Dual strategy to improve the oral bioavailability of efavirenz employing nanomicelles and curcumin as a bio-enhancer.

The present investigation was focused on the development of Soluplus®-based nanomicelles (NMs) (10 % w/v) loaded with Efavirenz (EFV) (5 mg/mL) and Curcumin (natural bio-enhancer) (CUR) (5, 10 and 15 mg/mL) to improve the oral bioavalability of EFV. Micellar formulations were obtained employing an acetone-diffusion technique. Apparent aqueous solubility was increased up to ∼1250-fold and 25,000-fold for EFV and CUR, respectively. Drug-loaded nanoformulations showed an excellent colloidal stability with unimodal size distribution and PDI values < 0.30. In vitro drug release was 41.5 % (EFV) and 2.6 % (CUR) from EFV-CUR-NMs over 6 h in simulated gastrointestinal fluids. EFV-CUR-loaded NMs resulted as safe nanoformulations according to the in vitro cytocompatibility assays in Caco-2 cells. Furthermore, CUR bio-enhancer activity was demonstrated for those nanoformulations. A CUR concentration of 15 mg/mL produced a significant (p < 0.05) increment (2.64-fold) of relative EFV oral bioavailability. Finally, the active role of the lymphatic system in the absorption process of EFV, after its oral administration was assessed in a comparative pharmacokinetic study in presence and absence of cycloheximide, a lymphatic transport inhibitor. Overall our EFV-CUR-NMs denoted their potential as a novel nanotechnological platform, representing a step towards an optimized "nano-sized" therapy for AIDS patients.

Formulation and Characterization of Ursodeoxycholic Acid Nanosuspension Based on Bottom-Up Technology and Box-Behnken Design Optimization.

BACKGROUND: Ursodeoxycholic acid (UDCA) is a therapeutic agent used for the treatment of cholestatic hepatobiliary diseases in pediatric patients. It is a bile acid that presents high lipophilicity, and it belongs to Class II of the Biopharmaceutical Classification System (BCS), which exhibits low water solubility and high intestinal permeability, which leads to poor oral absorption. The objective of this work was to design and optimize UDCA nanosuspensions by means of the precipitation-ultrasonication method to improve the solubility, dissolution, and oral bioavailability of UDCA. METHODS: A three-level, three-factor Box-Behnken design was used to optimize formulation variables and obtain uniform, small-particle-size UDCA nanosuspensions. The independent variables were: stabilizer percentage (X1), amplitude (X2), and sonication time (X3), and the dependent variable was the particle size (Y1). In the precipitation-ultrasonication method, UDCA was dissolved in acetone:PEG 400 (1:1 v/v) and quickly incorporated into the antisolvent (pre-cooled aqueous dispersion of HPMC E-15 0.3%), by means of intense sonication at 50 W for 5 min, controlling temperature through an ice water bath. The lyophilization efficacy was evaluated by means of a cryoprotective efficacy test, working with 10% maltose at -80 °C. The nanosuspensions were characterized by dynamic light scattering (DLS), X-ray diffraction, and scanning electron microscopy (SEM). The physicochemical stability was determined at 25 °C and 4 °C at 7, 14, 30, and 60 days, and the UDCA content was analyzed via HPLC-UV. An in vitro dissolution assay and an oral bioavailability study were performed in male Wistar rats. RESULTS: A significant impact was achieved in the optimized nanosuspension with 0.3% (stabilizer), 50 W (amplitude), and 5 min (sonication time), with a particle size of 352.4 nm, PDI of 0.11, and zeta potential of -4.30 mV. It presented adequate physicochemical stability throughout the study and the UDCA content was between 90% and 110%. In total, 86% of UDCA was dissolved in the in vitro dissolution test. The relative oral bioavailability was similar without significant statistical differences when comparing the lyophilized nanosuspension and the commercial tablet, the latter presenting a more erratic behavior. The pharmacokinetic parameters of the nanosuspension and the commercial tablet were Tmax (1.0 ± 0.9 h vs. 2.0 ± 0.8 h, respectively), Cmax (0.558 ± 0.118 vs. 0.366 ± 0.113 µM, respectively), ΔCmax (0.309 ± 0.099 vs. 0.232 ± 0.056, respectively), AUC (4.326 ± 0.471 vs. 2.188 ± 0.353 µg/mL.h, respectively, p < 0.02), and IAUC0-24h (2.261 ± 0.187 µg/mL.h vs. 1.924 ± 0.440 µg/mL.h, respectively). CONCLUSIONS: The developed nanosuspension presents an appropriate dosage and administration for pediatric patients. On the other hand, it exhibits an adequate absorption and UDCA oral bioavailability.

Characterization and bioavailability of a novel coenzyme Q10 nanoemulsion used as an infant formula supplement.

Supplementation with Coenzyme Q10 (CoQ10), in patients with its deficiency, has greater odds of success if the treatment is carried out early with an appropriate formulation. For neonatal CoQ10 deficiency, infant formula supplementation could be an attractive option. However, solid CoQ10 cannot be solubilized or dispersed in milk matrix leading to an inefficient CoQ10 dosage and poor intestinal absorption. We developed and characterized a high-dose CoQ10 oil-in-water (O/W) nanoemulsion suitable to supplement infant formula without modifying its organoleptic characteristics. CoQ10 powder and soy lecithin were solubilized in an oil phase consisted of Labrasol® and LabrafacTM. The aqueous phase was Tween 80, TPGS, methylparaben and propylparaben. O/W nanoemulsion was prepared by adding dropwise the oil phase to the aqueous phase under stirring to a final concentration of CoQ10 9.5 % w/w followed by ultrasonic homogenization. Pharmacotechnical parameters were determined. This formulation resulted to be easily to be dispersed in milk matrix, stable for at least 90 days, with no cytotoxicity in in vitro assays, and higher bioavailability than CoQ10 powder. CoQ10 nanoemulsion supplementation in the infant formula facilitates the individualized administration for the child with accurate dosage, overcome swallowing difficulties and in turn could increase the treatment adherence and efficacy.

New and developing pharmacotherapies for hypertension.

INTRODUCTION: Despite the significant contribution of hypertension to the global burden of disease, disease control remains poor worldwide. Considering this unmet clinical need, several new antihypertensive drugs with novel mechanisms of action are under development. AREAS COVERED: The present review summarizes the recent advances in the development of emerging pharmacological agents for the management of hypertension. The latest technological innovations in the design of optimized formulations of available antihypertensive drugs and the potential role of the modification of intestinal microbiota to improve blood pressure (BP) control are also covered. EXPERT OPINION: Significant efforts have been made to develop new antihypertensive agents with novel actions that target the main mechanisms involved in resistant hypertension. Sacubitril/valsartan may emerge as a potential first-line drug due to its superiority over renin angiotensin system inhibitors, and SGLT2 inhibitors can reduce BP in difficult-to-control hypertensive patients with type 2 diabetes. In addition, firibastat and aprocitentan may expand the therapeutic options for resistant hypertension by novel mechanism of actions. Since gut dysbiosis not only leads to hypertension but also causes direct target organ damage, prebiotics and probiotics could represent a potential strategy to prevent or reduce the development of hypertension and to contribute to BP control.

Efectos cardioprotectores de implantes subcutáneos de liberación crónica de carvedilol en ratas espontáneamente hipertensas / Cardioprotective Effects of Chronic Release Formulations of Subcutaneous Implants of Carvedilol in Spontaneously Hypertensive Rats

RESUMEN Introducción: En nuestro laboratorio hemos desarrollado implantes subcutáneos de carvedilol capaces de mantener niveles plasmáticos sostenidos del β-bloqueante durante 3 semanas. Objetivo: Evaluación de la liberación in vivo y la eficacia cardioprotectora de implantes subcutáneos de carvedilol desarrollados con poliepsilon- caprolactona (PCL) y Soluplus (SP) en ratas espontáneamente hipertensas (REH). Materiales y métodos: Se utilizaron 12 REH macho (250-300 g), a las cuales se colocó un implante subcutáneo cada 3 semanas de PCL: SP 100:50 mg (control, n = 6) o carvedilol: PCL:SP (100mg:100mg:50mg) (carvedilol, n = 6). Se evaluó el perfil plasmático y el efecto sobre la presión arterial sistólica (PAS) durante 62 días. Al final del tratamiento, se realizaron determinaciones ecocardiográficas y la medición de la PAS y. la presión arterial media (PAM) directas. Resultados: El grupo que recibió el implante conteniendo 100 mg de carvedilol presentó concentraciones plasmáticas del fármaco en el rango de 100- 500 ng/mL a lo largo de 62 días de tratamiento, luego del cual la PAS fue 20 mmHg menor que en el grupo control (217 ± 3 mmHg vs. 237 ± 6 mmHg; p <0,05). Las PAS y PAM directas fueron significativamente menores el grupo tratado que en el control. El implante de carvedilol 100 mg redujo la variabilidad de la presión arterial (VPA) de corto plazo en comparación con el control. Parámetros ecocardiográficos como la fracción de eyección del ventrículo izquierdo (FEVI), fracción de acortamiento, y relación E/A fueron significativamente mayores en las ratas tratadas. El peso del VI fue menor en las ratas que recibieron el implante con carvedilol. Conclusión: Los implantes conteniendo CAR/PCL/SP (100:100:50) mg aportan niveles plasmáticos terapéuticos de carvedilol y estables durante el transcurso del tratamiento, los cuales se correlacionan con una disminución significativa y sostenida de los valores de PA indirecta. El tratamiento con los implantes de carvedilol logró atenuar los valores de PA directa y su variabilidad en las REH. Se demostró que el tratamiento con los implantes ejerció un efecto cardioprotector evidenciado en el ecocardiograma y por una reducción de la hipertrofia ventricular izquierda.

ABSTRACT Background: In our laboratory, we have developed subcutaneous implants of carvedilol capable of maintaining stable concentrations of the β-blocker during 3 weeks. Objective: The aim of this study was to evaluate the in vivo release and the cardioprotective efficacy of subcutaneous implants of carvedilol developed with poly-epsilon-caprolactone (PCL) and Soluplus (SP) polymers in spontaneously hypertensive rats (SHR). Methods: Twelve spontaneously hypertensive male rats (250-300 g) underwent placement of subcutaneous implant of PCL:SP 100:50 mg (control group, n=6) or carvedilol:PCL:SP (100mg:100mg:50mg) (carvedilol group, n=6), every 3 weeks. The plasma profile of each implant and its effect on systolic blood pressure (SBP) was evaluated for 62 days. At the end of treatment, echocardiographic parameters were determined, and direct SBP and direct mean arterial pressure (MAP) were measured. Results: The group that received the implant containing 100 mg of carvedilol presented plasma concentrations of the drug in the range of 100- 500 ng/mL throughout 62 days of treatment, after which the SBP was 20 mmHg lower than in the control group (217±3 mm Hg vs. 237±6 mm Hg; p <0.05). Direct SBP and MAP were significantly lower in the treated group than in the control group. The implant loaded with carvedilol 100 mg reduced short-term blood pressure variability (BPV) in SHR compared with the control group. Echocardiographic parameters as left ventricular ejection fraction (LVEF), shortening fraction and E/A ratio were significantly greater in treated rats. Left ventricular weight was lower in the rats with carvedilol implant. Conclusion: Implants containing CAR/PCL/SP (100:100:50) mg provide therapeutic and stable plasma levels of carvedilol during treatment, which correlate with a significant and sustained decrease in indirect BP values. Treatment with carvedilol implants attenuated dirct BP values and blood pressure variability in SHR. Treatment with implant produced cardioprotective effects evidenced in the echocardiogram by a reduction in left ventricular hypertrophy.

Plasma membrane calcium ATPase downregulation in dopaminergic neurons alters cellular physiology and motor behaviour in Drosophila melanogaster.

The accumulation of Ca2+ and its subsequent increase in oxidative stress is proposed to be involved in selective dysfunctionality of dopaminergic neurons, the main cell type affected in Parkinson's disease. To test the in vivo impact of Ca2+ increment in dopaminergic neurons physiology, we downregulated the plasma membrane Ca2+ ATPase (PMCA), a pump that extrudes cytosolic Ca2+ , by expressing PMCARNAi in Drosophila melanogaster dopaminergic neurons. In these animals, we observed major locomotor alterations paralleled to higher cytosolic Ca2+ and increased levels of oxidative stress in mitochondria. Interestingly, although no overt degeneration of dopaminergic neurons was observed, evidences of neuronal dysfunctionality were detected such as increases in presynaptic vesicles in dopaminergic neurons and in the levels of dopamine in the brain, as well as presence of toxic effects when PMCA was downregulated in the eye. Moreover, reduced PMCA levels were found in a Drosophila model of Parkinson's disease, Parkin knock-out, expanding the functional relevance of PMCA reduction to other Parkinson's disease-related models. In all, we have generated a new model to study motor abnormalities caused by increments in Ca2+ that lead to augmented oxidative stress in a dopaminergic environment, added to a rise in synaptic vesicles and dopamine levels.

Nebivolol is more effective than atenolol for blood pressure variability attenuation and target organ damage prevention in L-NAME hypertensive rats.

ß-Adrenergic blockers are no longer recommended as first-line therapy due to the reduced cardioprotection of traditional ß-blockers compared with other antihypertensive drugs. It is unknown whether third-generation ß-blockers share the limitations of traditional ß-blockers. The aim of the present study was to compare the effects of nebivolol or atenolol on central and peripheral systolic blood pressure (SBP) and its variability and target organ damage (TOD) in N-nitro-L-arginine methyl ester (L-NAME) hypertensive rats. Male Wistar rats were treated with L-NAME for 8 weeks together with oral administration of nebivolol 30 mg/kg (n = 8), atenolol 90 mg/kg (n = 8), or vehicle (n = 8). The control group was composed of vehicle-treated Wistar rats. SBP and its variability, as well as echocardiographic parameters, were assessed during the last 2 weeks of treatment. Tissue levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and transforming growth factor ß (TGF-ß), and histopathological parameters were evaluated in the left ventricle and aorta. Nebivolol had a greater ability than atenolol to decrease central SBP and mid-term and short-term blood pressure variability (BPV) in L-NAME rats. Echocardiographic analysis showed that nebivolol was more effective than atenolol on E/A wave ratio normalization. Compared with atenolol treatment, nebivolol had a greater protective effect on different TOD markers, inducing a decrease in collagen deposition and a reduction in the proinflammatory cytokines IL-6 and TNF-α in the left ventricle and aorta. Our findings suggest that the adverse hemodynamic profile and the reduced cardiovascular protection reported with traditional ß-blockers must not be carried forward to third-generation ß-blockers.

Bioavailability of coenzyme Q10 loaded in an oleogel formulation for oral therapy: Comparison with a commercial-grade solid formulation.

Coenzyme Q10 (CoQ10) is essential in mitochondrial bioenergetics and is a potent endogenous antioxidant. Low CoQ10 levels are associated with neurodegenerative, metabolic, muscular and cardiovascular disorders. Early treatment with high doses (5-50 mg/kg/day) demonstrated to limit the onset and progression of neuropathology. Recently, we developed an oleogel matrix able to support a high dose of oil-dissolved CoQ10, easy to swallow by CoQ10-deficient patients who suffer from secondary dysphagia. In the present study, we evaluated the bioavailability of oleogel-dissolved CoQ10 and plasma antioxidant status in healthy adults in single-dose and repeated-dose studies. The single-dose study demonstrated that, in terms of CoQ10 bioavailability, 1 g CoQ10/5g oleogel-disk was equivalent to the solid form (1 g CoQ10/three 00-size-capsules), whereas the repeated-dose study (14-days-administration) demonstrated a significantly higher increase in plasma CoQ10 when administered through the oleogel, which could be compatible with the levels necessary to achieve an adequate therapeutic response. Also, a trend to a higher plasma apparent half-life (greater than24 h) was observed for the oleogel-loaded-CoQ10. In conclusion, the oleogel matrix does not compromise the oil-dissolved CoQ10 bioavailability and can prevent the non-adherence to this vital supplementation in patients with high CoQ10 requirements. No significant variation in the plasma antioxidant status (vitamins A, E and C, glutathione and TBARs) was observed.

Desarrollo de implantes subcutáneos de liberación controlada de Carvedilol para la reducción sostenida de la presión arterial en modelos experimentales de hipertensión arterial / Development of Subcutaneous Implants of Controlled Release Formulations of Carvedilol for Sustained Blood Pressure Reduction in Experimental Models of Hypertension

RESUMEN Objetivo: El objetivo del presente estudio fue el desarrollo y la evaluación farmacocinética y farmacodinámica de la liberación in vivo de implantes subcutáneos de carvedilol capaces de aportar niveles tisulares estables en modelos experimentales de hipertensión arterial. La incorporación del polímero hidrofílico SoluPlus (SP) en los implantes PCL:SP 150:150 y 50:250 favorece un incremento de la liberación de carvedilol dado que aporta concentraciones plasmáticas en el rango de 100-200 ng/mL durante 2 semanas, lo que tiene como resultado una reducción sostenida de la presión arterial sistólica indirecta en animales SHR. Material y métodos: Se prepararon implantes subcutáneos de poli (epsilon-caprolactona) (PCL) con diferentes proporciones del polímero hidrofílico SoluPlus (300:0; 250:50; 150:150 y 50:250 mg) cargados con 100 mg de carvedilol. Se evaluó el perfil plasmático y el efecto sobre la presión arterial sistólica (PAS) luego del implante de cada formulación en el tejido subcutáneo de ratas espontáneamente hipertensas (REH) macho. Resultados: Las formulaciones PCL:SP 50:250 y 150:150 aportaron niveles en el rango de 100-200 ng/mL. Las formulaciones PCL:SP 250:50 y 300:0 aportaron concentraciones inferiores de carvedilol comprendidas en el rango de los 0-100 ng/mL durante el transcurso del tratamiento. Los animales espontáneamente hipertensos tratados con PCL:SP 50:250 y 150:150 experimentaron un descenso significativo de la presión arterial sistólica (PCL:SP 50:250: DPAS: -36,6 ± 2,0 mmHg; PCL:SP150:150: 35,7 ± 2,2 mmHg; p <0,05 vs. basal). Conclusiones: La incorporación del polímero hidrofílico SoluPlus en los implantes PCL:SP 150:150 y 50:250 favorece un incremento de la liberación de carvedilol, ya que aporta concentraciones plasmáticas del β-bloqueante que aseguran una reducción sostenida de la PAS indirecta en animales espontáneamente hipertensos.

ABSTRACT Objective: The aim of this study was the development and pharmacokinetic/pharmacodynamic evaluation of the in vivo release of subcutaneous implants of carvedilol capable of providing stable tissue levels in experimental models of hypertension. Methods: The subcutaneous implants were prepared with poly (epsilon-caprolactone) (PCL) and different proportions of the SoluPlus (SP) hydrophilic polymer (300:0; 250:50; 150:150 and 50:250 mg) loaded with 100 mg carvedilol. The plasma profile and the effect on systolic blood pressure (SBP) after subcutaneous implantation of each formulation was evaluated in male spontaneously hypertensive rats (SHR). Results: The PCL:SP 50:250 and 150:150 formulations provided levels ranging from 100 to 200 ng/mL and the PCL:SP 250:50 and 300:0 formulations provided lower concentrations of carvedilol ranging from 0 to 100 ng/mL during the treatment period. Spontaneously hypertensive animals treated with the PCL:SP 50:250 y 150:150 implants presented a significant decrease in SBP (PCL:SP 50:250: DPAS: -36.6 ± 2.0 mm Hg; PCL:SP150:150: -35.7 ± 2.2 mmHg; p <0.05 vs. baseline values) Conclusions: The incorporation of the SoluPlus hydrophilic polymer in PC:SP 150:150 and 50:250 implants increases the release of carvedilol, since it provides plasma concentrations ranging from 100 to 200 ng/ml, resulting in a sustained reduction of indirect SBP in SHR.

Neurochemical, Behavioral, and Neurogenic Validation of a Hyposerotonergic Animal Model by Voluntary Oral Consumption of para-Chlorophenylalanine.

The inhibitor of tryptophan hydroxylase, para-chlorophenylalanine (PCPA), has been classically employed as a pharmacological tool to deplete serotonin (5-HT) in animal models and to evaluate whether this neurotransmitter is involved in the action of pharmacological compounds. PCPA is usually administrated by intraperitoneal (ip) injections, which are stressful and painful. To avoid ip injections, we designed and validated a protocol for PCPA oral administration. C57BL/6 elite male mice received PCPA during 7 days either ip or by giving the drug inside jelly cubes at an estimated dose of 500 mg/kg on days 1 and 2 and 250 mg/kg for the rest of the treatment. 5-HT levels decreased by 85% and 55% in the hippocampus of mice treated with oral or ip PCPA, respectively, whereas in the prefrontal cortex, 5-HT levels decreased by 65% (oral) and 50% (ip). Behavioral tests, like the forced swimming test (FST), the nestlet shredding test (NST), and the marble burying test (MBT), were performed. In the FST, mice received fluoxetine ip 30 min before the test. In mice with oral PCPA treatment, fluoxetine did not induce significant reductions of immobility, indicating that reduction of 5-HT levels was effective. No effect of ip or oral 5-HT depletion was observed in the NST nor in the MBT. In a second experiment, mice received oral PCPA for 8 weeks: again, serotonin levels were significantly decreased in both hippocampus and cortex, and effects on hippocampal neurogenesis replicated previous observations in hyposerotonergic mice. Therefore, neurochemical, behavioral, and neurogenic results allow us to validate this refined protocol for voluntary oral consumption of PCPA.

Effects of third-generation ß-blockers, atenolol or amlodipine on blood pressure variability and target organ damage in spontaneously hypertensive rats.

BACKGROUND: ß-blockers are no longer considered as first-line antihypertensive drugs due to their lower cardioprotection. METHOD: Considering the differences in the pharmacological properties of ß-blockers, the present work compared the effects of third-generation ß-blockers - carvedilol and nebivolol - with a first-line agent - amlodipine - on hemodynamic parameters, including short-term blood pressure variability (BPV), and their ability to prevent target organ damage in spontaneously hypertensive rats (SHR). SHR rats were orally treated with carvedilol, nebivolol, atenolol, amlodipine or vehicle for 8 weeks. Wistar Kyoto rats treated with vehicle were used as normotensive group. Echocardiographic evaluation, BP, and short-term BPV measurements were performed. Left ventricle and thoracic aorta were removed for histological evaluations and to assess the expression of transforming growth factor ß (TGF-ß), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). RESULTS: Carvedilol, nebivolol or amlodipine induced a greater reduction of carotid BP, short-term BPV and echocardiography parameters than atenolol in SHR rats. Carvedilol, nebivolol and amlodipine were more effective than atenolol in the prevention of cardiac hypertrophy, and cardiac and aortic collagen deposit. Carvedilol and nebivolol, but not atenolol, reduced the expressions of fibrotic and inflammatory biomarkers - TGF-ß, TNF-α and IL-6 - in SHR rats to a similar extent to that of amlodipine. CONCLUSION: Chronic treatment with carvedilol or nebivolol attenuates carotid BP and short-term BPV, and reduces target organ damage in SHR to a greater extent than atenolol. Our findings suggest that the lower cardiovascular protection of nonvasodilating ß-blockers, as atenolol, in hypertension must not be translated to third-generation ß-blockers.

Circadian modulation of motivation in mice.

Most living organisms have a circadian timing system adapted to optimize the daily rhythm of exposure to the environment. This circadian system modulates several behavioral and physiological processes, including the response to natural and drug rewards. Food is the most potent natural reward across species. Food-seeking is known to be mediated by dopaminergic and serotonergic transmission in cortico-limbic pathways. In the present work, we show evidence of a circadian modulation of motivation for food reward in young (4-months old) and aged (over 1.5 years old) C57BL/6 mice. Motivation was assayed through the progressive ratio (PR) schedule. Mice under a 12:12 light/dark (LD) cycle exhibited a diurnal rhythm in motivation, becoming more motivated during the night, coincident with their active phase. This rhythm was also evident under constant dark conditions, indicating the endogenous nature of this modulation. However, circadian arrhythmicity induced by chronic exposure to constant light conditions impaired the performance in the task causing low motivation levels. Furthermore, the day/night difference in motivation was also evident even without caloric restriction when using a palatable reward. All these results were found to be unaffected by aging. Taken together, our results indicate that motivation for food reward is regulated in a circadian manner, independent of the nutritional status and the nature of the reward, and that this rhythmic modulation is not affected by aging. These results may contribute to improve treatment related to psychiatric disorders or drugs of abuse, taking into account potential mechanisms of circadian modulation of motivational states.

Role of indoleamine 2,3-dioxygenase in testicular immune-privilege.

Male meiotic germ cell including the spermatozoa represent a great challenge to the immune system, as they appear long after the establishment of normal immune tolerance mechanisms. The capacity of the testes to tolerate autoantigenic germ cells as well as survival of allogeneic organ engrafted in the testicular interstitium have led to consider the testis an immunologically privileged site. Disruption of this immune privilege following trauma, tumor, or autoimmune orchitis often results in male infertility. Strong evidence indicates that indoleamine 2,3-dioxygenase (IDO) has been implicated in fetal and allograft tolerance, tumor immune resistance, and regulation of autoimmune diseases. IDO and tryptophan 2,3-dioxygenase (TDO) catalyze the same rate-limiting step of tryptophan metabolism along a common pathway, which leads to tryptophan starvation and generation of catabolites collectively known as kynurenines. However, the relevance of tryptophan metabolism in testis pathophysiology has not yet been explored. Here we assessed the in vivo role of IDO/TDO in experimental autoimmune orchitis (EAO), a model of autoimmune testicular inflammation and immunologically impaired spermatogenesis. EAO was induced in adult Wistar rats with testicular homogenate and adjuvants. Control (C) rats injected with saline and adjuvants and normal untreated rats (N) were also studied. mRNA expression of IDO decreased in whole testes and in isolated Sertoli cells during EAO. TDO and IDO localization and level of expression in the testis were analyzed by immunostaining and Western blot. TDO is expressed in granulomas from EAO rats, and similar protein levels were observed in N, C, and EAO groups. IDO was detected in mononuclear and endothelial cells and reduced IDO expression was detected in EAO group compared to N and C rats. This phenomenon was concomitant with a significant reduction of IDO activity in EAO testis measured by tryptophan and kynurenine concentrations (HPLC). Finally, in vivo inhibition of IDO with 1-methyl-tryptophan increased severity of the disease, demonstrating down regulation of IDO-based tolerance when testicular immune regulation was disrupted. We present evidence that an IDO-based mechanism is involved in testicular immune privilege.

Adenosine A1 receptors and mitochondria: targets of remote ischemic preconditioning.

Adenosine is involved in classic preconditioning in most species and acts especially through adenosine A1 and A3 receptors. The aim of the present study was to evaluate whether remote ischemic preconditioning (rIPC) activates adenosine A1 receptors and improves mitochondrial function, thereby reducing myocardial infarct size. Isolated rat hearts were subjected to 30 min of global ischemia and 60 min of reperfusion [ischemia-reperfusion (I/R)]. In a second group, before isolation of the heart, a rIPC protocol (3 cycles of hindlimb I/R) was performed. Infarct size was measured with tetrazolium staining, and Akt/endothelial nitric oxide (NO) synthase (eNOS) expression/phosphorylation and mitochondrial function were evaluated after ischemia at 10 and 60 min of reperfusion. As expected, rIPC significantly decreased infarct size. This beneficial effect was abolished only when 8-cyclopentyl-1,3-dipropylxanthine (adenosine A1 receptor blocker) and NG-nitro-l-arginine methyl ester (NO synthesis inhibitor) were administered during the reperfusion phase. At the early reperfusion phase, rIPC induced significant Akt and eNOS phosphorylation, which was abolished by the perfusion with an adenosine A1 receptor blocker. I/R led to impaired mitochondrial function, which was attenuated by rIPC and mediated by adenosine A1 receptors. In conclusion, we demonstrated that rIPC limits myocardial infarct by activation of adenosine A1 receptors at early reperfusion in the isolated rat heart. Interestingly, rIPC appears to reduce myocardial infarct size by the Akt/eNOS pathway and improves mitochondrial function during myocardial reperfusion. NEW & NOTEWORTHY Adenosine is involved in classic preconditioning and acts especially through adenosine A1 and A3 receptors. However, its role in the mechanism of remote ischemic preconditioning is controversial. In this study, we demonstrated that remote ischemic preconditioning activates adenosine A1 receptors during early reperfusion, inducing Akt/endothelial nitric oxide synthase phosphorylation and improving mitochondrial function, thereby reducing myocardial infarct size.

Factors influencing hepatic metabolism of antihypertensive drugs: impact on clinical response.

INTRODUCTION: Although main antihypertensive drugs are able to efficiently reduce blood pressure, only a third of treated hypertensive patients achieve optimal blood pressure control. Extensive interpatient variability on drug metabolism and oral disposition of blood pressure lowering drugs can contribute to this failure in hypertension management. Areas covered: The aim of the present review is to update the knowledge on the features of hepatic metabolism of the main antihypertensive agents, including ß-blockers, calcium channel blockers, angiotensin receptor blockers, and angiotensin converting enzyme inhibitors. The factors that contribute to the large interindividual variability of main antihypertensive drugs are also covered. Expert opinion: The variability of plasma concentration of antihypertensive drugs due to the involvement of hepatic metabolism can contribute to the inadequate control of blood pressure in the daily clinical practice. Genotype screening of specific hepatic drug-metabolizing enzymes may contribute to optimize dose selection and to increase the rate of blood pressure control in patients treated with specific ß-blockers, calcium channel blockers, and angiotensin receptor blockers.

Sociability deficits after prenatal exposure to valproic acid are rescued by early social enrichment.

Background: Autism spectrum disorder (ASD) is characterized by impaired social interactions and repetitive patterns of behavior. Symptoms appear in early life and persist throughout adulthood. Early social stimulation can help reverse some of the symptoms, but the biological mechanisms of these therapies are unknown. By analyzing the effects of early social stimulation on ASD-related behavior in the mouse, we aimed to identify brain structures that contribute to these behaviors. Methods: We injected pregnant mice with 600-mg/kg valproic acid (VPA) or saline (SAL) at gestational day 12.5 and evaluated the effect of weaning their offspring in cages containing only VPA animals, only SAL animals, or mixed. We analyzed juvenile play at PD21 and performed a battery of behavioral tests in adulthood. We then used preclinical PET imaging for an unbiased analysis of the whole brain of these mice and studied the function of the piriform cortex by c-Fos immunoreactivity and HPLC. Results: Compared to control animals, VPA-exposed animals play less as juveniles and exhibit a lower frequency of social interaction in adulthood when reared with other VPA mice. In addition, these animals were less likely to investigate social odors in the habituation/dishabituation olfactory test. However, when VPA animals were weaned with control animals, these behavioral alterations were not observed. Interestingly, repetitive behaviors and depression-related behaviors were not affected by social enrichment. We also found that VPA animals present high levels of glucose metabolism bilaterally in the piriform cortex (Pir), a region known to be involved in social behaviors. Moreover, we found alterations in the somatosensory, motor, and insular cortices. Remarkably, these effects were mostly reversed after social stimulation. To evaluate if changes in glucose metabolism in the Pir correlated with changes in neuronal activity, we measured c-Fos immunoreactivity in the Pir and found it increased in animals prenatally exposed to VPA. We further found increased dopamine turnover in the Pir. Both alterations were largely reversed by social enrichment. Conclusions: We show that early social enrichment can specifically rescue social deficits in a mouse model of ASD. Our results identified the Pir as a structure affected by VPA-exposure and social enrichment, suggesting that it could be a key component of the social brain circuitry.

Deficits in temporal processing in mice prenatally exposed to Valproic Acid.

Temporal processing in the seconds-to-minutes range, known as interval timing, is a crucial cognitive function that requires activation of cortico-striatal circuits via dopaminergic-glutamatergic pathways. In humans, both children and adults with autism spectrum disorders (ASD) present alterations in their estimation of time intervals. At present, there are no records of interval timing studies in animal models of ASD. Hence, the objective of the present work was to evaluate interval timing in a mouse model of prenatal exposure to valproic acid (VPA) - a treatment used to induce human-like autistic features in rodent models. Animals were assessed for their ability to acquire timing responses in 15-s and 45-s peak-interval (PI) procedures. Our results indicate that both female and male mice prenatally exposed to VPA present decreased timing accuracy and precision compared to control groups, as well as deviations from the scalar property. Moreover, the observed timing deficits in male VPA mice were reversed after early social enrichment. Furthermore, catecholamine determination by HPLC-ED indicated significant differences in striatal dopaminergic, but not serotonergic, content in female and male VPA mice, consistent with previously identified alterations in dopamine metabolism in ASD. These deficits in temporal processing in a mouse model of autism complement previous results in humans, and provide a useful tool for further behavioral and pharmacological studies.

Nicotine-induced molecular alterations are modulated by GABAB receptor activity.

It has been demonstrated that GABAB receptors modulate nicotine (NIC) reward effect; nevertheless, the mechanism implicated is not well known. In this regard, we evaluated the involvement of GABAB receptors on the behavioral, neurochemical, biochemical and molecular alterations associated with the rewarding effects induced by NIC in mice, from a pharmacological and genetic approach. NIC-induced rewarding properties (0.5 mg/kg, subcutaneously, sc) were evaluated by conditioned place preference (CPP) paradigm. CPP has three phases: preconditioning, conditioning and postconditioning. GABAB receptor antagonist 2-hydroxysaclofen (0.25, 0.5 and 1 mg/kg; intraperitoneally, ip) or the GABAB receptor agonist baclofen (3 mg/kg; ip) was injected before NIC during the conditioning phase. GABAB1 knockout (GABAB1 KO) mice received NIC during the conditioning phase. Vehicle and wild-type controls were employed. Neurochemical (dopamine, serotonin and their metabolites), biochemical (nicotinic receptor α4ß2, α4ß2nAChRs) and molecular (c-Fos) alterations induced by NIC were analyzed after the postconditioning phase by high-performance liquid chromatography (HPLC), receptor-ligand binding assays and immunohistochemistry, respectively, in nucleus accumbens (Acb), prefrontal cortex (PFC) and ventral tegmental area (VTA). NIC induced rewarding effects in the CPP paradigm and increased dopamine levels in Acb and PFC, α4ß2nAChRs density in VTA and c-Fos expression in Acb shell (AcbSh), VTA and PFC. We showed that behavioral, neurochemical, biochemical and molecular alterations induced by NIC were prevented by baclofen. However, in 2-hydroxysaclofen pretreated and GABAB1 KO mice, these alterations were potentiated, suggesting that GABAB receptor activity is necessary to control alterations induced by NIC-induced rewarding effects. Therefore, the present findings provided important contributions to the mechanisms implicated in NIC-induced rewarding effects.

What is the Real Efficacy of Beta-Blockers for the Treatment of Essential Hypertension?

OBJECTIVE: This review covers the pharmacokinetics and pharmacodynamic of ß-blockers, the rationale for their use, some recent controversies in its use for managing hypertension, as well as, the beneficial properties of the third-generation ß-blockers beyond hypertension. BACKGROUND: The efficacy and safety of ß-blockers in the treatment of hypertension and other cardiovascular diseases have been established during more than 50 years of clinical experience. Recent updates of clinical guidelines have downgraded the use of ß-blockers for the treatment of uncomplicated hypertension to second and third line therapy. It is a well-known fact that ß-blockers exhibit heterogeneous pharmacokinetic and pharmacodynamic properties that clearly influence their clinical efficacy and tolerability in the management of essential hypertension. Conventional nonvasodilating ß-blockers (atenolol and metoprolol) are inferior to first-line antihypertensive agents in terms of cardioprotection due to lower ability to reduce central blood pressure and its variability and the adverse effects on glycemic and lipid metabolism. CONCLUSION: New vasodilating ß-blockers, mainly carvedilol and nebivolol, show enhanced hemodynamic and metabolic properties, which probably result in a higher prevention of major cardiovascular events in hypertensive patients. Despite head-to-head clinical trials comparing the effects of vasodilating vs nonvasodilating ß-blockers on hard clinical endpoints are lacking, the current evidence suggests that third-generation ß-blockers are superior to conventional ß-blockers for the prevention of cardiovascular events in patients with essential hypertension. Moreover, beyond their antihypertensive properties, third-generation ß-blockers also have pleiotropic, antioxidant and antiinflammatory effects that warrant a "promissory new era" of this newly group.

Effects of carvedilol or amlodipine on target organ damage in L-NAME hypertensive rats: their relationship with blood pressure variability.

The aim of the study was to compare the effects of chronic oral treatment with carvedilol or amlodipine on blood pressure, blood pressure variability and target organ damage in N-nitro-l-arginine methyl ester (L-NAME) hypertensive rats. Wistar rats were treated with L-NAME administered in the drinking water for 8 weeks together with oral administration of carvedilol 30 mg/kg (n = 6), amlodipine 10 mg/kg (n = 6), or vehicle (n = 6). At the end of the treatment, echocardiographic evaluation, blood pressure, and short-term variability measurements were performed. Left ventricular and thoracic aortas were removed to assess activity of metalloproteinase 2 and 9 and expression levels of transforming growth factor ß, tumor necrosis factor α, and interleukin 6. Histological samples were prepared from both tissues. Carvedilol and amlodipine induced a comparable reduction of systolic and mean arterial pressure and its short-term variability in L-NAME rats. The expression of transforming growth factor ß, tumor necrosis factor α, and interleukin 6 decreased in both organs after carvedilol or amlodipine treatment and the activity of metalloproteinase was reduced in aortic tissue. Treatment with carvedilol or amlodipine completely prevented left ventricular collagen deposition and morphometric alterations in aorta. Oral chronic treatment with carvedilol or amlodipine significantly attenuates blood pressure variability and reduces target organ damage and biomarkers of tissue fibrosis and inflammation in L-NAME hypertensive rats.