Charlie Taylor, PhD CpTaylor Consulting Chelsea, MI, USA

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1 Contribution of Calcium Channel α 2 δ Binding Sites to the Pharmacology of Gabapentin and Pregabalin Charlie Taylor, PhD CpTaylor Consulting Chelsea, MI, USA

2 Disclosure Information Charlie Taylor, PhD Pfizer Full-time research Scientist until 2007 Pfizer Present: Consultant No discussion of unlabeled uses 2

Topology of α 2 -δ Subunit From EM: Wolf et. al. 2003 J. Mol.

sites) Post-translational cleavage of α 2 and delta; later

3 Topology of α 2 -δ Subunit From EM: Wolf et. al J. Mol. Biol. 332: kd protein, highly glycosylated (17 possible sites) Post-translational cleavage of α 2 and delta; later ligated VonWillebrand domain (ribbons) interacts with α 1 domain 3

4 The α 2 -δ Protein: Physiologic Function R A Accessory subunit of calcium channels Enhances channel function extracellular intracellular Speeds inactivation Slows calcium channel turnover away from cell membrane Arikkath & Campbell (2003) Curr Opin Neurobiol 3:

3) Water soluble > 30 mg/ml Zwitterion (double charge) MW = 160-170

5 Pregabalin & Gabapentin: Physicochemical Properties (S) Pregabalin Gabapentin Hydrophilic (logp = -1.3) Water soluble > 30 mg/ml Zwitterion (double charge) MW = Amino acids Cross blood-brain barrier Inactive at GABA receptors Pharmacology requires rightside chain 5

6 Transport of Pregabalin via System L Gut System L amino acid transporter Bloodstream System L Blood-brain barrier Brain extracellular Space System L Neuron Cytosol 6

[ 3 H]Gabapentin Binding Protein Purification 1 2 3 4 Pig cerebral cortex membranes Solubilized protein Q Sepharose Ca 2+ -IMAC kda 200 116 66 31 22 M 1 2 3 4 5 6

7 [ 3 H]Gabapentin Binding Protein Purification Pig cerebral cortex membranes Solubilized protein Q Sepharose Ca 2+ -IMAC kda M Mono Q 5 Wheat germ lectin 6.5 N-terminal sequencing Recombinant α 2 δ reproduces brain membrane binding Gee NS, Brown J, et al. J Biol Chem. 1996;271:

8 Mutations & Deletions of α 2 δ 1 protein Wang et al. (1999) Biochem J. 342: Deleted or mutated small regions Brown & Gee (1998) J Biol Chem 273: Deleted C-terminal deletion or RRR RRA abolished drug binding NH 2 deletions retained binding hydrophobic regions transmembrane region CO 2 H charged region +/- charged region deletion retained binding deletion abolished binding 8

9 R217A Mutation to α 2 -δ-1 Reduces [ 3 H]Gabapentin Binding Wild type α 2 δ type 1 protein (transiently expressed in COS7) R217A α 2 δ type 1 protein (transiently expressed in COS7) Z. Li et al., Pfizer Data on file, Pfizer Inc. (Z. Li) 9

10 Pregabalin Binding in the Nervous System

white matter Spinal cord ventral horn Bian et al.

11 [ 3 H]Pregabalin Autoradiography: Mouse Spinal Cord and Brain Spinal cord dorsal horn laminae I & II Mouse spinal cord Cortex Mouse brain (sagittal) Spinal cord white matter Spinal cord ventral horn Bian et al. (2006) Brain Res 1075: Hippocampus Cerebellum Hill et al., 1993 [ 3 H]gabapentin rat brain (horizontal) 11

12 Ca 2+ α 2 δ Immunostaining is dense in small-diameter primary sensory neurons Garrido et al. (Pfizer), Unpublished 12

13 Ca 2+ α 2 δ Immunostaining : Rat Olfactory Nucleus, Insula, Piriform & Amygdala Garrido et al. (Pfizer), Unpublished 13

14 Ca 2+ α 2 δ Immunostaining is concentrated in endings of certain glutamate-containing pathways Garrido et al. (Pfizer), Unpublished 14

10 µm 1 µm CA3 Pyramidal Neuron Mossy Fiber Endings α 2 δ

15 Dendritic Spines are a Major Site of Glutamate Synapses & Immunostain Presynaptic Terminals Dendrite Shaft & Spines 10 µm 1 µm CA3 Pyramidal Neuron Mossy Fiber Endings α 2 δ Type 1 Immunostain Richards et al PNAS 102:

16 The α 2 δ Subunit and Pregabalin Pharmacological Activity

R217A Mutant Mouse and Pregabalin Activity Mutation of position 217 (R217A) alters α 2 -δ-1 subunit: R217A mutant mice are like wildtype in other respects

17 R217A Mutant Mouse and Pregabalin Activity Mutation of position 217 (R217A) alters α 2 -δ-1 subunit: R217A mutant mice are like wildtype in other respects Pregabalin binding affinity at α 2 -δ-1 subunit is greatly reduced Homozygous Wildtype Homozygous R217A Mutant Bian et al. (2006) Brain Res 1075:

18 [ 3 H] Pregabalin Binding Significantly Reduced in R217A Mutant Mouse Spinal Cord* Wild Type R217A Mutant * Lumbar dorsal horn autoradiograph MJ Field et al. (2006) PNAS 103: ; F Bian et al. (2006) Brain Res 1075:

19 [ 3 H]Pregabalin Binding Is Reduced in Brain of R217A Mutant Mouse Superficial neocortex Wild Type Hippocampus R217A Mutant Hypothalamus Amygdala/ Pyriform Cortex Mammillary nucleus F Bian et al., Brain Res., 1075:

20 Pregabalin: INACTIVE in Chronic Sciatic Constriction in R217A Mutant Mice Withdrawal Threshold (g) ** Wild Type ** ** ** ** ** ** ** R217A Mutant Vehicle Pregabalin 30 mg/kg SC 100 mg/kg SC BL Time After Dose (hr) BL Time After Dose (hr) BL= baseline. **P<0.01 loose ligation of sciatic nerve von Frey hair static allodynia MJ Field et al. (2006) PNAS 103:

21 Amitriptyline: ACTIVE in Chronic Sciatic Constriction in R217A Mutant Mice 2 Wild Type 2 R217A Mutant Withdrawal Threshold (g) ** ** ** ** ** ** ** ** ** ** ** ** ** Vehicle Amitriptyline 4 (mg/kg,sc) 8 (mg/kg,sc) 12 (mg/kg,sc) ** BL BL Time After Dose (hr) Time After Dose (hr) BL= baseline. ** P<0.01 Loose ligation of sciatic nerve static allodynia MJ Field et al. (2006) Proc Nat Acad Sci 103:

22 The α 2 δ Subunit and Neurotransmitter Release

23 Neurotransmission at Excitatory CNS Synapse presynaptic Pre- Synaptic 2+ Ca glutamate Ca 2+ postsynaptic P/Q N α 2 δ Wheeler, Randall & Tsien, 1994 Post- Synaptic AMPA NMDA 23

24 Pregabalin Reduces Ca 2+ Influx in Depolarized Human Neocortex Synaptosomes 110 Ca 2+ fluorescence (% of control) * P < 0.05 vs. vehicle * * * * 50 Vehicle Pregabalin concentration (μm) Fink et al. (2002) Neuropharmacol 42:

25 Gabapentin Reduces Ca 2+ Currents in Mammalian Cells (recombinant channels) 40 hr Hendrich, Dolphin et al. (2008) PNAS 105:

26 Gabapentin & L-leu Reciprocally Alter Ca 2+ Channel Traffic to the Membrane Trafficking vesicle α 2 δ α 1 β Hendrich, Dolphin et al. (2008) PNAS 105: GBP L-leu Plasma membrane 26

27 Pregabalin Reduces Frequency of Glutamate Miniature Potentials in Rat Brain Note: Amplitude of minis are not altered by pregabalin Hypothesis: α 2 δ modulates asynchronous vesicle release (% of Control) mepsc Frequency * * * * Pregabalin Concentration (µm) Cunningham M, Jones R, et al. (2004) Eur. J. Neurosci. 20:

28 Substance P (% total / min) Pregabalin Reduces Substance P Release in Spinal Cord Slices Capsaicin alone Time (min) Ipsilateral to CFA Injection Capsaicin + pregabalin µm Pregabalin Time (min) Pregabalin reduces capsaicin-induced Substance P release Requires prior inflammation or activation of kinases Fehrenbacher et al. Pain. 2003;105:

29 α 2 δ Drugs Reduce Neurotransmitter Release Reduced presynaptic Ca 2+ current Reduced glutamate release from: spinal cord slices neocortex slices cultured hippocampal neurons Reduced Substance P, CGRP from spinal slices α 2 δ Drug effects are enhanced by: Sustained depolarization (+) Neuromodulators Inflammation Prolonged (>30 min) incubation 29

30 Summary/Conclusions: Voltage-gated Ca 2+ channels consist of subunits Ca 2+ channel α 2 δ type 1 is required for pregabalin & gabapentin actions α 2 δ type 1 is localized in spinal dorsal horn, and also in neocortex, entorhinal cortex, amygdala & other regions Reduced transmitter release by α 2 δ drugs causes pharmacology (analgesic, anticonvulsant, anxiolytic) Pregabalin and gabapentin comprise a novel drug class - α 2 δ ligands 30

The Nervous System. Anatomy of a Neuron

The Nervous System. Anatomy of a Neuron The Nervous System Chapter 38.1-38.5 Anatomy of a Neuron I. Dendrites II. Cell Body III. Axon Synaptic terminal 1 Neuron Connections dendrites cell body terminal cell body cell body terminals dendrites