Structure-activity relationships in 1,4-benzodioxan-related compounds. 7. Selectivity of 4-phenylchroman analogues for alpha(1)-adrenoreceptor subtypes

WB4101 (1)-related compounds 5-10 were synthesized, and their biological profile at alpha(1)-adrenoreceptor (AR) subtypes and 5-HT(1A) serotoninergic receptors was assessed by binding assays in Chinese hamster ovary and HeLa cell membranes expressing the human cloned receptors. Moreover, their receptor selectivity was further determined in functional experiments in isolated rat prostate (alpha(1A)), vas deferens (alpha(1A)), aorta (alpha(1D)), and spleen (alpha(1B)). In functional assays, compound 5 was the most potent at alpha(1D)-ARs with a reversed selectivity profile (alpha(1D) > alpha(1A) > alpha(1B)) relative to both prototype 1 and phendioxan (2) (alpha(1A) > alpha(1D) > alpha(1B)), whereas compound 8, bearing a carbonyl moiety at position 1, was the most potent at alpha(1A)-ARs with a selectivity profile similar to that of prototypes. The least potent of the series was the trans isomer 6, suggesting that optimum alpha(1)-AR blocking activity in this series is associated with a cis relationship between the 2-side chain and the 4-phenyl ring rather than a trans relationship as previously observed for the 2-side chain and the 3-phenyl ring in 2 and related compounds. Binding affinity results were not in complete agreement with the selectivity profiles deriving from functional experiments. Although a firm explanation was not available, neutral and negative antagonism and receptor dimerization were considered as two possibilities to account for the difference between binding and functional affinities. Finally, compound 5 was selected for a modeling study in comparison with 1, mephendioxan (3), and open phendioxan (4) to achieve information on the physicochemical interactions that account for its high affinity toward alpha(1d/D)-ARs.     

Oxygenated metabolites of anandamide and 2-arachidonoylglycerol: conformational analysis and interaction with cannabinoid receptors,membrane transporter,and fatty acid amide hydrolase

This study was aimed at finding structural requirements for the interaction of the acyl chain of endocannabinoids with cannabinoid receptors, membrane transporter protein, and fatty acid amide hydrolase (FAAH). To this end, the flexibility of the acyl chain was restricted by introduction of an 1-hydroxy-2Z,4E-pentadiene system in anandamide (N-arachidonoylethanolamine, AEA) and 2-arachidonoylglycerol (2-AG) at various positions using different lipoxygenases. This brought about selectivity and attenuated the binding potency of AEA and 2-AG. Although the displacement constants were modest, 15(S)-hydroxy-eicosa-5Z,8Z,11Z,13E-tetraenoyl-N-(2-hydroxyethyl)amine was found to bind selectively to the CB(1) receptor, whereas its 1-arachidonoyl-sn-glycerol analogue and 13(S)-hydroxy-octadeca-9Z,11E-dienoyl-N-(2-hydroxyethyl)amine could selectively bind to the CB(2) receptor. 11(S)-Hydroxy-eicosa-5Z,8Z,12E,14Z-tetraenoyl-N-(2-hydroxyethyl)amine did not bind to either receptor, whereas 12(S)-hydroxy-eicosa-5Z,8Z,10E,14Z-tetraenoyl-N-(2-hydroxyethyl)amine did bind to both CB receptors with an affinity similar to that of AEA. All oxygenated anandamide derivatives were good inhibitors of FAAH (low micromolar K(i)) but were ineffective on the AEA transporter. 2-AG rapidly isomerizes into 1(3)-arachidonoyl-sn-glycerol. Both 1- and 3-arachidonoyl-sn-glycerol did not bind to either CB receptor and did not interfere with AEA transport. Thus, after it is isomerized, 2-AG is inactivated, thereby decreasing effective concentrations of 2-AG. Analysis of (1)H NMR spectra revealed that chloroform did not induce notably different conformations in the acyl chain of 15(S)-hydroxy-eicosa-5Z,8Z,11Z,13E-tetraenoic acid as compared with water. Molecular dynamics (MD) simulations of AEA and its analogues in the presence of explicit water molecules revealed that a tightly folded conformation of the acyl chain is not the only requirement for CB(1) binding. Structural details of the C(2)-C(15) loop, such as an sp(2) carbon at position 11, are necessary for receptor binding. The MD simulations may suggest that the average orientations of the pentyl tail of AEA and 12(S)-hydroxy-eicosa-5Z,8Z,10E,14Z-tetraenoyl-N-(2-hydroxyethyl)amine are different from that of the low-affinity, inactive ligands.     

Adenosine A(1) receptor: analysis of the potential therapeutic effects obtained by its activation in the central nervous system

Adenosine modulates several physiological functions in the CNS and in peripheral tissues via membrane receptors which have been classified into four adenosine subtypes. A(1) activation produces neuronal depression: this inhibition allows A(1) agonists to produce ischemic tolerance and protection in neuronal tissue. In order to selectively reproduce these effects, several A(1) selective ligands have been synthesised and evaluated to understand how they interact with the adenosine A(1) receptor. The investigation methods include SAR studies using native and chemically modified A(1) receptors, molecular cloning of native and mutant adenosine A(1) receptors, molecular modeling and thermodynamic analysis of drug-receptor interaction. Despite the great quantity of information available on the adenosine A(1) receptor, no A(1) agonist has so far entered in clinical use against brain diseases in view of the side effects; moreover selective A(1) agonists appear to be poorly adsorbed into the brain and can be quickly degraded in vivo or in the whole blood. In an attempt to overcome these problems studies have been undertaken dealing with the use of partial agonists to inhibit side-effects and the employment of prodrugs to increase stability and diffusion through lipid barriers of A(1) ligands. Other attempts involve either the use of A(1) receptor enhancers as modulators able to locally enhance the action of endogenously produced adenosine, or the encapsulation of A(1)agonists in drug delivery systems targeted to the brain. In this review, these approaches will be described together with the effects of adenosine A(1) receptor ligands and their binding mechanisms on the central nervous system.     

Intravenous administration to rabbits of non-stealth and stealth doxorubicin-loaded solid lipid nanoparticles at increasing concentrations of stealth agent: pharmacokinetics and distribution of doxorubicin in brain and other tissues

The pharmacokinetics and tissue distribution of doxorubicin incorporated in non-stealth solid lipid nanoparticles (SLN) and in stealth solid lipid nanoparticles (SSLN) (three formulations at increasing concentrations of stearic acid-PEG 2000 as stealth agent) after intravenous administration to conscious rabbits have been studied. The control was the commercial doxorubicin solution. The experiments lasted 6 h and blood samples were collected at fixed times after the injections. In all samples, the concentration of doxorubicin and doxorubicinol were determined. Doxorubicin AUC increased as a function of the amount of stealth agent present in the SLN. Doxorubicin was still present in the blood 6 h after the injection of SLN or SSLN, while no doxorubicin was detectable after the i.v. injection of doxorubicin solution. Tissue distribution of doxorubicin was determined 30 min, 2 and 6 h after the administration of the five formulations. Doxorubicin was present in the brain only after the SLN administration. The increase in the stealth agent affected the doxorubicin transported into the brain; 6 h after injection, doxorubicin was detectable in the brain only with the SSLN at the highest amount of stealth agent. In the other rabbit tissues (liver, lungs, spleeen, heart and kidneys) the amount of doxorubicin present was always lower after the injection of any of the four types of SLN than after the commercial solution. In particular, all SLN formulations significantly decreased heart and liver concentrations of doxorubicin.     

Influence of monoamine oxidase A and serotonin receptor 2A polymorphisms in SSRI antidepressant activity

The aim of the present study was to test a possible effect of the monoamine oxidase A (MAOA) and serotonin receptor 2A (5-HT-2A) gene variants on the antidepressant activity of fluvoxamine and paroxetine in a sample of major (n = 248) and bipolar (n = 195) depressives, with or without psychotic features. A total of 443 in-patients were treated with 300 mg fluvoxamine (n = 307) or 20-40 mg paroxetine (n = 136) for 6 wk. The severity of depressive symptoms was assessed weekly with the Hamilton Rating Scale for Depression (HAMD). Allele variants were determined in each subject using a PCR-based technique. We observed a marginal association between 5-HT-2A variants and antidepressant response while MAOA genotypes were not associated. Possible stratification factors, such as sex, diagnosis, presence of psychotic features, HAMD scores at baseline, pindolol augmentation and SSRIs plasma levels did not significantly influence the observed results. The investigated MAOA and 5-HT-2A gene variants, therefore, do not seem to play a major role in SSRI antidepressant activity.     

Epitope identification and vaccine design for cancer immunotherapy

The development of processes for engineering multi-epitope vaccines based on the identification and selection of epitope packages, along with vaccine design optimization using epitope placements and spacers to optimize processing efficacy, are reviewed. The Epimmune Inc epitope identification process has been applied to numerous cancer types, but also applies to infectious diseases. Epitope-analog efforts in novel vaccine design have also been explored and their uses in prophylactic and therapeutic applications are eagerly anticipated.     

Brain-derived neurotrophic factor regulates expression of vasoactive intestinal polypeptide in retinal amacrine cells

Brain-derived neurotrophic-factor (BDNF) is expressed in the retina and controls the development of subtypes of amacrine cells. In the present study we investigated the effects of BDNF on amacrine cells expressing vasoactive intestinal polypeptide (VIP). Rats received three intraocular injections of BDNF on postnatal days (P) 16, 18, and 20. The animals were sacrificed on P22, P40, P60, P80, and P120, and VIP expression in their retinas was detected by immunohistochemistry (P22, P40) and by radioimmunoassay (RIA; P22, P40, P60, P80, P120) to assess the time course of BDNF effects on VIP. A significant increase in the density of VIP-positive amacrine cells was detected in BDNF-treated retinas, and VIP concentration was up-regulated by 150% both at P22 and at P40 with respect to untreated controls. VIP concentration then slowly declined in the treated retinas over a period of 3 months; however, a statistically significant increase of 50% was still detectable on P120. The impact of endogenous BDNF on the regulation of VIP expression in the retina was analyzed in mice homozygous for a targeted deletion of the BDNF gene locus (bdnf-/-). VIP immunohistochemistry revealed a marked reduction of VIP-positive amacrine cells and of VIP-immunopositive processes in the inner plexiform layer of the BDNF knockout mice. Mice lacking BDNF expressed only 5% of the VIP protein in their retinas compared with the retinas of wild-type mice as measured by RIA. Our data show that BDNF is a major regulator of VIP expression in retinal amacrine cells and exerts long-lasting effects on VIP content.     

Forty-years (1961-2001) of all-cause and coronary heart disease mortality and its determinants: the Corfu cohort from the Seven Countries Study

AIMS: This study examined risk factors in relation to 40-year all-cause and coronary heart disease mortality in the Corfu cohort of the Seven Countries Study. METHODS: The population studied in this analysis consisted of 529 rural middle-aged men enrolled in 1961. Multivariate analysis was performed using the proportional hazards Cox model with all-cause as well as coronary heart disease death as end points and age, blood pressure, serum total cholesterol, smoking, physical activity, body mass index, skinfold thickness, vital capacity and forced expiratory volume as predictors. RESULTS: The 40-year all-cause mortality rate was 87.1% (461 deaths/529 individuals at entry), while the CHD mortality rate was 22.7% (120 deaths/529 individuals at entry). The proportion of CHD deaths varied from 16 to 28.5% of all deaths during the period investigated. Age (hazard ratio (HR)=1.08, P<0.001), smoking (HR=1.40, P<0.01), diastolic blood pressure (HR=1.01, P<0.05), and forced expiratory volume (HR=0.97, P<0.05) were independently associated with 40-year all-cause mortality. Moreover, age (HR=1.093, P<0.001), smoking (HR=1.596, P<0.05), and body mass index (HR=1.05, P<0.05) were independently associated with 40-year CHD mortality. CONCLUSION: Among the investigated cardiovascular risk factors, age, smoking, physical activity, skinfold thickness, diastolic blood pressure, and forced expiratory volume seem to be associated with all-cause mortality, while age, smoking, and body mass index were consistently associated with 40-year CHD mortality.