Contribution of Calcium Channel α 2 δ Binding Sites to the Pharmacology of Gabapentin and Pregabalin Charlie Taylor, PhD CpTaylor Consulting Chelsea, MI, USA
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. Biol. 332:171 140 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
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 3:298-307 4
Pregabalin & Gabapentin: Physicochemical Properties (S) Pregabalin Gabapentin Hydrophilic (logp = -1.3) Water soluble > 30 mg/ml Zwitterion (double charge) MW = 160-170 Amino acids Cross blood-brain barrier Inactive at GABA receptors Pharmacology requires rightside chain 5
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 6 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;271:5768-5776. 7
Mutations & Deletions of α 2 δ 1 protein Wang et al. (1999) Biochem J. 342:313-320 Deleted or mutated small regions Brown & Gee (1998) J Biol Chem 273:25458-25465 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
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
Pregabalin Binding in the Nervous System
[ 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: 68-80 Hippocampus Cerebellum Hill et al., 1993 [ 3 H]gabapentin rat brain (horizontal) 11
Ca 2+ α 2 δ Immunostaining is dense in small-diameter primary sensory neurons Garrido et al. (Pfizer), Unpublished 12
Ca 2+ α 2 δ Immunostaining : Rat Olfactory Nucleus, Insula, Piriform & Amygdala Garrido et al. (Pfizer), Unpublished 13
Ca 2+ α 2 δ Immunostaining is concentrated in endings of certain glutamate-containing pathways Garrido et al. (Pfizer), Unpublished 14
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. 2005 PNAS 102:6166-6171 15
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 Pregabalin binding affinity at α 2 -δ-1 subunit is greatly reduced Homozygous Wildtype Homozygous R217A Mutant Bian et al. (2006) Brain Res 1075: 68-80 17
[ 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: 17537-17542; F Bian et al. (2006) Brain Res 1075: 68-80 18
[ 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., 2006. Brain Res., 1075: 68-80 19
Pregabalin: INACTIVE in Chronic Sciatic Constriction in R217A Mutant Mice Withdrawal Threshold (g) 2 1 0.4 0.16 0.08 0.04 0.02 ** Wild Type ** ** ** ** ** ** ** 2 1 0.4 0.16 0.08 0.04 0.02 R217A Mutant Vehicle Pregabalin 30 mg/kg SC 100 mg/kg SC 0.01 0.01 BL 0 1 2 3 4 Time After Dose (hr) BL 0 1 2 3 4 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: 17537-17542 20
Amitriptyline: ACTIVE in Chronic Sciatic Constriction in R217A Mutant Mice 2 Wild Type 2 R217A Mutant Withdrawal Threshold (g) 1 0.4 0.16 0.08 0.04 0.02 ** ** ** ** ** ** ** 1 0.4 0.16 0.08 0.04 0.02 ** ** ** ** ** ** Vehicle Amitriptyline 4 (mg/kg,sc) 8 (mg/kg,sc) 12 (mg/kg,sc) ** 0.01 0.01 BL 0 1 2 3 4 5 BL 0 1 2 3 4 5 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: 17537-17542 21
The α 2 δ Subunit and Neurotransmitter Release
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
Pregabalin Reduces Ca 2+ Influx in Depolarized Human Neocortex Synaptosomes 110 Ca 2+ fluorescence (% of control) 100 90 80 70 60 * P < 0.05 vs. vehicle * * * * 50 Vehicle 10 100 1000 Pregabalin concentration (μm) Fink et al. (2002) Neuropharmacol 42:229-36. 24
Gabapentin Reduces Ca 2+ Currents in Mammalian Cells (recombinant channels) 40 hr Hendrich, Dolphin et al. (2008) PNAS 105:3628-3633. 25
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 105:3628-3633. GBP L-leu Plasma membrane 26
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 100 80 60 40 * * * * 20 0 0 10 20 50 100 Pregabalin Concentration (µm) Cunningham M, Jones R, et al. (2004) Eur. J. Neurosci. 20:1566-1576. 27
Substance P (% total / min) Pregabalin Reduces Substance P Release in Spinal Cord Slices 0.75 0.50 0.25 0.00 Capsaicin alone 0 2 4 6 8 10 12 14 16 Time (min) Ipsilateral to CFA Injection 0.75 0.50 0.25 0.00 Capsaicin + pregabalin 0 2 4 6 8 10 12 14 16 10 µm Pregabalin Time (min) Pregabalin reduces capsaicin-induced Substance P release Requires prior inflammation or activation of kinases Fehrenbacher et al. Pain. 2003;105:133-141 28
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
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