Muscarinic subtype affinity and functional activity profile of 1-methyl-2-(2-methyl-1,3-dioxolan-4-yl)pyrrolidine and 1-methyl-2-(2-methyl-1,3-oxathiolan-5-yl)pyrrolidine derivatives

Starting from two previously studied muscarinic full agonists, characterized by a 1,3-dioxolane ((+)-1) and a 1,3-oxathiolane ((+)-2) cycle, two new series of muscarinic ligands were designed, obtained by the steric complication of the parent compounds produced by freezing the aminoalkyl chain into a pyrrolidine ring. Both tertiary amines and the corresponding iodomethyl derivatives were synthesised and studied, and several compounds of the series which behaved as muscarinic agonists have been selected, on the basis of preliminary binding experiments on rat cortex homogenates, for the present work. Results are presented obtained from testing the affinity of the selected compounds against cloned human muscarinic receptors expressed in CHO cells, in order to evaluate subtype selectivity. Their functional activity on classical models of M1-M4 receptors, in guinea pig and rabbit tissues is also reported. With respect to parent compounds, the new molecules present some selectivity toward hm2 receptors; fair M2 selectivity is also evident in functional studies, where these compounds behave as partial agonists. Among the other compounds of the series (2S, 4'R, 2'S)-1,1-dimethyl-2-(2-methyl-1,3-dioxolan-4-yl)pyrrolidinium iodide (-)-3 and (2R, 5'S, 2'S)-1-methyl-2-(2-methyl-1,3-oxathiolan-5-yl)pyrrolidine (+)-5 present a promising pharmacological profile. Compound (-)-3 shows modest hm2 selectivity in binding experiments but a clearcut M2 selectivity in functional tests, where it behaves as a weak antagonist on M1 and M4 subtypes, as a weak full agonist on the M3 subtype and as a potent partial agonist on M2 subtype. Tertiary amine (+)-5 presents a quite similar profile but appears more interesting since, lacking a permanent charge on the nitrogen atom, it may represent an interesting tool to study CNS muscarinic receptors. Our results confirm that sterical complication of parent compounds (+)-1 and (+)-2 produces more selective muscarinic agonists.     

Structural requisites of 2-(p-chlorophenoxy)propionic acid analogues for activity on native rat skeletal muscle chloride conductance and on heterologously expressed CLC-1

(1) The 2-(p-chlorophenoxy)propionic acid (CPP) modulates in a stereoselective manner the macroscopic chloride conductance (gCl), the electrical parameter sustained by the CLC-1 channel, of skeletal muscle. In order to determine the structural requirements for modulating native gCl and to identify high-affinity ligands, the effects of newly synthesised CPP analogues have been evaluated on gCl of rat EDL muscle fibres by means of the two-microelectrode current-clamp technique. (2) Each type of the following independent modification of CPP structure led to a three- to 10-fold decrease or to a complete lack of gCl-blocking activity: replacement of the electron-attractive chlorine atom of the aromatic ring, substitution of the oxygen atom of the phenoxy group, modification at the chiral centre and substitution of the carboxylic function with a phosphonate one. (3) The analogues bearing a second chlorophenoxy group on the asymmetric carbon atom showed a significant gCl-blocking activity. Similar to racemate CPP, the analogue with this group, spaced by an alkyl chain formed by three methylenic groups, blocked gCl by 45% at 100 micro M. (4) These latter derivatives were tested on heterelogously expressed CLC-1 performing inside-out patch-clamp recordings to further define how interaction between drug and channel protein could take place. Depending on the exact chemical nature of modification, these derivatives strongly blocked CLC-1 with K(D) values at -140 mV ranging from about 4 to 180 micro M. (5) In conclusion, we identified four molecular determinants pivotal for the interaction with the binding site on muscle CLC-1 channels: (a) the carboxylic group that confers the optimal acidity and the negative charge; (b) the chlorophenoxy moiety that might interact with a hydrophobic pocket; (c) the chiral centre that allows the proper spatial disposition of the molecule; (d) an additional phenoxy group that remarkably stabilises the binding by interacting with a second hydrophobic pocket.     

Silicosis and lung function decrements among female ceramic workers in Italy

It is well known that male ceramic workers have elevated risks of chronic silicosis. The objective of this study was to assess whether female ceramic workers also have an increased risk of silicosis and whether these women have decreased lung function related to silica exposure. Ceramic workers from Civitacastellana, Italy, were enrolled in health surveillance during the 1970s. A total of 642 women were under surveillance; a respiratory monitoring program was conducted from 1974 to 1987, with follow-up through 1991 that included annual chest radiography and measurement of lung function. Radiography findings were defined as silicosis if the chest films were > or =1/0 with small, rounded opacities. Multiple linear regression models for repeated measures (generalized estimating equations) were run to evaluate associations of forced vital capacity (FVC) and forced expiratory volume in 1 second (FEV(1)) with years of exposure and radiograph opacities. Nine cases of silicosis were identified on the basis of radiographic evidence. Silicosis risk was not associated with smoking but was related to employment before 1970 and demonstrated a dose-response gradient for years of exposure. FVC and FEV(1) both showed significant (p < 0.05) associations with duration of exposure and with positive radiography findings. The results for female ceramic workers are consistent with those for male employees regarding exposure to fibrogenic dusts.     

Platelet activation, inflammatory mediators and hypercholesterolemia

Atherogenic cofactors, such as altered cholesterol metabolism, may impact locally on inflammatory responses in atherosclerotic lesions. Blood levels of inflammatory markers (e.g., C-reactive protein, fibrinogen) have been associated with hypercholesterolemia and with overt atherothrombotic disorders. More recently. cytokines (e.g., interleukin-6, interleukin-1beta) and soluble adhesion molecules (e.g., selectins, intercellular adhesion molecule-1, vascular cell adhesion molecule-1) have been associated with both hypercholesterolemia and atherosclerotic disease, suggesting their use as potential therapeutic targets for the non-specific "anti-inflammatory" treatment of atherosclerosis. The inflammatory response associated with hypercholesterolemia involves not only the intrinsic cells of the artery wall. but also circulating cells. Platelets participate in this disease process through the release of a wide variety of biologically active substances. An imbalance of the hemostatic system and persistent in vivo platelet activation can be observed in hypercholesterolemia and may have pathophysiological implications in the development and progression of atherosclerotic plaques. Recent findings on the inflammatory actions of platelets have established the potential for a previously unrecognized biologic role for platelets in inflammation and vascular injury, and have opened new perspectives in the comprehension of the pathogenetic mechanism(s) of atherosclerosis. Stimulated platelets actively synthesize proinflammatory cytokines (e.g., CD40L, IL-1beta) and are able to release chemokines (i.e., platelet factor-4, RANTES) which have been all involved in the inflammatory process associated with hypercholesterolemia. This review will summarize the present understanding of the interplay between hypercholesterolemia, inflammation and platelet activation in the development and progression of atherosclerosis, and we also discuss the effects of lipid-lowering treatment on these phenomena.     

Highly chiral muscarinic ligands: the discovery of (2S,2'R,3'S,5'R)-1-methyl-2-(2-methyl-1,3-oxathiolan-5-yl)pyrrolidine 3-sulfoxide methyl iodide, a potent, functionally selective, M2 partial agonist

By further steric complication of previously studied highly chiral muscarinic agonists, we have obtained a small chiral library of enantiomeric and diasteromeric 1-methyl-2-(2-methyl-1,3-oxathiolan-5-yl)pyrrolidine 3-sulfoxides. Binding studies on cloned human muscarinic receptors expressed in CHO cells show that the introduction of a fourth stereogenic center gives undetectable affinity for hm1, hm3, hm4 and hm5 subtypes while leaving a quite modest affinity only for hm2 subtypes. However, functional studies on model M1-M4 muscarinic tissues have shown that three compounds of the series [(-)-5, (-)-7, (+)-8] are endowed with functional activity and behave as M2 selective partial agonists. Among them, compound (2S,2'R,3'S,5'R)-1-methyl-2-(2-methyl-1,3-oxathiolan-5-yl)pyrrolidine 3-sulfoxide methyl iodide [(+)-8] is particularly interesting, as it is a potent partial agonist on guinea pig atrium (force) (M2; pD2=7.65, alpha=0.41) while being a poor antagonist on M1, M3, and M4 model tissues (pKb<5).     

A novel model to study the biological effects of red wine at the molecular level

Several food items of plant origin, and in particular red wine, have been reported to protect from cardiovascular disease (CVD) development, thanks to their polyphenol components. Polyphenols undergo complex metabolic transformation during digestion and intestinal absorption. Here we report a novel model to study the effects of complex food matrices, applied to red wine, on gene expression in cultured primary human endothelial cells that takes into account the polyphenol metabolic transformation. Red wine was administered to human volunteers acting as 'bio-reactors'. Serum (RWS) obtained after 40 min was utilized to enrich endothelial cell culture media. The expression of specific genes involved in cell adhesion (vascular cell adhesion molecule (VCAM), intercellular adhesion molecule (ICAM) and monocytes chemoattractant protein (MCP-1)) and fibrinolysis (tissue-plasminogen activator (t-PA), plasminogen activator inhibitor-1 (PAI-1) and plasminogen activator inhibitor-2 (PAI-2)) was considered as a molecular marker of cell function and related to the effects of RWS. The gene expression profile determined by RWS incubation was significantly different from that observed after the addition of red wine. Data obtained by this approach indicate the importance of taking into account the complex metabolic transformation of polyphenols that occurs during absorption when studying their effect on human health.     

Potential of a Khaya ivorensis -Alstonia boonei extract combination as antimalarial prophylactic remedy

Ethnopharmacological relevance

The decoction of the combined stem barks of Khaya ivorensis A. Chev. (Meliaceae) and Alstonia boonei De Wild (Apocynaceae) has a history of use in traditional medicine of central Cameroon for malaria treatment but also for the prevention of the disease.

Aim of the study

The purpose of this investigation was to determine the antiplasmodial activity of Khaya ivorensis (K) and Alstonia boonei (A) preparations in the murine malaria model Plasmodium berghei/Anopheles stephensi, to estimate their prophylactic potential and to assess acute and sub-acute toxicity of the formulations prepared according to the traditional recipes.

Materials and methods

Aqueous extracts from the stem-bark of the two plants were prepared and tested separately and in combination. BALB/c mice were treated for 9 days and challenged on day 3 by exposure to mosquitoes infected with Plasmodium berghei. Treatment doses ranged between 200 and 400 mg/kg/day, corresponding approximately to the dosage applied by traditional healers to cure malaria patients or prevent the disease. Parasitemia reduction in treated animals was calculated from Giemsa smear counts, of two replicate experiments. To estimate acute toxicity in terms of median lethal dose (LD50), geometrically increasing doses were administered to mice. Sub-acute toxicity of the herbal combination (KA) was investigated by administering the same doses as in the antiplasmodial activity test for a period of 14 days, followed by 14 days of recovery observation. Locomotor activity (Open Field Test), body weight, liver and kidney morphology were monitored.

Results

The combination KA was found to exhibit antiplasmodial activity in the murine malaria model. In mice treated with the combination remedy at a dosage of 200 mg/kg/day, parasitemia values of 6.2% ± 1.7 and 6.5% ± 0.8 were recorded, compared to 10.8% ± 1.3 and 12.0% ± 4.0 in controls (p < 0.01). Doubling the dosage of the extracts did not significantly increase parasite suppression. When extracts of K and A were administered separately at a dosage of 400 mg/kg, a reduction in parasitemia was still obtained, but it did not reach statistical significance. Toxicity studies yielded comforting results: the LD50 was estimated to be greater than 2779.5 mg/kg. Moreover, mice exposed to the fourteen-day repeated-dose toxicity test (sub-acute toxicity test) did not display weight loss, liver or kidney morphological modifications, significant alterations in locomotor activity or any other sign of illness.

Conclusion

The antiplasmodial activity and the wide dose interval between the therapeutic dosage and the toxic dosage exhibited by the KA herbal combination in the murine malaria model argue in favor of its use as an antimalarial prophylactic remedy. It remains to be demonstrated by human clinical trials whether the combination remedy, when taken by inhabitants during malaria transmission season, can reduce parasite density and lead to a reduction of malaria episodes in the community.     

Association between total number of deaths, diabetes mellitus, incident cancers, and haplotypes in chromosomal region 8q24 in a prospective study

The 8q24 region is a gene desert, although chromosomal aberrations and somatic amplification involving this region, including translocations involving the protooncogene c-MYC, have been frequently reported in people with cancer. To investigate the role of variants in 8q24 region, the authors analyzed data from a prospective study (n = 10,372 participants who were followed for 11 years) in which a large number of health events (>1,500) occurred (1993-1998). They genotyped all subjects for 5 candidate single nucleotide polymorphisms (rs672888, rs1447295, rs9642880, rs16901979, and rs6983267) that were identified in previous genome-wide scans. Although significant associations with individual single nucleotide polymorphisms were small in magnitude, the authors observed higher increases in the risks of different types of cancer with specific haplotypes, particularly when subjects were homozygous for the haplotype: for breast cancer and homozygotes for haplotype CAGCT, hazard ratio = 3.40, 95% confidence interval: 1.24, 9.21; for prostate cancer and grouped rare haplotypes, hazard ratio = 7.43, 95% confidence interval: 3.00, 18.37; and for brain cancer and homozygotes for haplotype CGGCT, hazard ratio = 13.48, 95% confidence interval: 3.00, 59.53. Significant associations were also observed between haplotypes and deaths from cardiovascular diseases and cerebrovascular diseases; the most stable association was between homozygotes for haplotypes CGTCG and CAGCT and total deaths in men (hazard ratio = 3.5, 95% confidence interval: 1.8, 6.9, and hazard ratio = 2.8, 95% confidence interval: 1.3, 6.4, respectively). In conclusion, the authors have observed a strong pleiotropic effect of the 8q24 region in a large prospective study. This observation can shed light on the mechanisms underlying reported associations between 8q24 variants and disparate chronic diseases.     

Surgical treatment of pleural recurrence from thymoma.

A complete surgical resection represents the cornerstone of the therapy of the thymic tumours. Pleural disease may already be present at the diagnosis representing an advanced stage thymoma (Masaoka IVA) or it may appear during the follow-up, representing a pleural recurrence. The treatment of Stage IVA thymoma is quite examined in the medical literature; however just a few reports analysed the surgical treatment of thymoma recurrences, whose exclusive pleural recurrences represent more than 90%. Our aim was to review the literature laying the stress on the incidence, diagnosis, treatment options and prognosis of this highly selected group of patients with pleural recurrence from thymoma.     

体育运动对癫痫猝死症的影响：并非单纯运动

癫痫猝死（sudden unexpected deathin epilepsy，SUDEP）是癫痫患者最重要的直接与癫痫相关的死亡原因。其潜在病理机制尚未阐明，但癫痫发作时和发作间期的心律失常，电解质紊乱，致心律失常药物或癫痫电活动通过自主神经系统向心脏的传导，很可能与SUDEP的发病机理有关。引人注意的是，临床和实验数据都显示体力活动可以降低癫痫发作频率，并促进癫痫患者的心血管健康。基于上述情况，本文综述了体育锻炼可能预防SUDEP发生的相关文献内容。