Influence of transportation methods and pre-slaughter rest periods on cortisol level in lambs

This study aimed to verify the influence of the transport in open or closed compartments (0 h), followed by two resting periods (1 and 3 h) for the slaughter process on the levels of cortisol as a indicative of stress level. At the slaughterhouse, blood samples were taken from 86 lambs after the transport and before slaughter for plasma cortisol analysis. The method of transport influenced in the cortisol concentration (0 h; P < 0.01). The animals transported in the closed compartment had a lower level (28.97 ng ml(-1)) than the animals transported in the open compartment (35.49 ng ml(-1)). After the resting period in the slaughterhouse. there was a decline in the plasmatic cortisol concentration, with the animals subjected to 3 h of rest presenting the lower average cortisol value (24.14 ng ml(-1); P < 0.05) than animals subjected to 1 h of rest (29.95 ng ml(-1)). It can be inferred that the lambs that remained 3 h in standby before slaughter had more time to recover from the stress of the transportation than those that waited just 1 h. Visual access to the external environment during the transport of the lambs is a stressful factor changing the level of plasmatic cortisol, and the resting period before slaughter was effective in lowering stress, reducing the plasmatic cortisol in the lambs. (c) 2012 Elsevier B.V. All rights reserved.

Low-dose Enalapril Reduces Angiotensin II and Attenuates Diabetic-induced Cardiac and Autonomic Dysfunctions

Activation of renin-angiotensin system has been linked to cardiovascular and autonomic dysfunctions in diabetes. Experiments were performed to investigate the effects of angiotensin-converting enzyme inhibitor (ACEI), enalapril, on cardiac and autonomic functions in diabetic rats. Diabetes was induced by streptozotocin (50 mg/kg), and rats were treated with enalapril (1 mg.kg(-1).d(-1)). After 30 days, evaluations were performed in control, diabetic, and enalapril-treated groups. Cardiac function was evaluated by echocardiography and through cannulation of the left ventricle (at baseline and in response to volume overload). Heart rate and systolic blood pressure variabilities were evaluated in the time and frequency domains. Streptozotocin rats had left ventricular systolic and diastolic dysfunctions, expressed by reduced ejection fraction and increased isovolumic relaxation time. The ACEI prevented these changes, improved diastolic cardiac responses to volume overload and total power of heart rate variability, reduced the ACE1 activity and protein expression and cardiac angiotensin (Ang) II levels, and increased angiotensin-converting enzyme 2 activity, despite unchanged blood pressure. Correlations were obtained between Ang II content with systolic and diastolic functions and heart rate variability. These findings provide evidence that the low-dose ACEI prevents autonomic and cardiac dysfunctions induced by diabetes without changing blood pressure and associated with reduced cardiac Ang II and increased angiotensin-converting enzyme 2 activity.

Effects of aerobic exercise training on cardiac renin-angiotensin system in an obese Zucker rat strain

Objective: Obesity and renin angiotensin system (RAS) hyperactivity are profoundly involved in cardiovascular diseases, however aerobic exercise training (EXT) can prevent obesity and cardiac RAS activation. The study hypothesis was to investigate whether obesity and its association with EXT alter the systemic and cardiac RAS components in an obese Zucker rat strain. Methods: The rats were divided into the following groups: Lean Zucker rats (LZR); lean Zucker rats plus EXT (LZR+EXT); obese Zucker rats (OZR) and obese Zucker rats plus EXT (OZR+EXT). EXT consisted of 10 weeks of 60-min swimming sessions, 5 days/week. At the end of the training protocol heart rate (HR), systolic blood pressure (SBP), cardiac hypertrophy (CH) and function, local and systemic components of RAS were evaluated. Also, systemic glucose, triglycerides, total cholesterol and its LDL and HDL fractions were measured. Results: The resting HR decreased (, 12%) for both LZR+EXT and OZR+EXT. However, only the LZR+EXT reached significance (p, 0.05), while a tendency was found for OZR versus OZR+EXT (p = 0.07). In addition, exercise reduced (57%) triglycerides and (61%) LDL in the OZR+EXT. The systemic angiotensin I-converting enzyme (ACE) activity did not differ regardless of obesity and EXT, however, the OZR and OZR+EXT showed (66%) and (42%), respectively, less angiotensin II (Ang II) plasma concentration when compared with LZR. Furthermore, the results showed that EXT in the OZR prevented increase in CH, cardiac ACE activity, Ang II and AT2 receptor caused by obesity. In addition, exercise augmented cardiac ACE2 in both training groups. Conclusion: Despite the unchanged ACE and lower systemic Ang II levels in obesity, the cardiac RAS was increased in OZR and EXT in obese Zucker rats reduced some of the cardiac RAS components and prevented obesity-related CH. These results show that EXT prevented the heart RAS hyperactivity and cardiac maladaptive morphological alterations in obese Zucker rats.

Physical exercise improves cardiac autonomic modulation in hypertensive patients independently of angiotensin-converting enzyme inhibitor treatment

We investigated the influence of angiotensin-converting enzyme inhibitor (ACEi) treatment and physical exercise on arterial pressure (AP) and heart rate variability (HRV) in volunteer patients with hypertension. A total of 54 sedentary volunteers were divided into three groups: normotensive (NT Group), hypertensive (HT Group) and HT volunteers treated with ACEi (ACEi Group). All volunteers underwent an aerobic physical-training protocol for 15 weeks. HRV was investigated using a spectral analysis of a time series of R-R interval (RRi) that was obtained in a supine position and during a tilt test. Physical training promoted a significant reduction in the mean arterial pressure of the HT group (113 +/- 3 vs. 106 +/- 1 mm Hg) and the ACEi group (104 +/- 2 vs. 98 +/- 2 mm Hg). Spectral analysis of RRi in the supine position before physical training demonstrated that the NT and ACEi groups had similar values at low frequency (LF; 0.04-0.15 Hz) and high frequency (HF; 0.15-0.5 Hz) oscillations. The HT group had an increase in LF oscillations in absolute and normalized units and a decrease in HF oscillations in normalized units compared with the other groups. The HT group had the lowest responses to the tilt test during LF oscillations in normalized units. Physical training improved the autonomic modulation of the heart rate in the supine position only in the HT group. Physical training promoted a similar increase in autonomic modulation responses in the tilt test in all groups. Our findings show that aerobic physical training improves cardiac autonomic modulation in HT volunteers independently of ACEi treatment. Hypertension Research (2012) 35, 82-87; doi:10.1038/hr.2011.162; published online 29 September 2011

The effect of low-level laser irradiation (In-Ga-Al-AsP - 660 nm) on melanoma in vitro and in vivo

Abstract Background It has been speculated that the biostimulatory effect of Low Level Laser Therapy could cause undesirable enhancement of tumor growth in neoplastic diseases. The aim of the present study is to analyze the behavior of melanoma cells (B16F10) in vitro and the in vivo development of melanoma in mice after laser irradiation. Methods We performed a controlled in vitro study on B16F10 melanoma cells to investigate cell viability and cell cycle changes by the Tripan Blue, MTT and cell quest histogram tests at 24, 48 and 72 h post irradiation. The in vivo mouse model (male Balb C, n = 21) of melanoma was used to analyze tumor volume and histological characteristics. Laser irradiation was performed three times (once a day for three consecutive days) with a 660 nm 50 mW CW laser, beam spot size 2 mm2, irradiance 2.5 W/cm2 and irradiation times of 60s (dose 150 J/cm2) and 420s (dose 1050 J/cm2) respectively. Results There were no statistically significant differences between the in vitro groups, except for an increase in the hypodiploid melanoma cells (8.48 ± 1.40% and 4.26 ± 0.60%) at 72 h post-irradiation. This cancer-protective effect was not reproduced in the in vivo experiment where outcome measures for the 150 J/cm2 dose group were not significantly different from controls. For the 1050 J/cm2 dose group, there were significant increases in tumor volume, blood vessels and cell abnormalities compared to the other groups. Conclusion LLLT Irradiation should be avoided over melanomas as the combination of high irradiance (2.5 W/cm2) and high dose (1050 J/cm2) significantly increases melanoma tumor growth in vivo.

A comparison between diuretics and angiotensin-receptor blocker agents in patients with stage I hypertension (PREVER-treatment trial): study protocol for a randomized double-blind controlled trial

Abstract Background Cardiovascular disease is the leading cause of death in Brazil, and hypertension is its major risk factor. The benefit of its drug treatment to prevent major cardiovascular events was consistently demonstrated. Angiotensin-receptor blockers (ARB) have been the preferential drugs in the management of hypertension worldwide, despite the absence of any consistent evidence of advantage over older agents, and the concern that they may be associated with lower renal protection and risk for cancer. Diuretics are as efficacious as other agents, are well tolerated, have longer duration of action and low cost, but have been scarcely compared with ARBs. A study comparing diuretic and ARB is therefore warranted. Methods/design This is a randomized, double-blind, clinical trial, comparing the association of chlorthalidone and amiloride with losartan as first drug option in patients aged 30 to 70 years, with stage I hypertension. The primary outcomes will be variation of blood pressure by time, adverse events and development or worsening of microalbuminuria and of left ventricular hypertrophy in the EKG. The secondary outcomes will be fatal or non-fatal cardiovascular events: myocardial infarction, stroke, heart failure, evidence of new subclinical atherosclerosis and sudden death. The study will last 18 months. The sample size will be of 1200 participants for group in order to confer enough power to test for all primary outcomes. The project was approved by the Ethics committee of each participating institution. Discussion The putative pleiotropic effects of ARB agents, particularly renal protection, have been disputed, and they have been scarcely compared with diuretics in large clinical trials, despite that they have been at least as efficacious as newer agents in managing hypertension. Even if the null hypothesis is not rejected, the information will be useful for health care policy to treat hypertension in Brazil. Clinical trials registration number ClinicalTrials.gov: NCT00971165