A pilot study on the transcriptional response of androgen- and insulin-related genes in peripheral blood mononuclear cells induced by testosterone administration in hypogonadal men.

The aim of the present study is to determine whether testosterone (T) administration changes the expression profile of androgen- and insulin-related genes in peripheral blood mononuclear cells (PBMC). To this end, we evaluated the gene expression profile of 19 genes (AKT2, CCND1, GSK3ALPHA, IGF1, GSK3BETA, FOXO3, IL6, IGFBP2, UGT2B17, ARA55, CREBBP, CYP11A, HSD17B1, HSD17B7, UGT2B7, SELADIN 1, CLU, PGC1, AKR1C1) selected according their function in the androgen pathways, in a series of 11 hypogonadal men pharmacologically treated with T. We noted that 7 genes were differentially expressed, five of them were up-regulated (AKT2 FC=2.39, CREBBP FC=11.2, GSK3beta FC=5.6, UGT2B7 FC=4.49, UGT2B17 FC=2.88) and two were down-regulated (ARA55 FC= -2.0, CYP11A FC= -2.47). This experience suggests that androgen- and insulin-related genes can be considered useful blood genomic biomarkers for specific steroid drugs.

SOS1 over-expression in genital skin fibroblasts from hirsute women: a putative role of the SOS1/RAS pathway in the pathogenesis of hirsutism.

Hirsutism is the development of androgen-dependent terminal body hair in women in places in which terminal hair are normally not found. It is often associated with hyperandrogenemia and/or polycystic ovary syndrome (PCOS), but the existence of uncommom hirsutism forms that are not related to altered androgen plasma levels lead also to the definition of - idiopathic hirsutism. Although the pathophysiology of hirsutism has been linked to increasing 5-alpha reductase (SRD5A) activity and to an alteration of the androgen receptor (AR) transcriptional machinery, many aspects remain unclear. In particular, the relationships between androgens and local factors are poorly understood. In the present paper, we selected for a genital skin biopsy, 8 women affected with severe hirsutism (Ferriman-Gallway score greater than 25) but with normal plasma androgen levels, with the exception of slightly higher serum 3alpha-diol-glucuronide levels, and 6 healthy controls and analyzed their androgen- and insulin-specific transcriptional profile using a specific custom low density microarray (AndroChip 2, GPL9164). We identified the over-expression of the Son of Sevenless-1 (SOS1) gene in all of the hirsute skin fibroblast primary cell cultures compared to control healthy women. Since SOS1 is a guanine nucleotide exchange factor that couples receptor tyrosine kinases to the RAS signaling pathway that controls cell proliferation and differentiation, we further analyzed SOS1 expression, protein level and RAS signaling activation pathway in an in vitro model (NHDF, normal human dermal fibroblast cell line). NHDF treated for 24 h with different concentrations of DHT and T showed an increase in SOS1 levels (both mRNA and protein) and also an activation of the RAS pathway. Our in vivo and in vitro data represent a novel preliminary observation that factors activating SOS1 could act as local proliferative modulators linked to the androgen pathway in the pilosebaceous unit. SOS1 over-expression may play a role in the regulation of the RAS/mitogen-activated protein kinase pathway in the skin, in the hair follicle proliferation and cell cycle, suggesting new perspectives in understanding the pathogenesis of idiopathic hirsutism.

Androgen- and insulin-related gene signature using a specific low density oligoarray AndroChip 2 in peripheral blood mononuclear cells in agonists, recreational athletes and sedentary subjects.

The early detection of genomic biomarkers (e.g. RNAs) through analysis of circulating blood cells could have a substantial impact on biomedicine, particularly in monitoring clinical trials, drug toxicity and doping in athletes. To achieve this goal, it is essential to develop methods that are sufficiently sensitive to detect biomarker alterations during normal biologic processes, pathogenic processes, and or in response to therapeutic or other intervention. Using a low density microarray (AndroChip 2) we detected a transcriptional profiling signature of 190 genes related to androgen and insulin metabolism pathway, in peripheral blood mononuclear cell (PBMC) in subjects with different intensities of sports activities. We demonstrated that androgen and insulin gene transcriptional levels are independent to sports activity and therefore potentially suitable for drug monitoring and/or drug doping (such as anabolic androgen steroid AAS abuse) and or gene doping.

In silico pharmacology for drug discovery: applications to targets and beyond.

Computational (in silico) methods have been developed and widely applied to pharmacology hypothesis development and testing. These in silico methods include databases, quantitative structure-activity relationships, similarity searching, pharmacophores, homology models and other molecular modeling, machine learning, data mining, network analysis tools and data analysis tools that use a computer. Such methods have seen frequent use in the discovery and optimization of novel molecules with affinity to a target, the clarification of absorption, distribution, metabolism, excretion and toxicity properties as well as physicochemical characterization. The first part of this review discussed the methods that have been used for virtual ligand and target-based screening and profiling to predict biological activity. The aim of this second part of the review is to illustrate some of the varied applications of in silico methods for pharmacology in terms of the targets addressed. We will also discuss some of the advantages and disadvantages of in silico methods with respect to in vitro and in vivo methods for pharmacology research. Our conclusion is that the in silico pharmacology paradigm is ongoing and presents a rich array of opportunities that will assist in expediting the discovery of new targets, and ultimately lead to compounds with predicted biological activity for these novel targets.

In silico pharmacology for drug discovery: methods for virtual ligand screening and profiling.

Pharmacology over the past 100 years has had a rich tradition of scientists with the ability to form qualitative or semi-quantitative relations between molecular structure and activity in cerebro. To test these hypotheses they have consistently used traditional pharmacology tools such as in vivo and in vitro models. Increasingly over the last decade however we have seen that computational (in silico) methods have been developed and applied to pharmacology hypothesis development and testing. These in silico methods include databases, quantitative structure-activity relationships, pharmacophores, homology models and other molecular modeling approaches, machine learning, data mining, network analysis tools and data analysis tools that use a computer. In silico methods are primarily used alongside the generation of in vitro data both to create the model and to test it. Such models have seen frequent use in the discovery and optimization of novel molecules with affinity to a target, the clarification of absorption, distribution, metabolism, excretion and toxicity properties as well as physicochemical characterization. The aim of this review is to illustrate some of the in silico methods for pharmacology that are used in drug discovery. Further applications of these methods to specific targets and their limitations will be discussed in the second accompanying part of this review.

Outcome assessment in patients with chronic obstructive rhinitis CO2 laser treated.

Surgical lasers have been used to restore nasal flow in chronic obstructive rhinitis, with a significant improvement in symptoms having been reported in almost all cases. However, evidence supporting the efficacy at long-term, and studies on the assessment of quality of life remain limited. In the present study, efficacy at long term and improvement in the quality of life were assessed in patients with chronic obstructive rhinitis, treated with CO2 laser. A total of 308 patients with chronic obstructive rhinitis were enrolled. The primary outcome measure assessed was the change in score regarding specific and general symptoms, between baseline to 2-4.5 and 7.8 mean years follow-up. Laser turbinotomy restored nasal flow and induced a change in total score which was statistically significant, for specific and general symptoms at the first, second and third follow-up, p < 0.01. CO2 laser turbinate surgery improved symptoms and quality of life in patients with chronic obstructive rhinitis as observed at 2-4.5 and 7.8 mean years follow-up.

Conformational constraints on side chains in protein residues increase their information content.

Like all other complex biological systems, proteins exhibit properties not seen in free amino acids (i.e., emergent properties). The present investigation arose from the deduction that proteins should offer a good model to approach the reverse phenomenon, namely top-down constraints experienced by protein residues compared to free amino acids. The crystalline structure of profilin Ib, a contractile protein of Acanthamoeba castellanii, was chosen as the object of study and submitted to 2-ns molecular dynamics simulation. The results revealed strong conformational constraints on the side chain of residues compared to the respective free amino acids. A Shannon entropy (SE) analysis of the conformational behavior of the side chains showed in most cases a strong decrease in the SE of the chi1 and chi2 dihedral angles compared to free amino acids. This is equivalent to stating that conformational constraints on the side chain of residues increase their information content and hence recognition specificity compared to free amino acids. In other words, the vastly increased information content of a protein relative to its free monomers is embedded not only in the tertiary structure of the backbone, but also in the conformational behavior of the side chains. The postulated implication is that both backbone and side chains, by virtue of being conformationally constrained, contribute to the recognition specificity of the protein toward other macromolecules and ligands.

Avaliação da atividade IMAO e antibacteriana de extratos de Mikania glomerata Sprengel

Neste trabalho avaliou-se a atividade antibacteriana e IMAO de extratos de diferentes polaridades de Mikania glomerata. A atividade antibacteriana foi medida frente à cepa multiresistente de Staphylococus aureus PI57, através das técnicas de bioautografia e antibiograma. A atividade IMAO foi medida utilizando uma suspensão de mitocôndrias. Mikania glomerata mostrou conter no extrato hexânico substâncias antimicrobianas. Os extratos hexânico e CH2Cl2 foram ativos frente à MAO-B, sem apresentarem atividade de inibição da MAO-A, enquanto o extrato metanólico apresentou atividade de inibição da MAO-A e MAO-B, sem seletividade.

Antibacterial and IMAO inhibition activities of different polarities extracts of Mikania glomerata were evaluated. The antibacterial activity was assayed against a multiresistant strain of Staphyllococus aureus PI57. The IMAO activity was measured with a suspension of mitochondrion. In the hexanic extract of Mikania glomerata substances with antibacterial activity were detected. Hexanic and CH2Cl2 extracts showed MAO-B inhibition activity while MAO-A inhibition activity was not detected. The methanolic extract showed non-selective inhibition activity of MAO-A and MAO-B.

Physicochemical profiling in drug research: a brief survey of the state-of-the-art of experimental techniques.

This review begins with a general presentation of the new paradigm of drug discovery, with its emphasis on the rapid identification and elimination of compounds with unsuitable physicochemical and pharmacokinetic properties. The focus of the paper is on the various experimental methods used to determine such key physicochemical properties as ionization, lipophilicity and distribution in isotropic and anisotropic systems, solubility, and permeability across artificial membranes. Both traditional and high-throughput methods are presented and their limits highlighted. The text concludes with the trade-off between quantity/speed in high-throughput screening techniques versus greater data quality in the more labor-intensive methods.

In vitro biotransformation of the selective serotonin reuptake inhibitor citalopram, its enantiomers and demethylated metabolites by monoamine oxidase in rat and human brain preparations.

This study was conducted to identify enzyme systems eventually catalysing a local cerebral metabolism of citalopram, a widely used antidepressant of the selective serotonin reuptake inhibitor type. The metabolism of citalopram, of its enantiomers and demethylated metabolites was investigated in rat brain microsomes and in rat and human brain mitochondria. No cytochrome P-450 mediated transformation was observed in rat brain. By analysing H2O2 formation, monoamine oxidase A activity in rat brain mitochondria could be measured. In rat whole brain and in human frontal cortex, putamen, cerebellum and white matter of five brains monoamine oxidase activity was determined by the stereoselective measurement of the production of citalopram propionate. All substrates were metabolised by both forms of MAO, except in rat brain, where monoamine oxidase B activity could not be detected. Apparent Km and Vmax of S-citalopram biotransformation in human frontal cortex by monoamine oxidase B were found to be 266 microM and 6.0 pmol min(-1) mg(-1) protein and by monoamine oxidase A 856 microM and 6.4 pmol min(-1) mg(-1) protein, respectively. These Km values are in the same range as those for serotonin and dopamine metabolism by monoamine oxidases. Thus, the biotransformation of citalopram in the rat and human brain occurs mainly through monoamine oxidases and not, as in the liver, through cytochrome P-450.

The Hydrogen-Bond: computational approaches and applications to drug design.

This mini-review begins with a presentation of the hydrogen-bond in the context of other intermolecular recognition forces of significance in molecular biology and molecular pharmacology. This is followed by a survey of the various computational methods available to calculate the hydrogen-bonding capacity of compounds. Such methods use quantum mechanics, molecular mechanics, or algorithms based on experimental fragmental values. A recent and promising advance in the computation of H-bonding capacity is the development of specific molecular interaction fields (MIFs) known as molecular hydrogen-bonding potentials (MHBPs). Their interest in relating molecular properties to pharmacokinetic behaviour is highlighted with two examples, namely oral drug absorption and blood-brain barrier permeation.

The esterase-like activity of serum albumin may be due to cholinesterase contamination.

PURPOSE: The "esterase-like activity" of human serum albumin (HSA) is described in the literature, but a contamination of commercially available HSA preparations by plasma cholinesterase is conceivable in some cases. The purpose of the present work was to examine this hypothesis. METHODS: The hydrolytic activity of HSA and its inhibition by physostigmine were measured fluorimetrically by monitoring the hydrolysis of the ester substrate moxisylyte. Affinity chromatography was used to separate cholinesterase and HSA. The cholinesterase activity in the eluted fractions was assessed using Ellman's reagent and butyrylthiocholine as substrate. RESULTS: A significant variation in the esterase-like activity of different albumin batches was observed. This activity was strongly inhibited by physostigmine, a well-known inhibitor of cholinesterase. Affinity chromatography led to a complete separation between HSA and the esterase activity, which was found exclusively in the cholinesterase fraction. CONCLUSIONS: The apparent esterase-like activity of HSA toward moxisylyte and butyrylthiocholine was due to a contamination by cholinesterase. With these substrates, HSA showed a total lack of esterase-like activity.

Monoamine oxidase inhibitory activity of some Hypericum species native to South Brazil.

The total methanol crude extracts and petroleum ether, chloroform, and methanol fractions obtained from Hypericum species, H. caprifoliatum, H. carinatum, H. connatum, H. cordatum, H. myrianthum, H. piriai, H. polyanthemum and H. brasiliense, all native to South Brazil, were assayed for monoamine oxidase A (MAO A) and MAO B inhibitory activity in rat brain mitochondrial preparations at concentrations ranging from 1 to 20microg mL(-1). Three benzopyrans, HP1 (6-isobutyryl-5,7-dimethoxy-2,2-dimethylbenzopyran), HP2 (7-hydroxy-6-isobutyryl-5-methoxy-2,2-dimethylbenzopyran) and HP3 (5-hydroxy-6-isobutyryl-7-methoxy-2,2dimethylbenzopyran) isolated from H. polyanthemum were also tested at maximal concentrations of 150, 150 and 75/microM, respectively. The lipophilic extracts of H. polyanthemum, H. caprifoliatum and H. piriai displayed MAO A inhibitory activity greater than 50%. Among the benzopyrans, only HP3 showed significant activity, with an IC50 value of 22 microM. The total methanol crude extracts of aerial parts from H. carinatum, H. connatum, H. cordatum, H. polyanthemum and H. piriai were evaluated for antidepressant activity in the Porsolt's forced swimming test in Wistar rats (270 mg kg(-1) day(-1); i.p); however, none of them showed activity.

Generalization of ionic partition diagrams to lipophilic compounds and to biphasic systems with variable phase volume ratios.

The ionic partition diagram methodology has been generalized to address both hydrophilic and lipophilic compounds and to consider biphasic systems with variable phase volume ratios. With this generalized approach electrochemical measurements of ion transfer potentials afford the determination of the standard partition coefficients of all forms of ionizable molecules, including the neutral form, as well as the evaluation of the dissociation constant of monoprotic substances. An interesting consequence of this approach is the definition of an extraction pK(a,ext) which is the apparent pK(a) of neutral acids and bases when dissolved in the organic phase.

Management of chronic otitis media with effusion: the role of glutathione.

BACKGROUND: The inflammatory cells documented in chronic otitis media with effusion (OME) spontaneously release oxidants which can induce middle ear (ME) epithelial cell damage. Glutathione (GSH), a major extracellular antioxidant in humans, plays a central role in antioxidant defense. PURPOSE: To evaluate the effects of GSH treatment on chronic otitis media with effusion (OME). SUBJECTS AND INTERVENTION: Sixty children with chronic OME were enrolled, 30 of whom were randomly assigned to the treatment group and 30 to the placebo group. Patients in the treatment group received 600 mg glutathione in 4 mL saline per day subdivided into five 2-minute administrations given by nasal aerosol every 3 or 4 waking hours for 2 weeks. Patients in the control group received 4 mL saline per day following the same procedure as for GSH treatment. RESULTS: Three months after therapy improvement had occurred in 66.6% of patients in the GSH-treated group and in 8% of the control subjects (P <.01). CONCLUSION: On the basis of these results, GSH treatment could be considered for the nonsurgical management of chronic OME.

Development of molecular hydrogen-bonding potentials (MHBPs) and their application to structure-permeation relations.

Hydrogen bonds are major forces of recognition in biochemistry and molecular pharmacology; they are an essential component of intermolecular interactions and determine to a significant extent the 3D-structure of bio-macromolecules. To explore three-dimensional H-bonding properties, a new tool called Molecular Hydrogen-Bonding Potentials (MHBPs) was created. The development of this tool is based on a stepwise procedure similar to the one used successfully to generate the Molecular Lipophilicity Potential (MLP). First, a H-bonding fragmental system was developed starting from published solvatochromic parameters. An atomic H-bonding donor fragmental value (alpha) is associated to each hydrogen atom in a polar moiety. Similarly, an atomic H-bonding acceptor fragmental value (beta) is associated to each polar atom. A distance function and an angle function were defined to take into account variations of the MHBPs in space. The fragmental system and the geometric functions were then combined to generate the MHBPs. These are calculated at each point of an adequate molecular surface or on a three-dimensional grid. The MHBPs were compared with GRID interactions energies and correlated with success to oral drug absorption data. Available examples demonstrate that the MHBPs are a promising computational tool in drug design. Their combination with CoMFA and VolSurf is being studied.

Coumarins derivatives as dual inhibitors of acetylcholinesterase and monoamine oxidase.

A set of 17 coumarin and 2 chromone derivatives with known inhibitory activity toward monoamine oxidase (MAO) A and B were tested as acetylcholinesterase (AChE) inhibitors. All compounds inhibited AChE with values in the micromolar range (3-100 microM). A kinetic study showed that most compounds acted as noncompetitive AChE inhibitors. This finding may be of interest in the context of Alzheimer's disease because recent observations suggest that MAO and AChE inhibition might decrease beta-amyloid deposition.

Interaction of psychotropic drugs with monoamine oxidase in rat brain.

Literature observations indicate that some psychotropic drugs may have inhibitory activity towards monoamine oxidase (MAO). This study was undertaken to assess the potency, isozyme selectivity and mechanism of inhibition of representative first- and second-generation antidepressant drugs towards rat brain MAO-A and MAO-B. Five tricyclic antidepressants (imipramine, trimipramine, clomipramine, amitriptyline and doxepine) and three selective serotonin reuptake inhibitors (fluoxetine, fluvoxamine and citalopram) were examined. They showed inhibitory activity towards MAO-A and MAO-B, with clear selectivity for MAO-B (Ki in the micromolar range). Their mechanism of inhibition was competitive towards MAO-B and of a mixed competitive type towards MAO-A. The results suggest that some of the drugs examined might also contribute an MAO inhibitory effect in chronically treated patients.

The apparent lipophilicity of quaternary ammonium ions is influenced by galvani potential difference, not ion-pairing: a cyclic voltammetry study.

PURPOSE: This work examines whether ion-pairing contributes to the apparent lipophilicity of cations, which is seen by a shake-flask or titrimetic method to be influenced by the nature and concentration of counter-ions. METHODS: To solve this problem, the lipophilicity of several quaternary ammonium drugs was measured by cyclic voltammetry in the 1,2-dichloroethane/water system. The standard ionic partition coefficient values so obtained (log Pdce(o,C)) were correlated with log Poct values calculated by the CLOGP algorithm for the respective neutral molecules. RESULTS: The standard (i.e., intrinsic) lipophilicity values are shown to depend on a, the structure of the ion (nature, volume, charge), and b, on the Galvani potential difference at the ITIES (interface between two immiscible electrolyte solutions). CONCLUSIONS: The standard lipophilicity values were not influenced by counter-ions. In contrast, simulations showed that the increased apparent lipophilicity of cations, as measured by the shake-flask method in the presence of lipophilic anions, is fully accounted for by the resulting increase in the Galvani potential difference.