1. 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, 2008 Toxicity Pathways Mediated by Ion Channels

3. 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 Goals and Approach

4. Effects of Four Solvents on Behavior Benignus et al., in preparation

5. 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 Why Ion Channels?

6. 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) What are Ion Channels?

7. Voltage-gated: Sensitive to changes in electrical potential across membrane Types of Ion Channels Feldman et al., 1997

8. Ligand-gated channels Sensitive to neurotransmitters, drugs, and solvents Endogenous ligands – neurotransmitters Excitatory (depolarizing) transmitters Glutamate NMDA (N-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 Selected Types of Ion Channels

9. Representative Ligand-Gated Ion Channels Campagna et al., 2003

10. Effects of Anesthetics on Selected Ion Channels Modified from Dilger, 2002

11. Glutamate and GABA Pathways in the CNS Feldman et al., 1997 Glutamate (NMDA etc) Pathways GABA Pathways Cortex Thalamus Hippo- campus Brain Stem Spinal Cord

12. 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 Characteristics of Anesthesia Rudolf & Antkowiak, 2004

13. 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

14. “ Narcosis ” Pathways for Volatile Anesthetics nACh Primary Brain Region Behavioral Effects Hippocampus Light Sedation Amnesia Anxiolysis Ion Channel Receptor Agent Cellular Response GABA A Glycine NMDA Decreased channel-open time Reduced membrane current Increased channel-open time Increased duration of mIPSCs Tissue Response Reduced excitatory transmission Facilitated inhibitory transmission Cortex - Thalamus Brain Stem Spinal Cord Unconsciousness Loss of perceptual awareness Heavy Sedation Slow responses Immobility Loss of pain response Increasing Depth of Anesthesia KineticsDynamics

15. nACh Immobility Pathway for Isoflurane Primary Brain Region Behavioral Effects Hippocampus Light Sedation Amnesia Anxiolysis Ion Channel Receptor Agent Cellular Response GABA A Glycine NMDA Decreased channel-open time Reduced membrane current Increased channel-open time Increased duration of mIPSCs Tissue Response Reduced excitatory transmission Facilitated inhibitory transmission Cortex - Thalamus Brain Stem Spinal Cord Unconsciousness Loss of perceptual awareness Heavy Sedation Slow responses Immobility Loss of pain response Increasing Depth of Anesthesia KineticsDynamics

16. nACh Amnesia Pathway for Isoflurane Primary Brain Region Behavioral Effects Hippocampus Light Sedation Amnesia Anxiolysis Ion Channel Receptor Agent Cellular Response GABA A Glycine NMDA Decreased channel-open time Reduced membrane current Increased channel-open time Increased duration of mIPSCs Tissue Response Reduced excitatory transmission Facilitated inhibitory transmission Cortex - Thalamus Brain Stem Spinal Cord Unconsciousness Loss of perceptual awareness Heavy Sedation Slow responses Immobility Loss of pain response KineticsDynamics Increasing Depth of Anesthesia

17. 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 Light sedation Heavy sedation Unconsiousness Immobility

18. 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 2. Can specific sites or modes of action be identified for each pathway? Can these qualitative pathways be quantified?

19. Interactions between Anesthetics Hendrickx et al., 2008 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

20. Interactions Among Inhaled Anesthetics Hendrickx et al., 2008 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

21. 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?

22. 4. Comparative Molecular Field Analysis (Sewell et al., in press) Can these qualitative pathways be quantified? Correlate IC50 at NMDA receptor and MAC-I for 16 anesthetics, find poor relationship: Models predict observed IC50 and MAC-I data very well:

23. Can these qualitative pathways be quantified? Find partial overlap between the high-potency isocontour maps for IC50 and MAC-I derived by CoMFA: 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 Electrostatic Molecular bulk

24. 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?

25. 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)

26. References