Toxicity Pathways Mediated by Ion Channels

Transcription

1 Toxicity Pathways Mediated by Ion Channels Philip J. Bushnell Neurotoxicology Division National Health and Environmental Effects Research Laboratory Office of Research and Development, US EPA Research Triangle Park, NC McKim Conference on Predictive Toxicology September 18,

2 Goals and Approach Model the acute toxicity of organic solvents for purposes of extrapolation from experimental observations to exposures relevant to public health Explore potential toxicity pathways for the acute effects of these chemicals Use the large existing database of acute effects of anesthetic agents as source of mechanistic information Effects of Four Solvents on Behavior BEHAVIORAL EFFECT TOL TCA PERC TCE RAT, VEP HUMAN RAT, REW RAT ES/AV BEHAVIORAL EFFECT TOL TCA PERC TCE RAT, VEP HUMAN RAT, REW RAT ES/AV ESTIMATED BRAIN SOLVENT (µm) ESTIMATED BRAIN SOLVENT (µm) Benignus et al., in preparation 2

3 Why Ion Channels? Ion channels are a prominent target of nonpolar narcotics Organic solvents Anesthetic agents Inert gases Ion channels are pervasive in the nervous system and other excitable tissues Ion channels carry out essential homeostatic and signaling functions in the nervous system What are Ion Channels? Gated pores in membranes Control electrical potentials across membranes Allow ions (typically Na +, K +, Ca ++, Cl - ) to flow between intra- and extra-cellular fluids Activated by voltage changes and/or ligand binding Functions of ion channels in nerves Modulate electrical excitibility via membrane potential Propagate signals along axons and dendrites Release neurotransmitters at synapses Neuron neuron Neuron effector (e.g., muscle) 3

4 Types of Ion Channels Voltage-gated: Sensitive to changes in electrical potential across membrane Feldman et al., 1997 Selected Types of Ion Channels Ligand-gated channels Sensitive to neurotransmitters, drugs, and solvents Endogenous ligands neurotransmitters Excitatory (depolarizing) transmitters Glutamate NMDA ( -methyl-d-aspartate) Kainate AMPA (α-amino-3-methyl-4-isoxazole propionic acid) nach (Nicotinic acetylcholine) 5-HT 3 (5-Hydroxytryptamine type 3, or Serotonin) Inhibitory (hyperpolarizing) transmitters GABA-A (γ-amino butyric acid type A) Glycine 4

5 Representative Ligand-Gated Ion Channels Campagna et al., 2003 Effects of Anesthetics on Selected Ion Channels NMDA nachr a4b2 nachr a7 GABA-A Glycine V-G Na V-G Ca Excite Excite Excite Inhibit Inhibit Propagate Release NT Toluene Trichloroethylene Perchloroethylene 1,1,1-Trichloroethane Isoflurane 0 Halothane 0 Ether Cyclopropane 0 0 Butane 0 0 Urethane Short Alcs 5 Long Alcs >5 Nitrous Oxide Xenon Facilitation Barbiturates Ketamine 0 Propofol 0 0 No effect 0 Etomidate Steroids 0 Non-immobilizers 0 0 Suppression Modified from Dilger,

6 Glutamate and GABA Pathways in the CNS Cortex Thalamus Hippocampus Brain Stem Spinal Cord Glutamate (NMDA etc) Pathways GABA Pathways Feldman et al., 1997 Light Sedation Amnesia Anxiolysis Moderate Sedation Characteristics of Anesthesia Slowed responses Slurred speech Unconsciousness Loss of awareness No response to verbal commands Immobility Loss of response to pain Rudolf & Antkowiak,

7 Light Sedation Amnesia Anxiolysis Moderate Sedation Slowed responses Slurred speech Unconsciousness Loss of awareness No response to verbal commands Immobility Loss of response to pain Dose-Effect Relationships Campagna et al., 2003 Narcosis Pathways for Volatile Anesthetics Ion Channel Receptor Cellular Response Tissue Response Primary Brain Region Behavioral Effects Agent GABA A nach MDA Glycine Increased channel-open time Decreased channel-open time membrane current Increased duration of mipscs Facilitated inhibitory excitatory excitatory Facilitated inhibitory Cortex - Thalamus Hippocampus Brain Stem Spinal Cord Light Sedation Amnesia Anxiolysis Heavy Sedation Slow responses Unconsciousness Loss of perceptual awareness Immobility Loss of pain response Increasing Depth of Anesthesia Kinetics Dynamics 7

8 Ion Channel Receptor Immobility Pathway for Isoflurane Cellular Response Tissue Response Primary Brain Region Behavioral Effects Agent GABA A nach MDA Glycine Increased channel-open time Decreased channel-open time membrane current Increased duration of mipscs Facilitated inhibitory excitatory excitatory Facilitated inhibitory Cortex - Thalamus Hippocampus Brain Stem Spinal Cord Light Sedation Amnesia Anxiolysis Heavy Sedation Slow responses Unconsciousness Loss of perceptual awareness Immobility Loss of pain response Increasing Depth of Anesthesia Kinetics Dynamics Amnesia Pathway for Isoflurane Ion Channel Receptor Cellular Response Tissue Response Primary Brain Region Behavioral Effects Agent GABA A nach MDA Glycine Increased channel-open time Decreased channel-open time membrane current Increased duration of mipscs Facilitated inhibitory excitatory excitatory Facilitated inhibitory Cortex - Thalamus Hippocampus Brain Stem Spinal Cord Light Sedation Amnesia Anxiolysis Heavy Sedation Slow responses Unconsciousness Loss of perceptual awareness Immobility Loss of pain response Increasing Depth of Anesthesia Kinetics Dynamics 8

9 Can these qualitative pathways be quantified? 1. Do dose-effect relationships in vitro support the pathway scheme for anesthetic vapors? Depth of anesthesia depends on dose, but Receptor sensitivity does not predict the anesthetic depth: Isoflurane EC10 EC50 (um) GABA nach NMDA Glycine Light sedation Heavy sedation Unconsiousness Immobility Can these qualitative pathways be quantified? 2. Can specific sites or modes of action be identified for each pathway? Pharmacological analysis of drug interactions Injected anesthetics (e.g., benzodiazepines) acting at specific receptors (e.g. GABA) interact synergistically with agents acting at other receptors. Implication: Different modes / sites of action Inhaled anesthetics interact dose-additively Implication: inhalants act at a single site or by a single mode of action 9

10 Interactions between Anesthetics Review of anesthesia literature Two well-defined endpoints Hypnosis (Unconsciousness) Immobility Drugs grouped by receptor Inhalants listed separately Synergy? Yes between iv drug pairs Yes between iv drugs and inhalants No between inhalant pairs Implication: Inhalants act at a common site / common MOA Hendrickx et al., 2008 Interactions Among Inhaled Anesthetics Experimental studies Immobility to shock Additivity most frequent No case of synergy One case of infra-additivity Implication: Inhalants act at a common site / common MOA Hendrickx et al.,

11 Can these qualitative pathways be quantified? 3. However, analysis of dose-effect functions does not support a single MOA for volatile anesthetics Dose-effect curves for single agents have a very steep slope (Hill coefficient γ = 6 20) in vivo Effects of individual agents in vitro have shallow slopes (γ ~ 1) In linear circuits with multiple receptors, γ 1.5 at limit Thus a single site model with simple linear circuits cannot account for dose-effect relationships Can these qualitative pathways be quantified? 4. Comparative Molecular Field Analysis (Sewell et al., in press) Correlate IC50 at NMDA receptor and MAC-I for 16 anesthetics, find poor relationship: Models predict observed IC50 and MAC-I data very well: 11

12 Can these qualitative pathways be quantified? Find partial overlap between the high-potency isocontour maps for IC50 and MAC-I derived by CoMFA: Electrostatic Molecular bulk Implications: Potency at NMDA receptor contributes to anesthetic potency, but does not account for it Could similar models for other receptors reveal contributions of each receptor to each effect? IC50 MAC-I Can these qualitative pathways be quantified? The inevitable, likely possibility: Anesthesia involves amplification of effects on specific channels via dynamic interactions among inter-related CNS pathways Understanding those interactions is necessary for a complete mechanistic understanding of anesthesia Understading those interactions may not be necessary to make quantitative predictions of the potency and efficacy of untested compounds For example Could CoMFA be used to estimate the contributions of the major ion channels to various endpoints? 12

13 Conclusions Volatile solvents and anesthetics reversibly affect the nervous system Effects are Graded in severity and quality (anesthetic depth ) Mediated in large part by interactions with ion channels Qualitative pathways can be drawn that are consistent with known modes of action in relevant CNS structures Quantifying these pathways will require a great deal more knowledge about dynamic interactions among CNS structures and interconnections Structure-activity relationships can be developed at both the receptor and functional levels (e.g., CoMFA) References Eger, E. I., 2nd, D. M. Fisher, et al. (2001). "Relevant concentrations of inhaled anesthetics for in vitro studies of anesthetic mechanisms." Anesthesiology 94(5): Eger, E. I., 2nd, M. Tang, et al. (2008). "Inhaled anesthetics do not combine to produce synergistic effects regarding minimum alveolar anesthetic concentration in rats." Anesth Analg 107(2): Grasshoff, C., B. Drexler, et al. (2006). "Anaesthetic drugs: linking molecular actions to clinical effects." Curr Pharm Des 12(28): Hendrickx, J. F., E. I. Eger, 2nd, et al. (2008). "Is synergy the rule? A review of anesthetic interactions producing hypnosis and immobility." Anesth Analg 107(2): Sewell, J. C. and J. W. Sear (2004). "Derivation of preliminary three-dimensional pharmacophores for nonhalogenated volatile anesthetics." Anesth Analg 99(3): , table of contents. Sewell, J. C. and J. W. Sear (2004). "Derivation of preliminary three-dimensional pharmacophoric maps for chemically diverse intravenous general anaesthetics." Br J Anaesth 92(1): Sewell, J. C. and J. W. Sear (2006). "Determinants of volatile general anesthetic potency: a preliminary three-dimensional pharmacophore for halogenated anesthetics." Anesth Analg 102(3): Sewell, J.C., Raines, D.E., Eger II, E.I., Laster, M.J., Sear, J.W. "Comparison of the molecular bases for NMDA-receptor inhibition versus immobilizing activities of volatile aromatic anesthetics." Anesth. Analg., in press. Shafer, S. L., J. F. Hendrickx, et al. (2008). "Additivity versus synergy: a theoretical analysis of implications for anesthetic mechanisms." Anesth Analg 107(2): Sonner, J. M., J. F. Antognini, et al. (2003). "Inhaled anesthetics and immobility: mechanisms, mysteries, and minimum alveolar anesthetic concentration." Anesth Analg 97(3):