Optimization of Long-Acting Bronchodilator Dose Ratios Using Isolated Guinea Pig Tracheal Rings for Synergistic Combination Therapy in Asthma and COPD.

The co-administration of a long-acting ß2-agonist (LABA), and a long-acting muscarinic antagonist (LAMA), has been shown to be beneficial in the management of non-communicable chronic respiratory diseases, such as asthma and chronic obstructive pulmonary disease (COPD). The resulting relaxation of the airways can be synergistically enhanced, reducing symptoms and optimizing lung function. This provides an insight into more effective treatments. In this study, the LABAs formoterol fumarate dihydrate (FOR) and indacaterol maleate (IND) were each associated with tiotropium bromide monohydrate (TIO) to assess their synergistic potential. This was done using an appropriate ex vivo model of isolated perfused guinea pig tracheal rings, and pharmacological models of drug interaction. Among the dose ratios studied for both types of combination, a higher synergistic potential was highlighted for FOR/TIO 2:1 (w/w). This was done through three steps by using multiple additions of drugs to the organ baths based on a non-constant dose ratio and then on a constant dose ratio, and by a single addition to the organ baths of specific amounts of drugs. In this way, the synergistic improvement of the relaxant effect on the airways was confirmed, providing a basis for improving therapeutic approaches in asthma and COPD. The synergy found at this dose ratio should now be confirmed on a preclinical model of asthma and COPD by assessing lung function.

Polyphenolic and Methylxanthine Bioaccessibility of Cocoa Bean Shell Functional Biscuits: Metabolomics Approach and Intestinal Permeability through Caco-2 Cell Models.

Cocoa bean shell (CBS), a by-product with considerable concentrations of bioactive compounds and proven biofunctional potential, has been demonstrated to be a suitable ingredient for high-fiber functional biscuits adapted to diabetic consumers. In this work, the in vitro bioaccessibility and intestinal absorption of polyphenols and methylxanthines contained in these biscuits were evaluated, and the effect of the food matrix was studied. Biscuits containing CBS and the CBS alone underwent in vitro digestion followed by an intestinal permeability study. The results confirmed that compounds were less bioavailable in the presence of a food matrix, although the digestion contributed to their release from this matrix, increasing the concentrations available at the intestinal level and making them capable of promoting antioxidant and antidiabetic activities. After digestion, CBS biscuits were shown to possess α-glucosidase inhibition capacity comparable to that of acarbose. Moreover, the presence of the food matrix improved the stability of polyphenols throughout the digestion process. Intestinal absorption of flavan-3-ols seemed to be limited to a maximum threshold and was therefore independent of the sample, while procyanidin was not absorbed. Methylxanthine absorption was high and was boosted by the presence of the food matrix. The results confirmed the biofunctional potential of CBS-based biscuits.

Characterization of cytotoxic effects of aristolochic acids on the vascular endothelium.

Aristolochic acid nephropathy (AAN) is characterized by interstitial fibrosis, proximal tubular atrophy, and hypoxia. A correlation between a reduced peritubular capillary density and the severity of fibrosis has been demonstrated. As calcium, redox and energetic homeostasis are crucial in maintaining endothelial cell function and survival, we aimed to investigate AA-induced disturbances involved in endothelial cell injury. Our results showed a cytotoxic effect of AA on EAhy926 endothelial cells. Exposure of aortic rings to AA impaired vascular relaxation to Acetylcholine (ACh). Increased levels of intracellular reactive oxygen species (ROS) were observed in cells exposed to AA. Pre-treatment with antioxidant N-acetyl cysteine inhibited AA-induced cell death. Superoxide dismutase resulted in restoring ACh-induced relaxation. An increase in intracellular calcium level ([Ca2+]i) was observed on endothelial cells. Calcium chelators BAPTA-AM or APB, a specific inhibitor of IP3R, improved cell viability. Moreover, AA exposure led to reduced AMP-activated protein kinase (AMPK) expression. AICAR, an activator of AMPK, improved the viability of AA-intoxicated cells and inhibited the rise of cytosolic [Ca2+]i levels. This study provides evidence that AA exposure increases ROS generation, disrupts calcium homeostasis and decreases AMPK activity. It also suggests that significant damage observed in endothelial cells may enhance microcirculation defects, worsening hypoxia and tubulointerstitial lesions.

Vasorelaxant and antihypertensive effects of methanolic extracts from Hymenocardia acida Tul.

ETHNOPHARMACOLOGICAL RELEVANCE: In Congolese traditional medicine, decoctions of Hymenocardia acida root bark (HaRB) and trunk bark (HaTrB) are used for the treatment of conditions assumed to be hypertension. In this work, we propose to study the vasorelaxant effect of HaRB and HaTrB methanolic extracts on isolated rat thoracic aorta, to characterize the group of molecules responsible for the observed vasorelaxant activity, to evaluate the in vitro antioxidant activity of these extracts and to determine the antihypertensive activity of the HaRB extract on spontaneously hypertensive rats (SHR). MATERIALS AND METHODS: The vasorelaxant effect of the HaRB and HaTrB methanolic extracts was studied on endothelium-intact aortic rings pre-contracted with phenylephrine (PE, 1µM). The mechanism of this vasorelaxant effect was investigated on endothelium-denuded vessels and on endothelium-intact aortic rings in the presence of three inhibitors: l-N(G)-nitroarginine methyl ester (100µM), indomethacin (10µM) and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (10µM). To determine the nature of the compounds responsible for the vasorelaxant activity, we carried out a fractionation of the extracts and a thiolysis of the most active fraction followed by a liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) analysis. The extracts antioxidant activity was determined by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) colorimetric assay. In vivo anti-hypertensive activity of the HaRB extract was conducted on SHR. RESULTS: HaRB and HaTrB methanolic extracts produced a concentration-dependent vasorelaxation on intact aortic rings pre-contracted with PE (1µM). The vasorelaxant responses obtained were 95.3±1.5% (5µg/ml) and 100.6±3.0% (1µg/ml), respectively. The effect was markedly attenuated by removal of endothelium or pretreatment of aortic rings with all inhibitors except indomethacin. The LC/ESI-MS analysis of the thiolysis products indicated that the fraction which caused the most important vasorelaxation (97.9±2.5% at 3µg/ml) was a mixture of procyanidins and prodelphinidins, with a predominance of procyanidins. Both extracts and all fractions from HaRB extract showed a DPPH scavenging activity, ranging from 0.4 to 0.8 quercetin-equivalents. The HaRB methanolic extract reduced the systolic blood pressure in SHR (from 214±3mmHg to 194±4mmHg) after a 5-week treatment. CONCLUSIONS: The methanolic extracts of Hymenocardia acida root and trunk bark have vasorelaxant activity. The vasorelaxant effect observed is endothelium-dependent and seems mainly mediated through the NO-cGMP pathway. The COX pathway is not involved. The vasorelaxant activity appears to be due to polymeric procyanidins and prodelphinidins. These extracts also have an antioxidant effect. The extract of Hymenocardia acida root bark shows a significant but weak antihypertensive activity in SHR.

Vascular oxidative stress induced by diesel exhaust microparticles: synergism with hypertension.

BACKGROUND: Epidemiological and clinical studies have shown that traffic-related air pollution and, particularly, diesel exhaust particles (DEP) are strongly linked to cardiovascular mortality. METHODS: Vascular toxicity was studied by assessing vasomotor responses of aortas isolated from normotensive Wistar rats exposed in vitro to DEP (DEP suspension and aqueous DEP extract). In vivo experiments were performed on Wistar rats and spontaneously hypertensive rats (SHRs) exposed for 4 weeks via intratracheal instillation to either DEP or saline vehicle. After killing, vascular responses to acetylcholine (ACh) or sodium nitroprusside were assessed in vitro and the expression of p22phox, a major nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunit, was studied by real-time quantitative polymerase chain reaction. RESULTS: In aortas from Wistar rats, in vitro DEP incubation (both preparations) markedly inhibited the relaxations to ACh and slightly to sodium nitroprusside; this effect was reversed in the presence of superoxide dismutase. In contrast, in aortas from in vivo-exposed animals, ACh-induced relaxations were only significantly impaired in the SHR group, accompanied with a significant upregulation of p22phox and no change in systolic blood pressure. CONCLUSIONS: Although in vitro exposure to DEP produces a vascular oxidative stress, repeated in vivo exposures to DEP only impair vascular function in SHR, via an upregulation of p22phox. This suggests a synergistic effect on endothelial dysfunction between particulate air pollution and hypertension.

Vascular effects and antioxidant activity of two Combretum species from Democratic Republic of Congo.

ETHNOPHARMACOLOGICAL RELEVANCE: Combretum racemosum P. Beauv (Combretaceae) leaves (CrLv) and root bark (CrRB) and Combretum celastroides subsp. laxiflorum Welw (Combretaceae) leaves (ClLv) are used in Congolese traditional medicine for several therapeutic purposes, notably for the treatment of conditions consistent with hypertension. The present study aims to investigate the vasorelaxant and in vitro antioxidant activities of these plants polar extracts and to examine the in vivo antihypertensive effect of the extract which displays the most potent vasorelaxant effect. MATERIAL AND METHODS: The vasorelaxant effect of CrLv, CrRB and ClLv methanolic extracts was studied on rat aorta rings pre-contracted with phenylephrine (PE, 1 µM) in the presence or absence of the endothelium. In some experiments, prior to the addition of the extract, rings were incubated for 30 min with either L-N(G)-nitroarginine methyl ester (L-NAME; 100 µM), a nitric oxide synthase (NOS) inhibitor, indomethacin (10 µM), a cyclooxygenase inhibitor, or 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 10 µM), a guanylate cyclase inhibitor. The antioxidant activity was determined by the measurement of the scavenging ability of extracts towards the stable free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH). Blood pressure was measured on normotensive Wistar rats and spontaneously hypertensive rats (SHR) treated orally with a daily dose (40 mg/kg) of the CILv extract for 5 weeks. Tested extracts have been characterised by TLC profiles targeted at flavonoids. RESULTS: All tested extracts showed an important DPPH scavenging activity, ranging from 0.6 to 1.1 quercetin-equivalents. They caused a concentration-dependent vasorelaxation on intact aortic rings pre-contracted with PE (1 µM). The responses to CrRB and CrLv methanolic extracts reached 74.0±5.1% and 62.2±8.6% at a cumulative concentration of 50 µg/ml, respectively. The ClLv (10 µg/ml) extract was more active and, in the same conditions, relaxed aortic rings by 90.3±5.8%. The vasorelaxant activity of all extracts disappeared or was significantly attenuated by removal of the endothelium or after pretreatment with L-NAME or ODQ. Indomethacin only inhibited the activity of CrLv and CrRB extracts. The ClLv extract was able to lower the systolic blood pressure in SHR rats by 7% after a 5-week treatment. CONCLUSIONS: The present study shows that methanolic extracts from ClLv, CrRB and CrLv have an antioxidant activity and an endothelium-dependent vasorelaxant effect. ClLv induces the vasorelaxant effect through the NO-cGMP pathway while CrLv and CrRB extracts also act via a prostanoid pathway. ClLv extract demonstrated a modest but significant antihypertensive activity in SHR rats.