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PHM142 Fall 2016 Instructor: Dr. Jeffrey Henderson Biochemistry of Anesthetics Alex Seki Shi Yao (William) Lu Tom Wang Anesthesia Local Produces temporary & reversible loss of sensation in a local area No loss of consciousness General Produces temporary and reversible loss of sensation in whole body Muscle paralysis & loss of reflexes Loss of consciousness Local Anesthetics • Two classes: Ester and Amide • Ester: Eg. Tetracaine • Hydrolyzed by plasma pseudocholinesterases • Amide: Eg. Lidocaine • N-dealkylated and hydroxylated in liver by CYP1A2 & CYP3A4 Local Anesthetics mechanism of action They are generally weak bases and can be ionized to form cations In uncharged state they can cross cell membranes of neurons Once inside the cell they form a cation that interact with sodium channels Review of electrochemical potential In resting state, nongated K+ channels are open, but the gated Na+ channels are closed. Outward flow of K+ helps maintain the negative cytosolic face. During depolarization, Na+ channels open and influx of sodium causes the membrane potential to reverse. Fig. 7-32, Molecular Cell Biology 5th Edition – Lodish, Berk, Matsudaira, Kaiser, Krieger, Scott, Zipursky, Darnell Fig 5.4, Essentials of Regional Anesthesia – Kaye, Urman & Vadivelu The local Anesthetic (B) crosses the cell membrane in its uncharged form; becomes charged inside the cell cytoplasm (BH+). The charged cation (BH+) enters the open sodium channel, and binds, inhibiting Na+ entry, and preventing depolarization. Key properties to consider in Anesthetics pKa • needs to be neutral to get into cytosol, membranes & blood-brain barrier • needs to be cation to have an effect on sodium channel (local anesthetics) Lipid Solubility • More lipophilic = more able to penetrate the cell membrane • More lipophilic = greater sequestration in the myelin and cell membrane; serves as depot for slow release of anesthetic = slower onset, but prolonged action Fraction unbound • Increase in molecular weight correlates to increase in plasma & tissue proteins binding • High protein binding slow uptake and absorption & slow metabolism; again serving as a depot = longer duration of action, slow onset • Non-linear PK – some anesthetics bind to one uncommon blood protein (e.g. a1 acid glycoprotein) with high affinity, and albumin with low affinity. Initially, most anesthetic will bind the glycoprotein so no effect can be observed, but increasing dose will saturate glycoprotein and so doubling dose may result in disproportionate increase in unbound fraction General Anesthetics Administration Inhaled or Intravenous General properties Typically small lipophilic molecules General Anesthetic mechanisms of action Multiple Hypotheses Unitary Lipid theory of anesthesia Lipophilic anesthetics reside in the cell membrane, disrupting the membrane fluidity effects The Ion Channel/Protein binding hypothesis Lipophilic anesthetics bind to ion channels to disrupt ion flow effects Strong Correlation between solubility of a anesthetic in a hydrophobic solvent and its potency. Hypothesized that anesthetics act in an unitary mechanism involving perturbing the cell membrane to cause changes in ion channel conductance Effective Concentration Unitary lipid Theory Olive Oil: Gas partition Coefficient Modified from Fig 24.2, Anesthetic Pharmacology 2nd edition – Evers , Maze & Kharasch Problems with the Unitary lipid theory Certain hydrophobic drugs do not obey the Meyer-Overton correlation Enantiomeric selectivity exists even though lipophilicity is the same CF4 causes anesthesia, but C2F6 and larger perfluoroalkanes do not Isoflurane (+) isomer is twice as effective as (-) isomer in inducing anesthesia Many drugs that should be predicted to function as anesthetic instead triggered convulsions Polyhalogenated and perfluorinated compounds CCl2F2; CBrF2CF3, CBrF2CF2CF3 Ion Channel Theory evidence suggest Anesthetics interact with ligand gated ion channels and voltage gated ion channels Structure of NaChBac – Bacterial homologue of Mammalian Sodium Channel Isoflurane binding Fig. 25-7, Millers Anesthesia 8th Edition Vol. 1 – Miller, Cohen, Eriksson, Fleisher, Wiener-Kronish & Young Fig. 1 A & C, Raju et al. PLoS Comput Biol.:9(6) e1003090 Interactions with ligand gated ion channels Potentiates inhibitory GABAA and Glycine receptors on postsynaptic neurons This can come in the form of increasing the inhibitory post-synaptic current Inhibition of acetylcholine and glutamate receptors on the postsynaptic neurons This may come as reducing the excitatory post-synaptic current Halothane potentiates GABA binding and Chloride Current Fig 5-5, Clinical Anesthesia 7th edition – Barash, Stock, Cahalan, Stoelting, Cullen & Ortega, Figures originally published by Wakamori et al. J. Neurophysiol: 66(6) 2014-21 A) Enflurane (Enf) and Halothane (Hal) increase the chloride current of activated GABA receptors in Rat brainstem neurons (relative to control current) B) Halothane decreases the concentration of GABA needed to generate a given current strength *Note that Hexafluorodiethyl Ether (HFE) is a convulsant, and has the reverse effect on GABA mediated chloride currents. Voltage gated ion channels Inhibits excitatory Na+ channels Reducing the Sodium current Inhibition of Ca2+ channels Reducing the calcium current Combine to reduce neurotransmitter release in presynaptic neurons Figure 1A, Study Anesthesiology:81(1) 104-16 Ca2+ current in N-type Calcium Channel in rat hippocampal pyramidal neurons are depressed by isoflurane Reduction in calcium can lead to reduced neurotransmitter release in pre-synaptic neuron Na+ current in rat posterior pituitary nerve terminals are depressed by isoflurane Figure 2A, Ouyang et al. Mol Pharm.:64(2) 373-81 Summary Page Local anesthetics produces temporary & reversible loss of sensation in a local area General anesthetics produces temporary and reversible loss of sensation in whole body Two classes of local anesthetics: ester and amide Esters metabolized by pseudocholinesterases Amides are N-dealkylated and Hydroxylated by Cyp1A2 and Cyp3A4 Key Properties to consider pKa Neutral to cross membranes, Local anesthetics must be cation inside cell to have effect Lipophilicity ↑ lipophilic = easy to cross membranes, stuck in membrane slow onset, long duration ↓ lipophilic = hard to cross membranes, need more to have effect toxicity? Faster onset, short duration Fraction unbound only unbound fraction will have be able to act on tissue; many don’t have linear PK Mechanisms of Action Local anesthetics Cross cell as neutral compound, becomes cation inside cell, cation binds to sodium channel and blocks activity General anesthetics Lipid theory - anesthetic enters the lipid membrane and perturbs the ion channels reducing ion flow Ion channel theory – Anesthetics bind to epitopes on ion channels – either voltage gated or ligand gated. Potentiates inhibitory GABA and Glycine currents increased IPSP Inhibits excitatory ACh & Glutamate currents decreased EPSP Inhibits excitatory voltage-gated Na+, Ca2+ channels reduced neurotransmitter release Journal References: REFERENCES Franks, N., & Lieb, W. (1991). Stereospecific effects of inhalational general anesthetic optical isomers on nerve ion channels. Science, 254(5030), 427-430. Kalow, W. (1952). Hydrolysis of local Anesthetics by Human Serum Cholinesterase. Journal of Pharmacology and Experimental Therapeutics, 104(2), 122-134. Koblin, D. D., Chortkoff, B. S., Laster, M. J., Eger, E. I., II, Halsey, M. J., & Ionescu, P. (1994). Polyhalogenated and perfluorinated compounds that disobey the Meyer-Overton hypothesis. Anesthesia & Analgesia, 79(6), 1043-1048. Liu, J., Laster, M. J., Koblin, D. D., Eger, E. I., II, Halsey, M. J., Taheri, S., & Chortkoff, B. (1994). A Cutoff in Potency Exists in the Perfluoroalkanes. Anesthesia & Analgesia, 79(2), 238-244. Ouyang, W., Wang, G., & Hemmings, H., Jr. (2003). Isoflurane and Propofol Inhibit Voltage-Gated Sodium Channels in Isolated Rat Neurohypophysial Nerve Terminals. Molecular Pharmacology, 64(2), 373-381. Raju, S. G., Barber, A. F., Lebard, D. N., Klein, M. L., & Carnevale, V. (2013). Exploring Volatile General Anesthetic Binding to a Closed Membrane-Bound Bacterial Voltage-Gated Sodium Channel via Computation. PLos Computational Biology, 9(6). e1003090. Study, R. E. (1994). Isoflurane Inhibits Multiple Voltage-gated Calcium Currents in Hippocampal Pyramidal Neurons. Anesthesiology, 81(1), 104116. Wakamori, M., Ikemoto, Y., & Akaike, N. (1991). Effects of two volatile anesthetics and a volatile convulsant on the excitatory and inhibitory amino acid responses in dissociated CNS neurons of the rat. Journal of Neurophysiology, 66(6), 2014-2021. Textbook References: Barash, P. G., Stock, M. C., Cahalan, M. K., Stoelting, R. K., Cullen, B. F., & Ortega, R. (2013). Clinical Anesthesia (7th ed.). Chapter 5. Evers, A. S., Maze, M., & Kharasch, E. D. (2011). Anesthetic Pharmacology Basic Principles and Clinical Practice (2nd ed.). Chapter 24, 36. Kaye, A. D., Urman, R. D., & Vadivelu, N. (2012). Essentials of regional anesthesia. Chapter 5. Lodish, H. F., Berk, A., Matsudaira, P., Kaiser, C. A., Krieger, M., Scott, M. P., Zipursky, L., & Darnell, J. (2003). Molecular cell biology (5th ed.). Chapter 7. Miller, R. D., Cohen, N. H., Eriksson, L. I., Fleisher, L. A., Wiener-Kronish, J. P., & Young, W. L. (2015). Miller's Anesthesia (8th ed.). Chapter 25, 36.
