Jay Yang

Knock down of spinal NMDA receptors reduces NMDA and formalin evoked behaviors in rat.

Chronic pain remains a major health problem afflicting an estimated 70% of patients with advanced cancer and inflammatory disorders, and up to 94% of patients with spinal cord injuries. Although progress has been made in the pharmacotherapy of chronic pain management, such as usage of adjuvant drugs and more effective methods of drug delivery, the mainstay of clinical pain management still depends on opiates. NMDA receptor activation, at the level of the spinal cord has been shown to play an important role in the facilitation of nociception (pain) in several animal models. Unfortunately, potent NMDA receptor antagonists, such as MK-801 and APV, have toxic properties and low safety margins that preclude their clinical use. We present evidence which indicates that the use of antisense oligonucleotides targeted to the NMDA-R1 receptor subunit (AS-NMDA-R1), but not sense, abolishes NMDA and formalin induced behaviors. Moreover, we demonstrate that spinal administration of AS-NMDA-R1 results in the abolition of staining for immunoreactive NMDA-R1 in the spinal cord. These data provide novel evidence supporting the feasibility of the use of gene therapy approaches in the management of neuropathic pain.

Ketamine blockade of voltage-gated sodium channels: evidence for a shared receptor site with local anesthetics.

Background The general anesthetic ketamine is known to be an N-methyl-d-aspartate receptor blocker. Although ketamine also blocks voltage-gated sodium channels in a local anesthetic–like fashion, little information exists on the molecular pharmacology of this interaction. We measured the effects of ketamine on sodium channels. Methods Wild-type and mutant (F1579A) recombinant rat skeletal muscle sodium channels were expressed in Xenopus oocytes. The F1579A amino acid substitution site is part of the intrapore local anesthetic receptor. The effect of ketamine was measured in oocytes expressing wild-type or mutant sodium channels using two-electrode voltage clamp. Results Ketamine blocked sodium channels in a local anesthetic–like fashion, exhibiting tonic blockade (concentration for half-maximal inhibition [IC50] = 0.8 mm), phasic blockade (IC50 = 2.3 mm), and leftward shift of the steady-state inactivation; the parameters of these actions were strongly modified by alteration of the intrapore local anesthetic binding site (IC50 = 2.1 mm and IC50 = 10.3 mm for tonic and phasic blockade, respectively). Compared with lidocaine, ketamine showed greater tonic inhibition but less phasic blockade. Conclusions Ketamine interacts with sodium channels in a local anesthetic–like fashion, including sharing a binding site with commonly used clinical local anesthetics.

Gene therapy for the management of pain part I: methods and strategies

A RECENT report from the Recombinant DNA Advisory Committee lists 393 approved human gene therapy protocols (www.od.nih.gov/oba/documents.htm). Of these, 33 are for infectious diseases, 49 for monogenic diseases (mostly cystic fibrosis), 237 for cancer, 35 for other disorders, and the remainder for nontherapeutic trials. Despite the serious nature of pain, both in terms of human suffering and economic cost, there are no human gene therapy protocols that target it. Could it be that the biologic basis of pain is too ill defined to apply the state-of-art gene therapy to it? In recent years, significant progress has been made in our fundamental understanding of the neurobiology of pain. In parallel, detailed molecular biologic characterization of many of the receptors and ion channels playing significant roles in nociception have become available. Although in its infancy, gene therapy, or the translational clinical application of the molecular biologic revolution, has fundamentally changed the way we study basic biologic processes and clearly will impact our clinical management of diverse diseases, including pain. This review summarizes recent developments in gene therapy and demonstrates the possibility of gene therapy for the management of pain. This is a broad topic requiring expertise in diverse areas of science and medicine. As such, diverse literature spanning basic molecular biology, viral vectors, antisense oligonucleotide, and pain literature was reviewed. We have strived to create a review with a dual mission of serving as a didactic document and as a document that will serve as a reference for active investigators in this area of science. Most importantly, we hope that this review will stimulate the interest of the readers to further explore the genetic approach to pain management. In Part I, we begin with a discussion of strategies for gene therapy, followed by an introduction of the fundamental methods and technologies (both viral and nonviral) available for gene therapy and transfer. Part II of the review, to be published subsequently, will describe potential nociceptive targets for the methods and strategies of gene therapy described in this review. The pathways of nociception are not summarized; however, there are many excellent reviews summarizing recent developments in our understanding of pain mechanisms. An authoritative review covering neuronal pathways to molecules mediating pain was recently published. Our goal is to minimize overlap with these reviews by focusing on information pertinent for gene therapy considerations. Readers unfamiliar with the basic molecular biologic concepts are referred to an excellent primer on molecular biology recently published in ANESTHESIOLOGY or chapters in standard reference books. Specific protocols for routine molecular biologic techniques can be found in any of the laboratory manuals, and practical methods for creating genetransduction viral vectors can be found in references cited in table 1. Reviews by Eck and Wilson, Andersen, and Kay et al. provide excellent general overviews of gene therapy. Lastly, clinical trials using gene therapeutic agents are subject to full federal regulations, as with any new investigational drug. Moreover, gene therapy protocols are subject to an additional oversight by the federal recombinant DNA advisory committee. Further information and guidelines for developing a human clinical trial protocol, as well as an updated list of active gene therapy trials, can be found at the National Institutes of Health web site referenced above. *Assistant Professor, Department of Anesthesiology and Critical Care Medicine, The Johns Hopkins Hospital. †Assistant Professor, Department of Anesthesiology and Pain Management, University of Texas Southwestern Medical Center. ‡Senior Instructor, Department of Anesthesiology, §Associate Professor, Departments of Anesthesiology, Pharmacology, and Physiology, University of Rochester School of Medicine and Dentistry, Rochester, New York.

Gene therapy for the management of pain: Part II: Molecular targets

TREATMENT of chronic pain, particularly of neuropathic etiology, is extremely difficult and resistant to many available pharmacologic therapies. Current analgesic agents may be limited with regard to analgesic efficacy or side effects. Newer and experimental pharmacologic agents may also have significant limitations. By targeting a specific receptor or other specific protein targets, a gene therapy approach to the treatment of pain may provide greater analgesic efficacy without the limitations associated with current pharmacotherapy. Advances in the field of gene therapy, along with significant increases in our understanding of the neurobiology of nociception and knowledge of the fundamental genetic structure of many nociceptive targets, have made gene therapy for the management of pain a conceivable reality. In part I of this review, we introduced the basic concepts of gene therapy with an emphasis on the available tools (e.g., viral vectors and antisense oligonucleotides) and strategies for upregulating antinociceptive or downregulating pronociceptive targets. In part II, we summarize current knowledge regarding the nociceptive role, molecular biology, and antisense and knockout data of several novel nociceptive targets for gene therapy. We base our selection of the targets included in this review on the three aforementioned criteria. The targets selected are the best characterized and, in our opinion, most likely amenable to the gene therapeutic approach. A simple but feasible strategy and potential gene therapy targets for the management of pain are summarized in figure 1. However, the list is admittedly incomplete, and the readers are referred to other recent reviews cited in part I of this review for a broader perspective on potential targets for the management of pain.

Nondepolarizing neuromuscular blockers inhibit the serotonin-type 3A receptor expressed in xenopus oocytes

UNLABELLED Molecular cloning and sequence comparison indicates a high degree of structural homology between muscle nicotinic acetylcholine (nACh) and serotonin-type 3 (5-HT(3A)) receptors, both members of the direct ligand-gated family of ion channels. Because of the structural similarities and common evolutionary origin of these receptors, neuromuscular blockers (competitive nACh antagonists) may demonstrate pharmacologic cross talk and exhibit attributes of 5-HT(3A) receptor antagonists. We examined six clinically-used neuromuscular blockers for their ability to antagonize currents flowing through the 5-HT(3A) receptors in voltage clamped Xenopus oocytes. The neuromuscular blockers reversibly inhibited the 5-HT(3A) receptor-gated current in the rank order potency of (IC50 mean +/- SEM): d-tubocurarine (0.046 +/- 0.003 microM), atracurium (0.40 +/- 0.03 microM), mivacurium (15.1 +/- 2.93 microM), vecuronium (16.3 +/- 2.24 microM), and rocuronium (19.5 +/- 2.31 microM). Gallamine was essentially inactive as a 5-HT(3A) receptor antagonist with an extrapolated IC50 of 1170 microM. We demonstrate that drugs classically known as competitive nACh receptor antagonists also block 5-HT(3A) receptors. It is likely that certain neuromuscular blockers share pharmacological properties with 5-HT(3A) receptor antagonists, such as a reduction in postoperative nausea and vomiting. With careful drug selection, pharmacological cross talk could potentially be used to minimize polypharmacy and optimize patient management. IMPLICATIONS Muscle nicotinic acetylcholine and serotonin-type 3A (5-HT(3A)) receptors are similar. Therefore neuromuscular relaxants may block 5-HT(3A) receptors. Our pharmacological study demonstrates that neuromuscular relaxants, as with ondansetron, are 5-HT(3A) receptor antagonists. It is likely that certain neuromuscular relaxants exhibit ondansetron-like clinical properties, such as reduction in postoperative nausea and vomiting.