Functional topography of midbrain and pontine serotonergic systems: implications for synaptic regulation of serotonergic circuits

Rationale

Dysfunction of serotonergic systems is thought to play an important role in a number of neurological and psychiatric disorders. Recent studies suggest that there is anatomical and functional diversity among serotonergic systems innervating forebrain systems involved in the control of physiologic and behavioral responses, including the control of emotional states.

Objective

Here, we highlight the methods that have been used to investigate the heterogeneity of serotonergic systems and review the evidence for the unique anatomical, hodological, and functional properties of topographically organized subpopulations of serotonergic neurons in the midbrain and pontine raphe complex.

Conclusion

The emerging understanding of the topographically organized synaptic regulation of brainstem serotonergic systems, the topography of the efferent projections of these systems, and their functional properties, should enable identification of novel therapeutic approaches to treatment of neurological and psychiatric conditions that are associated with dysregulation of serotonergic systems.     

Different drug-susceptibilities of long-term potentiation in three input systems to the CA3 region of the guinea pig hippocampus in vitro

The susceptibilities to several drugs of long-term potentiations in the three input systems (mossy, commissural/associational and fimbrial fibres) to CA3 pyramidal neurones were investigated in hippocampal slices from the guinea pig. D-2-Amino-5-phosphonovalerate (D-APV), a selective antagonist at N-methyl-D-aspartate (NMDA) receptors, blocked the long-term potentiations in the commissural/associational fibre- and fimbrial fibre-CA3 systems, but did not significantly affect that in the mossy fibre-CA3 system. The latter was suppressed by kynurenate, a non-selective glutamate receptor antagonist. On the other hand, naloxone, an opioid antagonist, inhibited and bifemelane, which improves metabolism in brain and has an anti-amnesic action, augmented long-term potentiation in mossy fibre-CA3 system but did not influence those in commissural/associational fibre- and fimbrial fibre-CA3 systems. These findings suggest that the mechanisms, relevant to production of long-term potentiation in the mossy fibre-CA3 system, are different from those in the commissural/associational fibre- and fimbrial fibre-CA3 systems. N-Methyl-D-aspartate receptors are involved in the latter systems, while non-NMDA receptors for L-glutamate and opioid receptors are involved in the former. Further, the mossy fibre-CA3 system is more susceptible to a drug, having an anti-amnesic action, than are the other two systems.     

Three-dimensional type I collagen cell culture systems for the study of bone pathophysiology

Three-dimensional (3-D) type I collagen cell culture systems composed of reconstituted collagen fibres are able to support short- and long-term growth of various cell types, including cancer cell lines, endothelial cells, endometrial cells, hepatocytes, osteoblasts and fibroblasts and to sustain or even enhance cell differentiation, in vitro. In addition, 3-D culture systems have been successfully used in the investigation of complex biological processes, such as angiogenesis, wound healing, tumour invasion and metastasis. The latter suggested that 3-D culture systems have the potential to simulate cell-cell interactions, which take place in tissues under physiological and pathophysiological conditions. This review focuses on the investigational use of 3-D collagen cell culture systems in bone physiology and the pathophysiology of skeletal metastasis.     

Co-delivery of Vascular Endothelial Growth Factor and Angiopoietin-1 Using Injectable Microsphere/Hydrogel Hybrid Systems for Therapeutic Angiogenesis

Purpose

We hypothesized that combined delivery of vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang-1) using microsphere/hydrogel hybrid systems could enhance mature vessel formation compared with administration of each factor alone.

Methods

Hybrid delivery systems composed of alginate hydrogels and poly(D,L-lactic-co-glycolic acid) (PLGA) microspheres containing angiogenic factors were prepared. The release behavior of angiogenic factors from hybrid systems was monitored in vitro. The hybrid systems were injected into an ischemic rodent model, and blood vessel formation at the ischemic site was evaluated.

Results

The sustained release over 4 weeks of both VEGF and Ang-1 from hybrid systems was achieved in vitro. Co-delivery of VEGF and Ang-1 was advantageous to retain muscle tissues and significantly induced vessel enlargement at the ischemic site, compared to mice treated with either VEGF or Ang-1 alone.

Conclusions

Sustained and combined delivery of VEGF and Ang-1 significantly enhances vessel enlargement at the ischemic site, compared with sustained delivery of either factor alone. Microsphere/hydrogel hybrid systems may be a promising vehicle for delivery of multiple drugs for many therapeutic applications.     

An in vitro approach to detect metabolite toxicity due to CYP3A4-dependent bioactivation of xenobiotics

Many adverse drug reactions are caused by the cytochrome P450 (CYP) dependent activation of drugs into reactive metabolites. In order to reduce attrition due to metabolism-mediated toxicity and to improve safety of drug candidates, we developed two in vitro cell-based assays by combining an activating system (human CYP3A4) with target cells (HepG2 cells): in the first method we incubated microsomes containing cDNA-expressed CYP3A4 together with HepG2 cells; in the second approach HepG2 cells were transiently transfected with CYP3A4. In both assay systems, CYP3A4 catalyzed metabolism was found to be comparable to the high levels reported in hepatocytes. Both assay systems were used to study ten CYP3A4 substrates known for their potential to form metabolites that exhibit higher toxicity than the parent compounds. Several endpoints of toxicity were evaluated, and the measurement of MTT reduction and intracellular ATP levels were selected to assess cell viability. Results demonstrated that both assay systems are capable to metabolize the test compounds leading to increased toxicity, compared to their respective control systems. The co-incubation with the CYP3A4 inhibitor ketoconazole confirmed that the formation of reactive metabolites was CYP3A4 dependent. To further validate the functionality of the two assay systems, they were also used as a "detoxification system" using selected compounds that can be metabolized by CYP3A4 to metabolites less toxic than their parent compounds. These results show that both assay systems can be used to screen for metabolic activation, or de-activation, which may be useful as a rapid and relatively inexpensive in vitro assay for the prediction of CYP3A4 metabolism-mediated toxicity.     

Influence of body weight and type of chow on the sensitivity of rats to the behavioral effects of the direct-acting dopamine-receptor agonist quinpirole

Rationale  

Amount and type of food can alter dopamine systems and sensitivity to drugs acting on those systems.     

Glycogen synthase kinase-3 (GSK3) in psychiatric diseases and therapeutic interventions

Glycogen synthase kinase-3 (GSK3) has recently been linked to mood disorders and schizophrenia, and the neurotransmitter systems and therapeutic treatments associated with these diseases. GSK3 is a widely influential enzyme that is capable of phosphorylating, and thereby regulating, over forty known substrates. Four mechanisms regulating GSK3 (phosphorylation, protein complexes, localization, and substrate phosphorylation) combine to provide substrate-specific regulation of the actions of GSK3. Several intracellular signaling cascades converge on GSK3 to modulate its activity, and several neurotransmitter systems also regulate GSK3, including serotonergic, dopaminergic, cholinergic, and glutamatergic systems. Because of changes in these neurotransmitter systems and the actions of therapeutic drugs, GSK3 has been linked to the mood disorders, bipolar disorder and depression, and to schizophrenia. Inhibition of GSK3 may be an important therapeutic target of mood stabilizers, and regulation of GSK3 may be involved in the therapeutic effects of other drugs used in psychiatry. Dysregulated GSK3 in bipolar disorder, depression, and schizophrenia could have multiple effects that could impair neural plasticity, such as modulation of neuronal architecture, neurogenesis, gene expression, and the ability of neurons to respond to stressful, potentially lethal, conditions. In part because of these key actions of GSK3 and its associations with mood disorders and schizophrenia, much research is currently being devoted to identifying new selective inhibitors of GSK3.     

Basal level insulin delivery: in vitro release, stability, biocompatibility, and in vivo absorption from thermosensitive triblock copolymers

The major goal of this study was to develop the biodegradable and biocompatible thermosensitive polylactic acid-polyethylene glycol-polylactic acid triblock copolymer-based delivery systems for controlled release of basal level insulin for a longer duration after single subcutaneous injection. Insulin was dispersed into aqueous copolymer solutions to prepare the delivery system. The in vitro release profile of insulin from delivery systems was studied at 37°C in phosphate-buffered saline. Stability of released insulin was investigated using circular dichroism, differential scanning calorimetry, and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and skin histology were used to determine the in vitro and in vivo biocompatibility of the delivery systems, respectively. Streptozotocin-induced diabetic rat model was used to study the in vivo absorption and bioactivity of insulin. In vitro release studies indicated that the delivery systems released insulin over 3 months in structurally stable form. The delivery systems were biocompatible in vitro and in vivo. In vivo absorption and bioactivity studies demonstrated elevated insulin level and corresponding decreased blood glucose level in diabetic rats. Thus, the delivery systems released insulin at a controlled rate in vitro in conformationally and chemically stable form and in vivo in biologically active form up to 3 months.     

Folate-Targeted Nanoparticles Based on Albumin and Albumin/Alginate Mixtures as Controlled Release Systems of Tamoxifen: Synthesis and In Vitro Characterization

Purpose

Preparation and in vitro characterization of tamoxifen (TMX)-loaded folate-targeted nanoparticles based on disulfide bond reduced bovine serum albumin (BSA-SH) and BSA-SH/alginate-cysteine (BSA-SH/ALG-CYS) mixtures as drug delivery systems.

Methods

Folate-nanoparticles were characterized in terms of folate content, morphology, size, zeta potential, TMX load and drug release kinetics. Additionally, cell viability and cellular uptake of nanoparticles were determined using different cancer cell lines.

Results

Folic acid (FOL) was successfully attached to nanoparticles (ranging between 79 and170 μmol folate/g NP). Nanoparticles with 76–417 nm mean size were obtained and loaded with TMX (4.2–7.7 μg/mg NP). Zeta potential and drug extraction revealed major superficial placement of the drug, especially in the case of BSA/ALG-FOL systems. Drug release studies in the presence of surfactant showed a gradual release of the drug between 4–7 h. In general, low cytotoxicity of unloaded systems was found. Internalization of the systems was achieved and mediated by folate receptor, especially in the case of BSA NP-FOL. The administration of 10 μM TMX by TMX-FOL NP showed their efficacy as controlled TMX release systems.

Conclusions

Promising anticancer action of these new TMX-loaded folate-targeted systems was demonstrated, allowing a new administration route to be studied in further in vivo studies in order to improve current TMX therapy.     

Selective effects of activation of protein kinase C isozymes on cyclic AMP accumulation.

Activation of protein kinase C (PKC) in intact cells can induce significant changes, either facilitatory or inhibitory, in cyclic AMP accumulation elicited either by receptor activation or by the activator of adenylate cyclase, forskolin. Such interaction represents an example of "cross-talk" between second messenger systems and may underlie the biochemical basis of synchronization between external stimuli and biological responses. PKC is now known to comprise a variety of subspecies. Although differences among the PKC subspecies are apparent in terms of their enzymological properties, no functional differences among them have been described. In PC12 cells, where both alpha and gamma isozymes of PKC are present, activation of PKC causes enhancement of the responses of cyclic AMP-generating systems. In NCB20 cells and NIH 3T3 cells, where only the alpha isozyme is expressed, activation of PKC causes inhibition of cyclic AMP-generating systems. In NIH 3T3 cells after transfection of gamma-PKC, activation of the enzyme was no longer inhibitory; instead, a facilitation of cyclic AMP accumulation was observed. Thus, the alpha and gamma isozymes of PKC appear to have opposite actions, facilitatory for gamma-PKC and inhibitory for alpha-PKC, on the responses of cyclic AMP-generating systems in NIH 3T3 cells. Such opposing actions represent a remarkable functional distinction between two PKC subspecies.     

GABAA positive modulator and NMDA antagonist-like discriminative stimulus effects of isoflurane vapor in mice

Rationale  

Several neurotransmitter systems have been hypothesized to be involved in the in vivo effects of volatile anesthetics. Drug discrimination may represent a novel procedure to explore the neurochemical systems underlying the sub-anesthetic behavioral effects of these compounds.     

Two-level delivery systems based on CaCO3 cores for oral administration of therapeutic peptides

Abstract

Two-level systems for oral delivery of therapeutic peptides were developed; the carriers consist of CaCO₃ cores included into alginate granules. Such systems were first used for the delivery of low molecular weight drugs. It was shown that efficiency of encapsulation of peptides depends on their pI value, hydrophobicity, characteristics of the compounds used for doping CaCO₃ cores, their surface potential and the techniques employed for loading peptides into the first-level carriers. Doping CaCO₃ cores with dextran sulphate save their viability compared to the pristine CaCO₃ cores, but ensures delivery of the desired quantity of peptide when using a smaller amount of delivery systems. Introducing the inhibitor of peptidases leads to an increase in the concentration of peptide in rat blood after intragastric administration of the developed delivery systems. Scanning electron microscopy and energy-dispersive X-ray spectroscopy demonstrated the presence of fragments of destructed first-level carriers in blood and plasma of experimental animals.     

Application of drug-related problem (DRP) classification systems: a review of the literature

Background

For over 20 years, researchers wanting to record, discuss and compare drug-related problems (DRPs) have had the task of choosing between a multiplicity of classification systems offering a variable number of categories identified as causes of DRPs and DRPs.

Objective

To characterise studies which have reported DRPs through the use of a DRP classification system to determine types of classification systems chosen, factors influencing their choice, and methodological issues that may have affected their application.

Method

A systematic search of MEDLINE, CINAHL, International Pharmaceutical Abstracts (Ovid), EMBASE and PubMed was performed. All English language studies reporting DRPs through the use of a classification system published between January 2000 and July 2013 were reviewed, with no limitation on the type of study.

Results

Of 2,774 articles screened, 268 met our inclusion criteria. We identified the use of 20 different types of DRP classification systems. Three quarters of studies modified an existing classification system or developed their own. Few studies stated reasons for choice of system. We identified issues such as variability in skills of data collectors, selective choice of patients and missing data, affecting application of classification systems and limiting quality, analysis and comparison of studies.

Conclusion

There appeared to be no consensus on preference or structure of classification systems. Future studies should consider addressing or acknowledging the methodological issues identified. Through identification and discussion of these problems, recommendations for future studies and for practice have been made.     

Carbamazepine: a 'blind' assessment of CVP-associated metabolism and interactions in human liver-derived in vitro systems

1. The ability of various in vitro systems for CYP enzymes (computer modelling, human liver microsomes, precision-cut liver slices, hepatocytes in culture, recombinant enzymes) to predict various aspects of in vivo metabolism and kinetics of carbamazepine (CBZ) was investigated. 2. The study was part of the EUROCYP project that aimed to evaluate relevant human in vitro systems to study drug metabolism. 3. CBZ was given to the participating laboratories without disclosing its chemical nature. 4. The most important enzyme (CYP3A4) and metabolic route (10,11-epoxidation) were predicted by all the systems studied. 5. Minor enzymes and routes were predicted to a different extent by various systems. 6. Prediction of a clearance class, i.e. slow clearance, was correctly predicted by microsomes, slices, hepatocytes and recombinant enzymes (CYP3A4). 7. The 10,11-epoxidation of CBZ by the recombinant CYP3A4 was enhanced by the addition of exogenous cytochrome-b5, leading to a considerable over-prediction. 8. Induction potency of CBZ was predicted in cultured hepatocytes in which 7-ethoxycoumarin O-deethylase was used as an index activity. 9. It seems that for a principally CYP-metabolized substance such as CBZ, all liver-derived systems provide useful information for prediction of metabolic routes, rates and interactions.     

Targeting Tamoxifen to Breast Cancer Xenograft Tumours: Preclinical Efficacy of Folate-Attached Nanoparticles Based on Alginate-Cysteine/Disulphide-Bond-Reduced Albumin

Purpose

In vivo evaluation of tamoxifen (TMX)-loaded folate-targeted nanoparticles prepared from a mixture of disulphide bond reduced bovine serum albumin (BSA-SH) and alginate-cysteine (ALG-CYS) as targeted delivery systems of TMX to tumour tissues.

Methods

TMX in solution, TMX included into folate-nanoparticles and their non-targeted analogues were intravenously administered to nude mice carrying xenograft MCF-7 tumours. The antitumor activity of these systems was characterized in terms of tumour growth rate, histological and immunohistochemical analysis of tumour tissues and TMX biodistribution.

Results

TMX-folate-attached nanoparticles caused tumour remission whereas free TMX or TMX-non-targeted nanoparticles could only stop the tumour development. The histological evaluation of tumour tissues showed that those treated with folate-conjugated systems presented the most quiescent and disorganized structures. Additionally, the lowest concentrations of TMX accumulated in non-targeted organs were also found after administration of the drug using this formulation.

Conclusions

This study demonstrated that TMX-loaded folate-targeted systems were capable of reaching tumour sites, so enhancing the in vivo anticancer action of TMX, and allowing a new administration route to be applied and some of the current TMX therapy problems to be overcome.     

Antioxidant principles of<Emphasis Type="Italic">Nelumbo nucifera</Emphasis> stamens

In our ongoing study to identity antioxidants from natural sources, the antioxidant activity of Nelumbo nucifera stamens was evaluated for their potential to scavenge stable 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radicals, inhibit total reactive oxygen species (ROS) generation, in kidney homogenatas using 2',7'-dichlorodihydrofluorescein diacetate (DCHF-DA), and scavenge authentic peroxynitrites (ONOO-). A methanol (MeOH) extract of the stamens of N. nucifera showed strong antioxidant activity in the ONOO- system, and marginal activity in the DPPH and total ROS systems, so were therefore fractionated with several organic solvents, such as dichloromethane (CH2Cl2), ethyl acetate (EtOAc) and n-butanol (n-BuOH). The EtOAc soluble fraction, which exhibited strong antioxidant activity in all the model systems tested, was further purified by repeated silica gel and Sephadex LH-20 column chromatographies. Seven known flavonoids [kaempferol (1), kaempferol 3-O-beta-D-glucuronopyranosyl methylester (2), kaempferol 3-O-beta-D-glucopyranoside (3), kaempferol 3-O-beta-D-galactopyranoside (4), myricetin 3',5'-dimethylether 3-O-beta-D-glucopyranoside (5), kaempferol 3-O-alpha-L-rhamnopyranosyl-(1-->6)-beta-D-glucopyranoside (6) and kaempferol 3-O-beta-D-glucuronopyranoside (7)], along with beta-sitosterol glucopyranoside (8), were isolated. Compound 1 possessed good activities in all the model systems tested. Compounds 2 and 7 showed scavenging activities in the DPPH and ONOO- tests, while compounds 3 and 4 were only active in the ONOO- test. Conversely, compound 8 showed no activities in any of the model systems tested.     

Development of three parallel cytochrome P450 enzyme affinity detection systems coupled on-line to gradient high-performance liquid chromatography.

A high resolution screening (HRS) technology is described, in which gradient high-performance liquid chromatography (HPLC) is connected on-line to three parallel placed bioaffinity detection systems containing mammalian cytochromes P450 (P450s). The three so-called enzyme affinity detection (EAD) systems contained, respectively, liver microsomes from rats induced by beta-naphthoflavone (CYP1A activity), phenobarbital (CYP2B activity), and dexamethasone (CYP3A activity). Each P450-EAD system was optimized for enzyme, substrate, and organic modifier (isopropyl alcohol, methanol, and acetonitrile) in flow injection analysis mode. Characteristic P450 ligands were used to validate the P450-EAD systems. IC(50) values of the ligands were measured and found to be similar to those obtained with conventional microtiter plate reader assays. Detection limits (n = 3; signal-to-noise ratio = 3) of potent inhibitors ranged from 1 to 3 pmol for CYP1A activity, 4 to 17 pmol for CYP2B activity, and 4 to 15 pmol for CYP3A activity. The three optimized P450-EAD systems were subsequently coupled to gradient HPLC and used to screen compound mixtures for individual ligands. Finally, to increase analysis efficiency, a HRS system was constructed in which all three P450-EAD systems were coupled on-line and in parallel to gradient HPLC. The triple parallelized P450-EAD system was shown to enable rapid profiling of individual components in complex mixtures for inhibitory activity to three different P450s.     

Correlation of inhibitory potencies of putative antagonists for sigma receptors in brain and spleen

The putative sigma receptor antagonists, haloperidol, HR 375, BMY 14802 and BW 234U potently inhibited both [3H]d-N-allylnormetazocine binding to sigma receptors in brain homogenates and [3H]haloperidol binding to sigma receptors in spleen homogenates. An excellent correlation of inhibitory potencies in the two assay systems was obtained. The results support the view that [3H]d-N-allylnormetazocine and [3H]haloperidol both label the same receptor populations, and suggest that sigma antagonists may be useful in elucidating physiological role(s) of sigma receptors in the nervous and immune systems.     

医院药学计算机网络系统现状与展望

医院药学计算机网络管理是医院管理信息系统（HMIS）的重要组成部分，HMIS主要采用3＋以太网和NOVELL以太网，以NOVELL网为主，体系结构以微机网络＋文件服务器系统为主。传统的小型数据库管理系统如dBase、Foxbase、FoxPro等已越来越不能适应复杂的HMIS的需要，但MsSQLServer等大型数据库是一种企业级关系型数据库，能很好地实现网络计算.将是今后网络的发展方向。