Cerebrovascular ß-amyloid deposition and associated microhemorrhages in a Tg2576 Alzheimer mouse model are reduced with a DHA-enriched diet.

Cerebral amyloid angiopathy (CAA) is a major contributor to Alzheimer's disease (AD) pathogenesis. Like AD, CAA is often accompanied by marked inflammation, aggravating associated vasculopathies. No evidence-based prevention or treatment strategies are available. Here, we evaluate the possible beneficial effect of a diet enriched with docosahexaenoic acid (DHA), which is known to attenuate inflammation in CAA. Tg2576 mice, a transgenic model of AD/CAA, were fed a DHA-enriched diet starting at 2 mo of age and ending at 10, 14, or 18 mo of age. ß-Amyloid (Aß)-peptide deposition and bleeding were visualized by immunohistochemistry or histochemistry on coronal sections of the brain. DHA, arachidonic acid, and eicosanoid levels were measured by liquid chromatography/mass spectrometry or GC-MS. DHA-enriched diet throughout aging limits the accumulation of vascular Aß peptide deposits as well as the likelihood of microhemorrhages. There is a strong correlation between systemic 12-hydroxyeicosatetraenoic acid (HETE) levels and the size of the area affected by both vascular amyloid deposits and hemorrhages. The lowest levels of 12-HETE, a lipid-derived proinflammatory product of 12-lipoxygenase (LOX), were found in DHA-fed mice. In vitro experiments performed on amyloid vascular smooth muscle cells showed that a 12-LOX inhibitor almost completely blocked the Aß1-40 peptide-induced apoptosis of these cells. This study yet again highlights the important role of inflammation in CAA pathogenesis and identifies potential new targets for preventive care.-Hur, J., Mateo, V., Amalric, N., Babiak, M., Béréziat, G., Kanony-Truc, C., Clerc, T., Blaise, R., Limon, I. Cerebrovascular ß-amyloid deposition and associated microhemorrhages in a Tg2576 Alzheimer mouse model are reduced with a DHA-enriched diet.

Characterization of V0162, a new long-acting antagonist at human M3 muscarinic acetylcholine receptors.

The anticholinergic properties of the mequitazine enantiomer V0162 make it a drug candidate for the treatment of chronic obstructive airway diseases. Here, we compared V0162's in vitro pharmacological activity at recombinant human M3 muscarinic acetylcholine receptors (hM3Rs) with that of other anticholinergics, using (i) a radioligand binding assay, (ii) a functional reporter gene assay and (iii) a bronchoconstriction inhibition assay on human bronchial preparations. V0162 had high affinity for hM3Rs, with a pKi varying from 9.01 after a 2 h incubation to 9.21 after 23 h. The other mequitazine enantiomer (V0114) was less potent. V0162 displayed rapid off-kinetics and a biphasic time course of binding. V0162 was found to be an antagonist behaving as an inverse agonist for hM3R-mediated reporter gene activation, with much the same efficacy as atropine, ipratropium and tiotropium. However, in contrast to ipratropium and atropine, V0162's inhibitory potency was only slightly affected by compound washout. V0162 antagonized acetylcholine-mediated contractions in a human bronchial preparation; the pA2 values increased with the incubation time (up to 2 h). Moreover, there was a progressive increase in V0162's ability to inhibit electrically-induced contractions, which persisted after compound washout. In conclusion, V0162 is the most active mequitazine enantiomer at hM3Rs and shows a complex pattern of binding to the membrane compartment. These particular features may be of therapeutic value when persistent antagonism at hM3Rs is required.

V0162 a new long-acting bronchodilator for treatment of chronic obstructive lung diseases: preclinical and clinical results.

BACKGROUND: Long acting bronchodilators are the standard of care in the management of chronic obstructive pulmonary disease (COPD). The aim of this study was to investigate the efficacy and safety of V0162, a novel anticholinergic agent with bronchodilator properties, in preclinical models and in patients with COPD. METHODS: Guinea pigs were used to evaluate the impact of V0162 on the acetylcholine or histamine-induced bronchoconstriction. V0162 was also investigated in an allergic asthma model on ovalbumin-sensitized guinea pig. For clinical investigations, healthy volunteers were included in a dose-escalation, randomized, placebo-controlled phase I study to determine the maximal tolerated dose, followed by a randomized, placebo-controlled, cross-over phase II study in patients with COPD. V0162 was given via inhalation route. The objectives of the phase I/II study were to assess the safety and efficacy of V0162, in terms of bronchodilation and reduction in hyperinflation. RESULTS: Preclinical results showed that V0162 was able to prevent bronchoconstriction induced either by acetylcholine or histamine. V0162 reversed the bronchoconstriction and airway inflammation caused by ovalbumin challenge in sensitized guinea pigs. In the healthy volunteers study, 88 subjects were enrolled: 66 received V0162 and 22 received placebo. No particular safety concerns were raised. The maximal tolerated dose was not reached and the dose escalation was stopped at 2400 µg. A total of 20 patients with COPD were then enrolled. All patients received a single-dose of V0162 1600 µg and of placebo in two alternating periods. In COPD patients, V0162 demonstrated a significant increase in FEV1 compared with placebo (148 ± 137 ml vs. 36 ± 151 ml, p = 0.003). This bronchodilatory effect was corroborated by a reduction in hyperinflation. There was a trend toward dyspnea relief (change in visual analog scale at 22 h, -15.1 ± 26.0 mm vs.- 5.3 ± 28.8 mm with placebo, p = 0.054). No serious adverse events (AEs) were reported. Most common AEs were productive and non-productive cough, dyspnea and pruritus. CONCLUSIONS: V0162 improved pulmonary function and tended to improve dyspnea in patients with COPD over more than 24 h. The slight plasmatic exposure observed might support the good safety profile. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT01348555.

In vitro and in vivo methotrexate disposition in alveolar macrophages: comparison of pharmacokinetic parameters of two formulations.

MTX-liposomes, prepared with a polymerised core (LSP), were administered in anaesthetised rats by pulmonary instillation versus free drug. No toxicological effects were macroscopically observed. After each time point: 15, 30, 60 and 90 min, animals were humanely killed and analyses of radio-signal were done. This approach allowed recovery of MTX or breakdown products within biological samples. Previously, kinetics of MTX cellular uptake was performed to identify the cytotoxic concentration of drug formulation for human macrophage. Flow cytometry was set-up to characterise liposomal uptake by ex vivo pulmonary macrophage. Cells were isolated by bronchioloalveolar washes from animals. Results have shown clear different pharmacokinetic parameters between free MTX and the liposomal form of MTX. Unlike classical liposomes, which are mainly taken up by the reticulo-endothelial system, LSP-MTX was not targeted to spleen or kidney. The route of administration could be an explanation of this phenomenon. In addition, LSP-MTX was more retained by the lung tissue. Moreover, free form of the drug reaches easily lymph node. This latest result should be taken into consideration for neoplasic disease and more specifically when lymph nodes are a way for pulmonary metastasis. Finally, LSP-MTX should be tested in physio-pathological model of lung cancer to evaluate the influence of the variation of liposomal formulation pharmacokinetic parameters on the drug efficacy.

Synthesis of new covalently bound kappa-carrageenan-AZT conjugates with improved anti-HIV activities.

This paper describes the first covalent synthesis of kappa-carrageenan-3'-azido-3'-deoxythymidine (AZT) conjugates. A succinate diester spacer was used to covalently couple AZT onto kappa-carrageenan, resulting in a tripartite prodrug. Two methods (UV and radioactive counting) are described and validated to determine the AZT loading onto the kappa-carrageenan carrier. This polymeric carrier, through its own intrinsic anti-HIV activity, is expected to act not only as a drug delivery agent but also as an anti-HIV agent. Synergism between the two drugs (kappa-carrageenan and AZT) was demonstrated when MT-4 cells were preincubated with the kappa-carrageenan-AZT conjugate prior to HIV-1-infection. A threshold of AZT loaded onto the kappa-carrageenan was required to achieve this synergistic effect. Such kappa-carrageenan-AZT conjugates could be of great therapeutic interest because these conjugates, which contain a low AZT concentration, present improved anti-HIV activities relative to free AZT. Moreover, kappa-carrageenan is a well-tolerated biopolymer, already used in the food industry.