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Rodent Test of Attention and Impulsivity: The 5-Choice Serial Reaction Time Task.
Authors: Higgins GA, Silenieks LB Abstract The 5-choice serial reaction time task (5-CSRTT) is employed extensively to measure attention in rodents. The assay involves animals trained to respond to a brief, unpredictable visual stimulus presented in one of five locations. The effects of experimental manipulations on response speed and choice accuracy are measured, and each related to attentional performance. The 5-CSRTT is also used to measure motor impulsivity. Adapted from a human task, the 5-CSRTT can be employed with rodents or primates, highlighting its translational value. Another strength of this procedure i...
Source: Current Protocols in Pharmacology - September 12, 2017 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research

Development and Applications of Patient-Derived Xenograft Models in Humanized Mice for Oncology and Immune-Oncology Drug Discovery.
Authors: Verma B, Ritchie M, Mancini M Abstract With the recent approval of four novel immune oncology agents for the treatment of various cancers, the emerging power of this drug class has been substantiated. However, the full potential of such agents is yet to be realized, with only a fraction of the patient population responding to these drugs. A more advanced pre-clinical and translational research platform may increase our understanding of the mechanisms associated with immune-mediated cancer cell death, thereby facilitating the design and development of more generally efficacious agents and drug regimens. Des...
Source: Current Protocols in Pharmacology - September 12, 2017 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research

All-Optical Electrophysiology for Disease Modeling and Pharmacological Characterization of Neurons.
Authors: Werley CA, Brookings T, Upadhyay H, Williams LA, McManus OB, Dempsey GT Abstract A key challenge for establishing a phenotypic screen for neuronal excitability is measurement of membrane potential changes with high throughput and accuracy. Most approaches for probing excitability rely on low-throughput, invasive methods or lack cell-specific information. These limitations stimulated the development of novel strategies for characterizing the electrical properties of cultured neurons. Among these was the development of optogenetic technologies (Optopatch) that allow for stimulation and recording of membrane ...
Source: Current Protocols in Pharmacology - September 12, 2017 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research

Murine Retrovirally-Transduced Bone Marrow Engraftment Models of MLL-Fusion-Driven Acute Myelogenous Leukemias (AML).
Authors: Stubbs MC, Krivtsov AV Abstract MLL-rearranged leukemia represents approximately 5% to 10% of adult acute myelogenous leukemia (AML) and nearly half of all infant/pediatric acute leukemia cases. These leukemias have a poor prognosis, and there are no approved therapeutic options. The rearrangement in the MLL gene leads to aberrant expression of MLL-fusion proteins. These are transforming in murine bone marrow and, in particular, on stem cells and myeloid progenitors derived from bone marrow or fetal liver. The commonality of the MLL fusions is the in-frame fusion of 8 to 11 N-terminal exons of MLL1 (KMT2a)...
Source: Current Protocols in Pharmacology - September 12, 2017 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research

Label-Free Dynamic Mass Redistribution and Bio-Impedance Methods for Drug Discovery.
Authors: Grundmann M Abstract Label-free biosensors are increasingly employed in drug discovery. Cell-based biosensors provide valuable insights into the biological consequences of exposing cells and tissues to chemical agents and the underlying molecular mechanisms associated with these effects. Optical biosensors based on the detection of dynamic mass redistribution (DMR) and impedance biosensors using cellular dielectric spectroscopy (CDS) capture changes of the cytoskeleton of living cells in real time. Because signal transduction correlates with changes in cell morphology, DMR and CDS biosensors are exquisitel...
Source: Current Protocols in Pharmacology - June 24, 2017 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research

Overview of Transgenic Mouse Models of Myeloproliferative Neoplasms (MPNs).
Authors: Dunbar A, Nazir A, Levine R Abstract Myeloproliferative neoplasms (MPNs) are a class of hematologic diseases characterized by aberrant proliferation of one or more myeloid lineages and progressive bone marrow fibrosis. In 2005, seminal work by multiple groups identified the JAK2V617F mutation in a significant fraction of MPN patients. Since that time, murine models of JAK2V617F have greatly enhanced the understanding of the role of aberrant JAK-STAT signaling in MPN pathogenesis and have provided an in vivo pre-clinical platform that can be used to develop novel therapies. From early retroviral transductio...
Source: Current Protocols in Pharmacology - June 24, 2017 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research

The Ussing Chamber Assay to Study Drug Metabolism and Transport in the Human Intestine.
Authors: Kisser B, Mangelsen E, Wingolf C, Partecke LI, Heidecke CD, Tannergren C, Oswald S, Keiser M Abstract The Ussing chamber is an old but still powerful technique originally designed to study the vectorial transport of ions through frog skin. This technique is also used to investigate the transport of chemical agents through the intestinal barrier as well as drug metabolism in enterocytes, both of which are key determinants for the bioavailability of orally administered drugs. More contemporary model systems, such as Caco-2 cell monolayers or stably transfected cells, are more limited in their use compared to...
Source: Current Protocols in Pharmacology - June 24, 2017 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research

Biomarkers-A General Review.
Authors: Aronson JK, Ferner RE Abstract A biomarker is a biological observation that substitutes for and ideally predicts a clinically relevant endpoint or intermediate outcome that is more difficult to observe. The use of clinical biomarkers is easier and less expensive than direct measurement of the final clinical endpoint, and biomarkers are usually measured over a shorter time span. They can be used in disease screening, diagnosis, characterization, and monitoring; as prognostic indicators; for developing individualized therapeutic interventions; for predicting and treating adverse drug reactions; for identifyi...
Source: Current Protocols in Pharmacology - March 18, 2017 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research

The Quantitative Characterization of Functional Allosteric Effects.
Authors: Kenakin T Abstract Seven-transmembrane receptors (7TMRs or GPCRs [G protein-coupled receptors]) are nature's prototypic allosteric proteins in that they mediate the interaction between ligand binding to the receptor and the receptor interacting with another cell signaling protein. A growing class of potential drugs acting through 7TMRs are allosteric in nature in that they bind to a separate site on the receptor protein to modify the interactions between the receptor, natural binding/orthosteric ligand, and signaling proteins. Two main allosteric compound categories are those that increase (positive allost...
Source: Current Protocols in Pharmacology - March 18, 2017 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research

Assessment of Ex Vivo Transport Function in Isolated Rodent Brain Capillaries.
Authors: Chan GN, Cannon RE Abstract The blood-brain barrier plays an important role in neuroprotection; however, it can be a major obstacle for drug delivery to the brain. This barrier primarily resides in the brain capillaries and functions as an interface between the brain and peripheral blood circulation. Several anatomical and biochemical elements of the blood-brain barrier are essential to regulate the permeability of nutrients, ions, hormones, toxic metabolites, and xenobiotics into and out of the brain. In particular, high expression of ATP-driven efflux transporters at the blood-brain barrier is a major ob...
Source: Current Protocols in Pharmacology - March 18, 2017 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research

Logical Experimental Design and Execution in the Biomedical Sciences.
Authors: Holder DJ, Marino MJ Abstract Lack of reproducibility has been highlighted as a significant problem in biomedical research. The present unit is devoted to describing ways to help ensure that research findings can be replicated by others, with a focus on the design and execution of laboratory experiments. Essential components for this include clearly defining the question being asked, using available information or information from pilot studies to aid in the design the experiment, and choosing manipulations under a logical framework based on Mill's "methods of knowing" to build confidence in puta...
Source: Current Protocols in Pharmacology - March 18, 2017 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research

Visualization of 5-HT Receptors Using Radioligand-Binding Autoradiography.
Authors: Cortés R, Vilaró MT, Mengod G Abstract Described in this unit are techniques to visualize the majority of serotonin (5-hydroxytryptamine, 5-HT) receptor subtypes in sections of frozen brain tissue using receptor autoradiography. Protocols for brain extraction and sectioning, radioligand exposure, autoradiogram generation, and data quantification are provided, as are the optimal incubation conditions for the autoradiographic visualization of receptors using agonist and antagonist radioligands. © 2016 by John Wiley & Sons, Inc. PMID: 27960027 [PubMed - in process] (Source: Current ...
Source: Current Protocols in Pharmacology - December 14, 2016 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research

Overview on Research and Clinical Applications of Optogenetics.
Authors: Towne C, Thompson KR Abstract Optogenetics is a method that uses light to control cells in living tissue, typically neurons, that have been modified to express light-sensitive ion channels and pumps. The approach facilitates neuromodulation in brain preparations and freely moving animals with unmatched spatial and temporal resolution. This optogenetics overview describes the vast array of light-sensitive proteins available and the methods used to deliver them to tissue and modulate them with light. How these methods have so far enhanced our knowledge of fundamental neuroscience and psychiatric disease will...
Source: Current Protocols in Pharmacology - December 14, 2016 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research

Stability of Drugs, Drug Candidates, and Metabolites in Blood and Plasma.
Authors: Reed GA Abstract Determination of drug or drug metabolite concentrations in biological samples, particularly in serum or plasma, is fundamental to describing the relationships between administered dose, route of administration, and time after dose for achieving the optimal clinical response. While a well-characterized, accurate analytical method is needed to define these parameters, it must also be established that the analyte concentration in the sample at the time of analysis is identical to the concentration at sample acquisition. This is necessitated by the fact that drugs and their metabolites are sus...
Source: Current Protocols in Pharmacology - December 14, 2016 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research

Detection of Receptor Heteromerization Using In Situ Proximity Ligation Assay.
Authors: Gomes I, Sierra S, Devi LA Abstract Although G protein-coupled receptor (GPCR) heteromerization has been extensively demonstrated in vitro using heterologous cells that overexpress epitope-tagged receptors, their presence in endogenous systems is less well established. This is because a criterion to identify receptor heteromerization is the demonstration that the two interacting receptors are present not only in the same cell but also in the same subcellular compartment in close enough proximity to allow for direct receptor-receptor interaction. This has been difficult to study in native tissues due to a l...
Source: Current Protocols in Pharmacology - December 14, 2016 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research

Functional Studies of Sodium Channels: From Target to Compound Identification.
Authors: Bertrand D, Biton B, Licher T, Chambard JM, Lanneau C, Partiseti M, Lefevre IA Abstract Over the last six decades, voltage-gated sodium (Nav ) channels have attracted a great deal of scientific and pharmaceutical interest, driving fundamental advances in both biology and technology. The structure and physiological function of these channels have been extensively studied; clinical and genetic data have uncovered their implication in diseases such as epilepsy, arrhythmias, and pain, bringing them into focus as current and future drug targets. While different techniques have been established to record the act...
Source: Current Protocols in Pharmacology - December 14, 2016 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research

Measurement of Receptor Signaling Bias.
Authors: Kenakin T Abstract G protein-coupled receptors (GPCRs) are often pleiotropically linked to numerous cellular signaling mechanisms in cells, and it is now known that many agonists differentially activate some signaling pathways at the expense of others. The mechanism for this effect is the stabilization of different active receptor states by different agonists, and it leads to varying qualities of efficacy for different agonists. Agonist bias is a powerful mechanism to amplify beneficial signals and diminish harmful signals, and thus improve the overall profile of agonist ligands. This unit describes a meth...
Source: Current Protocols in Pharmacology - September 18, 2016 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research

A Mouse Model of Furosemide-Induced Overactive Bladder.
Authors: Saporito MS, Zuvich E, DiCamillo A Abstract Detailed in this unit is a mouse model of overactive bladder and urinary incontinence based on diuretic stress-induced urination. The procedure involves the use of a unique, highly sensitive, and automated urine capturing method to measure urinary latency, frequency, and void volume. Although this method was first described and validated using an anti-muscarinic drug used for treating overactive bladder, subsequent work has shown that effective non-cholinergic agents can be detected. These findings indicate good predictive value for this model regarding the possi...
Source: Current Protocols in Pharmacology - September 18, 2016 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research

In Vitro Drug Metabolism Using Liver Microsomes.
Authors: Knights KM, Stresser DM, Miners JO, Crespi CL Abstract Knowledge of the metabolic stability of newly discovered drug candidates eliminated by metabolism is essential for predicting the pharmacokinetic (PK) parameters that underpin dosing and dosage frequency. Further, characterization of the enzyme(s) responsible for metabolism (reaction phenotyping) allows prediction, at least at the qualitative level, of factors (including metabolic drug-drug interactions) likely to alter the clearance of both new chemical entities (NCEs) and established drugs. Microsomes are typically used as the enzyme source for the m...
Source: Current Protocols in Pharmacology - September 18, 2016 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research

Overview of Critical Parameters for the Design and Execution of a High-Throughput Screen for Allosteric Ligands.
Authors: Alt A Abstract Allosteric ligands modulate the activity of receptor targets by binding to sites that are distinct from the orthosteric (native agonist) binding site. Allosteric modulators have potential therapeutic advantages over orthosteric agonists and antagonists, including improved selectivity, and maintenance of the spatial and temporal fidelity of native signaling patterns. The identification of allosteric ligands presents unique challenges because of the requirement for screening in the presence of an orthosteric agonist, the small signal window that is produced by many allosteric modulators, the p...
Source: Current Protocols in Pharmacology - September 18, 2016 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research

Mammalian Cell Tissue Culture Techniques.
Authors: Phelan K, May KM Abstract Cultured tissues and cells are used extensively in physiological and pharmacological studies. In vitro cultures provide a means of examining cells and tissues without the complex interactions that would be present if the whole organism were studied. A number of special skills are required in order to preserve the structure, function, behavior, and biology of cells in culture. This unit describes the basic skills required to maintain and preserve cell cultures: maintaining aseptic technique, preparing media with the appropriate characteristics, passaging, freezing and storage, reco...
Source: Current Protocols in Pharmacology - June 3, 2016 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research

Genetically Engineered Mouse Models of Pancreatic Cancer: The KPC Model (LSL-Kras(G12D/+) ;LSL-Trp53(R172H/+) ;Pdx-1-Cre), Its Variants, and Their Application in Immuno-oncology Drug Discovery.
Authors: Lee JW, Komar CA, Bengsch F, Graham K, Beatty GL Abstract Pancreatic ductal adenocarcinoma (PDAC) ranks fourth among cancer-related deaths in the United States. For patients with unresectable disease, treatment options are limited and lack curative potential. Preclinical mouse models of PDAC that recapitulate the biology of human pancreatic cancer offer an opportunity for the rational development of novel treatment approaches that may improve patient outcomes. With the recent success of immunotherapy for subsets of patients with solid malignancies, interest is mounting in the possible use of immunotherapy ...
Source: Current Protocols in Pharmacology - June 3, 2016 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research

Assays for Determination of Protein Concentration.
Authors: Olson BJ Abstract Biochemical analysis of proteins relies on accurate quantification of protein concentration. Detailed in this appendix are some commonly used methods for protein analysis, e.g., Lowry, Bradford, bicinchoninic acid (BCA), UV spectroscopic, and 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde (CBQCA) assays. The primary focus of this report is assay selection, emphasizing sample and buffer compatibility. The fundamentals of generating protein assay standard curves and of data processing are considered, as are high-throughput adaptations of the more commonly used protein assays. Also included ...
Source: Current Protocols in Pharmacology - June 3, 2016 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research

Overview of Transgenic Glioblastoma and Oligoastrocytoma CNS Models and Their Utility in Drug Discovery.
Authors: Chen F, Becker A, LoTurco J Abstract Many animal models have been developed to investigate the sources of central nervous system (CNS) tumor heterogeneity. Reviewed in this unit is a recently developed CNS tumor model using the piggyBac transposon system delivered by in utero electroporation, in which sources of tumor heterogeneity can be conveniently studied. Their applications for studying CNS tumors and drug discovery are also reviewed. © 2016 by John Wiley & Sons, Inc. PMID: 26995546 [PubMed - as supplied by publisher] (Source: Current Protocols in Pharmacology)
Source: Current Protocols in Pharmacology - March 21, 2016 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research

Overview of Genetically Engineered Mouse Models of Distinct Breast Cancer Subtypes.
Authors: Usary J, Darr DB, Pfefferle AD, Perou CM Abstract Advances in the screening of new therapeutic options have significantly reduced the breast cancer death rate over the last decade. Despite these advances, breast cancer remains the second leading cause of cancer death among women. This is due in part to the complexity of the disease, which is characterized by multiple subtypes that are driven by different genetic mechanisms and that likely arise from different cell types of origin. Because these differences often drive treatment options and outcomes, it is important to select relevant preclinical model syst...
Source: Current Protocols in Pharmacology - March 21, 2016 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research

Methods of Inducing Inflammatory Bowel Disease in Mice.
Authors: Bang B, Lichtenberger LM Abstract Animal models of experimentally induced inflammatory bowel disease (IBD) are useful for understanding more about the mechanistic basis of the disease, identifying new targets for therapeutic intervention, and testing novel therapeutics. This unit provides detailed protocols for five widely used mouse models of experimentally induced intestinal inflammation: chemical induction of colitis by dextran sodium sulfate (DSS), hapten-induced colitis via 2,4,6-trinitrobenzene sulfonic acid (TNBS), Helicobacter-induced colitis in mdr1a(-/-) mice, the CD4(+) CD45RB(hi) SCID transfer ...
Source: Current Protocols in Pharmacology - March 21, 2016 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research

Models of Inflammation: Carrageenan Air Pouch.
Authors: Duarte DB, Vasko MR, Fehrenbacher JC Abstract The subcutaneous air pouch is an in vivo model that can be used to study the components of acute and chronic inflammation, the resolution of the inflammatory response, the oxidative stress response, and potential therapeutic targets for treating inflammation. Injection of irritants into an air pouch in rats or mice induces an inflammatory response that can be quantified by the volume of exudate produced, the infiltration of cells, and the release of inflammatory mediators. The model presented in this unit has been extensively used to identify potential anti-inf...
Source: Current Protocols in Pharmacology - March 21, 2016 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research

Drug Combinations: Tests and Analysis with Isoboles.
Authors: Tallarida RJ Abstract Described in this unit are experimental and computational methods to detect and classify drug interactions. In most cases, this relates to two drugs or compounds with overtly similar effects, e.g., two analgesics or two anti-hypertensives. From the dose-response data of the individual drugs, it is possible to generate a curve, the isobole, which defines all dose combinations that are expected to yield a specified effect. The theory underlying the isobole involves the calculation of doses of drug A that are effectively equivalent to doses of drug B with that equivalence determining whe...
Source: Current Protocols in Pharmacology - March 21, 2016 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research

Sigma Receptor Binding Assays.
Authors: Chu UB, Ruoho AE Abstract Sigma receptors, both Sigma-1(S1R) and Sigma-2 (S2R), are small molecule-regulated, primarily endoplasmic reticulum (ER) membrane-associated sites. A number of drugs bind to sigma receptors, including the antipsychotic haloperidol and (+)-pentazocine, an opioid analgesic. Sigma receptors are implicated in many central nervous system disorders, in particular Alzheimer's disease and conditions associated with motor control, such as Amyotrophic Lateral Sclerosis (ALS). Described in this unit are radioligand binding assays used for the pharmacological characterization of S1R and S2R. ...
Source: Current Protocols in Pharmacology - December 12, 2015 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research

In Vitro Binding of [(3) H]PSB-0413 to P2Y12 Receptors.
Authors: Dupuis A, Heim V, Ohlmann P, Gachet C Abstract The P2Y12 /ADP receptor plays a central role in platelet activation. Characterization of this receptor is mandatory for studying disorders associated with a P2Y12 receptor defect and for evaluating P2Y12 receptor agonists and antagonists. In the absence of suitable anti-P2Y12 antibodies, radioligand binding assays are the only way to conduct such studies. While various radioligands were employed in the past for this purpose, none were found to be suitable for routine use. Described in this unit are protocols for quantitatively and qualitatively assessing P2Y12...
Source: Current Protocols in Pharmacology - December 12, 2015 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research

A Human Induced Pluripotent Stem Cell-Derived Cardiomyocyte (hiPSC-CM) Multielectrode Array Assay for Preclinical Cardiac Electrophysiology Safety Screening.
Authors: Harris K Abstract Cardiotoxicity is a leading cause of compound attrition during drug development. Preclinical models used to assess the risk for compound-induced effects on cardiac electrophysiology largely rely on animals that can differ in terms of sensitivity and specificity to the targeted clinical response. There is currently no in vitro human cardiomyocyte model for routine preclinical compound screening, as adult human cardiac tissue is unsuitable for such screening. The commercial availability of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) makes possible the development ...
Source: Current Protocols in Pharmacology - December 12, 2015 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research

Measurement of Glucose Uptake in Cultured Cells.
Authors: Yamamoto N, Ueda-Wakagi M, Sato T, Kawasaki K, Sawada K, Kawabata K, Akagawa M, Ashida H Abstract Facilitative glucose uptake transport systems are ubiquitous in animal cells and are responsible for transporting glucose across cell surface membranes. Evaluation of glucose uptake is crucial in the study of numerous diseases and metabolic disorders such as myocardial ischemia, diabetes mellitus, and cancer. Detailed in this unit are laboratory methods for assessing glucose uptake into mammalian cells. The unit is divided into five sections: (1) a brief overview of glucose uptake assays in cultured cells; (2)...
Source: Current Protocols in Pharmacology - December 12, 2015 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research

In vitro isolated tissue functional muscarinic receptor assays.
Authors: Pulido-Rios MT, Steinfeld T, Armstrong S, Watson N, Choppin A, Eglen R, Hegde SS Abstract Muscarinic receptor (mAChRs) subtypes are viable targets for the design of novel agents for use in a number of central and peripheral disorders. In vitro isolated tissue functional assays for muscarinic receptor subtypes have played an invaluable role in basic research and drug discovery. The availability of biological assays for generation of quantitative estimates of affinity and potency of ligands allows evaluation of the contribution of a given mAChR to the functional end organ response and also enables drug disco...
Source: Current Protocols in Pharmacology - November 18, 2015 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research

Mouse models of human bladder cancer as a tool for drug discovery.
Authors: Seager C, Puzio-Kuter AM, Cordon-Cardo C, McKiernan J, Abate-Shen C Abstract Muscle-invasive bladder cancer is a deadly condition in dire need of effective new treatments. This unit contains a description of mouse models suitable for the evaluation of potential new therapies. Included is a genetically engineered mouse model of bladder cancer generated by the delivery of an adenovirus expressing Cre recombinase into the bladder lumen. Also described is an orthotopic mouse model created by the instillation of human bladder tumor cells into the bladder lumen of immune deficient mice. Protocols are also provid...
Source: Current Protocols in Pharmacology - November 18, 2015 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research

Cyclic nucleotide phosphodiesterase assay technology.
Authors: MacKenzie SJ, Hastings SF, Wells C Abstract Because of their critical role in modulating cellular cyclic nucleotide levels, phosphodiesterases (PDEs) are involved in many disease-related signaling pathways. The PDE family is large and diverse, with members having different tissue distribution, sub-cellular localizations, and substrate specificities. Because of these characteristics, the PDEs represent a broad group of potential drug targets. Described in the present unit are the assay development and validation procedures needed to establish a high-throughput screening system for these important enzymes. T...
Source: Current Protocols in Pharmacology - November 18, 2015 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research

Novel object recognition in the rat: a facile assay for cognitive function.
Authors: Mathiasen JR, DiCamillo A Abstract The rat novel object recognition (NOR) assay is a relatively high-throughput, robust, and sensitive procedure for evaluating compounds for cognition-enhancing activity. For the test, rats are given the opportunity to explore two identical objects for a predetermined period of time. After a delay, the animals are then presented with two objects to explore, one of which is the same as in the first exploration trial, the other a new object. Depending on the length of the delay between the two trials, the rats will either explore the novel object for a greater time period, in...
Source: Current Protocols in Pharmacology - November 18, 2015 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research

Rodent models of depression: forced swim and tail suspension behavioral despair tests in rats and mice.
Authors: Castagné V, Moser P, Roux S, Porsolt RD Abstract The development of antidepressants requires simple rodent behavioral tests for initial screening before undertaking more complex preclinical tests and clinical evaluation. Presented in the unit are two widely used screening tests used for antidepressants, the forced swim (also termed behavioral despair) test in the rat and mouse, and the tail suspension test in the mouse. These tests have good predictive validity and allow rapid and economical detection of substances with potential antidepressant-like activity. The behavioral despair and the tail susp...
Source: Current Protocols in Pharmacology - November 18, 2015 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research

Pharmacological applications of baculovirus-mediated protein expression in mammalian cells.
Authors: Condreay JP, Watson CA Abstract The development of cell-based assays for cellular receptors, ion channels, and transporters requires the delivery and expression of transgenes. Viral-mediated gene delivery is a particularly attractive approach for this purpose because of its efficiency and potential to deliver genes to a wide variety of cell types. Recombinant baculoviruses, long used to deliver genes to insect cells for overexpression, also effectively transfer genes to mammalian cells. The only required modification to the virus for this purpose is the addition of transgene expression cassettes controlled...
Source: Current Protocols in Pharmacology - November 18, 2015 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research

Overview on the Rule of Five.
Authors: Pollastri MP Abstract In the mid- to late 1990 s, because of the drug discovery paradigm shift from phenotypic screens to combinatorial chemistry and high-throughput screening, the physicochemical properties of exploratory drug molecules displayed a dramatic shift toward higher molecular weight and lipophilicity. In response, Lipinski and coworkers reported an analysis of compounds that successfully navigated Phase I and entered into Phase II clinical studies, and correlated the computed physicochemical properties of these molecules to their aqueous solubility, permeability, and oral bioavailability. In do...
Source: Current Protocols in Pharmacology - November 18, 2015 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research

Xenograft and Transgenic Mouse Models of Epithelial Ovarian Cancer and Non Invasive Imaging Modalities to Monitor Ovarian Tumor Growth In situ -Applications in Evaluating Novel Therapeutic Agents.
Authors: Connolly DC, Hensley HH Abstract Epithelial ovarian cancer (EOC) is the most commonly fatal gynecologic malignancy in developed countries. Most EOC patients are diagnosed at advanced stage when disease has spread beyond the ovary. While many patients initially respond to surgery and chemotherapy, the long term prognosis is generally unfavorable, with recurrence and development of drug resistant disease. There is a critical need to identify new therapeutic agents that prolong disease-free intervals and effectively manage recurrent disease. Murine models of ovarian carcinoma are excellent models to study tum...
Source: Current Protocols in Pharmacology - November 18, 2015 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research

Static biofilm cultures of Gram-positive pathogens grown in a microtiter format used for anti-biofilm drug discovery.
Authors: Kwasny SM, Opperman TJ Abstract An in vitro assay is presented for culturing staphylococcal biofilms and biofilms of nonmotile Gram-positive bacteria under static conditions in microtiter assay plates, and for the quantification of biofilm growth, using a simple staining procedure that measures amounts of bacterial cells and extracellular matrix. This basic assay can be adapted readily to study several aspects of biofilm formation, for high-throughput screening to identify small molecule inhibitors of biofilm formation or biofilm-defective mutants, and for quantifying the anti-biofilm activity of biofilm i...
Source: Current Protocols in Pharmacology - November 18, 2015 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research

Evaluation of compound activity against hepatitis C virus in replicon systems.
Authors: Yang G, Huang M Abstract Described in this unit are protocols for evaluation of new chemical entities for activity against hepatitis C virus (HCV) using HCV replicon systems. While agents designed to target NS3 protease, NS3 helicase, and NS5B RNA polymerase can be evaluated in biochemical assays, it is important to confirm their inhibitory effect on HCV RNA replication using HCV replicon systems, especially since replication involves many components besides these enzymes. Screening of compound libraries using replicon systems has led to the discovery of replication inhibitors, which act through different ...
Source: Current Protocols in Pharmacology - November 18, 2015 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research

Studies on hepatitis C virus resistance to inhibitors in replicon systems.
Authors: Zhao Y, Huang M Abstract Viruses evolve under selection pressure from a particular antiviral agent, resulting in the emergence of organisms that are not susceptible to the drug. This process is referred to as "virus resistance induction." While conventional in vitro resistance studies are conducted using infectious viruses, the lack of a robust hepatitis C virus (HCV) infection system in cell culture makes such an approach impossible in this case. Instead, cell lines harboring a self-replicating HCV RNA (or HCV replicon) are used for this purpose. The protocols detailed in this unit describe meth...
Source: Current Protocols in Pharmacology - November 18, 2015 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research

Label-free imaging and temporal signature in phenotypic cellular assays: a new approach to high-content screening.
Authors: Martin J Abstract Some drug targets are not amenable to screening because of the lack of a practical or validated biological assay. Likewise, some screening assays may not be predictive of compound activity in a more disease-relevant scenario, or assay development may demand excessive allocation of resources (i.e., time, money or personnel) with limited knowledge of the actual tractability of the target. Label-free methodologies, implemented in microtiter plate format, may help address these issues and complement, simplify, or facilitate assays. Label-free biosensors, based on grating resonance or electric...
Source: Current Protocols in Pharmacology - November 18, 2015 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research

Measuring receptor target coverage: a radioligand competition binding protocol for assessing the association and dissociation rates of unlabeled compounds.
Authors: Sykes DA, Dowling MR, Charlton SJ Abstract The kinetics of the ligand-receptor interaction is an important feature in lead optimization for new drug candidates. The protocol described in this unit is a kinetic radioligand competition binding assay that makes possible the determination of both the association and dissociation rates of unlabeled receptor ligands. PMID: 22294377 [PubMed - indexed for MEDLINE] (Source: Current Protocols in Pharmacology)
Source: Current Protocols in Pharmacology - November 18, 2015 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research

Pre-clinical mouse models of human prostate cancer and their utility in drug discovery.
Authors: Park SI, Kim SJ, McCauley LK, Gallick GE Abstract In vivo animal experiments are essential to current prostate cancer research, and are particularly critical to studying interactions between tumor cells and their microenvironment. Numerous pre-clinical animal models of prostate cancer are currently available, including transgenic mouse models and human prostate cancer xenograft mouse models. In contrast to transgenic mouse models producing more heterogeneous cohorts of tumors, xenograft mouse models provide more controlled approaches. This unit describes the detailed procedures necessary to establish sever...
Source: Current Protocols in Pharmacology - November 18, 2015 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research

Establishment, maintenance and in vitro and in vivo applications of primary human glioblastoma multiforme (GBM) xenograft models for translational biology studies and drug discovery.
Authors: Carlson BL, Pokorny JL, Schroeder MA, Sarkaria JN Abstract Development of clinically relevant tumor model systems for glioblastoma multiforme (GBM) is important for advancement of basic and translational biology. One model that has gained wide acceptance in the neuro-oncology community is the primary xenograft model. This model entails the engraftment of patient tumor specimens into the flank of nude mice and subsequent serial passage of these tumors in the flank of mice. These tumors are then used to establish short-term explant cultures or intracranial xenografts. This unit describes detailed procedures ...
Source: Current Protocols in Pharmacology - November 18, 2015 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research

The fentanyl/etomidate-anesthetized beagle (FEAB) model in safety pharmacology assessment.
Authors: van der Linde HJ, Deuren BV, Somers Y, Teisman A, Gallacher DJ Abstract This unit describes a procedure for performing safety studies in the anesthetized beagle dog. Detailed are the anesthetic regime, the surgical procedure, and all materials needed to perform cardiovascular, central nervous system, and respiratory safety studies in these animals. An overview of all parameters that can be measured and calculated is provided, as are experimental protocols. Endpoints discussed include hemodynamic, electrocardiological, respiratory, arterial blood, and electroencephalogical parameters. Also presented are a f...
Source: Current Protocols in Pharmacology - November 18, 2015 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research

Preparation of HCV NS3 and NS5B proteins to support small-molecule drug discovery.
Authors: Hung M, Wang R, Liu X Abstract Production of high-quality, well-characterized recombinant proteins facilitates screening of compound libraries. The protocols detailed in this unit are used to purify three recombinant enzymes that are widely used in HCV research: the HCV NS3 protease domain, the helicase domain as an NS3+NS4A complex, and the NS5B RNA-dependent RNA polymerase. The active enzymes are purified to homogeneity by two-column chromatography to support a screening program for HCV inhibitors. PMID: 21898331 [PubMed - indexed for MEDLINE] (Source: Current Protocols in Pharmacology)
Source: Current Protocols in Pharmacology - November 18, 2015 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research

Biochemical evaluation of HCV NS3 protease inhibitors.
Authors: Schultz B, Yang H, Delaney WE Abstract This unit describes assays for characterizing the potency and mechanism of action of NS3 protease inhibitors. Determination of IC(50) values is described using in vitro expressed and purified NS3 protease. This assay can also be used for the rapid exploration of structure-activity relationships. Another protocol describes using the full-length NS3/4A complexes expressed in HCV replicon cell lines for a rapid alternative method for assessing protease activity without requiring conventional protein expression and purification. A method is then provided for determination...
Source: Current Protocols in Pharmacology - November 18, 2015 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research