Current Protocols in Pharmacology

WHAT'S NEW AND COMING

March, 2002

RECENTLY PUBLISHED:

UNITS 5.25 & 5.26 Guinea Pig Models of Asthma (Stuart B. Mazzone and Brendan J. Canning, The Johns Hopkins Medical Institute, Baltimore, Maryland). Asthma is an inflammatory disease characterized by airways obstruction, airways hyperresponsiveness, excessive mucous secretion, and cough. Guinea pig airways display many anatomical, physiological and pharmacological attributes of human airways, making this species ideal for modeling the asthmatic condition. UNIT 5.25 provides an overview of animal models of asthma, including definitions, descriptions of available animal models, and discussion of numerous critical issues to consider before designing a model to study this complex disease. The aim of UNIT 5.26 is to describe methods for establishing guinea pig models of asthma. Detailed descriptions are provided so that investigators can establish preparations in which bronchoconstriction, airway hyperresponsiveness, cough, and airway inflammation can be studied.

UNIT 1.26 Characterization of Cannabinoid Receptors (Richard F. Cox and Vincent Rash, GlaxoSmithKline, Research Triangle Park, North Carolina). This unit describes the use of cannabinoid radioligands in competitive binding assays for determining affinity parameters (IC₅₀, K_i) of unlabeled compounds at cloned CB1 and CB2 receptors expressed in cells.

UNIT 5.27 Models of Ischemia-Reperfusion Injury in Dogs and Rats (Garrett J. Gross, Medical College of Wisconsin, Milwaukee). A marked reduction in coronary blood flow produces a reduction in myocardial function, electrocardiographic abnormalities, and eventually a myocardial infarction if the ischemic episode persists for more than 20 to 30 min. There have been numerous studies in animal models in search for a 'magic bullet' or drug that can ameliorate these symptoms and result in a reduction in infarct size, improvement in the recovery of contractile function, and abrogation of malignant ventricular arrhythmias in man. This unit describes two animal models of myocardial ischemia/reperfusion injury which are used to evaluate pharmacological agents that may eventually demonstrate cardioprotective activity in a clinical setting.

UNIT 1.24 Characterization of Chemokine Receptors (Alexander Scheer and Amanda E. I. Proudfoot, Serono Pharmaceutical Research Institute, Plan-les-Ouates, Switzerland). This unit describes the procedures for measuring binding of a radiolabeled chemokine to chemokine receptors in cells or cell membranes. The whole-cell binding assay can be used for both purified leukocytes as well as transfected cell lines expressing chemokine receptors. Iodinated chemokines ([¹²⁵I]-labeled) are used as radioligands, and the procedures for measuring the agonist-stimulated binding of [³⁵S]GTPgS to chemokine cell membranes allows the discrimination between agonist and antagonist properties of chemokines or compounds.

UNIT 12.4 Cellular Assays of Chemokine Receptor Activation (Amanda E. I. Proudfoot, Christine A. Power, Dennis J. Church, Dulce Soler and Matthias Mack, Serono Pharmaceutical Research Institute, Plan-les-Ouates, Switzerland). This unit presents methods for measuring the interaction of a chemokine with its receptor, both in primary cells and in cell lines stably transfected with a chemokine receptor. The protocols describe methods for analyzing the chemoattractant properties of these proteins, termed chemotaxis, as well as two methods for measuring the ability of a chemokine to activate its receptor(s) through G protein coupling, resulting in the mobilization of intracellular calcium stores, and, lastly, the property shared by all 7TM receptors, receptor down-regulation upon ligand activation.

UNIT 3.9 Cytochrome P450 Assays (Enock Delaporte and A. David Rodrigues, Merck Research Labs, West Point, Pennsylvania). Cytochrome P450s (CYP) play a major role in drug detoxification, and inhibition of CYP-mediated metabolism may lead to accumulation of toxic drug levels in the plasma. To prevent adverse drug-drug interactions, new drug candidates are routinely tested for their ability to inhibit these enzymes. This unit describes a variety of protocols for evaluating new chemical entities as inhibitors of CYP activity. An example protocol illustrates a high-throughput screening format using a fluorogenic probe. A method for evaluating a test compound as a time-dependent inhibitor of CYP is also described.

UNIT 5.24 Classic In Vivo Cancer Models: Three Examples of Mouse Models Used in Experimental Therapeutics (Anna Kruczynski and Bridget T. Hill, Centre de Recherche Pierre Fabre, Cedex, France). Transplantable animal tumors have been associated with the discovery of most clinically active anticancer agents. They are still useful today in conducting detailed evaluations of new candidate anticancer drugs. Three protocols relating to transplantable experimental tumors are described in this unit. Included are the intravenously-implanted murine P388 leukemia, the subcutaneously-implanted murine B16 melanoma, and two examples of subcutaneously-implanted human tumor xenografts, the LX-1 (lung) and MX-1 (breast) tumors.

UNIT 7.3 Overview of Drug Product Development (Kathleen M. Lee, Scios Inc.). Drug product development, also called formulation chemistry, is the process of developing formulations for use in toxicology and clinical studies and for commercial supply. In this overview, the development process is described, including dosage forms for various delivery routes, toxicology formulation development, ingredient selection, and manufacturing process development.

UNIT 11.3 The Action Potential of the Purkinje Fiber: An In Vitro Model for Evaluation of the Proarrhythmic Potential of Cardiac and Non-Cardiac Drugs (René Rouet and Joffrey Ducroq, Université de Caen; and Sandra Picard, Porsolt and Partners Pharmacology, Boulogne-Billancourt, France). The proarrhythmic potential of new drugs can be investigated using in vitro electrophysiological techniques measuring the cardiac action potential in isolated Purkinje fibers. Different types of arrhythmias may occur, such as early after-depolarizations (EADs), which are favored by action potential duration lengthening and bradycardia, or delayed after-depolarizations (DADs), which are facilitated by tachycardia. The effects of a test compound on the occurrence of these arrhythmias, known to induce in vivo torsades de pointes (or triggered activities), can be studied following the experimental protocols described in this unit.

UNIT 3.7 Characterization of Matrix Metalloproteinase Inhibitors: Enzymatic Assays (Patrick A. Marcotte and Steven K. Davidsen, Abbott Laboratories, Abbott Park, Illinois). Matrix metalloproteinase (MMP) inhibitors may be beneficial in the treatment of disease states involving excessive catabolism of extracellular matrix components of the cell—e.g., cancer, arthritis, and macular degeneration. Enzyme inhibition potency for a range of different MMPs can be assessed by the measurement of rates of cleavage of a fluorogenic substrate. This unit provides a convenient method for measuring the potency and selectivity of test compounds.

UNIT 5.23 Characterization of Matrix Metalloproteinase Inhibitors: Angiogenesis and Tumor Models (Daniel H. Albert and Steven K. Davidsen, Abbott Laboratories, Abbott Park, Illinois). Since the matrix metalloproteinases (MMPs) have an essential role in the process of tumor growth, invasion, and metastasis, small-molecule MMP inhibitors have the ability to modulate tumor progression in animals and the potential to be of therapeutic benefit to cancer patients. The antiangiogenic properties of MMP inhibitors can be assessed by the measurement of hemoglobin content of Matrigel plugs containing angiogenic growth factors introduced into the flanks of mice. A flank-tumor growth model using B16 murine melanoma cells provides a useful means of determining the antitumor effects of MMP inhibitors and so correlates efficacy with the concentration of drug in blood.

FORTHCOMING:

UNIT 11.4 Using Electrophysiological Methods in the Study of Recombinant and Native Neuronal GABA_A Receptors (Philip Thomas and Trevor G. Smart, University of London, United Kingdom). This unit is geared towards investigators wishing to study the effects of externally and internally applied agents on the behavior of ligand-gated ion channels (LGICs, specifically the GABA_A receptor). The reader is taken through a number of electrophysiological techniques applied to whole cell and excised patch recordings of recombinant and native GABA_A receptor subtypes used in the generation and analysis of a variety of pharmacological parameters. These data interpretations form the basis for the analysis of potentially novel pharmacological agents active at the GABA_A receptor target, but could equally be applied to other LGICs. The analysis is extended to incorporate post-synaptic inhibitory events in hippocampal neurons. Complementary descriptions of how tissues for such studies are prepared from recombinant and native receptor preparations are included. Attention is given to the physiological phenomena most relevant to everyday scientific literature.

UNIT 12.5 In Vivo Measurement of Blood-Brain Barrier Permeability (Kathleen M.K. Boje, School of Pharmacy, University at Buffalo, New York). This unit describes various protocols for the in vivo quantitation of drug permeability across the rodent blood-brain barrier. Methods for the measurement of drug influx and efflux are described, and support protocols are provided for determining intravascular capillary volume and cerebral perfusion flow. An in situ perfusion technique is also provided for assessing whether transport of a test compound occurs by carrier-mediated or saturable transport.

UNIT 1.27 Characterization of the NMDA Receptor Glycine Binding Site (Scott Perschke, NovaScreen Bioscience Corporation, Hanover, Maryland; and John Ferkany, Virtual Drug Discovery, Inc. Avon, Connecticut). This unit describes a competitive binding assay for the glycine binding site on the NMDA subtype of glutamate receptors in rat brain homogenates. Agonists of the NMDA receptor–associated glycine binding site have been proposed as potential therapeutics in cognitive disorders. Conversely, antagonists may be useful in a variety of disorders associated with excessive activation of EAA receptors, including Parkinson's, Huntington's and Alzheimer's diseases, and neuropathic pain, among others.

UNIT 5.28 Electrocardiographic Toxicity in the Guinea Pig (Pierre Lacroix, Porsolt and Partners Pharmacology, Boulogne Billancourt, France). Abnormalities of cardiac rhythm are one of the most common clinical problems in cardiology and arise as the result of either disorders of cardiac impulse formation or conduction, or a combination of both. It has been established that some classes of drugs, such as tricyclic antidepressants (e.g., imipramine), cardiac glycosides (e.g., digoxin), and Class I or Class III antiarrhythmic drugs (e.g., quinidine or amiodarone) can produce electrocardiographic toxicity in humans. It is therefore highly advisable to assess the effect of any new compound in this respect, during the early phases of drug development. This unit presents a protocol to detect the electrocardiographic toxicity of compounds in the anesthetized guinea pig.

UNIT 10.8 Patch-Clamp Studies of Human Cardiac Ion Channels in the Evaluation of Cardiac Electrophysiological Effects of Drugs (William J. Crumb, Zenas Technologies L.L.C., New Orleans, Louisiana; and Icilio Cavero, Bonneuil sur Marne, France). Drugs prolonging the QT interval appear to consistently inhibit the outward, rapid delayed rectifier K⁺ current (I_Kr) conveyed by the HERG channel. Hence, for determining whether a new drug candidate blocks the latter channel, this unit presents a basic electrophysiology protocol to conduct patch clamp studies in single cell preparations expressing heterologously cloned HERG channels. An additional protocol details the isolation of myocytes from specimens of human atria which are used in the study of native cardiac ion currents (I_Na, I_Ca, I_to, I_sus, I_K1). The results of these tests are useful for determining whether drug candidates have the desired cardiac safety profile for human use.

UNIT 11.5 Characterization of Cloned Calcium-Activated Potassium Channels by Patch Clamp (Rikke L. Schrøder, NeuroSearch A/S, Ballerup, Denmark). The family of calcium-activated potassium channels (K_Ca) are divided into three groups according to their single channel conductance properties: large conductance (BK), intermediate conductance (IK), and small conductance (SK) channels. A unifying characteristic of all K_Ca channels is that the open-state probability is augmented by physiological increases in intracellular free Ca²⁺ concentrations in the nanomolar to micromolar range. K_Ca channels are ubiquitously distributed in both excitable and nonexcitable cells, and have diverse functions including shaping of the action potential, spike frequency adaptation, regulation of smooth muscle excitability, T lymphocyte proliferation and secretion from secretory epithelia. This unit presents protocols for the electrophysiological characterization of cloned K_Ca channels stably expressed in mammalian cell lines (i.e., HEK293) cells. The unit describes how to determine macroscopic currents as a function of voltage and time, [Ca²⁺]_i, the effect of pharmacological modulators, ionic selectivity, and rectification.

UNIT 6.9 Tissue Expression Profiling Using Real-Time PCR (Jay C. Strum, Kevin M. Carrick, Joan S. Stuart, Shelby A. Martensen, GlaxoSmithKline, Research Triangle Park, North Carolina). The recent development of real-time PCR allows for the rapid and accurate quantitation of gene expression in cells and tissues. Real-time PCR instrumentation is designed for the simultaneous quantitation of gene expression from a few samples up to 384 samples. The normal tissue-expression profile of a gene can provide useful insights into its potential role in normal physiological processes. When combined with the tissue expression profile of the gene in diseased tissues, information concerning the potential role in pathological processes can be determined. This unit describes a protocol to determine the relative abundance of mRNA in a panel of human tissues using real-time PCR.

UNIT 4.19 5-Hydroxytryptamine Receptor Assays (Katherine W. Figueroa, R. W. Johnson Pharmaceutical Research Institute, La Jolla, California; Graeme R. Martin, Telik, Inc., South San Francisco, California; Magda F. Morton, Clodagh E. Prendergast, and Nigel P. Shankley, R. W. Johnson Pharmaceutical Research Institute, La Jolla, California). Nine in vitro bioassays are described for studying 5-HT_1A, 5-HT_1B, 5-HT_1D, 5-HT_2A, 5-HT_2B, 5-HT_2C, 5-HT₃, 5-HT₄, and 5-HT₇ receptors in isolated intact guinea pig, rat and rabbit tissues. The 5-HT_1A, 5-HT₃, and 5-HT_1A assays are based on preparations of the ileum. 5-HT_1B, 5-HT_1D, 5-HT_2A, and 5-HT_2C receptor assays are all based on vascular preparations, whereas the 5-HT_2B receptor is best studied in stomach fundic muscle and the 5-HT₄ in esophageal tissue.

UNIT 5.29 Animal Models for Evaluating Anti-Neoplastic Agents (Melinda Hollingshead, National Cancer Institute, Frederick, Maryland). Evaluation of compounds for in vivo antineoplastic activity can be achieved in a variety of animal models. Two models commonly employed for these studies are described in this unit: the intraperitoneal challenge survival model and the subcutaneous tumor implant model. The challenge survival model involves intraperitoneal tumor inoculation, test compound treatment, and monitoring for survival. This assay can be modulated for strenuousness by altering the treatment dose, route and schedule. The second model, employing subcutaneous tumor implants, is more strenuous as the test compound must cross physiological barriers to reach the target tumor. Additionally, the subcutaneous model is more labor-intensive to set up, monitor and evaluate.

UNIT 7.4 Determination of Plasma Protein Binding (Natasha Dow, Ricerca LLC, Concord, Ohio). The pharmocokinetic and pharmacodynamic properties of a compound are profoundly affected by the extent of its binding to plasma proteins. Consequently, the determination of the plasma protein binding of a compound is essential during drug development and is increasingly required during lead prioritization. This unit describes the use of ultrafiltration and equilibrium dialysis techniques for determination of plasma protein binding.

UNIT 11.6 Electrophysiological Analysis of Heterologously Expressed Kv and SK/IK Potassium Channels (Neil A. Castle, Alan D. Wickenden, and Anruo Zou, ICAGEN Inc., Durham, North Carolina). This unit describes protocols to aid investigators in determining the electrophysiological and pharmacological profile of heterologously expressed voltage or calcium-activated potassium channels belonging to the Kv1.x and SK/IK gene families. Protocols for data acquisition as well as analysis are provided.