Attacking pain at its source: new perspectives on opioids

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1 Attacking pain at its source: new perspectives on opioids Christoph Stein, Michael Schäfer & Halina Machelska The treatment of severe pain with opioids has thus far been limited by their unwanted central side effects. Recent research promises new approaches, including opioid analgesics acting outside the central nervous system, targeting of opioid peptide containing immune cells to peripheral damaged tissue, and gene transfer to enhance opioid production at sites of injury. The first step in pain treatment is usually pharmacologic, along with cognitive and behavioral approaches in more chronic stages. Opioids are the most powerful drugs for severe acute and chronic pain, but their widespread use is hampered by side effects such as depression of breathing, nausea, clouding of consciousness, constipation, addiction and tolerance 1.A new generation of opioid drugs is now emerging. This class selectively activates opioid receptors outside the central nervous system, thus avoiding all centrally-mediated, unwanted effects 2. Endogenous ligands of these peripheral opioid receptors are produced within skin and immunocytes 3.This has led to new directions of research, such as the selective targeting of opioid peptide containing cells to sites of painful injury, and the augmentation of opioid synthesis by gene transfer. Acute and chronic pain are frequently associated with inflammation as a result of tissue destruction, abnormal immune reactivity or nerve injury. Within damaged peripheral tissue, primary sensory neurons transduce mechanical, chemical or heat stimuli into action potentials. These sensory neurons have myelinated (Aδ) or small-diameter unmyelinated axons (C-fibers, or nociceptors ). The latter are particularly sensitive to the vanniloid receptor-1 ligand and the neurotoxin capsaicin, and are considered the dominant fibers in clinical pain. Fibers normally transferring innocuous touch (Aβ) can also contribute to pain after nerve damage. After synaptic transmission and modulation within the spinal cord, nociceptive signals reach supraspinal and cortical sites, where they can be perceived as painful. Peripheral and central opioid receptors Three cdnas and their genes have been identified, encoding the µ-, δ- and κ-opioid receptors (MOR, DOR and KOR, respectively) 1.All three receptors mediate pain inhibition and are found throughout the nervous system, in somatic and visceral sensory neurons, spinal cord projection and interneurons, midbrain and cortex. The locations of different opioid receptors on functionally distinct cell types within local circuits of the brain and spinal cord can result in additive or sometimes opposing interactions between receptor types (for example, KOR can have antianalgesic actions) 1,4. Klinik für Anaesthesiologie und Operative Intensivmedizin, Freie Universität Berlin, Klinikum Benjamin Franklin, Hindenburgdamm 30, Berlin, Germany. Correspondence should be addressed to C.S. (christoph.stein@medizin.fu-berlin.de). Recently, opioid receptors have been identified on peripheral processes of sensory neurons (Fig. 1). The cell bodies of these neurons in dorsal root ganglia express all three mrnas and receptor proteins (reviewed in ref. 5). Opioid receptors are intra-axonally transported into the neuronal processes 6 10 and are detectable on peripheral sensory nerve terminals in animals 9,11 13 and humans 14.Colocalization studies confirmed the presence of opioid receptors on C- and A-fibers 15,on vanniloid receptor-1 positive visceral fibers 16 and on neurons expressing isolectin B4, substance P or calcitonin gene related peptide 13,17,18, consistent with the nociceptor phenotype. Sympathetic neurons and immune cells can also express opioid receptors but their functional role is unclear 8,19,20.The binding characteristics of peripheral and central opioid receptors are similar 8,21 but the molecular mass of peripheral and central MORs seems to be different 9.Ifthese findings are confirmed, a search for selective ligands at such distinct receptors may be warranted. There is no dispute that peripheral, spinal and supraspinal opioid receptors mediate analgesic effects. However, the relative contribution of each site to a given analgesic effect after the systemic (intravenous or oral) administration of an opioid agonist has not been examined thoroughly. Recent studies suggest that systemically 22,23 and even centrally (intracerebroventricularly) injected opioid agonists may act predominantly through peripheral opioid receptors. This finding was attributed to the active transport of opioids from the brain to the blood by the saturable P-glycoprotein pump within endothelial cells of the bloodbrain-barrier 24. Finally, tissue damage stimulates the expression of peripheral opioid receptors, which likely leads to a concomitant increase of their function. Opioid receptor signaling in sensory neurons All three types of opioid receptors inhibit high-voltage activated calcium currents in cultured primary afferent neurons (reviewed in ref. 5). These effects are transduced by G-proteins (G i or G o or both) 5,18,25 and may be relatively resistant to desensitization 26 or tolerance development. Although it is well known that opioids induce membrane hyperpolarization as a result of increased potassium currents in central neurons, this has not yet been detected in dorsal root ganglion neurons 27.Thus, the modulation of calcium channels seems to be the primary mechanism for the inhibitory effects of opioids on peripheral sensory neurons. In addition, opioids, through inhibition of adenylyl cyclase, suppress tetrodotoxin-resistant sodium-selective and nonselective cation cur- NATURE MEDICINE VOLUME 9 NUMBER 8 AUGUST

2 Zeichnung Christine Voigts-Grafik/UKBF Berlin rents stimulated by the inflammatory agent prostaglandin E 2 (refs. 28,29). Tetrodotoxin-resistant sodium channels are selectively expressed in nociceptors, where they have an important role in impulse initiation and action potential conductance. These channels mediate spontaneous activity in sensitized nociceptors 30 and accumulate at the site of injury in damaged nerves, leading to ectopic impulse generation 31.The latter observations may explain the notable efficacy of peripheral opioids in inflammatory and neuropathic pain 10,20,32,33.Consistent with their effects on ion channels, opioids attenuate the excitability of peripheral nociceptor terminals, the propagation of action potentials, the release of excitatory proinflammatory neuropeptides (substance P, calcitonin gene-related peptide) from peripheral sensory nerve endings, and the vasodilatation evoked by stimulation of C-fibers (reviewed in ref. 5). All of these mechanisms result in analgesia or anti-inflammatory actions (Fig. 1). Peripheral opioid receptors and tissue injury Peripheral opioid analgesia is augmented under conditions of tissue injury such as inflammation, neuropathy or bone damage (reviewed in refs. 5,32,34). One underlying mechanism is an increased number of peripheral opioid receptors. In neuronal cell cultures, MOR transcription is upregulated by the cytokine interleukin (IL)-4 through the binding of STAT-6 transcription factors to the MOR gene promoter 35.This promoter also responds to activator protein-1 (ref. 36). In dorsal root ganglia, the synthesis and expression of opioid receptors can be increased by peripheral tissue inflammation 21,37. Subsequently, the axonal transport of opioid receptors is greatly enhanced 8,9,38, leading to Figure 1 Opioid receptor transport and signaling in primary afferent neurons. Opioid receptors and neuropeptides (such as substance P) are synthesized in the dorsal root ganglion and transported along intra-axonal microtubules into central and peripheral processes of the primary afferent neuron. At the terminals, opioid receptors are incorporated into the neuronal membrane and become functional receptors. Upon activation by exogenous or endogenous opioids, opioid receptors couple to inhibitory G-proteins. This leads to direct or indirect (through decrease of cyclic adenosine monophosphate) suppression ( ) of Ca 2+ or Na + currents, and subsequent attenuation of substance P release. The permeability of the perineurium is increased within inflamed tissue. OR, opioid receptor; sp, substance P; EO, exogenous opioids; OP, endogenous opioid peptides; G i/o, inhibitory G proteins; camp, cyclic adenosine monophosphate. their upregulation and enhanced agonist efficacy at peripheral nerve terminals 19.IL-1β can stimulate this axonal transport 38.In addition, the specific milieu (low ph, prostanoid release) of inflamed tissue may increase opioid agonist efficacy by enhanced G-protein coupling and by increased neuronal cyclic adenosine monophosphate (camp) levels 21,28,39.Inflammation also increases the number of sensory nerve terminals ( sprouting ) and disrupts the perineural barrier, thus facilitating the access of opioid agonists to their receptors 40.Clinical studies indicate that the perineural application of opioid agonists along uninjured nerves (such as the axillary plexus 41,42, intrapleural and intercostal nerves 43, and mandibular nerve 44 ) does not reliably produce analgesic effects, supporting the notion that inflammation promotes accessibility and efficient coupling of opioid receptors in primary afferent neurons. In fact, it is unknown whether opioid receptor coupling normally occurs in axons or only in nerve terminals. Finally, the secretion of endogenous opioid ligands within inflamed tissue may produce additive or synergistic interactions at peripheral opioid receptors. Tolerance at peripheral opioid receptors An important question is whether tolerance a loss of analgesic efficacy after recurring application of agonists also develops at peripheral opioid receptors. Peripheral tolerance has been observed in animal models using repeated opioid pretreatment in the absence of persistent inflammation However, because the number, affinity and coupling efficacy of opioid receptors are enhanced under inflammatory conditions, these studies do not permit conclusions regarding tolerance in pathological situations. In other models, peripheral opioid analgesia is resistant to the development of tolerance 49,50, and clinical studies suggest a Figure 2 Migration of opioid-producing cells and opioid secretion within inflamed tissue. P-selectin, ICAM-1 and PECAM-1 are upregulated on vascular endothelium. L-selectin is coexpressed by immune cells producing opioid peptides. L- and P-selectin mediate rolling of opioid-containing cells along the vessel wall. ICAM-1 mediates their firm adhesion and diapedesis. Adhesion molecules interact with their respective ligands. In response to stress or releasing agents (such as CRH or IL-1), the cells secrete opioid peptides. CRH and IL-1 elicit opioid peptide release by activating CRH receptors and IL-1 receptors, respectively. Opioid peptides or exogenous opioids bind to opioid receptors on primary afferent neurons, leading to analgesia (see Fig. 1). The immune cells, depleted of opioids, then migrate to regional lymph nodes. OP, opioid peptides; CRHR, CRH receptor; IL-1R, IL-1 receptor; EO, exogenous opioids. Zeichnung Christine Voigts-Grafik/UKBF Berlin 1004 VOLUME 9 NUMBER 8 AUGUST 2003 NATURE MEDICINE

3 lack of cross-tolerance between peripheral exogenous and endogenous opioids in synovial inflammation 14.Clearly, more investigations will be needed to define the differences in peripheral opioid receptor function between noninjured neurons, nerve damage and persistent inflammation, and to elucidate underlying mechanisms such as changes in receptor affinity, phosphorylation, G-protein coupling and internalization 51. From the clinician s viewpoint, the induction of tolerance by opioid pretreatment in the absence of painful tissue injury is not illustrative because patients usually do not consume opioids when they are not in pain (except in the case of opioid abuse). Endogenous ligands of peripheral opioid receptors Three families of opioid peptides are well characterized; the endorphins, enkephalins and dynorphins. They bind to all three opioid receptors. Each family derives from a distinct gene; their respective precursors are pro-opiomelanocortin (POMC), proenkephalin and prodynorphin. Additional selective µ-ligands, the endomorphins, have been isolated but their precursors are not yet known 1. Immune cells are the most extensively examined source of opioids interacting with peripheral opioid receptors 3.Transcripts and peptides derived from POMC and proenkephalin, as well as the prohormone convertases PC-1/3 and PC-2, which are necessary for post-translational Table 1 Peripheral opioid analgesia: comparison of experimental and clinical studies processing, were detected in immune cells 52. The expression of immune-derived opioids is stimulated by viruses, endotoxins, cytokines, corticotropin-releasing hormone (CRH) and adrenergic agonists 52.In conditions of painful inflammation, POMC mrna, β-endorphin, metenkephalin and dynorphin-a are found in circulating cells and lymph nodes 53,54.In the injured tissue, these peptides are upregulated in lymphocytes, monocytes, macrophages and granulocytes 9,14, Migration of opioid-producing cells to injured tissue Adhesion molecules direct the migration of circulating leukocytes to injured tissue. The initial step, rolling, is mediated by selectins on leukocytes (L-selectin) and endothelia (P- and E-selectin). The rolling leukocytes are exposed to tissue-derived chemokines that upregulate the avidity of integrins, which mediate the firm adhesion of cells to endothelia by interacting with immunoglobulin superfamily members such as intercellular adhesion molecule-1 (ICAM-1). Finally, the cells migrate through the vessel wall, directed by platelet endothelial cell adhesion molecule-1 (PECAM-1) and other immunoglobulin ligands. Interruption of this cascade can block immunocyte extravasation 58. These events also govern the homing of opioid-producing cells (Fig. 2). In inflamed tissue, leukocytes containing β-endorphin coexpress L- selectin 59.Opioid-producing cells, vascular P-selectin, ICAM-1 and Findings Experimental studies Clinical studies Comments Refs. Comments Refs. Peripheral versus central mechanism of action FCA inflammation; ipl. versus s.c. fentanyl 95 Postoperative pain; i.a. versus i.v. morphine 96 Thermal hyperalgesia; i.pl. versus s.c. fentanyl, EKC 97 Thermal hyperalgesia; morphine, isolated forearm technique 98 Dose dependency FCA inflammation; µ-, δ-, κ-selective agonists 74, 19 Postoperative pain; i.a. morphine 99 Opioid antagonism, stereospecificity i.pl. (+) versus ( ) naloxone with i.pl. fentany 95 i.a. naloxone with i.a. morphine 96 i.pl. µ-, δ-, κ-selective antagonists; i.pl. (+) versus 74 Local naloxone with morphine in tooth pain 100 ( ) morphine Identification of peripheral opioid receptors By anti-idiotypic antibody, on sensory nerves in 11 By autoradiography, on sensory nerves in synovia 14 subcutaneous tissue Efficacy of peripherally restricted opioids κ-agonist asimadoline effective in inflammation 68 Asimadoline ineffective in UVB-hyperalgesia 90 Asimadoline, early analgesia but late hyperalgesia 75 Asimadoline ineffective in postoperative pain 75 in FCA inflammation µ-agonist loperamide effective in thermal hyperalgesia 48 κ-opioid peptides, analgesic and anti-inflammatory effects 73 κ-agonist effective in pancreatitis pain 72 Inhibition of bone pain Bone damage elicited hyperalgesia:morphine applied to 81 Iliac bone graft: morphine applied to harvest site 85 bone marrow cavity Inhibition of thermal pain Hot-plate injury: local loperamide effective 48 UVB irradiation: local morphine effective 98 Burn injury: local morphine effective 101 Inhibition of visceral pain Bowel inflammation: κ-opioids effective 80 Intravesical morphine effective 87 Tubal ligation: local sufentanil effective 88 Inhibition of chronic arthritic pain Chronic polyarthritis: κ-opioids effective 102 Osteoarthritis: i.a. morphine effective 82 Rheumatoid and osteoarthritis: i.a. morphine and 83 dexamethasone equally effective Inhibition of neuropathic pain Chronic nerve constriction injury: asimadoline effective 33 Not tested Topical activity Thermal injury: loperamide effective 48 Skin ulcers: topical morphine gel 103 DMSO-pretreated rat tail: topical µ-opioids effective 46 Corneal abrasion: morphine eye drops effective 86 Anti-inflammatory activity Chronic polyarthritis: κ-opioids effective 79 Chronic arthritis: i.a. morphine reduces inflammatory cells 83 Paw inflammation: peripherally selective κ-opioids effective 73 Lack of tolerance Bradykinin-induced pain: no tolerance to i.pl. morphine 50 Postoperative pain: no cross-tolerance of i.a. morphine to 14 endogenous opioids Comparison of experimental and clinical studies investigating pharmacologic criteria of peripheral versus central mechanisms of analgesia, opioid receptor specificity of drug actions (naloxone antagonism, dose dependency, stereospecificity), anatomical and biochemical identification of peripheral opioid receptors, peripherally restricted drugs, and analgesic efficacy in different types of pain. FCA, Freund s complete adjuvant; i.pl, intraplantar; i.a., intra-articular; s.c., subcutaneous; i.v., intravenous; EKC, ethylketocyclazocine (κ-agonist); UVB, ultraviolet-b; DMSO, dimethylsulfoxide. (+) and ( ) indicate stereoisomers. NATURE MEDICINE VOLUME 9 NUMBER 8 AUGUST

4 PECAM-1 are simultaneously upregulated 59,60.Blocking the selectins or ICAM-1 reduces the number of opioid-producing cells 60,61.Thus, circulating opioid-producing immune cells home to damaged tissue to secrete the opioids and then travel to the regional lymph nodes 53,54 (Fig. 2). Opioid secretion and peripheral pain control As in the pituitary, the release of opioids from immunocytes is regulated by IL-1β and CRH (Fig. 2). This release is receptor-specific and calciumdependent and is mimicked by elevated extracellular potassium, consistent with a regulated secretory pathway as in neurons and endocrine cells 53,54. In vivo, small, systemically inactive doses of CRH and cytokines elicit opioid-mediated analgesia within injured tissues 3,62. Furthermore, environmental stress and endogenous CRH are triggers for local opioid release 55,63.The potency of this intrinsic pain inhibition is proportional to the number of opioid-producing immunocytes 56.An effective central blockade of pain decreases the migration of such cells to injured sites, indicating a reduced need for peripheral pain-control cells 64.Both CRH- and stress-induced analgesia can be extinguished by immunosuppression 3,11,62 and by blocking the extravasation of opioidcontaining leukocytes 60,61.In patients undergoing knee surgery, opioidproducing lymphocytes and macrophages accumulate in the inflamed synovium. Blockade of intra-articular opioid receptors increases postoperative pain 55,apparently without inducing cross-tolerance to locally administered morphine 14. Other opioid sources include the pituitary and adrenal glands, but neither seems to be involved in peripheral pain inhibition 65. Keratinocytes may release opioids upon stimulation by endothelin-1 (ref. 66). Finally, sensory neurons produce opioid peptides 57 and can be stimulated to overexpress by gene transfer 67. Preclinical studies on peripheral opioid analgesics This basic research has stimulated the development of new opioid ligands acting exclusively in the periphery without central side effects 2.A common approach is the use of hydrophilic compounds with minimal capability to cross the blood-brain barrier 68,69.Penetration of the blood-brain barrier may not be entirely precluded at high doses, however, and the polarizing residues may interfere with the affinity at opioid receptors. Among the first compounds tested were loperamide (originally known as an antidiarrheal drug) 70 and asimadoline 68. Peripheral restriction was also achieved with newly developed arylacetamide 71,72 and peptidic KOR agonists 73 by replacing 3,4-dichloro substituents on the phenyl acetyl group by trifluoromethylphenyl acetyl in the former. Although early attempts to demonstrate peripheral opioid analgesia in normal tissue failed, there was more success with models of pathological pain (Table 1; reviewed in ref. 32). In chronic inflammation of the rat paw, local injection of low, systemically inactive doses of µ-, δ- and κ- agonists produces analgesia that is dose dependent, stereospecific and reversible by selective antagonists, indicating the activation of peripheral MOR, DOR and KOR 74.Asimadoline, a κ-opioid agonist, elicits immediate analgesia and delayed proinflammatory effects in this model 68,75, whereas the peptidic KOR agonists produce both peripheral analgesic and anti-inflammatory effects 73.Possible underlying mechanisms of the latter include reduced release of proinflammatory neuropeptides 76,77 or cytokines 78,or diminished expression of adhesion molecules 79.Potent antinociception was also shown in models of nerve damage 10,20,33 and visceral 80, thermal 46,48 and bone pain 81.Thus, peripheral opioids are effective in a wide variety of animal models, including visceral and neuropathic pain, which are often resistant to conventional drug treatment in humans (Table 1). Clinical studies on peripheral opioid analgesics Numerous controlled studies have demonstrated significant analgesic effects following local application of opioids at sites of injury (Table 1; reviewed in refs. 5,34). Pain relief can be measured by reductions in subjective pain intensity scores, by extended time intervals to the patient s first request for additional pain medication, by a decreased number of patients asking for supplemental analgesic drugs, or by diminished total consumption of supplemental analgesics. Intra-articular administration of the µ-agonist morphine is the best examined clinical application 34.After knee surgery, it reduces pain scores or supplemental analgesic consumption (or both), in a dosedependent fashion, by a peripheral mechanism of action and without side effects 5,34.Intra-articular morphine is active even in the presence of opioid-containing inflammatory cells 14 and in chronic rheumatoid and osteoarthritis 82,83.Its effect is similar to a standard intra-articular local anesthetic or steroid injection and is surprisingly long lasting (up to 7 days) 83,84.This may be the result of morphine s anti-inflammatory activity, as evidenced by a reduced number of synovial inflammatory cells 83.A limitation of its use in chronic arthritis is that repeated injections carry a risk of infection and cannot be easily applied to more than one joint. Other controlled clinical trials examined patients undergoing spinal fusion surgery, who reported postoperative analgesia from a local morphine injection at the iliac bone harvest site. Even one year later, the incidence of chronic donor site pain was still significantly reduced 85.In chronic inflammatory tooth pain, the submucous injection of morphine resulted in long-lasting pain relief after dental sugery 44.Similar to the animal studies, the analgesic effect was absent in patients without preexisting inflammation 44.In unilateral corneal abrasions, topical morphine attenuated pain on the lesioned, but not the intact, side. Importantly, no detrimental effects on wound healing were found 86.In postoperative visceral pain, locally injected opioids produced analgesia in the urinary bladder 87 and after laparoscopic tubal ligation 88.These findings may be of growing importance with the increasing numbers of endoscopic procedures and with the heightened awareness for the need to pre-empt the development of chronic pain. In contrast, several studies have found no peripheral effects of opioids 41,42.The majority of those trials examined the injection of agonists into the noninflamed environment along nerve trunks. This suggests that intra-axonal opioid receptors may be in transit and may not be available as functional receptors at the membrane. In addition, many negative studies were flawed by methodological shortcomings such as lack of study sensitivity (very little pain in the placebo group), small sample sizes, lack of tissue inflammation, inadequately low doses or the superimposition of general or local anesthetic effects 34,41,42,89. New peripherally restricted opioids have recently entered human trials. Asimadoline was not successful in experimental pain 90 or in patients undergoing arthroscopy 75.This was attributed to its low potency and partial ability to cross the blood-brain barrier in humans, resulting in central side effects at higher doses. However, a second-generation κ-agonist markedly reduced visceral pain in patients with chronic pancreatitis without severe side effects 72. Perspectives These findings provide new insights into intrinsic mechanisms of pain control, and suggest innovative strategies for developing drugs and alternative approaches to pain treatment. Immunocompromised patients (such as those with AIDS, cancer and diabetes) frequently suffer from painful neuropathies that can be associated with intra- and perineural inflammation, with reduced intraepidermal nerve fiber density and low CD4 + lymphocyte counts 91. Thus, it may be useful to investigate 1006 VOLUME 9 NUMBER 8 AUGUST 2003 NATURE MEDICINE

5 intraepidermal opioid receptor density, opioid production and release, and migration of opioid-containing immune cells in these patients. The important role of selectins and ICAM-1 in the migration of opioid-containing cells to injured tissue indicates that antiadhesion strategies for the treatment of inflammatory diseases may in fact carry the risk of exacerbating pain. It would be desirable to identify adhesion molecules, chemokines or other stimulating factors that selectively attract opioidproducing cells to damaged tissue. Augmenting the synthesis or secretion of opioid peptides within injured tissue may be accomplished by growth factors, stem-cell or gene therapy: in sensory neurons, the synthesis, transport and peripheral release of enkephalins can be enhanced by herpesvirus-derived vectors containing proenkephalin cdna, leading to a decrease in chronic pain and inflammation in polyarthritic rats 67.Another study showed successful transfection of rat skin with POMC cdna delivered by a nonviral gene-gun system, resulting in increased tissue levels of β-endorphin and a tendency to decrease acute inflammatory pain 92. In view of the ongoing debate about the potential for abusing centrally acting opioids, it is crucial to promote the development of new, peripherally selective opioid drugs. Beyond the absence of central side effects, these compounds may offer advantages such as anti-inflammatory effects, lack of tolerance, lack of constipation (in the case of KOR agonists 72 ), lack of gastrointestinal, renal and thromboembolic complications associated with nonsteroidal anti-inflammatory drugs, and efficacy in neuropathic pain. Locally administered opioids are already used in clinical practice 34,but new drugs applicable by systemic routes are needed. A prototype has been successfully applied intravenously in patients with pain from chronic pancreatitis 72, and an orally available, peripherally restricted opioid antagonist was recently introduced for the treatment of postoperative ileus 93.Many efforts are currently being undertaken to develop peripherally acting analgesics by aiming at individual excitatory receptors or channels on sensory neurons 94.The major advantage of targeting opioid receptors is their mechanism of action: the inhibition of calcium (and possibly sodium) ion channels renders the nociceptor unexcitable to the plethora of stimulating molecules expressed in damaged tissue. Thus, peripherally acting opioids can prevent and reverse the action of multiple excitatory agents simultaneously, instead of blocking only a single noxious stimulus. Uncovering mechanisms that can enhance the availability of endogenous opioids within injured tissue, the signal transduction of peripheral opioid receptors and the exclusion of novel opioid drugs from the central nervous system will open exciting possibilities for pain research and therapy. ACKNOWLEDGMENTS We thank C. Voigts for the preparation of the illustrations, and B. Oldörp and C. Wiegand-Löhnert for their critical comments on the manuscript. The authors are supported by the Deutsche Forschungsgemeinschaft (KFO 100, GRK 276, STE 477), the International Anesthesia Research Society (Frontiers in Anesthesia Research Program), the Bundesministerium für Bildung und Forschung (01GZ0311) and the Freie Universität Berlin (Research Program Inflammatory Diseases). Published online 31 July 2003; doi: /nm Kieffer, B.L. & Gaveriaux-Ruff, C. Exploring the opioid system by gene knockout. Prog. Neurobiol. 66, (2002). 2. Brower, V. New paths to pain relief. Nat. Biotechnol. 18, (2000). 3. 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