Analysis Of Drug Effects Research Articles

Multiplexed Analysis for Anti-Epidermal Growth Factor Receptor Tumor Cell Growth Inhibition Based on Quantum Dot Probes.

Quantum dot (QD) multicolor analysis was achieved by labeling the epidermal growth factor and labeling the antibody against the epidermal growth factor receptor (anti-EGFR) using different QDs. On the basis of the fluorescence intensity of the two types of QDs, the amount of epidermal growth factors (EGFs) in the cells and anti-EGFRs bound to EGFRs were analyzed. The functions of anti-EGFR in preventing HeLa cells from engulfing EGF and inhibiting overproliferation of the HeLa cells were also studied. Meanwhile, parallel analysis was conducted to analyze the heterogeneity of cells at the single-cell level using a single-cell array. This provides a novel approach for the multiplexed analysis for the anti-EGFR functions in cells together with the cell heterogeneity. It also lays a foundation for parallel analysis and detection, using various fluorescence probes, simultaneous tracking, and detection of multiple targets at an overall level or individual level, and multichannel analysis of drug effects.

An integrative genome-wide transcriptome reveals that candesartan is neuroprotective and a candidate therapeutic for Alzheimer's disease.

BackgroundAlzheimer’s disease is the most frequent age-related dementia, and is currently without treatment. To identify possible targets for early therapeutic intervention we focused on glutamate excitotoxicity, a major early pathogenic factor, and the effects of candesartan, an angiotensin receptor blocker of neuroprotective efficacy in cell cultures and rodent models of Alzheimer’s disease. The overall goal of the study was to determine whether gene analysis of drug effects in a primary neuronal culture correlate with alterations in gene expression in Alzheimer’s disease, thus providing further preclinical evidence of beneficial therapeutic effects.MethodsPrimary neuronal cultures were treated with candesartan at neuroprotective concentrations followed by excitotoxic glutamate amounts. We performed genome-wide expression profile analysis and data evaluation by ingenuity pathway analysis and gene set enrichment analysis, compared with alterations in gene expression from two independent published datasets identified by microarray analysis of postmortem hippocampus from Alzheimer’s disease patients. Preferential expression in cerebrovascular endothelial cells or neurons was analyzed by comparison to published gene expression in these cells isolated from human cortex by laser capture microdissection.ResultsCandesartan prevented glutamate upregulation or downregulation of several hundred genes in our cultures. Ingenuity pathway analysis and gene set enrichment analysis revealed that inflammation, cardiovascular disease and diabetes signal transduction pathways and amyloid β metabolism were major components of the neuronal response to glutamate excitotoxicity. Further analysis showed associations of glutamate-induced changes in the expression of several hundred genes, normalized by candesartan, with similar alterations observed in hippocampus from Alzheimer’s disease patients. Gene analysis of neurons and cerebrovascular endothelial cells obtained by laser capture microdissection revealed that genes up- and downregulated by glutamate were preferentially expressed in endothelial cells and neurons, respectively.ConclusionsOur data may be interpreted as evidence of direct candesartan neuroprotection beyond its effects on blood pressure, revealing common and novel disease mechanisms that may underlie the in vitro gene alterations reported here and glutamate-induced cell injury in Alzheimer’s disease. Our observations provide novel evidence for candesartan neuroprotection through early molecular mechanisms of injury in Alzheimer’s disease, supporting testing this compound in controlled clinical studies in the early stages of the illness.Electronic supplementary materialThe online version of this article (doi:10.1186/s13195-015-0167-5) contains supplementary material, which is available to authorized users.

Quantitative analysis of drug effects at the whole-body level: a case study for glucose metabolism in malaria patients

We propose a hierarchical modelling approach to construct models for disease states at the whole-body level. Such models can simulate effects of drug-induced inhibition of reaction steps on the whole-body physiology. We illustrate the approach for glucose metabolism in malaria patients, by merging two detailed kinetic models for glucose metabolism in the parasite Plasmodium falciparum and the human red blood cell with a coarse-grained model for whole-body glucose metabolism. In addition we use a genome-scale metabolic model for the parasite to predict amino acid production profiles by the malaria parasite that can be used as a complex biomarker.

Mechanisms of astrocytic K(+) clearance and swelling under high extracellular K(+) concentrations.

In response to the elevation of extracellular K(+) concentration ([K(+)]out), astrocytes clear excessive K(+) to maintain conditions necessary for neural activity. K(+) clearance in astrocytes occurs via two processes: K(+) uptake and K(+) spatial buffering. High [K(+)]out also induces swelling in astrocytes, leading to edema and cell death in the brain. Despite the importance of astrocytic K(+) clearance and swelling, the underlying mechanisms remain unclear. Here, we report results from a simulation analysis of astrocytic K(+) clearance and swelling. Astrocyte models were constructed by incorporating various mechanisms such as intra/extracellular ion concentrations of Na(+), K(+), and Cl(-), cell volume, and models of Na,K-ATPase, Na-K-Cl cotransporter (NKCC), K-Cl cotransporter, inwardly-rectifying K(+) (KIR) channel, passive Cl(-) current, and aquaporin channel. The simulated response of astrocyte models under the uniform distribution of high [K(+)]out revealed significant contributions of NKCC and Na,K-ATPase to increases of intracellular K(+) and Cl(-) concentrations, and swelling. Moreover, we found that, under the non-uniform distribution of high [K(+)]out, KIR channels localized at synaptic clefts absorbed excess K(+) by depolarizing the equivalent potential of K(+) (E K) above membrane potential, while K(+) released through perivascular KIR channels was enhanced by hyperpolarizing E K and depolarizing membrane potential. Further analysis of simulated drug effects revealed that astrocyte swelling was modulated by blocking each of the ion channels and transporters. Our simulation analysis revealed controversial mechanisms of astrocytic K(+) clearance and swelling resulting from complex interactions among ion channels and transporters.

Data of a fluorescent imaging-based analysis of anti-cancer drug effects on three-dimensional cultures of breast cancer cells

Three-dimensional (3D) cell culture is a powerful tool to study cell growth under 3D condition. To perform a simple test for anti-cancer drugs in 3D culture, visualization of non-proliferated cells is required. We propose a fluorescent imaging-based assay to analyze cancer cell proliferation in 3D culture. We used a pulse-labeling technique with a photoconvertible fluorescent protein Kaede to identify non-proliferated cells. This assay allows us to observe change in cell proliferation in 3D culture by simple imaging. Using this assay, we obtained the data of the effects of anti-cancer drugs, 5-fluorouracil and PD0332991 in a breast cancer cell line, MCF-7.

Direct Estimation of CaV1.2 Gating Parameters: Quantification of Voltage Sensor - Pore Transductions and their Modulation by FLP 64176.

Calcium agonists such as FPL 64174 increase macroscopic calcium channel currents and induce substantial changes in current kinetics. Their molecular mechanism of action is currently unknown. Here we propose a technique enabling the estimation of FPL 64174 effects on rate constants of the voltage sensing machinery and pore transitions from macroscopic CaV1.2 current kinetics making use of a hybrid stochastic-deterministic optimization procedure. Current traces of wild type CaV1.2, a channel construct with neutralized segment IIS4 (IIS4N) and a pore mutant (A780T) were fitted to a circular four-state (rest, activated, open, deactivated) channel model in control and FPL 64174 (1 µM). The estimated rate constants provided novel insights in how structural elements of the voltage sensing unit and the channel pore influence the action of FPL 64174. The new approach may be applicable for the analysis of drug effects on other ion channels as well as for quantification of VS transitions and rate constants of pore gating from macroscopic current kinetics.

Abstract 1211: Metabolite assays to illuminate cellular energy networks

Abstract Every organism requires cellular energy metabolism to meet developmental needs. When cells change from a resting to a proliferative state, the metabolic pathways are reprogrammed to meet the demands of proliferation. Diseases such as cancer can occur when changes in the metabolic pathways support unwanted functions such as uncontrolled proliferation. Assays for analyzing metabolites and metabolic enzymes are needed to understand cellular metabolic requirements and energy network regulation under normal and disease conditions. We exploited the robustness, sensitivity and broad dynamic range of bioluminescence detection to develop assays for the key metabolites lactate, glucose, glutamate and glucose-6-phosphate. The technology involves using specific dehydrogenases to couple metabolite levels to NAD(P)H production. In the presence of NAD(P)H, a novel proluciferin is converted to a substrate for luciferase resulting in light signals proportional to the starting metabolite concentration. Samples (e.g. mammalian cells, culture media, tissues, 3D microtissues) are added to the metabolite detection reagent in a 1:1 ratio. An inactivation solution added to complex biological samples lowers the background and increases sensitivity by lysing the cells, inactivating cellular enzymes, and destroying endogenous NAD(P)H. The sensitivity (1-5 pmol/sample) and broad assay window (maximum signal-to-background > 100 fold) of the assays allows simultaneous detection of multiple metabolites from the same set of samples. The wide assay window allows detection of very small changes in metabolite concentration, an advantage over conventional fluorescent and colorimetric detection methods. The assays adapt to various formats (96, 384-well plates) and require no instrumentation other than a luminometer. The assays can be used to measure the levels of critical metabolites and to monitor the effects of inhibitors and activators of glycolysis. The shift from oxidative phosphorylation to glycolysis can be monitored by measuring lactate production and secretion as well as glucose consumption from the same set of samples. The assays easily multiplex with cell viability assays enabling analysis of drug effects on cell proliferation, cell metabolism and mitochondria function. Citation Format: Mary Sobol, Jolanta Vidugiriene, Donna Leippe, Gediminas Vidugiris, Wenhui Zhou, James Cali. Metabolite assays to illuminate cellular energy networks. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1211. doi:10.1158/1538-7445.AM2015-1211

Galahad: a web server for drug effect analysis from gene expression.

Galahad (https://galahad.esat.kuleuven.be) is a web-based application for analysis of drug effects. It provides an intuitive interface to be used by anybody interested in leveraging microarray data to gain insights into the pharmacological effects of a drug, mainly identification of candidate targets, elucidation of mode of action and understanding of off-target effects. The core of Galahad is a network-based analysis method of gene expression. As an input, Galahad takes raw Affymetrix human microarray data from treatment versus control experiments and provides quality control and data exploration tools, as well as computation of differential expression. Alternatively, differential expression values can be uploaded directly. Using these differential expression values, drug target prioritization and both pathway and disease enrichment can be calculated and visualized. Drug target prioritization is based on the integration of the gene expression data with a functional protein association network. The web site is free and open to all and there is no login requirement.

Verbal learning impairment in euthymic bipolar disorder: BDI v BDII

ObjectivesCognitive impairment is known to occur in bipolar disorder (BD), even in euthymic patients, with largest effect sizes often seen in Verbal Learning and Memory Tasks (VLT). However, comparisons between BD Type-I and Type-II have produced inconsistent results partly due to low sample sizes. MethodsThis study compared the performance of 183 BDI with 96 BDII out-patients on an adapted version of the Rey Verbal Learning Task. Gender, age, years of education, mood scores and age at onset were all used as covariates. Current medication and a variety of illness variables were also investigated for potential effects on VLT performance. ResultsBDI patients were significantly impaired relative to BDII patients on all five VLT outcome measures after controlling for the other variables [Effect Sizes=.13–.17]. The impairments seem to be unrelated to drug treatment and largely unrelated to illness variables, although age of onset affected performance on three outcome measures and number of episodes of mood elevation affected performance on one. LimitationsThis study used historical healthy controls. Analysis of potential drug effects was limited by insufficient participants not being drug free. Cross-sectional nature of the study limited the analysis of the potential effect of illness variables. ConclusionsThis study replicates earlier findings of increased verbal learning impairment in BDI patients relative to BDII in a substantially larger sample. Such performance cannot be wholly explained by medication effects or illness variables. Thus, the cognitive impairment is likely to reflect a phenotypic difference between bipolar sub-types.

In vivo imaging of cell proliferation for a dynamic, whole body, analysis of undesired drug effects.

Noninvasive in vivo imaging offers a novel approach to preclinical studies opening the possibility of investigating biological events in the spatiotemporal dimension (eg, in any district of the body in time). Toxicological analysis may benefit from this novel approach through precise identification of the time and the target organs of toxicity manifestations, and assessment of the reversibility of toxic insults. The current limitation for routine application of this technology is the lack of appropriate surrogate markers for imaging toxicological events. Here, we demonstrate that in vivo imaging of a proliferation marker is capable of measuring the reduction of cell proliferation due to genotoxic/apoptotic agents, γ rays or antineoplastic drugs, or the increased proliferation associated with the inflammatory and regenerative reactions occurring after a toxic insult. A number of tools are currently available for imaging proliferation in preclinical and clinical settings, however our data provide a novel way to translate the evidence of toxic effects obtained in preclinical animal studies, by the direct, noninvasive measure of dividing cells in humans.

Drug-target based cross-sectional analysis of olfactory drug effects.

Drug effects on the human sense of smell attract increasing interest, yet systematic evidence from controlled studies is sparse. The present cross-sectional approach to olfactory drug effects made use of the recent developments in informatics, knowledge discovery, and data mining allowing connecting drug-related information from humans with underlying molecular drug targets. In this prospective cross-sectional study, n = 1008 outpatients at a general practitioner were enrolled. All currently taken medications were obtained, and olfactory function was assessed by means of a clinically established 12-item odor identification test. The association between the patients' sense of smell and the administered medications was based (i) on the active pharmacological substances and (ii) on the molecular targets queried from the publicly accessible DrugBank database. Of the 168 different substances, six were taken sufficiently often to be analyzed. The administration of levothyroxine was associated with a higher olfactory score (p = 0.033). For the 168 drugs, 323 different targets could be queried. Thirty-one gene products were addressed sufficiently often to be analyzed. Besides agonistic targeting of thyroid hormone receptors (genes THRA1, THRB1) agreeing with the above result, antagonistically targeting the adrenoceptor alpha 1A (gene ADRA1A) by several unrelated medications was associated with a significantly higher olfactory score (p = 0.012). The identified drug effects on olfaction are both biologically plausible based on supportive information from basic science studies. The novel molecular target-based approach suggested clear advantages over the classical drug or drug class-based approach. It increased the analyzable data volume fivefold and provided plausible hypotheses about mechanistic drug effects opening possibilities for drug discovery and repurposing.

Radio sensitization of Colon Cancer Cells Mediated by Gemcitabine through Cell Cycle Synchronization

We evaluated gemcitabine together with ionizing radiation for improved cell growth inhibition with respect to that by radiation alone in the human colon carcinoma cell lines SW620, HT-29 and SNU-C4. To this end, cells were exposed for 24 h to gemcitabine and then assessed for growth response with the sulphorhodamine B assay. The cell lines, as well as exposed to ionizing radiation and a combination of gemcitabine and ionizing radiation for 24 h, 48 h and 72 h and the radiosensitivity was assessed using a clonogenic assay. Multiple drug effect analysis was used to evaluate the synergistic effect, which was then related to the cell cycle phase distribution. The SNU-C4 cell line showed a greater sensitivity to gemcitabine in comparison to the other two cell lines, while the SW620 cells was more sensitive to damage induced by radiation. Furthermore, gemcitabine increased by 50% the effect of ionizing radiation after 24 h in SW620 cell line, while in the others cell lines, this effect was observed only after 72 h. Moreover, gemcitabine associated with ionizing radiation was synergistic in SW620, HT-29 and SNU-C4 cells. Increased in S phase fraction was seen in gemcitabine treatment in all cell lines studied. While, ionizing radiation only induced an accumulation on G2/M in SW620 and HT-29 cell lines, indicating that SNU-C4 is less sensitive to radiation effect. A significant accumulation of cells in S phase after treatment with gemcitabine followed by radiation was observed in all cell lines. In summary our data indicate that gemcitabine increases the radiosensitivity to radiation in cell lines derived from human colon cancer, and that this effect seems to be associated with the ability of gemcitabine to synchronize cells in S phase of the cell cycle.

Abstract 3134: Crizotinib inhibits tumor growth and metabolic inactivation of gemcitabine in new patient derived orthotopic pancreatic tumors with c-Met overexpression: A step toward personalized treatment in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) remains a major unsolved health problem.Most drugs that pass preclinical tests fail in patient, emphasizing the need of improved preclinical models. This study aimed to (1) develop orthotopic models employing primary PDAC cells genetically engineered to express Firefly- and Gaussia- luciferase (Fluc/Gluc), enabling monitoring of tumor growth, and (2) evaluate the therapeutic potential of c-Met inhibitors.Four primary PDAC-cultures were successfully transduced with lentiviral vectors containing Fluc and Gluc, and then injected orthotopically into pancreas. The bioluminescence intensities increased overtime in all the models. Histopathological and immunohistochemical analyses were carried out on paraffin-embedded slices of pancreas, as well as on metastatic lesions in liver, lungs and lymph nodes, showing that the xenografts recapitulated the phenotype of human PDAC. Genetic characteristics of the xenografts were similar to their originator tumor/cells, as assessed by array-CGH. Genomic and immunohistochemical analysis revealed c-Met increased copy-number and overexpression in one of our models (PDAC3). Therefore we explored several c-Met inhibitors in vitro and in vivo. Crizotinib emerged as the most active inhibitor of cell growth, with an IC50 of 0.5 μM in the PDAC3. Median drug-effect analysis revealed strong synergistic interaction of crizotinib with gemcitabine. The mice receiving this combination had significantly lower BLI signals compared to mice receiving either gemcitabine or crizotinib alone (e.g., at day 30, Fluc was 7x108 vs. 1x108 photon/second/cm2 in gemcitabine and combination group, respectively), which was reflected in a significant longer survival of mice treated with combination. Importantly, we observed a significant increase of gemcitabine in the combination group with respect to the mice treated with gemcitabine alone (6.3 vs. 2.5nm/mg, as measured by LC/MS-MS). Correspondingly, crizotinib reduced the activity of cytidine deaminase both in tissue specimens, and blood samples. Since intact c-Met signaling is a critical factor in the protection against excessive generation of endogenous ROS, we demonstrated that ROS induction caused c-Met inhibition was responsible for CDA degradation after exposure to crizotinib, resulting in the increased stabilization of gemcitabine in the PDAC-3 cells. Our orthotopic imaging models showed genetic, histopathological and metastatic features similar to their originator tumors and enabled the identification of c-Met as a potential therapeutic target. Crizotinib and gemcitabine are a promising drug combination against this target, acting synergistically also through the increase of drug delivery by CDA modulation, and warranting further investigation for the treatment of PDAC patients. Citation Format: Amir Avan, Viola Caretti, Niccola Funel, Elena Galvani, Mina Maftouh, Richard J. Honeywell, Tonny Lagerweij, Daniela Campani, Henk M. Verheul, Gerrit Jan Schuurhuis, Ugo Boggi, Godefridus J. Peters, Thomas Würdinger, Elisa Giovannetti. Crizotinib inhibits tumor growth and metabolic inactivation of gemcitabine in new patient derived orthotopic pancreatic tumors with c-Met overexpression: A step toward personalized treatment in pancreatic cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3134. doi:10.1158/1538-7445.AM2014-3134

474P - Tivantinib-Gemcitabine: Pharmacological Rational for a New Combination in Pancreatic Cancer

ABSTRACT Background: A trial on the combination of tivantinib with gemcitabine demonstrated manageable toxicity and anticancer activity (20% partial response and 46% stable disease), in different tumor types, including lung and pancreatic cancer (1). However, pharmacokinetics showed no differences in the concentration of tivantinib or gemcitabine, while circulating VEGF, HGF and c-Met failed to correlate with response. Our in vitro and in vivo studies on the combination of another c-Met inhibitor (crizotinib) with gemcitabine showed increased blood, cellular, and tissue concentrations of gemcitabine, through a cytidine deaminase (CDA)-mediated mechanism. Here we evaluated whether the tivantinib-gemcitabine combination might also affect this enzyme. Furthermore we evaluated microtubule disruption as a possible pharmacodynamic biomarker. Methods: Four primary pancreatic cancer cell cultures were obtained from radically-resected patients. C-Met and phospho-c-Met were evaluated in the originator tumors and in the cells using specific monoclonal anti-human c-Met and anti-phospho-Y1003-c-Met antibodies. Cell growth inhibitory effects of tivantinib and gemcitabine were determined by SRB assay. CDA activity was evaluated by HPLC. Finally, perturbed microtubule dynamics was studied by FACS analysis. Results: Cells and tissues showed a variable expression of c-Met, and cells with the highest c-Met levels were more sensitive to tivantinib and tivantinib-gemcitabine combination. Median drug-effect analysis revealed strong synergistic interaction. However this synergism was not associated to the increase in the accumulation of gemcitabine-nucleotides, as observed for crizotinib-gemcitabine. Furthermore no modulation of CDA activity was determined by direct interaction with tivantinib. Conversely, we observed a significant inhibition of tubulin polymerization both after tivantinib and gemcitabine-tivantinib exposure. Conclusions: Tivantinib displays synergistic cytotoxic activity with gemcitabine, via molecular mechanisms that are independent from CDA, but affect tubulin polymerization. These notions might have an impact on the selection of patients for future trials. Disclosure: All authors have declared no conflicts of interest.

Fluorescence Microscopy Analysis of Drug Effect on Autophagosome Formation

The autophagy protein, LC3 represents a reliable characteristic marker for autophagosomal structures. The initial LC3 is processed by the cysteine protease autophagy-related gene 4 (Atg4) at its C terminus in order to create LC3-I generally localized in the cytoplasm. Afterwards LC3-I is conjugated with phosphatidylethanolamine (PE) to become LC3-PE or LC3-II predominantly localised on the autophagosomal membranes (outer and inner). Autolysosomal content of LC3-II is very low as upon autophago/lysosomal fusion it is either cleaved off from the outer membrane by Atg4 or degraded together with the inner membrane by the lysosomal activity. Therefore GFP-LC3 and mCherry-GFP-LC3 might be visualized by conventional or confocal fluorescence microscopy (FM). In this situation mCherry-GFP-LC3 or GFP-LC3 cytoplasmic pool is visualized as a homogeneously dispersed signal and mCherry-GFP-LC3-II or GFP-LC3-II containing autophagosomes are detected as punctae formations. The number of punctae may be used as marker of autophagosomal abundance. In general we recommend counting the average number of GFP-LC3 punctae per cell.

Polydatin, a natural precursor of resveratrol, induces cell cycle arrest and differentiation of human colorectal Caco-2 cell

BackgroundHuman colon adenocarcinoma cells are resistant to chemotherapeutic agents, such as anthracyclines, that induce death by increasing the reactive oxygen species. A number of studies have been focused on chemo-preventive use of resveratrol as antioxidant against cardiovascular diseases, aging and cancer. While resveratrol cytotoxic action was due to its pro-oxidant properties. In this study, we investigate whether the Resveratrol (trans-3,5,49-trihydroxystilbene) and its natural precursor Polydatin (resveratrol-3-O-b-mono- D-glucoside, the glycoside form of resveratrol) combination, might have a cooperative antitumor effect on either growing or differentiated human adenocarcinoma colon cancer cells.MethodsThe polydatin and resveratrol pharmacological interaction was evaluated in vitro on growing and differentiated Caco-2 cell lines by median drug effect analysis calculating a combination index with CalcuSyn software. We have selected a synergistic combination and we have evaluated its effect on the biological and molecular mechanisms of cell death.ResultsSimultaneous exposure to polydatin and resveratrol produced synergistic antiproliferative effects compared with single compound treatment. We demonstrated that polydatin alone or in combination with resveratrol at 3:1 molar ratio synergistically modulated oxidative stress, cell cycle, differentiation and apoptosis. Worthy of note treatment with polydatin induced a nuclear localization and decreased expression of heat shock protein 27, and vimentin redistributed within the cell.ConclusionsFrom morphological, and biochemical outcome we obtained evidences that polydatin induced a transition from a proliferative morphology to cell-specific differentiated structures and caused human CaCo-2 cell death by induction of apoptosis. Our data suggest the potential use of polydatin in combination chemotherapy for human colon cancer.

Innovations in studying in vivo cell behavior and pharmacology in complex tissues – microvascular endothelial cells in the spotlight

Many studies on the molecular control underlying normal cell behavior and cellular responses to disease stimuli and pharmacological intervention are conducted in single-cell culture systems, while the read-out of cellular engagement in disease and responsiveness to drugs in vivo is often based on overall tissue responses. As the majority of drugs under development aim to specifically interact with molecular targets in subsets of cells in complex tissues, this approach poses a major experimental discrepancy that prevents successful development of new therapeutics. In this review, we address the shortcomings of the use of artificial (single) cell systems and of whole tissue analyses in creating a better understanding of cell engagement in disease and of the true effects of drugs. We focus on microvascular endothelial cells that actively engage in a wide range of physiological and pathological processes. We propose a new strategy in which in vivo molecular control of cells is studied directly in the diseased endothelium instead of at a (far) distance from the site where drugs have to act, thereby accounting for tissue-controlled cell responses. The strategy uses laser microdissection-based enrichment of microvascular endothelium which, when combined with transcriptome and (phospho)proteome analyses, provides a factual view on their status in their complex microenvironment. Combining this with miniaturized sample handling using microfluidic devices enables handling the minute sample input that results from this strategy. The multidisciplinary approach proposed will enable compartmentalized analysis of cell behavior and drug effects in complex tissue to become widely implemented in daily biomedical research and drug development practice.

PKPD modelling of the interrelationship between mean arterial BP, cardiac output and total peripheral resistance in conscious rats

The homeostatic control of arterial BP is well understood with changes in BP resulting from changes in cardiac output (CO) and/or total peripheral resistance (TPR). A mechanism-based and quantitative analysis of drug effects on this interrelationship could provide a basis for the prediction of drug effects on BP. Hence, we aimed to develop a mechanism-based pharmacokinetic-pharmacodynamic (PKPD) model in rats that could be used to characterize the effects of cardiovascular drugs with different mechanisms of action (MoA) on the interrelationship between BP, CO and TPR. The cardiovascular effects of six drugs with diverse MoA, (amlodipine, fasudil, enalapril, propranolol, hydrochlorothiazide and prazosin) were characterized in spontaneously hypertensive rats. The rats were chronically instrumented with ascending aortic flow probes and/or aortic catheters/radiotransmitters for continuous recording of CO and/or BP. Data were analysed in conjunction with independent information on the time course of drug concentration using a mechanism-based PKPD modelling approach. By simultaneous analysis of the effects of six different compounds, the dynamics of the interrelationship between BP, CO and TPR were quantified. System-specific parameters could be distinguished from drug-specific parameters indicating that the model developed is drug-independent. A system-specific model characterizing the interrelationship between BP, CO and TPR was obtained, which can be used to quantify and predict the cardiovascular effects of a drug and to elucidate the MoA for novel compounds. Ultimately, the proposed PKPD model could be used to predict the effects of a particular drug on BP in humans based on preclinical data.

Dynamical modeling of drug effect using hybrid systems.

Drug discovery today is a complex, expensive, and time-consuming process with high attrition rate. A more systematic approach is needed to combine innovative approaches in order to lead to more effective and efficient drug development. This article provides systematic mathematical analysis and dynamical modeling of drug effect under gene regulatory network contexts. A hybrid systems model, which merges together discrete and continuous dynamics into a single dynamical model, is proposed to study dynamics of the underlying regulatory network under drug perturbations. The major goal is to understand how the system changes when perturbed by drugs and give suggestions for better therapeutic interventions. A realistic periodic drug intake scenario is considered, drug pharmacokinetics and pharmacodynamics information being taken into account in the proposed hybrid systems model. Simulations are performed using MATLAB/SIMULINK to corroborate the analytical results.

Immunotoxicity, Flow Cytometry, and Chemoinformatics: Review, Bibliometric Analysis, and New QSAR Model of Drug Effects Over Macrophages

Bibliometric methods for analyzing and describing research output have been supported internationally by the establishment and operation of organizations such as the Institute for Scientific Information (ISI) or Scimago Ranking Institutions (SRI). This study provides an overview of the research performance of major World countries in the field cytokines, Citometric bead assays and QSAR, the most important journals in which they published their research articles, and the most important academic institutions publishing them. The analysis was based on Thomson Scientific's Web of Science (WoS), and Scimago group calculated bibliometric indicators of publication activity and actual citation impact. Studying the time period 2005-2010, and shows the visibility of Medicinal Chemistry Bioorganic in this thematic noting that the visibility of a journal must take into account not only the impact factor, but the prestige, popularity and representativeness of the theme that addresses the same making a comprehensive assessment of bibliometric indicators.