WO2005092306A1 - Methods and compositions for modulating opioid tolerance and chronic pain - Google Patents

Abstract

The invention relates to products and methods for the prevention and/or treatment of opioid tolerance and/or chronic pain. The methods of the invention are useful to prevent and treat chronic pain associated with traumatic injury, surgical injury, or disease, including neuropathic pain. The methods of the invention are useful to inhibit opioid tolerance arising from administration of opioid compounds such as morphine.

Description

METHODS AND COMPOSITIONS FOR MODULATING OPIOID TOLERANCE AND CHRONIC PAIN

Related Applications This application claims priority under 35 U.S.C. §119 from U.S. provisional application serial number 60/552,432, filed March 11, 2004, the entire content of which is incorporated herein in its entirety.

Government Support This invention was made in part with government support under US PHS RO1 grants numbers DA08835 and NS42661 from the National Institutes of Health. The government may have certain rights in this invention.

Field of the Invention The invention relates to methods and products for preventing conditions such as opioid tolerance and chronic pain. The methods include the use of compounds and methods to modulate glucocorticoid receptor activity and expression. The methods of the invention are also useful in combination with additional therapeutic methods to prevent and/or treat opioid tolerance and chronic pain.

Background of the Invention Pain is a perceived experience of real or potential tissue damage, which is a major clinical symptom signifying many disease entities. Millions of people suffer from a variety of pain conditions and unalleviated pain is a major clinical concern. Although there have been descriptions of methods of treating clironic pain, as yet no satisfactory method of pain reduction has been identified. Pain management is a broad and widespread issue in the medical industry. There is ongoing research to identify methods of preventing and/or treating chronic pain, but there remains a pressing need for effective methods of reducing or eliminating chronic pain. One factor that reduces the efficacy and usefulness of some pain treatments is the development of opioid tolerance in patients under treatment with opioid compounds. Commonly used treatments for chronic pain include the administration of opioids, which may result in the development of pharmacological opioid tolerance, particularly in long-term opioid treatment regimens. Opioid analgesics are highly effective for treating many forms of acute and chronic pain. The development of opioid analgesic tolerance is a pharmacological phenomenon that hampers the clinical use of opioid analgesics. Several lines of recent research including studies on β-arrestin (Bohn, L. M. et al,

Nature 408: 720-723, 2000), μ-opioid receptor oligomerization (He, L. et al., Cell 108: 271- 282, 2002), and mechanisms of cellular adaptation through the intracellular adenosine 3',5'- monophosphate (cAMP) and cAMP-dependent-protein inase A (PKA) pathway (Nestler and Aghajanian, Science 278(5335) 58-63 ,1997) have shed light on the neurobiology of opioid tolerance. In addition, activation of N-methyl-D-aspartate receptors (NMDARs) and protein kinase C (PKC) as well as regulation of glutamate transporters also has been implicated in the mechanisms of opioid tolerance (Trujillo, K. A. et al., Science 251: 85-87, 1991; Zeitz, K. P. et al, Pain 94: 245-253, 2002; Mao, J. et al, J. Neurosci. 22: 8312-8323, 2002; Xu, N. J. et al., J. Neurosci. 23: 4775-4784, 2003), suggesting a possible link between neural plasticity resembling a memory process and the cellular mechanisms of opioid tolerance. In animal models, the development of opioid tolerance is routinely examined following a single cycle of opioid administration. It is not known whether reminiscences from a previous opioid exposure would be induced and retained at the cellular level and influence the development of opioid tolerance upon a subsequent exposure, an issue that has considerable importance in clinical opioid therapy and substance abuse (Mao, J. et al., J. Neurosci. 22: 8312-8323, 2002). The development of opioid tolerance is a pharmacological phenomenon associated with repeated administration of opioids and results in the need to administer increasing amounts of opioid doses to maintain the equipotent analgesic effects. Thus, the use of standard opioid treatments for chronic pain is hampered by the development of tolerance to the medications, and the need exists for methods to reduce or eliminate opioid tolerance in patients undergoing pain therapies.

Summary of the Invention We have discovered that compounds that directly or indirectly reduce the activity of glucocorticoid receptors (GR) are surprisingly useful to reduce chronic pain and to reduce opioid tolerance. We have found that GR antagonists and other compounds that reduce GR expression and/or activity can be used unexpectedly to treat opioid tolerance and chronic pain. The administration of a compound that reduces GR activity inhibits tolerance to opioids (e.g. morphine). We have also found that pain conditions such as hyperalgesia or allodynia can be reduced, unexpectedly, when a compound that reduces GR activity administered after the onset of the respective pain condition. Thus, we have discovered that agents that reduce the activity of glucocorticoid receptors, either directly or indirectly, can be used either alone or in combination with opioids or other analgesics, for the prevention and/or treatment of opioid tolerance or chronic pain. According to one aspect of the invention, methods for reducing or preventing opioid tolerance in a subject are provided. The methods include administering to the subject in need of such treatment an amount of a compound that reduces activity of a glucocorticoid receptor effective to reduce or prevent opioid tolerance in the subject. In some embodiments,, the method further includes one or more additional administrations of the compound that reduces activity of a glucocorticoid receptor. In some embodiments, the method further includes administering an opioid to the subject. In certain embodiments, the compound that reduces activity of a glucocorticoid receptor is administered prior to, substantially in conjunction with, or after administration of the opioid. In some embodiments, the opioid is morphine, meperidine, butorphanol, oxymorphone, methadone, propoxyphene, codeine, heroin, hydromorphone, oxycodone, and hydrocodone, fentanyl, sufentanil, nalbuphrine, bupreno hine, or tramadol. In some embodiments, the subject is human. In some embodiments, the subject has a pain condition. In certain embodiments, the pain condition is injury pain, neuropathic pain, surgical pain, disease-associated pain, phantom pain, or inflammation-associated pain. In some embodiments, the injury pain is nervous system injury pain. In some embodiments, the subject is undergoing pain treatment. In certain embodiments, the compound that reduces activity of a glucocorticoid receptor is linked to a targeting molecule. In some embodiments, the targeting molecule's target is a neuronal cell. In some embodiments, the compound that reduces activity of a glucocorticoid receptor is a glucocorticoid receptor antagonist or an antibody or antigen-binding fragment thereof. In certain embodiments, the glucocorticoid receptor antagonist is RU38486, RU486, or RU39305. In some embodiments, the compound that reduces activity of a glucocorticoid receptor is an antisense molecule, an siRNA molecule, or an RNAi molecule. In some embodiments, the mode of administration of the compound that reduces activity of a glucocorticoid receptor is implantation, mucosal administration, intrathecal administration, epidural administration, intravenous administration, inhalation, or oral administration. In certain embodiments, the compound that reduces activity of a glucocorticoid receptor is administered in combination with an opioid or non-opioid analgesic. In some embodiments, the non-opioid analgesic is acetaminophen, ibuprophen, gabapentin, tramadol, a tricyclic antidepressant, a sodium channel blocker, or a muscle relaxant. In certain embodiments, the antidepressant is nortriptyline. In some embodiments, the sodium channel blocker is lamotrigene. In certain embodiments, the muscle relaxant is cyclobenzaprine or tizanidine. In some embodiments, the method further includes administering an agent that inhibits adenylyl cyclase (AC) activity or protein kinase A (PKA) activity. In some embodiments, the agent that inhibits adenylate cyclase (AC) activity is 2', 5'-dideoxyadenosine (ddA). h certain embodiments, the agent that inhibits PKA activity is N-[2-(p-bromocinnamylamino) ethyl]-5-isoqumoline sulfonamide (H89). In some embodiments, the methods also include administering an agent that inhibits activation of glutamate receptors, and/or intracellular activation of protein kinases. In some embodiments, the agent that inhibits activation of glutamate receptors and/or intracellular activation of protein kinases is dextromethorphan, ketamine, amentadine, mementine, methadone, or a tricyclic antidepressant. In certain embodiments, tricyclic antidepressant is nortriptyline or amitriptyline. In some embodiments, the compound that reduces activity of a glucocorticoid receptor is formulated for sustained release. According to another aspect of the invention, opioid compositions are provided. The opioid compositions include an opioid and a compound that reduces activity of a glucocorticoid receptor, wherein administration of the opioid composition to a subject induces less opioid tolerance than the administration of the opioid to the subject in the absence of the compound that reduces activity of a glucocorticoid receptor. In some embodiments, the opioid is morphine, meperidine, butorphanol, oxymorphone, methadone, propoxyphene, codeine, heroin, hydromorphone, oxycodone, hydrocodone, fentanyl, sufentanil, nalbuphrine, buprenorphine, or tramadol. In certain embodiments, the compound that reduces activity of a glucocorticoid receptor is a glucocorticoid receptor antagonist or an antibody or antigen-binding fragment thereof. In some embodiments, the glucocorticoid receptor antagonist is RU38486, RU486, or RU39305. In some embodiments, the compound that reduces activity of a glucocorticoid receptor is an antisense molecule, an siRNA molecule, or an RNAi molecule. In certain embodiments, the compound that reduces activity of a glucocorticoid receptor is linked to a targeting molecule. In some embodiments, the targeting molecule's target is a neuronal cell. In certain embodiments, the opioid composition further includes a non-opioid drug for treating pain. In some embodiments, the non-opioid analgesic is acetaminophen, ibuprophen, gabapentin, tramadol, a tricyclic antidepressant, a sodium channel blocker, or a muscle relaxant. In some embodiments, the antidepressant is nortriptyline. In some embodiments, the sodium channel blocker is lamotrigene. In some embodiments, the muscle relaxant is cyclobenzaprine or tizanidine. In certain embodiments, the opioid composition further includes an agent that inhibits adenylyl cyclase (AC) activity or protein kinase A (PKA) activity. In some embodiments, the agent that inhibits adenylate cyclase (AC) activity is 2', 5'-dideoxyadenosine (ddA). hi some embodiments, the agent that inhibits PKA activity is N-[2-(p-bromocinnamylamino) ethyl] -5- isoquinoline sulfonamide (H89). In certain embodiments, the opioid composition further includes an agent that inhibits activation of glutamate receptors, and/or intracellular activation of protein kinases. In some embodiments, the agent is dextromethoφhan, ketamine, amentadine, mementine, methadone, or a tricyclic antidepressant. In certain embodiments, the tricyclic antidepressant is nortriptyline or amitriptyline. In some embodiments, the compound that reduces activity of a glucocorticoid receptor is formulated for sustained release. According to another aspect of the invention, methods for reducing or preventing chronic pain in a subject are provided. The methods include administering to the subject in need of such treatment an amount of compound that reduces activity of a glucocorticoid receptor effective to reduce or prevent the chronic pain in the subject. In some embodiments, the method further includes one or more additional administrations of the compound that reduces activity of a glucocorticoid receptor. In certain embodiments, the compound that reduces activity of a glucocorticoid receptor is a glucocorticoid receptor antagonist or an antibody or antigen-binding fragment thereof. In some embodiments, the glucocorticoid receptor antagonist is RU38486, RU486, or RU39305. In some embodiments, the compound that reduces activity of a glucocorticoid receptor is an antisense molecule, an siRNA molecule, or an RNAi molecule. In some embodiments, the compound that reduces activity of a glucocorticoid receptor is linked to a targeting molecule. In some embodiments, the targeting molecule's target is a neuronal cell. In certain embodiments, the compound that reduces activity of a glucocorticoid receptor is administered prior to, concurrently with, or subsequent to the onset of pain. In some embodiments, the method further includes administering one or more opioid and/or non-opioid analgesics. In some embodiments, the opioid analgesic is morphine, meperidine, butorphanol, oxymorphone, methadone, propoxyphene, codeine, heroin, hydromorphone, oxycodone, hydrocodone, fentanyl, sufentanil, nalbuphrine, buprenorphine, or tramadol. In some embodiments, the non-opioid analgesic is acetaminophen, ibuprophen, gabapentin, tramadol, a tricyclic antidepressant, a sodium channel blocker, or a muscle relaxant. In certain embodiments, the antidepressant is nortriptyline. In some embodiments, the sodium channel blocker is lamotrigene. In some embodiments, the muscle relaxant is cyclobenzaprine or tizanidine. In some embodiments, the method also includes administering an agent that inhibits adenylyl cyclase (AC) activity or protein kinase A (PKA) activity. In some embodiments, the agent that inhibits adenylate cyclase (AC) activity is 2', 5'-dideoxyadenosine (ddA). IN some embodiments, the agent that inhibits PKA activity is N-[2-(p-bromocinnamylamino) ethyl]-5-isoquinoline sulfonamide (H89). In some embodiments, the subject is human. In some embodiments, the chronic pain condition is injury pain, neuropathic pain, surgical pain, disease-associated pain, phantom pain, or inflammation-associated pain. In some embodiments, the injury pain is nervous system injury pain. In some embodiments, the subject is undergoing pain treatment. In certain embodiments, the mode of administration is implantation, mucosal administration, intrathecal administration, epidural administration, intravenous administration, inhalation, or oral administration. In some embodiments, the methods also include administrating an agent that inhibits activation of glutamate receptors, and/or intracellular activation of protein kinases. In some embodiments, the agent is dextxomethorphan, ketamine, amentadine, mementine, methadone, or a tricyclic antidepressant. In certain embodiments, the tricyclic antidepressant is nortriptyline or amitriptyline. In some embodiments, the compound that reduces activity of a glucocorticoid receptor is formulated for sustained release. According to yet another aspect of the invention, kits for treating a subject in accordance with the method of the aforementioned methods of the invention are provided. The kits include a package housing at least one first container containing at least one dose of a compound that reduces activity of a glucocorticoid receptor, and instructions for using the compound that reduces activity of a glucocorticoid receptor for the prevention and/or treatment of chronic pain and/or opioid tolerance. In some embodiments, the compound that reduces activity of a glucocorticoid receptor is linked to a targeting molecule. In certain embodiments, the targeting molecule's target is a neuronal cell. In some embodiments, the compound that reduces activity of a glucocorticoid receptor is a glucocorticoid receptor antagonist or an antibody or antigen-binding fragment thereof. In some embodiments, the glucocorticoid receptor antagonist is RU38486, RU486, or RU39305. In certain embodiments, the compound that reduces activity of a glucocorticoid receptor is an antisense molecule, an siRNA molecule, or an RNAi molecule. In some embodiments, the kit also includes an additional container containing at least one dose of an opioid or non-opioid analgesic. In some embodiments, the opioid analgesic is morphine, meperidine, butorphanol, oxymorphone, methadone, propoxyphene, codeine, heroin, hydromorphone, oxycodone, hydrocodone, fentanyl, sufentanil, nalbuphrine, buprenorphine, or tramadol. In certain embodiments, the non-opioid analgesic is acetaminophen, ibuprophen, gabapentin, tramadol, a tricyclic antidepressant, a sodium channel blocker, or a muscle relaxant. In some embodiments, the antidepressant is nortriptyline. In some embodiments, the sodium channel blocker is lamotrigene. In certain embodiments, the muscle relaxant is cyclobenzaprine or tizanidine. In some embodiments, the kit also includes a container containing an agent that inhibits adenylyl cyclase (AC) activity or protein kinase A (PKA) activity. In certain embodiments, the agent that inhibits adenylate cyclase (AC) activity is 2', 5'- dideoxyadenosine (ddA). In some embodiments, the agent that inhibits PKA activity is N-[2- (p-bromocinnamylamino) ethyl]-5-isoquinoline sulfonamide (H89). In some embodiments, the chronic pain condition is injury pain, neuropathic pain, surgical pain, disease-associated pain, phantom pain, or inflammation-associated pain. In certain embodiments, the injury pain is nervous system injury pain. In some embodiments, the kit also includes a container containing an agent that inhibits activation of glutamate receptors, and or intracellular activation of protein kinases. In some embodiments, the agent is dextromethorphan, ketamine, amentadine, mementine, methadone, or a tricyclic antidepressant. In certain embodiments, the tricyclic antidepressant is nortriptyline or amitriptyline. In some embodiments, the compound that reduces activity of a glucocorticoid receptor is formulated for sustained release. The invention also includes in some aspects, the use of the foregoing compositions in the preparation of a medicament, particularly a medicament for prevention and/or treatment of chronic pain and/or opioid tolerance. These and other objects of the invention will be described in further detail in connection with the detailed description of the invention.

Brief Description of the Drawings Fig. 1 shows a schematic morphine administration scheme and graphs of results obtained through the schemes. Fig. 1A displays three cycles of morphine treatment, two recovery periods (RP), and various test days (solid arrowheads) in each cycle. Fig. 1, B, D, and F are graphs demonstrating the onset of complete tolerance during the first (Fig. IB), second (Fig. ID) and third (Fig. IF) cycle of moφhine (MS) treatment. Note that 1) a progressively shortened onset following each cycle and 2) a full response to moφhine at the beginning of each treatment cycle indicating the recovery from pharmacological tolerance. * PO.05 and ** P< 0.01, as compared to day 1. % MPAE: percent of maximal possible antinociceptive effect. Fig. 1 C, E, and G are graphs of results indicating the rightward shift of dose-response curves in the MS group as compared to the saline group on day 7 (Fig. 1C), day 5 (Fig. IE), and day 3 (Fig. 1G) of the first, second, and third treatment cycle, respectively.

Fig. 2 shows digital images of Western blots and spinal cord dorsal horn sections. Fig. 2 A shows the time course of GR (95 kDa) upregulation during the first (1st) and second (2nd) cycle of 10 μg moφhine (GR-M) or saline (GR-S) treatment, β-actin (Actin, 42kDa) is a loading control. RD: relative gray density of Western blot bands calculated by normalizing each band with the corresponding loading control band. Only relative densities from the moφhine treatment group were shown in the panel. * P< 0.05, as compared to the baseline on day 1 of the MS group. Fig. 2B and C show topographic GR distribution in spinal cord dorsal horn sections taken from representative rats treated with saline (Fig. 2B) or moφhine (Fig. 2C) twice daily for six days. Figs. 2, D, E, and F show co-localization of GR (Fig. 2D) and the μ-opioid receptor (Fig. 2E) in the spinal cord dorsal horn, as shown in the merged image (Fig. 2F). Figs. 2G, H, and I show colocalization of GR (Fig. 2G) and NeuN (a neuronal marker)(Fig. 2H) in the spinal cord dorsal horn, as shown in the emerged image (Fig. 21). Scale bars: 100 μm in Figs. B and C and 60 μm in Figs. E-I. "DL" in the upper right corner of (Fig. 2C) indicates the dorsolateral part of the spinal cord dorsal horn and all images shown are in the same orientation. Fig. 3 shows digital image of a Western blot and graphs. Fig. 3 A illustrates that co- administration with moφhine and naloxone (M/N; 10 μg each, i.t.) twice daily for six days prevented the GR upregulation seen in the moφhine alone (M) group as compared to the saline alone (S) group. * P< 0.05 as compared to the saline group. See Figure 2 for additional legend. Fig. 3B shows that upregulation of GRs following 10 μg moφhine (twice daily x 6 days) was blocked by the co-administration of moφhine with the AC inhibitor ddA (1 μg) or PKA inhibitor N-[2-(p-bromocinnamylamino) ethyl]-5-isoquinoline sulfonamide (H89) (10 μg). ddA or H89 alone did not affect the baseline GR expression. While the AC activator forskolin induced an upregulation of GRs in naϊve rats similar to that following the moφhine administration, the combined treatment of moφhine and forskolin resulted in an increased GR expression as compared to the moφhine alone group. S: saline; M: moφhine; M/D: moφhine plus ddA; D: ddA alone; M/F: moφhine plus forskolin; F: forskolin alone; M/H: moφhine plus H89; H:H89 alone. * P< 0.05, as compared to the saline group; + P< 0.05, as compared to both saline and moφhine alone group. Fig. 3C is a graph of results obtained when examined on day 7, there was a rightward shift of the moφhine dose-response curve in those naϊve rats treated with forskolin (lOμg) twice daily for six days as compared to the naϊve rats treated with a vehicle. Fig. 4 shows digital images of Western blots and spinal cord sections. Fig. 4A and B shows upregulation of NMDARs (Fig. 4A) or PKCγ (Fig. 4B) following 10 μg moφhine (twice daily x 6 days) was blocked by the co-administration of moφhine and 1 μg RU38486 (M/R), but not of moφhine and 3 μg spironolactone (M/SRL). RU38486 (R) alone did not change the level of NMDAR (100 kDa) or PKCγ (80 kDa). Figs. 4C, D, and E show co- localization of 26GR (Fig. 4C) and the NMDAR (Fig. 4D) in the spinal cord dorsal horn, as shown in the emerged image (Fig. 4E). Figs. 4F, G, and H show co-localization of GR (Fig. 4F) and PKCγ (Fig. 4G) in the spinal cord dorsal horn, as shown in the emerged image (Fig. 4H). Scale bars: 60 μm. Figs. 41 and J show upregulation of NMDARs or PKCγ following 10 μg moφhine (twice daily x 6 days) was blocked by the coadministration of moφhine with the AC inhibitor ddA (1 μg) or PKA inhibitor H89 (1 Oμg). ddA or H89 alone did not affect the baseline NMDAR or PKCγ expression. While the AC activator forskolin induced an upregulation of NMDARs in naϊve rats similar to that following the moφhine administration, the combined treatment with moφhine and forskolin resulted in an increased NMDAR expression as compared to the moφhine alone group. * P< 0.05, as compared to the saline group; + P< 0.05, as compared to both saline and moφhine alone group.

Fig. 5 shows graphs of the development of opioid tolerance. Figs. 5 A and B show the development of tolerance was prevented in rats treated with moφhine (MS) and RU38486 (RU) but not spironolactone (SRL) during the first cycle. Note the differences in the onset of complete tolerance (Fig. 5 A) and the rightward shift of dose-response curves (Fig. 5B) among groups. * P<0.05 and ** P< 0.01, as compared to day 1. % MPAE:(see Fig. 1). Fig. 5C shows graph of results indicating that a single injection of 1 μg RU38486 (i.t, 30 min AFTER) did not reverse tolerance, as shown following a probe moφhine dose (10 μg) on day 7 (BEFORE), in those rats made tolerance to moφhine (i.t. 10 μg) twice daily for six days. Fig. 6 shows graphs and a digital image of a Western blot. Figs. 6 A and B are graphs indicating the shortened onset of complete tolerance (Fig. 6 A) and the rightward shift of the dose-response curve (Fig. 6B) during the second cycle of moφhine alone were prevented in those rats treated with moφhine plus RU38486 (MS+RU), but not with moφhine plus saline (MS), during the first treatment cycle. Fig. 6C shows that the co-administration with 10 μg moφhine and 1 μg RU38486 (M/R) twice daily for six days prevented the GR upregulation induced by moφhine (M) alone. RU38486 (1 μg, R) alone did not change the GR level as compared to the saline (S) control. *P< 0.05 as compared to the saline group. Fig. 6D shows that U38486 (i.t. 1 μg, MS+RU), but not saline (MS+SAL), given twice daily during the recovery period following the first cycle of moφhine treatment prevented the shortened onset of complete moφhine tolerance (7 days for MS+RU vs. 5 days for MS+SAL) in the second cycle of moφhine alone. In Fig. 6A and D, * PO.05 and ** P< 0.01, as compared to day 1. % MPAE: (see Fig. 1).

Fig. 7. is a graph of results indicating the time course of the effect of RU38486 on hyperalgesia. PWL = paw-withdrawal latency. Fig. 8. is a graph of results indicating the time course of the effect of RU38486 on allodynia.

Fig. 9 is a graph of results indicating the dose-response effect of RU38486 on hyperalgesia.

Fig. 10 is a graph of results indicting the dose-response effect of RU38486 on allodynia. Fig. 11 is a graph of results indicating that RU38486 reversed hyperalegsia.

Fig. 12 is a graph of results indicating that RU38486 reverses allodynia.

Detailed Description of the Invention We have discovered that opioid tolerance and/or chronic pain can be ameliorated by the administration of the compounds of the invention. The compositions of the invention include compounds that reduce glucocorticoid receptor (GR) activity in cells, tissues and/or subjects, thereby inhibiting opioid tolerance and reducing pain and their clinical manifestations. The GR activity-reducing compounds of the invention can be administered prophylatically to prevent the onset of opioid tolerance or pain and/or can be administered to a subject who has opioid tolerance or pain, as a treatment for the subject. The methods of the invention involve the administration of one or more compounds that reduce glucocorticoid receptor (GR) activity. As used herein the term "modulate" means enhance or inhibit. Preferred compositions of the invention include compounds that reduce GR activity. The GR activity-reducing methods and compositions of the invention are useful for preventing or reducing opioid tolerance and/or for preventing and/or treating chronic pain. Compositions of the invention include compounds that reduce opioid tolerance, for example, opioid tolerance that develops in a subject undergoing opioid treatment. Compositions of the invention also include compounds that reduce pain, including, but not limited to chronic pain. The methods of the invention involve the administration of a GR activity-reducing compound for the inhibition of opioid tolerance and/or the treatment of pain in a subject. Opioid tolerance is a decrease in the effect of an opioid drug administered in repeated doses or by continuous infusion. Opioid tolerance can arise due to chronic opioid administration or after an acute opioid administration. Opioid tolerance results in a diminishing analgesic effect of opioid therapy, and the decrease in analgesia may result an escalation of opioid administration in order to restore the analgesic effects. Opioids administered to subjects for the treatment of pain conditions include, but are not limited to: moφhine (e.g., in MS-Contin®), meperidine, butoφhanol, oxymoφhone, methadone, propoxyphene, codeine, heroin, hydromoφhone (e.g., in Dilaudid®), oxycodone (e.g. in Percodan® and Percocet®), hydrocodone, fentanyl, sufentanil, nalbuphrine, and buprenoφhine. As used herein, the term "opioid" includes opioid compounds and also includes non-opioid compounds with opioid-agonist activity. In some embodiments of the invention, the non-opioid compounds with opioid-agonist activity may be administered as non-opioid analgesics. Examples of compounds with opioid-agonist activity include, but are not limited to, tramadol, (e.g. in Ultram® and Ultracet®). As used herein, the term "pain condition" means the presence of pain in the subject. Pain conditions include, but are not limited to: injury pain, neuropathic pain, surgical pain, disease-associated pain, phantom pain, and inflammation-associated pain. The pain condition may be a chronic pain condition or may be episodic in nature. In some embodiments of the invention, the pain condition is nervous system injury pain. In some embodiments of the invention, a subject has more than one type of pain condition. The diagnosis and assessment of opioid tolerance and/or pain in a subject are known to those of skill in the art, and may include monitoring clinical features of opioid tolerance and/or pain for assessment of GR activity modulating compounds. Such assessment can be done with methods known to one of ordinary skill in the art, such as behavioral testing, verbal assessment, etc. As used herein, the term "subject" means any mammal that may be in need of treatment with a GR modulating compound of the invention. Subjects include but are not limited to: humans, non-human primates, cats, dogs, sheep, pigs, horses, cows, rodents such as mice, rats, etc. In some embodiments of the invention, a subject has a pain condition and is undergoing a treatment for the pain condition. The treatment may include the administration of one or more opioids. In the methods of the invention, one or more GR activity modulating compounds may be administered prior to, substantially in conjunction with, or after administration of an opioid compound. As used herein, the term glucocorticoid receptor (GR) modulating compound, means a compound that modulates the activity of a GR. As will be understood by those of ordinary skill in the art, the activity of a GR can be modulated with the methods and compounds of the invention either directly or indirectly. As used herein, "direct" modulation by a compound means that a compound that has a direct physical interaction with a GR and affects GR activity. For example, a direct-acting GR activity-reducing compound may be a GR antagonist, an antibody, or another molecule that interferes with the activity of a GR. In contrast, an indirect-acting GR activity-reducing compound, includes all other compounds that have an effect on GR activity, but don't have a direct physical interaction with the GR receptor. An example of an indirect-acting GR activity-reducing compound is a compound that reduces expression of a GR, for example, an antisense, siRNA, or RNAi molecule. An indirect-acting GR activity-reducing compound is a compound that reduces the level of GR activity, but does not directly physically interact with a GR polypeptide. GR activity-reducing compounds of the invention include, but are not limited to: GR antagonists and compounds that reduce expression of GR. Examples of GR antagonists that are useful in the methods of the invention are RU38486 and RU486 (mifepristone), although other GR antagonists, including, but not limited to RU39305, may be used in the methods and compositions of the invention. The methods of the invention also include the use of molecules, such as antibodies or antigen-binding fragments thereof that interfere with GR activity. Antibodies may interfere by specifically binding to a GR (direct acting) or to a GR agonist or other molecule involved with GR activity (indirect acting), and reducing the activity of a GR. Compounds that reduce expression of a GR (indirect acting) are also useful in the methods and compositions of the invention and include, but are not limited to: antisense, siRNA, and RNAi oligonucleotides. GR activity-reducing compounds useful in some aspects of the invention include GR antagonists. The general effects of receptor antagonists on receptor activity are well known in the art to include the reduction or elimination of the target receptor activity. An antagonist may compete with the agonist to bind and/or interact with the receptor, thus, the normal activity of the receptor is reduced in the presence of an antagonist. As described herein, RU486, RU38486, and RU39305 are examples of GR antagonists that are useful in the methods of the invention. The methods of the invention may also include the administration of one or more non- opioid analgesics. Examples of non-opoiod analgesics that can be used in the methods of the invention include, but are not limited to: acetaminophen, ibuprophen, gabapentin, tramadol, tricyclic antidepressants such as nortriptyline, sodium channel blockers such as lamotrigene muscle relaxants such as cyclobenzaprine (e.g. Flexeril®), and tizanidine, which may be administered with or without opioids. i some embodiments of the invention, non-opioid analgesics may be administered prior to, substantially in conjunction with, or after administration of a non-opioid analgesic compound. In some embodiments of the invention, the method of treating a subject with a GR activity-reducing compound of the invention also includes administering to the subject an agent that inhibits adenylyl cyclase (AC) activity or protein kinase A (PKA) activity. In some embodiments of the invention, an agent that inhibits AC activity or PKA activity may be administered prior to, substantially in conjunction with, and/or after administration of a GR activity-reducing compound of the invention. An example of an agent that inhibits AC activity, although not intended to be limiting, is 2', 5'-dideoxyadenosine (ddA). An example of an agent that inhibits PKA activity, although not intended to be limiting, is N-[2-(p- bromocinnamylamino) ethyl]-5-isoquinoline sulfonamide (H89). In some embodiments of the invention, the method of treating a subject with a GR activity-reducing compound of the invention also includes administering to the subject an agent that inhibits activation of glutamate receptors and/or intracellular activation of protein kinases. In some embodiments of the invention, an agent that inhibits activation of glutamate receptors and/or intracellular activation of protein kinases may be administered prior to, substantially in conjunction with, and/or after administration of a GR activity-reducing compound of the invention. Agents that inhibit activation of glutamate receptors and/or intracellular activation of protein kinases, and that are useful in the methods of the invention may include, but are not limited to, dextromethoφhan, ketamine, amentadine, mementine, methadone, and tricyclic antidepressants. The tricyclic antidepressants useful in the invention include, but are not limited to, nortriptyline and amitriptyline. With respect to GR modulation, the term "modulate" means either inhibit or enhance. As used herein, a "GR activity modulator" may be a compound that inhibits or enhances GR activity. As used herein, the term "compound that reduces GR activity" means a compound that acts directly or indirectly lower the activity of a GR. The compounds of the invention may be used to decrease the level of opioid tolerance and/or reduce the level of pain in a subject to a level or amount that is statistically significantly less than a control level of opioid tolerance and/or pain. In some cases, the decrease in the level of opioid tolerance and/or pain means the level of opioid tolerance and/or pain is reduced from an initial level to a level significantly lower than the initial level level. In some cases this reduced level may be zero. In some embodiments, a GR activity modulator may be a compound that increases GR activity. Such a compound may be useful for making cell or animal models of chronic pain or opioid tolerance. The GR activity-increasing compounds of the invention may be used to increase the level of opioid tolerance and/or pain in a subject to a level or amount that is statistically significantly more than a control level of opioid tolerance and/or pain. In some cases, the increase in the level of opioid tolerance and/or pain means the level of opioid tolerance and/or pain is raised from zero to a level above zero, in other cases an increase in opioid tolerance and/or pain means an increase from a level that is above zero to a level significantly higher than that original or baseline level of activity. The enhancement of opioid tolerance and/or pain using the methods of the invention that increase GR activity may be used for research tools or in models (e.g. animal models) for the study of opioid tolerance and/or pain in subjects. For example, such models may be useful for the identification and/or analysis of methods to prevent or treat opioid tolerance and/or chronic pain. A control level of opioid tolerance and/or pain is the level that represents the normal level of opioid tolerance and/or pain in a subject. In some instances, a control level will be the level in a subject under treatment with an opioid or other analgesic, and may be useful, for example, to monitor a change in the level of opioid tolerance in the subject. In some instances, a control level will be the level of pain in a subject not under treatment with an opioid or other analgesic, and may be useful, for example, to monitor the reduction in pain using the methods of the invention. Strategies for selecting control levels and conditions are well known in the art. The control levels are useful in assays to assess the efficacy of a GR activity-reducing compound of the invention to reduce opioid tolerance and/or to reduce pain in a subject. The invention relates in some aspects to the administration of a level of a GR activity- reducing compound of the invention in an amount effective to treat or prevent the opioid tolerance and/or chronic pain in a subject. GR activity-reducing compounds of the invention include analogs, derivatives, and variants of the GR activity-reducing compounds. For example, analogs, derivatives, and variants of the GR activity-reducing compounds described herein can be made, for example, to enhance GR activity-reducing function or another property of a compound, such as stability, bioavailability, reduced toxicity, etc. Analogs, derivatives, and variants of the GR activity-reducing compounds described herein may also be made to provide a novel activity or property to the compound. In some embodiments of the invention, modifications to a GR activity-reducing compound of the invention, can be made to the structure or side groups of the compound and can include deletions, substitutions, and additions of atoms, or side groups. Alternatively, modifications can be made by addition of a linker molecule, addition of a detectable moiety, such as biotin or a fluorophore, chromophore, enzymatic, and/or radioactive label, and the like. Analogs, derivatives, and variants of the GR activity-reducing compounds that retain some or all of the GR activity-reducing activity of the compounds described herein, can be used in accordance with the invention, hi some embodiments, an analog of a molecule may have a higher level of GR activity modulating activity than that molecule. Chemical groups that can be added to or substituted in the molecules include: hydrido, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycyl, aryl, heteroaryl, alkoxy, aryloxy, sulfoxy, acyl, amino, acyloxy, acylamino, carboalkoxy, carboxyamido, halo, and thio groups. Substitutions can replace one or more chemical groups or atoms on the molecules. Molecular terms, when used in this application, have their common meaning unless otherwise specified. The term "hydrido" denotes a single hydrogen atom (H). The term "acyl" is defined as a carbonyl radical attached to an alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl or heteroaryl group, examples of such radicals being acetyl and benzoyl. The term "amino" denotes a nitrogen radical containing two substituents independently selected from the group consisting of hydrido, alkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl. The term "acyloxy" denotes an oxygen radical adjacent to an acyl group. The term "acylamino" denotes a nitrogen radical adjacent to an acyl group. The term "carboalkoxy" is defined as a carbonyl radical adjacent to an alkoxy or aryloxy group. The term "carboxyamido" denotes a carbonyl radical adjacent to an amino group. The term "carboxy" embraces a carbonyl radical adjacent to an alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl or heteroaryl group. The term "halo" is defined as a bromo, chloro, fluoro or iodo radical. The term "thio" denotes a radical containing a substituent group independently selected from hydrido, alkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl, attached to a divalent sulfur atom, such as, methylthio and phenylthio. The term "alkyl" is defined as a linear or branched, saturated radical having one to about ten carbon atoms unless otherwise specified. Preferred alkyl radicals are "lower alkyl" radicals having one to about five carbon atoms. One or more hydrogen atoms can also be replaced by a substitutent group selected from acyl, amino, acylamino, acyloxy, carboalkoxy, carboxy, carboxyamido, cyano, halo, hydroxy, nitro, thio, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, sulfoxy, and formyl. Examples of alkyl groups include methyl, tert-butyl, isopropyl, and methoxymethyl. The term "alkenyl" embraces linear or branched radicals having two to about twenty carbon atoms, preferably three to about ten carbon atoms, and containing at least one carbon- carbon double bond. One or more hydrogen atoms can also be replaced by a substituent group selected from acyl, amino, acylamino, acyloxy, carboalkoxy, carboxy, carboxyamido, cyano, halo, hydroxy, nitro, thio, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, sulfoxy, and formyl. Examples of alkenyl groups include ethylenyl or phenyl ethylenyl. The term "alkynyl" denotes linear or branched radicals having from two to about ten carbon atoms, and containing at least one carbon-carbon triple bond. One or more hydrogen atoms can also be replaced by a substituent group selected from acyl, amino, acylamino, acyloxy, carboalkoxy, carboxy, carboxyamido, cyano, halo, hydroxy, nitro, thio, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, sulfoxy, and formyl. Examples of alkynyl groups include propynyl. The term "aryl" denotes aromatic radicals in a single or fused carbocyclic ring system, having from five to twelve ring members. One or more hydrogen atoms may also be replaced by a substituent group selected from acyl, amino, acylamino, acyloxy, carboalkoxy, carboxy, carboxyamido, cyano, halo, hydroxy, nitro, thio, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, sulfoxy, and formyl. Examples of aryl groups include phenyl, naphthyl, biphenyl, and teφhenyl. "Heteroaryl" embraces aromatic radicals which contain one to four hetero atoms selected from oxygen, nitrogen and sulfur in a single or fused heterocychc ring system, having from five to fifteen ring members. One or more hydrogen atoms may also be replaced by a substituent group selected from acyl, amino, acylamino, acyloxy, carboalkoxy, carboxy, carboxyamido, cyano, halo, hydroxy, nitro, thio, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, sulfoxy, and formyl. Examples of heteroaryl groups include pyridinyl, thiazolyl, thiadiazoyl, isoquinolinyl, pyrazolyl, oxazolyl, oxadiazoyl, triazolyl, and pyrrolyl groups. The term "cycloalkyl" is defined as a saturated or partially unsaturated carbocyclic ring in a single or fused carbocyclic ring system having from three to twelve ring members. One or more hydrogen atoms may also be replaced by a substituent group selected from acyl, amino, acylamino, acyloxy, carboalkoxy, carboxy, carboxyamido, cyano, halo, hydroxy, nitro, thio, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, sulfoxy, and formyl. Examples of a cycloalkyl group include cyclopropyl, cyclobutyl, cyclohexyl, and cycloheptyl. The term "heterocyclyl" embraces a saturated or partially unsaturated ring containing zero to four hetero atoms selected from oxygen, nitrogen and sulfur in a single or fused heterocychc ring system having from three to twelve ring members. One or more hydrogen atoms may also be replaced by a substituent group selected from acyl, amino, acylamino, acyloxy, carboalkoxy, carboxy, carboxyamido, cyano, halo, hydroxy, nitro, thio, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, sulfoxy, and formyl. Examples of a heterocyclyl group include moφholinyl, piperidinyl, and pyrrolidinyl. The term "alkoxy" denotes oxy-containing radicals substituted with an alkyl, cycloalkyl or heterocyclyl group. Examples include methoxy, -fert-butoxy, benzyloxy and cyclohexyloxy. The term "aryloxy" denotes oxy-containing radicals substituted with an aryl or heteroaryl group. Examples include phenoxy. The term "sulfoxy" is defined as a hexavalent sulfur radical bound to two or three substituents selected from the group consisting of oxo, alkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl, wherein at least one of said substituents is oxo. The GR activity modulating compounds of the invention also include, but are not limited to any pharmaceutically acceptable salts, esters, salts of an ester of the compound, or solvates. Examples of salts that may be used, which is not intended to be limiting include: chloride, acetate, hydrochloride, methansulfonate or other salt of a compound of the invention or an analog, derivative, variant, or fragment of the compound. Derivatives of the compounds of the invention include compounds which, upon administration to a subject in need of such administration, deliver (directly or indirectly) a pharmaceutically active GR activity modulating compound as described herein. An example of pharmaceutically active derivatives of the invention includes, but is not limited to, pro- drugs. A pro-drug is a derivative of a compound that contains an additional moiety that is susceptible to removal in vivo yielding the parent molecule as a pharmacologically active agent. An example of a pro-drug is an ester that is cleaved in vivo to yield a compound of interest. Pro-drugs of a variety of compounds, and materials and methods for derivatizing the parent compounds to create the pro-drugs, are known to those of ordinary skill in the art and may be adapted to the present invention. Analogs, variants, and derivatives of the GR activity modulating compounds of the invention may be identified using standard methods known to those of ordinary skill in the art. Useful methods involve identification of compounds having similar chemical structure, similar active groups, chemical family relatedness, and other standard characteristics. For the puφoses of this invention, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics 75^th Ed., inside cover, and specific functional groups are defined as described therein. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivity, are described in "Organic Chemistry", Thomas Sorrell, University Science Books, Sausalito. 1999, the contents of which are incoφorated herein by reference in their entirety. Using the structures of the compounds disclosed herein, one of ordinary skill in the art is enabled to make predictions of structural and chemical motifs for analogs, variants, and/or derivatives that possess similar functions of the GR activity modulating compounds disclosed herein. Using structural motifs as search, evaluation, or design criteria, one of ordinary skill in the art is enabled to identify classes of compounds (analogs, derivatives and/or variants of the GR activity-modulating compounds) that possess the modulatory function of the compounds disclosed herein. These compounds may be synthesized using standard synthetic methods and tested for activity as described herein. The methods of the invention also include, in some aspects, the administration of compounds that reduce the expression of GR. For example, compositions of the invention may include a molecule that reduces transcription of GR, including nucleic acids that bind to other nucleic acids, [e.g. antisense, RNAi, or small interfering RNA (siRNA) methods]. For example, the methods of the invention may include the administration of molecules that are antisense of the nucleic acids that encode GR. The methods of the invention include in some embodiments, the use of RNAi and/or siRNA to inhibit GR expression and activity. As used herein, a "siRNA molecule" is a double-stranded RNA molecule (dsRNA) consisting of a sense and an antisense strand, which are complementary (Tuschl, T. et al., 1999, Genes & Dev., 13: 3191-3197; Elbashir, S.M. et al., 2001, EMBOJ., 20: 6877-6888). hi one embodiment the last nucleotide at the 3' end of the antisense strand may be any nucleotide and is not required to be complementary to the region of the target gene. The siRNA molecule may be 19-23 nucleotides in length in some embodiments. In other embodiments, the siRNA is longer but forms a haiφin structure of 19-23 nucleotides in length. In still other embodiments, the siRNA is formed in the cell by digestion of double- stranded RNA molecule that is longer than 19-23 nucleotides. The siRNA molecule preferably includes an overhang on one or both ends, preferably a 3 ' overhang, and more preferably a two nucleotide 3' overhang on the sense strand. In another preferred embodiment, the two nucleotide overhang is thymidine-thymidine (TT). The siRNA molecule corresponds to at least a portion of a target gene. In one embodiment the siRNA molecule corresponds to a region selected from a cDNA target gene beginning between 50 to 100 nucleotides downstream of the start codon. In a preferred embodiment the first nucleotide of the siRNA molecule is a purine. Many variations of siRNA and other double- stranded RNA molecules useful for RNAi inhibition of gene expression will be known to one of ordinary skill in the art. The siRNA molecules can be plasmid-based. In a preferred method, a polypeptide encoding sequence of a GR is amplified using the well-known technique of polymerase chain reaction (PCR). The use of the entire polypeptide encoding sequence is not necessary; as is well known in the art, a portion of the polypeptide encoding sequence is sufficient for RNA interference. For example, the PCR fragment can be inserted into a vector using routine techniques well known to those of skill in the art. The insert can be placed between two promoters oriented in opposite directions, such that two complementary RNA molecules are produced that hybridize to form the siRNA molecule. Alternatively, the siRNA molecule is synthesized as a single RNA molecule that self-hybridizes to form a siRNA duplex, preferably with a non-hybridizing sequence that forms a "loop" between the hybridizing sequences. Combinations of the foregoing can be expressed from a single vector or from multiple vectors introduced into cells. In one aspect use of the invention a vector comprising any of the nucleotide coding sequences of the invention is provided, preferably one that includes promoters active in mammalian cells. Non-limiting examples of vectors are the pSUPER RNAi series of vectors (Brummelkamp, T.R. et al., 2002, Science, 296: 550-553; available commercially from OligoEngine, Inc., Seattle, WA). In one embodiment a partially self-complementary nucleotide coding sequence can be inserted into the mammalian vector using restriction sites, creating a stem-loop structure. In a preferred embodiment, the man nαalian vector comprises the polymerase-III HI -RNA gene promoter. The polymerase-III HI -RNA promoter produces a RNA transcript lacking a polyadenosine tail and has a well-defined start of transcription and a termination signal consisting of five thymidines (T5) in a row. The cleavage of the transcript at the termination site occurs after the second uridine and yields a transcript resembling the ends of synthetic siRNAs containing two 3' overhanging T or U nucleotides. Other promoters useful in siRNA vectors will be known to one of ordinary skill in the art. Vector systems for siRNA expression in mammalian cells include pSUPER RNAi system described above. Other examples include but are not limited to pSUPER.neo, pSUPER.neo+gfp and pSUPERpuro (OligoEngine, Inc.); BLOCK-iT T7-TOPO linker, pcDNAl .2/V5-GW/lacZ, pENTR/U6, pLenti6-GW/U6-laminshnιa and pLenti6/BLOCK-iT- DEST (Invitrogen). These vectors and others are available from commercial suppliers. One set of embodiments of the invention includes the use of antisense molecules or nucleic acid molecules that reduce expression of genes via RNA interference (RNAi or siRNA). One example of the use of antisense, RNAi or siRNA in the methods of the invention is their use to decrease the level of GR expression. The antisense oligonucleotides, RNAi, or siRNA nucleic acid molecules used for this puφose may be composed of "natural" deoxyribonucleotides, ribonucleotides, or any combination thereof. That is, the 5' end of one native nucleotide and the 3' end of another native nucleotide may be covalently linked, as in natural systems, via a phosphodiester intemucleoside linkage. These oligonucleotides may be prepared by art-recognized methods, which may be carried out manually or by an automated synthesizer. They also may be produced recombinantly by vectors. In some embodiments of the invention, the antisense or siRNA oligonucleotides also may include "modified" oligonucleotides. That is, the oligonucleotides may be modified in a number of ways, which do not prevent them from hybridizing to their target but which enhance their stability or targeting or which otherwise enhance their therapeutic effectiveness. The term "modified oligonucleotide" as used herein describes an oligonucleotide in which (1) at least two of its nucleotides are covalently linked via a synthetic internucleoside linkage (i.e., a linkage other than a phosphodiester linkage between the 5' end of one nucleotide and the 3' end of another nucleotide) and/or (2) a chemical group not normally associated with nucleic acids has been covalently attached to the oligonucleotide. Preferred synthetic internucleoside linkages are phosphorothioates, alkylphosphonates, phosphorodithioates, phosphate esters, alkylphosphonothioates, phosphoramidates, carbamates, carbonates, phosphate triesters, acetamidates, carboxymethyl esters and peptides. The term "modified oligonucleotide" also encompasses oligonucleotides with a covalently modified base and/or sugar. For example, modified oligonucleotides include oligonucleotides having backbone sugars that are covalently attached to low molecular weight organic groups other than a hydroxyl group at the 3' position and other than a phosphate group at the 5' position. Thus, modified oligonucleotides may include a 2'-O- alkylated ribose group. In addition, modified oligonucleotides may include sugars such as arabinose instead of ribose. In some aspects of the invention, GR activity-reducing compounds include, but are not limited to polypeptides that reduce the activity of a GR. Such GR activity -reducing polypeptides include, but are not limited to antibodies or antigen-binding fragments thereof. GR activity-reducing compounds of the invention also include, but are not limited to, GR activity-reducing compounds that are variants of GR agonists that are not functional or are not fully functional. Such variants may compete with the functional endogenous versions of agonists in a cell, tissue, or subject, and thereby reduce the GR activity and opioid tolerance and/or pain in a subject The antibodies of the present invention may be prepared by any of a variety of methods, including administering protein, fragments of protein, cells expressing the protein or fragments thereof and the like to an animal to induce polyclonal antibodies. The production of monoclonal antibodies is according to techniques well known in the art. Antibodies also may be coupled to specific labeling agents, for example, for imaging of cells and tissues according to standard coupling procedures. Labeling agents include, but are not limited to, fluorophores, chromophores, enzymatic labels, radioactive labels, etc. Other labeling agents useful in the invention will be apparent to one of ordinary skill in the art. Significantly, as is well known in the art, only a small portion of an antibody molecule, the paratope, is involved in the bindiog of the antibody to its epitope (see, in general, Clark, W. R. (1986) The Experimental Foundations of Modern Immunology, Wiley & Sons, Inc., New York; Roitt, I. (1991) Essential Immunology, 7th Ed., Blackwell Scientific Publications, Oxford). The pFc' and Fc regions, for example, are effectors of the complement cascade but are not involved in antigen binding- An antibody from which the pFc' region lias been enzymatically cleaved, or which has been produced without the pFc' region, designated an F(ab')2 fragment, retains both of the antigen binding sites of an intact antibody. Similarly, an antibody from which the Fc region has been enzymatically cleaved, or which has been produced without the Fc region, designated an Fab fragment, retains one of the antigen binding sites of an intact antibody molecule. Proceeding further, Fab fragments consist of a covalently bound antibody light chain and a portion of the antibody heavy chain denoted Fd. The Fd fragments are the major determinant of antibody specificity (a single Fd Fragment may be associated with up to ten different light chains without altering antibody specificity) and Fd fragments retain epitope-binding ability in isolation. Within the antigen-binding portion of ao. antibody, as is well known in the art, there are complementarity determining regions (CDRLs) that directly interact with the epitope of the antigen, and framework regions (FRs), which maintain the tertiary structure of the paratope (see, in general, Clark, W. R. (1986) The Experimental Foundations of Modern Immunology, Wiley & Sons, Inc., New York; Roitt, I. (1991) Essential Immunology, 7th Ed., Blackwell Scientific Publications, Oxford), hi both the heavy chain Fd fragment and the light chain of IgG immunoglobulins, there are four framework regions (FR1 through FR4) separated respectively by three complementarity determiixing regions (CDR1 through CDR3). The CDRs, and in particular the CDR3 regions, and more particularly the heavy chain CDR3, are largely responsible for antibody specificity. It is now well established in the art that the non-CDR regions of a mammalian antibody may be replaced with similar regions of conspecific or heterospecific antibodies while retaining the epitopic specificity of the original antibody. This is most clearly manifested in the development and use of "hurα nized" antibodies in which non-human CDRs are covalently joined to human FR and/or Fc/pFc' regions to produce a functional antibody. See, e.g., U.S. patents 4,816,567, 5,225,539, 5,585,089, 5,693,762 and 5,859,205. Thus, for example, PCT International Publication Number WO 92/04381 teaches the production and use of murine RSN antibodies in which at least a portion of the murine FR regions have been replaced by FR regions of human origin. Such antibodies, including fragments of intact antibodies with antigen-binding ability, are often referred to as "chimeric" antibodies. Fully human monoclonal antibodies also can be prepared by immunizing mice transgenic for large portions of human immunoglobulin heavy and light chain loci. Following immunization of these mice (e.g., XenoMouse (Abgenix), HuMAb mice (Medarex/GenPharm)), monoclonal antibodies can be prepared according to standard hybridoma technology. These monoclonal antibodies will have human immunoglobulin amino acid sequences and therefore will not provoke human anti-mouse antibody (HAMA) responses^' when administered to humans. Thus, as will be apparent to one of ordinary skill in the art, the present invention also provides for F(ab')2, Fab, Fv and Fd fragments; chimeric antibodies in which the Fc and/or FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; chimeric F(ab')2 fragment antibodies in which the FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; chimeric Fab fragment antibodies in which the FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; and chimeric Fd fragment antibodies in which the FR and/or CDR1 and/or CDR2 regions have been replaced by homologous human or nonhuman sequences. The present invention also includes so-called single chain antibodies, or intrabodies. As used herein, antibodies of the invention include single chain antibodies (e.g., scFvs), and single domain antibodies (e.g. NLs). As described herein, the antibodies of the present invention may be prepared by starting with any of a variety of methods, including administering protein, fragments of protein, cells expressing the protein or fragments thereof and the like to an animal to induce polyclonal antibodies. The production of monoclonal antibodies is well known in the art. As detailed herein, such antibodies or antigen-binding fragments thereof may be used in the preparation of scFvs, and VLs and variants thereof. Additional steps in the production of antibodies of the invention may include directed antibody evolution and affinity engineering. Directed evolution includes the use of DΝA shuffling and error-prone PCR to generate mutations int. antibody sequences followed by the testing for affinity the antibody for the target protein. The invention involves polypeptides of numerons size and type that bind specifically to a GR, specific regions of a GR, and/or to agonists of^" GR. The polypeptides may be derived also from sources other than antibody technology. For example, such polypeptide- binding agents can be provided by degenerate peptide libraries, which can be readily prepared in solution, in immobilized form or as phage display libraries. Combinatorial libraries also can be synthesized of peptides containing one or more amino acids. Libraries further can be synthesized of peptoids and non-peptide synthetic moieties. The invention also involves methods for deterrrxining the functional activity of GR activity-reducing compounds described herein. The function or status of a compound as a GR activity-reducing compound can be determined according to assays known in the art or described herein. For example, cells can be contacted with a candidate GR activity-reducing compound under conditions that normally produce GR activity (e.g. a control), and standard procedures can be used to determine whether GR activity is reduced by the compound and/or whether the opioid tolerance and/or chronic pain level are reduced by the compound. Such methods may also be utilized to determine the status o analogs, variants, and derivatives as inhibitors of GR activity and as compounds that reduce opioid tolerance and/or chronic pain. Although not intended to be limiting, an example of a method with which the ability of a GR activity modulating compound to modulate GR activity can be tested in vivo or in vitro and the ability of GR activity-reducing compounds to reduce opioid tolerance and/or chronic pain levels can be tested, in an in vivo assay system provided herein in the Examples section. Using such assays the level of GR activity and/or opioid tolerance and/or chronic pain can be measured in the system both before and after contacting the system with a candidate GR activity modulating compound as an indication of the effect of the compound on the level of GR activity and/or opioid tolerance and/or pain. Secondary screens may further be used to verify the compounds identified as enhancers or inhibitors of GR activity and/or opioid tolerance and/or effective for treating chronic pain. In addition, analogs, derivative, and/or variants of GR activity-reducing compounds can be tested for their GR activity-reducing ability and/Or their efficacy in reducing opioid tolerance and/or pain levels by using an activity assay (see Examples). An example of an assay method to determine the efficacy of a compound "to modulate GR activity, although not intended to be limiting, is contacting a tissue or cell sample with a. GR activity-modulating compound and determining whether the compound increases, decreases or does not alter GR activity. Contacting a similar cell or tissue sample with an analog., derivative, and/or variant of the GR activity-modulating compound, determining its activity, and then comparing the two activity results can be used as a measure of the efficacy of the analog's GR activity modulating activity. In addition to the in vitro assays described above, an in viv& assay may be used to determine the functional activity of GR activity-reducing compounds described herein, hi such assays, animal models of opioid tolerance and/or pain conditions can be treated with a GR activity-reducing compound of the invention, and the efficacy of the compound in the reduction of opioid tolerance and/or pain in the subject can be determined. Opioid tolerance and/or pain levels may be assayed using methods described herein-, and standard methods known in the art, which may include behavioral testing. For exarr ple, animals with and without treatment with a GR activity-reducing compound can be examined for behavior and/or physiological effects as an indication of the effectiveness and/or efficacy of the compounds. Behavior and/or physiological effects may be assessed by examination of symptoms and manifestations of opioid tolerance and/or pain as known in the art and as described herein. These measurements can then be compared to corresponding measurements in control animals. For example, test and control animals may be examined following administration of a GR activity-modulating compound (enhancer or inhibitor) of the invention, hi some embodiments, test animals are administered a GR activity-reducing compound of the invention and control animals are not. Any resulting change in opioid tolerance and/or level of pain can then be determined for each type of animal using known methods in the art and as described herein. Such assays may be used to compare levels of opioid tolerance and/or pain in animals administered the candidate GR activity-reducing compounds to control levels of opioid tolerance and/or pain in animals not administered the GR activity-reducing compounds as an indication that the putative; GR activity-reducing compound is effective to prevent or reduce opioid tolerance and/or pain. A GR activity-reducing compound of the invention may be delivered to a cell, tissue, or subject using standard methods known to those of ordinary skill in the art. Various techniques maybe employed for introducing GR activity-reducing compounds of the invention to cells, depending on whether the compounds are introduced in vitro or in vivo in a subject. When administered, the GR activity-reducing compounds (also referred to herein as therapeutic compounds and/or pharmaceutical compounds) of the present invention are administered in pharmaceutically acceptable preparations. Such preparations may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, and optionally other therapeutic agents. The term "pharmaceutically acceptable" means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients. The characteristics of the carrier will depend on the route of administration. The therapeutics of the invention can be administered by any conventional route, including injection or by gradual infusion over time. The administration may, for example, be oral, intravenous, intrathecal, epidural, via implantation, intraperitoneal, intramuscular, intranasal, intracavity, subcutaneous, intradermal, or transdermal. The therapeutic compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing the compounds into association with a carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing the therapeutic agent into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product. Compositions suitable for parenteral administration conveniently comprise a sterile aqueous preparation of the therapeutic agent, which is preferably isotonic with the blood of the recipient. This aqueous preparation may be formulated according to known methods using those suitable dispersing or wetting agents and suspending agents. Preparations for parenteral admimstration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are 1, 3 -butane diol, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this pvu ose any bland fixed oil may be employed including synthetic mono or di-glycerides. h addition, fatty acids such as oleic acid find use in the preparation of injectables. Carrier formulations suitable for oral, subcutaneous, intravenous, intr-amuscular, etc. can be found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. Intravenous vehicles include fluid and nutrient replenishers, electrolyse replenishers (such as those based on Ringer's dextrose), and the like. Preservativ&s and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like. Compositions suitable for oral administration may be presented as discrete; units such as capsules, cachets, tablets, or lozenges, each containing a predetermined amount of the therapeutic agent. Other compositions include suspensions in aqueous liquors or non-aqueous liquids such as a syrup, an elixir, or an emulsion. The methods of the invention, in some aspects, include administration of GR activity- reducing compounds that preferentially target neuronal cells and/or tissues. The compounds of the invention can be specifically targeted to neuronal tissue (e.g. neuronal cells^) using various delivery methods, including, but not limited to: administration to neuronal tissue, the addition of targeting molecules to direct the compounds of the invention to neuronal tissues (e.g. neuronal cells), etc. Additional methods to specifically target molecules and compositions of the invention to spinal cord cells and tissue and/or other neuronal cells and tissues are known to those of ordinary skill in the art. hi some embodiments, the molecules and compositions of the invention are linked to a targeting molecule. As used herein, the term "linked to a targeting molecule" means attached or in association with a targeting molecule. For example, molecules and compositions of the invention can be linked to targeting molecule via covalent or non-covalent methods or can be prepared in coxyunction with a liposome or vesicle for targeted delivery using art-known methods. h some embodiments of the invention, a GR activity-reducing compound of the invention may be delivered in the form of a delivery complex. The delivery complex may deliver the GR activity-reducing compound into any cell type, or may be associated with a molecule for targeting a specific cell type. Examples of delivery complexes include a GR activity-reducing compound of the invention associated with: a sterol (e.g., cholesterol), a lipid (e.g., a cationic lipid, virosome or liposome), or a target cell specific binding; agent (e.g., an antibody, including but not limited to monoclonal antibodies, or a ligand recog nized by target cell specific receptor). Some complexes may be sufficiently stable in vivo to prevent significant uncoupling prior to internalization by the target cell. However, the complex can be cleavable under appropriate conditions within the cell so that the GR activity-reducing compound is released in a functional form. An example of a targeting method, although not intended to be limiting, is the use of liposomes to deliver a GR activity-reducing compound of the invention into a cell. Liposomes may be targeted to a particular tissue, such neuronal cells, (e.g. spinal cord neurons, etc) by coupling the liposome to a specific ligand such as a monoclonal antibody, sugar, glycolipid, or protein. Such proteins include proteins or fragments thereof specific for a particular cell type, antibodies for proteins that undergo internalization in cycling, proteins that target intracellular localization and enhance intracellular half life, and the like. Such a targeting molecule can be bound to or incoφorated within the GR activity modulating compound delivery vehicle. Where liposomes are employed to deliver the GR activity- reducing compounds of the invention, proteins that bind to a surface membrane protein associated with endocytosis may be incoφorated into the liposome formulation for targeting and/or to facilitate uptake. Liposomes are commercially available from Invitrogen, for example, as LIPOFECTiN™ and LIPOFECTACE™, which are formed of cationic lipids such as N-[l- (2,3 dioleyloxy)-propyl]-N, N, N-lrimethylammonium chloride (DOTMA) and dimethyl dioctadecylammonium bromide (DDAB). Methods for making liposomes are well known in the art and have been described in many publications. Methods of targeting cells to deliver nucleic acid constructs, for intracellular expression of the antibodies (i.e., as "intrabodies"), are known in the art. hi these applications, single chain antibodies are generally used, and the size of the antibody (or fragment) is kept to a minimum to facilitate translocation into the cell. The antibody polypeptide sequence can also be delivered into cells by providing a recombinant protein fused with peptide carrier molecules. These carrier molecules, which are also referred to as protein transduction domains (PTDs), and methods for their use, are known in the art. Examples of PTDs, though not intended to be limiting, are tat, antennapedia, and synthetic poly-arginine; nuclear localization domains also can be included in the antibody molecules. These delivery methods are known to those of skill in the art and are described in US patent 6,080,724, and US patent 5,783,662, the entire contents of which are hereby incoφorated by reference. The invention provides a composition of the above-described agents for use as a medicament, methods for preparing the medicament and methods for the sustained release of the medicament in vivo. Delivery systems can include time-release, delayed release or sustained release delivery systems. Such systems can avoid repeated administrations of the therapeutic agent of the invention, increasing convenience to the subject and the physician. Many types of release delivery systems are available and known to those of ordinary skill in the art. They include polymer-based systems such as polylactic and polyglycolic acid, poly(lactide-glycolide), copolyoxalates, polyanhydrides, polyesteramides, polyorthoesters, polyhydroxybutyric acid, and polycaprolactone. Microcapsules of the foregoing polymers containing drugs are described in, for example, U.S. Pat. No. 5,075,109. Nonpolymer systems that are lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono-, di- and tri-glycerides; phospholipids; hydrogel release systems; silastic systems; peptide based systems; wax coatings, compressed tablets using conventional binders and excipients, partially fused implants and the like. Specific examples include, but are not limited to: (a) erosional systems in which the polysacchari.de is contained in a form within a matrix, found in U.S. Patent Nos. 4,452,775, 4,675,189, and 5,736,152, and (b) diffusional systems in which an active component permeates at a controlled rate from a polymer such as described in U.S. Patent Nos. 3,854,480, 5,133,974 and 5,407,686. In addition, pump-based hardware delivery systems can be used, some of which are adapted for implantation. In one particular embodiment, the preferred vehicle is a biocompatible microparticle or implant that is suitable for implantation into the mammalian recipient. Exemplary bioerodible implants that are useful in accordance with this method are described in PCT International application no. WO 95/24929, entitled "Polymeric Gene Delivery System" describes a biocompatible, preferably biodegradable polymeric matrix for containing an exogenous gene under the control of an appropriate promoter. The polymeric matrix is used to achieve sustained release of the exogenous gene in the patient. In accordance with the instant invention, the compound(s) of the invention is encapsulated or dispersed within the biocompatible, preferably biodegradable polymeric matrix disclosed in WO 95/24929. The polymeric matrix preferably is in the form of a microparticle such as a microsphere (wherein the compound is dispersed throughout a solid polymeric matrix) or a microcapsule (wherein the compound is stored in the core of a polymeric shell). Other forms of the polymeric matrix for containing the compounds of the invention include films, coatings, gels, implants, and stents. The size and composition of the polymeric matrix device is selected to result in favorable release kinetics in the tissue into which the matrix device is implanted. The size of the polymeric matrix device further is selected according to the method of delivery which is to be used. The polymeric matrix composition can be selected to have both favorable degradation rates and also to be formed of a material which is bioadhesive, to further increase the effectiveness of transfer when the device is administered to a vascular surface. The matrix composition also can be selected not to degrade, but rather, to release by diffusion over an extended period of time. Both non-biodegradable and biodegradable polymeric matrices can be used to deliver agents and compounds of the invention of the invention to the subject. Biodegradable matrices are preferred. Such polymers may be natural or synthetic polymers. Synthetic polymers are preferred. The polymer is selected based on the period of time over which release is desired, generally in the order of a few hours to a year or longer. Typically, release over a period ranging from between a few hours and three to twelve months is most desirable. The polymer optionally is in the form of a hydro gel that can absorb up to about 90% of its weight in water and further, optionally is cross-linked with multi-valent ions or other polymers. In general, the agents and/or compounds of the invention are delivered using the bioerodible implant by way of diffusion, or more preferably, by degradation of the polymeric matrix. Exemplary synthetic polymers which can be used to form the biodegradable delivery system include: polyamides, polycarbonates, polyalkylenes, polyalkylene glycols, polyalkylene oxides, polyalkylene terepthalates, polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, polyvinyl halides, polyvinylpyrrolidone, polyglycolides, polysiloxanes, polyurethanes and co-polymers thereof, alkyl cellulose, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitro celluloses, polymers of acrylic and methacrylic esters, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxy-propyl methyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxylethyl cellulose, cellulose triacetate, cellulose sulphate sodium salt, poly(methyl methacrylate), poly(ethyl methacrylate), poly(butylmethacrylate), poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl acrylate), polyethylene, polypropylene, polyethylene glycol), poly(ethylene oxide), polyethylene terephthalate), poly(vinyl alcohols), polyvinyl acetate, poly vinyl chloride, polystyrene and polyvinylpyrrolidone. Examples of non-biodegradable polymers include ethylene vinyl acetate, poly(meth) acrylic acid, polyamides, copolymers and mixtures thereof. Examples of biodegradable polymers include synthetic polymers such as polymers of lactic acid and glycolic acid, polyanhydrides, poly(ortho)esters, polyurethanes, poly(butic acid), poly( valeric acid), and poly(lactide-cocaprolactone), and natural polymers such as alginate and other polysaccharides including dextran and cellulose, collagen, chemical derivatives thereof (substitutions, additions of chemical groups, for example, alkyl, alkylene, hydroxylations, oxidations, and other modifications routinely made by those skilled in the art), albumin and other hydrophilic proteins, zein and other prolamines and hydrophobic proteins, copolymers and mixtures thereof. In general, these materials degrade either by enzymatic hydrolysis or exposure to water in vivo, by surface or bulk erosion. Bioadhesive polymers of particular interest include bioerodible hydro gels described by H. S. Sawhney, C. P. Pathak and J. A. Hubell in Macromolecules, 1993, 26, 581-587, the teachings of which are incoφorated herein by reference, polyhyaluronic acids, casein, gelatin, glutin, polyanhydrides, polyacrylic acid, alginate, chitosan, poly(methyl methacrylates), poly(ethyl methacrylates), poly(butylmethacrylate), poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecyl acrylate). Use of a long-term sustained release implant may be particularly suitable for treatment of subjects with a need for long-term treatment with the methods of the invention, e.g, chronic pain, or a condition suitable for prolonged opioid treatment. "Long-term" release, as used herein, means that the implant is constructed and arranged to deliver therapeutic levels of the active ingredient for at least 7 days, and preferably 30-60 days, and in some embodiments for months or years. The implant may be positioned at or near the site or area of the brain or nervous system affected by or involved in the pain condition.

Long-term sustained release implants are well known to those of ordinary skill in the art and include some of the release systems described above. The preparations of the invention are administered in effective amounts. An effective amount is that amount of a pharmaceutical preparation that alone, or together with further doses, stimulates the desired response. In the case of preventing or treating opioid tolerance, the desired response is reducing the onset or level of the opioid tolerance in the subject. In the case of preventing or treating pain in a subject, the desired response is reducing the level of pain in the subject or preventing the onset of the pain in the subject. An effective amount for preventing and/or treating opioid tolerance and/or pain in a subject is that amount that reduces the amount or level of opioid tolerance and/or pain, when the subject is a subject with a pain undergoing opioid treatment or has a pain condition, with respect to that amount that would occur in the absence of the active compound. In other embodiments of the invention, (e.g. for making animal models), an effective amount of the pharmaceutical compound is that amount effective to enhance GR activity, and opioid tolerance and/or pain. Such enhancements can be determined using standard assays as described above herein. Measurements of GR activity, opioid tolerance and/or pain are provided herein and are known to those of ordinary skill in the art. The pharmaceutical compound dosage may be adjusted by the individual physician or veterinarian, particularly in the event of any complication. A therapeutically effective amount typically varies from 0.001 mg/kg to about 500 mg/kg, preferably from about 0.01 mg/kg to about 200 mg/kg, and most preferably from about 0.02 mg/kg to about 20 mg/kg, in one or more dose administrations daily, for one or more days. The absolute amount will depend upon a variety of factors, including the material selected for administration, whether the administration is in single or multiple doses, and individual subject parameters including age, physical condition, size, weight, and the stage of the disease or disorder. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. The pharmaceutical compounds of the invention maybe administered alone, in combination with each other, and/or in combination with other drug therapies that are administered to subjects with pain conditions or opioid tolerance. Additional drug therapies (for treatment and/or prophylaxis) that may be administered with pharmaceutical compounds of the invention include, but are not limited to: both opioid and non-opioid analgesics. Additional drug therapies may also include therapies to prevent or treat opioid tolerance, which include, but are not limited to: o2-adrenergic receptor agonists such as clonidine; mixed opioid receptor agonist/antagonist such as buphrenoφhine, butoφhanol and nalbuphine. An example of a therapy to reduce opioid tolerance is the use of opioid "vacations", which are periods of time when administration of opioid administration is stopped to reduce the development and presence of opioid tolerance. Other drug therapies (for treatment and/or prophylaxis) that may be administered with pharmaceutical compounds of the invention include agents that inhibit activation of glutamate receptors and/or intracellular activation of protein kinases, as described above herein. The opioid and non-opioid analgesic drug therapies and drug administration strategies to inhibit or reduce opioid tolerance are known to those of ordinary skill in the art. For example, opioid vacations are utilized by those of skill in the art to reduce opioid tolerance in patients. The therapeutic strategies (e.g. opioid vacations) are utilized in a manner that is effective to achieve the physiological goals (to reduce opioid tolerance in a subject), in combination with the pharmaceutical compounds of the invention. Thus, it is contemplated that the drug therapies may be administered, and strategies undertaken in amounts and manner that are not capable of preventing or reducing the physiological consequences of the opioid tolerance or pain when the drug therapies or therapeutic strategies are administered or utilized alone, but which are capable of preventing or reducing the physiological consequences of opioid tolerance or pain when administered or utilized in combination with the GR activity modulating compounds of the invention. Diagnostic tests known to those of ordinary skill in the art may be used to assess the level of opioid tolerance and/or pain in a subject and to evaluate a therapeutically effective amount of a pharmaceutical compound administered. Examples of diagnostic tests are set forth below. A first determination of opioid tolerance and/or pain patient may be obtained using one of the methods described herein (or other methods known in the art), and a second, subsequent determination of the level of opioid tolerance and/or pain may be done. A comparison of the level of opioid tolerance and/or pain may be used to assess the effectiveness of administration of a pharmaceutical compound of the invention as a prophylactic or a treatment of opioid tolerance and/or pain. Family history or prior occurrence of an opioid tolerance or pain condition, even if the opioid tolerance or pain condition is absent in a subject at present, may be an indication for prophylactic intervention by administering a pharmaceutical compound described herein to reduce or prevent the occurrence of opioid tolerance and/or pain. The invention also provides a pharmaceutical kit comprising one or more containers comprising one or more of the GR activity-reducing compounds of the invention and/or formulations of the invention. The kit may also include instructions for the use of the one or more GR activity-reducing compounds or formulations of the invention for the treatment of opioid tolerance and/or pain. The kits of the invention may also comprise additional drugs for preventing and/or treating opioid tolerance and/or pain, including one or more of the compounds and agents described herein. The invention also relates in some aspects to the identification and testing of candidate GR activity-modulating compounds. The GR activity-modulating compounds can be screened for modulating (enhancing or inhibiting) opioid tolerance and/or pain using the same type of assays as described herein (e.g., in the Example section). In vitro assays can be used to test GR activity modulating effects of candidate compounds and in vivo assays can also be used to test the GR activity modulating, opioid tolerance modulating, and or pain modulating effects of candidate agents. Using such assays, the GR activity modulating compounds that have the best inhibitory activity can be identified. It is understood that any mechanism of action described herein for the GR activity modulating compounds is not intended to be limiting, and the scope of the invention is not bound by any such mechanistic descriptions provided herein. The invention further provides efficient methods of identifying pharmacological agents or lead compounds for agents and compounds that modulate GR activity, and opioid tolerance and/or pain. Generally, the screening methods involve assaying for compounds which modulate (enhance or inhibit) the level of GR activity. As will be understood by one of ordinary skill in the art, the screening methods may measure the level of GR activity directly, e.g., screening methods described herein, addition, screening methods may be utilized that measure a secondary effect of GR activity, for example the level of opioid tolerance and/or pain in a subject. A wide variety of assays for pharmacological agents can be used in accordance with this aspect of the invention, including, GR translation assays, GR activity assays, pain assays, opioid tolerance assays, etc. As used herein, the term "pharmacological agent" means GR activity modulating compounds. An example of such an assay that is useful to test candidate GR activity modulating compounds is provided in the Examples section. In such assays, the assay mixture comprises a candidate pharmacological agent. Typically, a plurality of assay mixtures is run in parallel with different agent concentrations to obtain a different response to the various concentrations. Typically, one of these concentrations serves as a negative control, i.e., at zero concentration of agent or at a concentration of agent below the limits of assay detection. Candidate compounds useful in accordance with the invention encompass numerous chemical classes, although typically they are organic compounds. Preferably, the candidate pharmacological agents are small organic compounds, i.e., those having a molecular weight of more than 50 Da yet less than about 2500 Da, preferably less than about 1000 Da and, more preferably, less than about 500 Da. Candidate compounds comprise functional chemical groups necessary for structural interactions with proteins and/or nucleic acid molecules. The candidate compounds can comprise cyclic carbon or heterocychc structure and/or aromatic or polyaromatic structures substituted with one or more of the above- identified functional groups. Candidate compounds also can be biomolecules such as peptides, saccharides, fatty acids, sterols, isoprenoids, purines, pyrimidines, derivatives or structural analogs of the above, or combinations thereof and the like. Where the compound is a nucleic acid molecule, the agent typically is a DNA or RNA molecule, although modified nucleic acid molecules are also contemplated. It is contemplated that cell-based assays as described herein can be performed using cell samples and/or cultured cells. Biopsy cells and tissues as well as cell lines grown in culture are useful in the methods of the invention. Candidate compounds are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides, synthetic organic combinatorial libraries, phage display libraries of random peptides, and the like. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural and synthetically produced libraries and compounds can be readily be modified through conventional chemical, physical, and biochemical means. Further, known pharmacological compounds may be subjected to directed or random chemical modifications such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs of the compounds. Candidate compounds also include analogs, derivatives, and/or variants of the GR activity modulating compounds described herein. A variety of other reagents also can be included in the mixture. These include reagents such as salts, buffers, neutral proteins (e.g., albumin), detergents, etc. which may be used to facilitate optimal binding, or to reduce non-specific or background interactions of the reaction components. Other reagents that improve the efficiency of the assay such as protease inhibitors, nuclease inhibitors, antimicrobial agents, and the like may also be used. An exemplary GR activity modulating compound assay is described herein, which may be used to identify candidate compounds that modulate GR activity. The order of addition of components, incubation temperature, time of incubation, and other parameters of the assay may be readily determined. Such experimentation merely involves optimization of the assay parameters, not the fundamental composition of the assay. Incubation temperatures typically are between 4°C and 40°C. Incubation times preferably are minimized to facilitate rapid, high throughput screening, and typically are between 0.1 and 10 hours. After incubation, the level of GR activity may be detected by any convenient method available to the user. Detection may be effected in any convenient way for cell-based assays. For cell-based assays, one of the components usually comprises, or is coupled to, a detectable label. A wide variety of labels can be used, such as those that provide direct detection (e.g., radioactivity, luminescence, optical or electron density, etc.) or indirect detection (e.g., epitope tag such as the FLAG epitope, enzyme tag such as horse-radish peroxidase, etc.). A variety of methods may be used to detect the label, depending on the nature of the label and other assay components. Labels may be directly detected through optical or electron density, radioactive emissions, nonradiative energy transfers, etc. or indirectly detected with antibody conjugates, strepavidin-biotin conjugates, etc. Methods for detecting the labels are well known in the art.

The invention will be more fully understood by reference to the following examples. These examples, however, are merely intended to illustrate the embodiments of the invention and are not to be construed to limit the scope of the invention. Examples Example 1

Background Opioid analgesic tolerance is a pharmacological phenomenon involving mechanisms of cellular adaptation. However, it was not known whether reminiscences from a previous opioid exposure would be induced and retained at the cellular level and influence opioid analgesic effects upon a subsequent exposure. We have shown that the onset and degree of analgesic tolerance to a subsequent moφhine exposure were substantially exacerbated in those rats made tolerant to but later recovered behaviorally from a previous moφhine exposure. This process was mediated through upregulation and activation of spinal neuronal glucocorticoid receptors (GRs) that were preceded by activation of cyclic AMP and protein kinase A and followed by the GR-regulated downstream upregulation of N-methyl-D- aspartate receptors and protein kinase Cγ. These findings reveal a previously unknown cellular mechanism of moφhine tolerance that involves a neuronal GR-mediated memory process induced by cycles of moφhine exposure and provides insight into clinical opioid therapy and substance abuse. Glucocorticoid receptors (GRs) bind endogenous glucocorticoid hormones such as cortisol and serve as an active regulator in inflammatory responses through interactions with intracellular elements such as activating protein- 1 as well as transcriptional and translational regulation (Neeck, G. et al., Cytokines Cell Mol. Ther. 7: 61-69, 2002). However, GRs also are present in spinal cord dorsal horn neurons excitable by nociceptive stimulation (De Nicola, A., F. et al., Cell Mol. Neurobiol. 9: 179-192, 1989; Cintra, A. et al., Brain Res. 632: 334-338, 1993) and activation of neuronal GRs contributes to neural plasticity related to neuronal injury (Cameron, S. A. et al., J. Physiol. 518: 151-158, 1999) and the process of learning and memory (Quirarte, G. L. et al., Proc. Natl. Acad. Sci. USA 94: 14048-14053, 1997; Oitzl, M. S. et al, Eur. J. Neurosci. 10: 3759-3766, 1998; Roosendaal, B. et al., Eur. J. Neurosci. 11: 1317-1323, 1999). Importantly, activation of GRs has been shown to modulate moφhine-induced antinociception (Pieretti, S. et al., Gel. Pharmacol. 22: 929-933, 1991; Capasso, A. et al., LifeSci. 51: PL139-143, 1992), locomotor activity (Spanagel, B. et al., J. NeuroendocrinoL 8: 93-97, 1996), and dopamine-dependent responses (Schoffelmeer, A. N. et al., Neurochem. Res. 21: 1417-1423, 1996; Marinelli, M. et al, 1998 Proc. Natl. Acad. Sci. USA 95: 7742-7747, 1998). Utilizing a rat model of repeated cycles of moφhine exposure, we examined the hypotheses that neuronal GRs would be upregulated and activated following a previous exposure to moφhine mediated through the mtracellular cAMP and PKA pathway and that this cellular process would lead to the retained memory elements of moφhine exposure in part through regulation of NMDARs and PKCγ and contribute to the development of moφhine tolerance following a subsequent exposure.

Experimental Procedures Experimental Animals Adult male Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA) weighing 300-350 g were used. Animals were housed in cages with water and food pellets available ad libitum. The animal room was artificially illuminated from 7:00 to 19:00 hr. The experimental protocol was approved by our Institutional Animal Care and Use Committee.

Intrathecal (It.) catheter implantation and drug delivery An i.t. catheter (PE 10) was implanted in each rat according to our previously published method (Mao, J. et al., J. Neurosci. 22: 8312-8323, 2002). Those animals exhibited neurological deficits after i.t. catheter implantation were excluded from the experiments. Drugs were delivered via an i.t. catheter in a total volume of 10 μl followed by a saline flush. The following drugs were purchased from Sigma-Aldrich (St. Louis, MO): RU38486, moφhine, spironolactone, 2',5'-dideoxyadenosine (ddA), H89, and forskolin. Naloxone was purchased from Tocris (Ellisville, MO). Moφhine and naloxone were dissolved in normal saline, and other drugs were dissolved in 10% ethanol solution.

Induction of morphine tolerance and behavioral test Tolerance to the antinociceptive effect of moφhine was induced using an i.t. treatment regimen in that lOμg moφhine was given twice daily for various days. Differences in moφhine antinociception among treatment groups were assessed at various days using the tail-flick test by either testing at 30 min after a probe dose of lOμg moφhine (i.t.) or the generation of cumulative dose-response curves in that incremental log doses of moφhine were given to the same rats until no additional analgesia was demonstrated or the cut-off time was reached in response to a higher dose (Mao, J. et al., Pain 61: 353-364, 1995). The routine tail-flick test was used with baseline latencies of 4-5 sec and a cut-off time of 10 sec. At least two trials were made for each rat with an inter-trial interval of 1 min and with changes of the tail position receiving radiant heat stimulation at each trial. The percent of maximal possible antinociceptive effect (% MPAE) was determined by comparing the tail-flick latency before (baseline, BL) and after a drug injection (TL) using the equation: % MPAE = [(TL-BL)/(10-BL)] x 100% (the constant 10 refers to the cut-off time). The data was analyzed by using two-way ANONA to detect overall differences among treatment groups withpost-hoc Νewman-Keuls' tests. For the dose-response data analysis the analgesic dose that results in 50% pain reduction (AD_SQ values) and 95% confidence intervals were generated using a computerized regression model.

Immunocytochemical staining Routine immunocytochemical staining was used to detect GRs (1:1000, Santa Cruz

Biotechnology Inc., Santa Cruz, CA), μ-opioid receptor (1:1000, Santa Cruz Biotechnology Inc.), NMDARs (1:500, Novus Biological Inc, Littleton, CO), and PKCγ (1:500, Santa Cruz Biotechnology). Lumbar spinal cord sections were blocked with 1% goat serum in 0.3% triton for 1 hr at room temperature (RT) and incubated overnight at 4°C with a primary antibody. The sections were then incubated for 1 hr at RT with a corresponding FITC- or CY3-conjugated secondary antibody (1:300, Chemicon International, Temecula, CA). For double staining, a second primary antibody was added after the incubation with the first primary antibody for the same procedure as described above. Four to six nonadjacent spinal sections were randomly selected, analyzed using an Olympus fluorescence microscope, recorded using a digital camera, and processed using Adobe Photoshop.

Western blot For Western blotting, rats were rapidly (<1 min) killed in a CO₂ chamber and the dorsal horn of the lumbar spinal cord segments were removed and homogenized in SDS sample buffer containing a cocktail of proteinase inhibitors (Sigma-Aldrich). The lumbar segments were harvested because the i.t. drug delivery was aimed at this site. Protein samples were separated on SDS-PAGE gel (4-15% gradient gel, Bio-Rad Laboratories, Hercules, CA) and transferred to PNDF filters (Millipore Coφ., Billerica, MA). The filters were blocked with 3% milk and incubated overnight at 4°C with a primary antibody (GR: 1:1000; PKCy. 1:400, both from Santa Cruz Biotechnology; ΝMDAR: 1:2000; Νovus Biological Inc.) and 1 hr at RT with HRP-conjugated secondary antibody (Amersham Biosciences, Piscataway, ΝJ, 1 :700). The blots were then visualized in ECL solution (ΝEΝ, PerkinElmer, Boston, MA) for 1 min and exposed onto hyperfilms (Amersham Biosciences) for 1-10 min. The blots were then incubated in a stripping buffer (67.5 mM Tris, pH 6.8, 2% SDS, and 0.7%) β-mercaptoethanol) for 30 min at 50°C and reprobed with a polyclonal rabbit anti-β-actin antibody (1:20000; Alpha Diagnostic International, San Antonio, TX) as loading controls. The Western analysis was made in triplicates. The density of specific bands was measured with a computer-assisted imaging analysis system and normalized against a loading control. Differences were compared using AΝOVA followed by post-hoc Νewman-Keuls' tests.

Results

Evidence for the retained memory elements of morphine exposure To examine whether a previous moφhine exposure would influence the development of moφhine tolerance following a subsequent exposure, three repeated cycles of an intrathecal (i.t.) moφhine (10 μg) or saline treatment regimen were administered twice daily to two groups of rats (n=6-8, Fig. 1 A). No specific cues or test contexts were programmed and both groups were exposed to the same environment and handling throughout the experiment period to make valid comparisons (Grisel, J. E. et al., Psychopharmacol Berl 128: 248-255, 1996). Following the development of moφhine tolerance in each treatment cycle, rats were allowed to recover naturally from tolerance, as confirmed by the full antinociceptive effect in response to a probe moφhine dose (10 μg) before the beginning of the next cycle (Fig. 1 A). The recovery period lasted about seven days after each cycle of moφhine exposure and no withdrawal signs, such as diarrhea, weight loss, teeth chattering, and jumping, were observed during each recovery period. Under these circumstances, the onset of moφhine tolerance in each cycle of the same moφhine regimen was progressively shortened such that complete moφhine tolerance occurred on day 7, 5, and 3 of the first, second, and third moφhine treatment cycle, respectively (Fig. IB, D, F, P< 0.05). Moreover, there was an increased rightward shift of the moφhine antinociceptive dose-response curve at the end of the second cycle as compared to that of the first cycle (Fig 1C, E, Table 1), indicating an increased degree of tolerance as well in response to a subsequent cycle of moφhine treatment. Moreover, the degree of rightward shift of the dose-response curve similar to that seen in the second cycle occurred on day 3 of the third cycle as compared to day 5 during the second cycle (Fig. IE, G, Table 1). Thus, a shortened onset and enhanced degree of moφhine tolerance occurred in the same rats during repeated cycles of the same moφhine regimen, despite the apparent recovery from the behavioral manifestation of pharmacological tolerance before the beginning of each subsequent treatment cycle (Fig. IB, D, F). Since pharmacokinetic issues were unlikely to be involved due to the intrathecal regimen and there were no differences in the moφhine antinociceptive effects among three cycles in saline-treated rats with the same handling and testing as moφhine-treated rats (Fig. 1C, E, G), these results, for the first time, provide evidence indicating that memory elements of moφhine exposure (MEME) were induced and retained after a cycle of moφhine exposure, which exacerbated the development of moφhine tolerance following a subsequent exposure.

Table 1 Moφhine Tolerance in Each Cycle

Note that only the baseline AD₅₀ value on day one of the first treatment cycle is shown in the table, as there were no significant differences in the baseline moφhine dose-response on day one among three cycles. AD₅₀ moφhine: Dl, 3, 7: day 1, 3, 5 of the treatment; cycle 1, 2, 3: the first, second, and third moφhine treatment cycle. Expression of spinal neuronal GRs following morphine exposure To explore the cellular mechanisms underlying the induction of MEME, we first examined whether the spinal GR level was altered following moφhine exposure. The moφhine regimen that induced antinociceptive tolerance resulted in a significant increase in the spinal GR level using Western blot, when examined on day 5 and 7, but not day 3, of the first treatment cycle (Fig. 2 A, 1st cycle, P< 0.01, n=5). The saline treatment did not affect the spinal GR level over time (Fig. 2A, P>0.05, n=5). Topographically, GRs were located primarily within the superficial laminae of the spinal cord dorsal horn (Fig. 2B, C) as displayed by immunohistochemistry, where a high density of μ-opioid receptors exists. Consistently, there was considerable co-localization of GR-immunoreactivity (GR-ir) with the μ-opioid receptor in the spinal cord dorsal horn (Fig. 2D-F) and the vast majority of GR- positive cell profiles were identified as neuronal cell profiles because of their co-localization with NeuN-ir, a marker for a nuclear protein (Fig. 2G-I). Moreover, when the generic opioid receptor antagonist naloxone was co-administered with moφhine (10 μg each, i.t.) for six days, naloxone prevented the GR upregulation (Fig. 3 A, P>0.05, n=5), indicating that the GR upregulation was an opioid receptor-mediated cellular response.

Role of cAMP and PKA in the expression of neuronal GRs Previous studies have indicated an enhanced activity of the cAMP and cAMP dependent PKA pathway following chronic exposure to moφhine (Nestler and Aghajanian, Science 278(5335)58-63, 1997). To examine the possible intracellular mediators of GR upregulation following moφhine exposure, we asked whether inhibition of adenylyl cyclase (AC), the enzyme responsible for the cAMP production, or PKA would prevent the moφhine induced GR upregulation. Spinal cord dorsal horn samples were taken from five groups of rats (n=4-5) each treated with 10 μg moφhine plus vehicle, 10 μg moφhine plus 1 μg 2',5'- dideoxyadenosine (ddA, a broad AC inhibitor)(Aley, K.O. et al., J. Neurosci. 17: 8018-8023, 1997), 10 μg moφhine plus 10 μg H89 (a selective PKA inhibitor) (Jolas, T. et al., Neuroscience 95: 433-443, 2000), or vehicle alone twice daily for six days. The upregulation of GRs in the moφhine plus vehicle group was effectively blocked by the co-administration of moφhine with ddA or H89 (Fig. 3B, P< 0.05). ddA or H89 alone did not affect the expression of GRs (Fig. 3B, P> 0.05), indicating specific effects of diminishing cAMP production and inhibiting PKA activity on neuronal GR expression induced by a clironic moφhine exposure. The effect of cAMP on moφhine-induced GR upregulation was further examined in naϊve rats after i.t. administration of 10 μg forskolin (a broad AC activator) (Jolas et al., Neuroscience 95:433-443, 2000) or vehicle twice daily for six days. Forskolin, but not vehicle, induced the upregulation of spinal GRs in naϊve rats that mimicked that following moφhine exposure (Fig. 3B, P< 0.05, n=4-5). Of significance is that forskolin (10 μg) also enhanced the upregulation of GRs induced by moφhine (10 μg) when forskolin was coadministered with moφhine twice daily for six days (Fig. 3B, P< 0.05, n=4-5). Moreover, the moφhine antinociceptive dose-response curve was shifted to the right in those naϊve rats treated with forskolin as compared to the vehicle control when examined on day 7 (Fig. 3C, n=4-5). Taken together with the data from the AC inhibitor ddA and PKA inhibitor H89, these results indicate that cAMP and cAMP-dependent PKA are likely to he upstream mediators leading to the upregulation of GRs following chronic exposure to moφhine.

Role of GR activation in the expression of NMDARs and PKCγ To explore intracellular downstream events following the GR upregulation, we examined whether activation of GRs would regulate the spinal expression of NMDARs and PKCγ, two known contributors to the mechanisms of opioid tolerance (Trujillo, K. A. et al., Science 251: 85-87, 1991.; Zeitz, K. P. et al., Pain 94: 245-253, 2002.; Mao, J. et al., J. Neurosci. 22: 8312-8323, 2002). Spinal cord dorsal horn samples were taken from five groups of rats (n=4-5) each treated with 10 μg moφhine plus vehicle, 10 μg moφhine plus the GR antagonist RU38486 (1 μg), 10 μg moφhine plus the mineralocorticoid receptor antagonist spironolactone (3 μg) (Marinelli, M. et al., Proc. Natl. Acad. Sci. US A 95: 7742- 7747, 1998), 1 μg RU38486 alone, or a vehicle twice daily for six days. Both NMDARs and PKCγ were upregulated in the moφhine plus vehicle group as compared to the vehicle control (Fig.4A, B, P <0.05), which was blocked by the co-administration of moφhine with RU38486 but not spironolactone (Fig. 4A, B, P< 0.05). RU38486 alone did not affect the expression of NMDARs or PKCγ (Fig. 4A, B, P>0.05). Moreover, irrimuriohistochemistry revealed substantial co-localization of GR-ir and NMDAR-ir (Fig. 4C-E) as well as of GR-ir and PKCγ-ir (Fig. 4F-H) within the spinal cord dorsal horn, illustrating a cellular basis for the functional interaction between GRs, NMDARs, and PKCγ. Of significance to note is that the AC inhibitor ddA or the PKA inhibitor H89 also blocked the upregulation of NMDARs and PKCγ in those rats receiving the combined treatment with ddA (1 μg) or H89 (10 μg) and moφhine (10 μg) twice daily for six days(Fig. 41, J, P< 0.05, n=5). Consistently, forskolin (10 μg) but not vehicle, given alone to naϊve rats twice daily for six days, induced the upregulation of NMDARs and PKCγthat was similar to that induced by the moφhine treatment (Fig. 41, J, P< 0.05, n=4-5). Furthermore, the combined treatment with moφhine and forskolin resulted in an increased NMDAR expression as compared to the moφhine alone group (Fig. 41, P<0.05). These results further support a link between the cAMP and PKA pathway, GR upregulation and activation, and the expression of NMDARs and PKCγ following chronic exposure to moφhine.

Role of GR activation in morphine tolerance The role of GR activation in the development of moφhine tolerance following a single treatment cycle was investigated following i.t. co-administration of the GR antagonist RU38486 (0.5 or 1 μg) or vehicle with moφhine (10 μg) for six days (n=5-6). The rightward shift of the dose-response curve seen on day 7 in the moφhine plus vehicle group was effectively prevented in the moφhine plus RU38486 groups (Fig. 5A, B, table 2). RU38486 alone (1 μg, n=5) did not change the baseline tail-flick latency indicating a specific effect of RU38486 on the development of moφhine tolerance. In contrast, intrathecal co- administration of 10 μg moφhine with 3 μg spironolactone for six days did not prevent the development of moφhine tolerance (Fig. 5A, B, n=5, table 2), indicating a selective role of GRs in this process.

Table 2: Effect of RU38486 on Moφhine Tolerance

MS-3 μg SPL (D7) 6.7 5.6-7.1

NEH: vehicle; RU: RU38486; SPL: spironolactone; see Table 1 for additional legend.

The effect of RU38486 on moφhine tolerance was not due to masking the behavioral expression of moφhine tolerance, because a single dose of RU38486 (1 μg) failed to reverse moφhine tolerance on day 7 in those rats made tolerant to 10 μg moφhine given twice daily for six days (Fig. 5C, P> 0.05, n=5). Thus, RU38486 blocked a cellular process leading to the development of moφhine tolerance through inhibition of GR activity, suggesting a critical role of spinal neuronal GRs in the cellular mechanisms of moφhine tolerance.

Role of GR upregulation and activation in the induction of MEME In order to examine whether moφhine-induced GR upregulation and activation would contribute to the induction of MEME, we first examined the level of GR upregulation from the rats made tolerant to moφhine after the first treatment cycle but recovered pharmacologically from tolerance after a seven-day recovery period. In these rats (n=5), the spinal GR level remained elevated when examined before the beginning of the second treatment cycle and this GR level was moderately higher than the GR level at the end of the first treatment cycle (Fig. 2A, 2nd cycle, P< 0.05). Upon exposure to the second cycle of moφhine treatment, however, the spinal GR level was further elevated when examined on day 3 and 5 such that the GR level was significantly higher in response to the second cycle of moφhine treatment than to the first treatment cycle at the same time point (Fig. 2A, 2nd cycle, P< 0.05). Thus, a previous moφhine exposure induced a long-lasting upregulation of spinal GRs despite the recovery from the behavioral manifestation of pharmacological tolerance, which was followed by an expedited and further enhanced upregulation of spinal GRs upon a subsequent exposure to moφhine. Since these rats showed a full recovery from pharmacological tolerance while spinal GRs remained upregulated, the data suggest that the GR upregulation maybe an underlying cellular mechanism of MEME. If the upregulation of spinal GRs were contributory to the induction of MEME, preventing GR upregulation and activation would be expected to prevent the induction of MEME. To examine this possibility, RU38486 or saline was co-administered with moφhine during the first treatment cycle, but only moφhine was administered during the second cycle (n=5). Both groups underwent the same recovery period after the first treatment cycle. The onset of complete moφhine tolerance would be the same between the first and second cycle of moφhine exposure, should RU38486 block the induction of MEME during the first treatment cycle. Indeed, the onset of complete moφhine tolerance in response to the second cycle of moφhine treatment was no longer shortened in those rats receiving moφhine (10 μg) plus RU38486 (0.5 or 1 μg) during the first cycle (Fig. 6 A, B). hi contrast, the onset of complete tolerance was expectedly shortened (day 5) during the second cycle in those rats receiving moφhine plus vehicle during the first cycle (Fig. 6 A, B). Moreover, the combined treatment with moφhine and RU38486 during the first cycle prevented the upregulation of spinal GRs in a separate group of rats when examined at the beginning of the second treatment cycle (Fig. 6C, P< 0.05, n=4). Thus, both behavioral and Western blot data strongly indicate that the GR upregulation and activation mediated the induction of MEME.

Role of GR activation in the maintenance of MEME To examine the possibility that activation of GRs would be required for the maintenance of MEME, we asked whether RIT38486 would reverse MEME after its establishment using the following experimental paradigm. Following the first treatment cycle with moφhine alone that produced moφhine tolerance and MEME, RU38486 (lμg) or saline alone (n=5) was given during the recovery period. Both groups then received the second cycle of moφhine alone to assess the development of moφhine tolerance. The results demonstrated that 1) RU38486 significantly shortened the recovery period to five days as compared to seven days in the saline group and 2) during the second treatment cycle the onset of complete moφhine tolerance was no longer shortened in these rats treated with RU38486 during the recovery period in that complete tolerance developed on day 7 in the RU38486 group as compared to day 5 in the vehicle group (Fig. 6D). These results indicate that

RU38486 disrupted the established MEME and activation of GRs also was contributory to the maintenance of MEME.

Discussion Our results indicate a link between a previous moφhine exposure and the exacerbated development of moφhine tolerance upon a subsequent exposure, indicating that MEME can be induced and maintained through a cellular mechanism mediated through neuronal GRs despite the recovery from the behavioral manifestation of pharmacological tolerance. A possible mechanism underlying this process is that clironic moφhine treatment leads to upregulation and activation of neuronal GRs mediated through the cAMP and PKA pathway that is known to be upregulated following chronic moφhine exposure (Nestler and Aghajanian, Science 278(5335)58-63, 1997). GRs may be activated by circulatory corticosteroids and/or locally produced neurosteroids (Compagnone, N.A. et al, Front NeuroendocrinoL 21: 1-56, 2000; Plassart-Schiess, E. et al., Brain Res. Rev. 37: 133-140, 2001; Vallee, M. et al., Int. Rev. Neurobiol. 46: 273-320, 2001). Consequently, lasting upregulation and activation of neuronal GRs contribute to a cellular process that mediates the induction and maintenance of MEME and exacerbates the development of moφhine tolerance upon its subsequent exposure. At least one of the downstream responses to GR upregulation and activation is the regulation of NMDAR and PKCγ expression, two known contributors to the cellular mechanisms of neural plasticity related to learning and memory as well as opioid tolerance (Olds, J. L et al., Science 245: 866-869, 1989; CoUingridge, G. L. et al., Trends. Pharmacol. Sci. 11: 290-296, 1990; Madison, D. V. et al., Annu. Rev. Neurosci. 14: 379-397, 1991; Trujillo, K. A. et al., Science 251: 85-87, 1991; Zeitz, K. P. et al., Pain 94: 245-253, 2002; Mao, J. et al., J. Neurosci. 22: 8312-8323, 2002). Activation of GRs requires the formation of a GR homodimer after the dissociation from its cytosolic complex consisting of such elements as heat shock proteins (Drouin, J. et al., Mol. Endocrinol. 6: 1299-1309, 1992). A GR homodimer binds to specific nuclear DNA responsive elements to activate gene transcription and translation for a variety of cellular elements (Drouin, J. et al, Mol. Endocrinol. 6: 1299-1309, 1992). Indeed, activation GRs has been linked to 1) NMDAR- dependent long-term depression (Coussens, C. M. et al, J. Neurophysiol. 78: 1-9, 1997) and elevated intracellular Ca++ (Takahashi, T. et al., J. Neurochem. 83: 1441-1451, 2002) in the hippocampal neurons, 2) modulation of NMDAR functions (Nair, S. M. et al., J. Neurosci. 18: 2685-2696, 1998), 3) potentiated responses to NMDA in dopamine-sensitive neurons in the ventral tegmental area (Cho, K. et al., Neuroscience 88: 837-845, 1999), and 4) neuronal apoptosis mediated through the intracellular mitogen-activated protein kinase (Diem, R. et al, J. Neurosci. 23 : 6993-7000, 2003). The GR-mediated regulation of NMDAR and PKCγ expression demonstrated in the present study is consistent with the GR-mediated transcriptional and translational regulation and is critical to the mechanism of moφhine tolerance, because inhibition of GRs with RU38486 blocked both downstream responses to GR activation, i.e., upregulation of NMDARs and PKCγ, as well as the development of moφhine tolerance. Another consequence of GR upregulation and activation is the induction and maintenance of MEME. This role of GRs is indicated by 1) the lasting GR upregulation at the end of a recovery period from tolerance and 2) the prevention o_f exacerbated moφhine tolerance to a second cycle of moφhine exposure when RU38486 was given repeatedly during the recovery period following the first cycle of moφhine exposure. Although a known mechanism of GR actions is through interactions between GRs and other cellular protein elements (Refojo, D. et al., Immunol. Cell Biol. 79: 385-94, 2001; eeck, G. et al., Cytokines Cell Mol. Ther. 7: 61-69, 2002), the effect of GR activation on ME1ME is unlikely to be due to direct interactions between GRs and μ-opioid receptors for the following reasons. First, a single injection with the GR antagonist RU38486 failed to reverse the established tolerance to moφhine analgesia, a finding that is in agreement with the lack of effect of the NMDAR antagonist MK-801 on the reversal of an established moφhine tolerrance (Trujillo, K. A. et al., Science 251: 85-87, 1991). Second, the established MEME was reversed by RU38486, but only when RU48386 was given repeatedly during the recovery period between the first and second treatment cycle. Collectively, these data demonstrate a previously unknown cellular mechanism that links the role of cAMP/PKA and NMDARs/ PKCγ in the mechanisms of moφhine tolerance through upregulation and activation of neuronal GRs. This study may have considerable clinical implications. First, previous exposure to opioid analgesics or other substances of abuse such as heroin may >e a significant and yet poorly recognized factor of exacerbated opioid tolerance necessitating earlier and possibly higher dose escalation in clinical opioid therapy (Mao, J. et al., J. Weurosci. 22: 8312-8323, 2002). Second, the GR mediated induction of memory elements in response to a substance of abuse may predispose a subject to drug relapse and confound the rehabilitation effort for drug addicts. This possibility is particularly relevant to heroin abuse, sin ce heroin through its metabolites (6-monoacytalmoφhine or moφhine) does indeed inteiract with opioid receptors (Sim-Selley, L. J. et al., J. Neurosci. 20: 4555-4562, 2000). A recent report on the role of GRs in cocaine abuse is in agreement with this concept (Deroche-Gramonet,N. et al., J Neurosci 23: 4785-4790, 2003). Third, the present results suggest a potential role of the hypothalamic-pituitary-adrenal (HP A) axis in the mechanisms of opioid tolerance and substance abuse via activation of neuronal GRs. Conceivably, those factors that would activate the HPA axis (e.g., stress or emotional disturbance) could lead to the upregulation and activation of neuronal GRs feeding into the cellular mechanisms of opioid tolerance. Under each of these circumstances, however, the use of GR inhibitors such as RU38486 or agents blocking the upregulation of GRs, NMDARs, or PKC may prevent the development of opioid tolerance and restore the opioid analgesic efficacy by disrupting the induction and maintenance of MEME.

Example 2 Introduction We investigated a novel method of treating chronic pain, particularly pain resulting from injury to the nervous system, through the use of a glucocorticoid receptor (GR) antagonist such as RU38486, an agent that blocks the upregulation of central GRs in response to nerve injury and inflammation, and/or a combination of such agents and a GR antagonist. Despite several decades of extensive effort in search of new effective pain treatment, chronic pain remains difficult to treat. This method provides a novel alternative to improve chronic pain management for a variety of clinical pain conditions. The invention could be applied to millions of patients suffering from a variety of pain conditions both inside and outside of the United States. Pain is a perceived experience of real or potential tissue damage, which is a major clinical symptom signifying many disease entities. Millions of people suffer from a variety of pain conditions and unalleviated pain is a major clinical challenge. Chronic pain is one of the most disabling causes leading to loss of work force and increasing healthcare costs, hi spite of several decades of intense research efforts, clinical pain management remains largely unsatisfactory particularly for those suffering from chronic pain conditions. Injury to the nervous system may result in the development of pain hypersensitivity (neuropathic pain), a major clinical pain syndrome that remains difficult to treat. The central glutamatergic system including activation of the N-methyl-D-aspartate receptor has been a main focus of research interest over years in understanding the mechanisms of neuropathic pain. However, inflammatory cytokines including interleukin (IL)-lβ, IL-6, EL- 10, and tumor necrosis factor-α may be produced at peripheral and/or central loci in response to nerve injury and play a pivotal role in regional inflammatory responses through intracellular mediators. Inflammatory cytokines have been suggested to play a role in the development of neuropathic pain, but the central mechanisms by which inflammatory cytokines contribute to neuropathic pain remain unknown. Glucocorticoid receptors (GRs) play an active role in inflammatory responses and exist in spinal neurons excitable by nociceptive stimulation. To date, there is a lack of effective treatment for chronic pain, particularly pain resulting from injury to the nervous system and inflammation. We now describe a novel method we have identified that involves the use of a clinically available GR antagonist, an agent that blocks the upregulation of central GRs in response to nerve injury and inflammation, and/or a combination of such agents and a GR antagonist for the treatment of chronic pain including pain resulting from nerve injury and inflammation.

Methods

Animal model of peripheral nerve injury Adult male Sprague-Dawley rats weighing 275-325 g (Charles River Lab, Wilmington, MA) were used. The animal room was artificially lighted from 0700 to 1900 h. The experimental protocol was approved by our Institutional Animal Care and Use Committee. CCI rats were produced by loosely ligating the common sciatic nerve according to the method of Bennett, G. J. et al., Pain 33: 87-107, 1988. Sham rats were made following the same surgical procedure except for nerve ligation. An intrathecal (i.t.) catheter was implanted in each rat under the same surgical condition and a PE10 catheter was inserted onto the level of the lumbar enlargement (about 8.5 cm from the incision site for this rat age group) according to the method described previously (Mao, J. et al., J. Neurosci. 22: 8312- 8323, 2002). Those rats exhibiting postoperative neurological deficits (e.g., paralysis) or poor grooming were excluded from the experiments as described previously (Mao, J. et al., J. Neurosci. 22: 8312-8323, 2002). RU38486 was purchased from Sigma (St. Louis, MO) and dissolved in 10% ethanol solution with normal saline. Behavioral tests and statistical analysis Animals were habituated to the test environment daily (a 60 min session) for two da ^s before baseline testing. The testing procedure for thermal hyperalgesia was carried out. The temperature was set to have the baseline latency at about 10-12 sec and a cut-off of 20 sec. Mechanical allodynia was examined by applying a von Frey filament to the plantar surface of each hind paw. The cut-off force was 20 gm. Data from both thermal hyperalgesia (withdrawal latency in seconds) and mechanical allodynia (threshold bending force in grams.) tests were analyzed by using two-way analysis of variance (ANON A) repeated across testing time points to detect overall differences among treatment groups followed by the Waller- Duncan K-ratio t test. The statistical significant level was set at P< 0.05.

Time course of the effect of RU38486 on hyperalgesia The paw- withdrawal latency (PWL) of the mice was tested. The development of thermal hyperalgesia in CCI (chronic constriction nerve injury) rats was prevented by the GR antagonist RU38486 (2 μg, CCI+RU), but not vehicle (CCI+NEH), given intrathecally twice daily for the first six postoperative days. RU38486 alone did not affect the behavioral responses in sham rats. *P< 0.05, as compared to sham rats. See Fig. 7.

Time course of the effect of RU38486 on allodynia The development of mechamcal allodynia in CCI rats was prevented by the GR antagonist RU38486 (2 μg, CCI+RU), but not vehicle (CCI+NEH), given twice daily for the; first six postoperative days. RU38486 alone did not affect the behavioral responses in sham rats. *P< 0.05, as compared to sham rats. See Fig. 8.

The dose-response effect of RU38486 on hyperalgesia The dose-response effects of RU38486 on thermal hyperalgesia were examined on postoperative day 7 in CCI rats. The mineralocorticoid receptor antagonist spironolactone (3 μg, C+S) did not affect hyperalgesia in CCI rats. C+N: CCI+vehicle; C+R-0.5 to C+R-4: CCI rats treated with 0.5-4 μg RU38486. *P< 0.05, as compared to sham rats. See Fig. 9.

The dose-response effect of RU38486 on allodynia The dose-response effects of RU38486 on mechanical allodynia were examined on postoperative day 7 in CCI rats. The mineralocorticoid receptor antagonist spironolactone (3 μg, C+S) did not affect allodynia in CCI rats. C+N: CCI+vehicle; C+R-0.5 to C+R-4: CCI rats treated with 0.5-4 μg RU38486. *P< 0.05, as compared to sham rats. See Fig. 10.

RU38486 reversed hyperalgesia A single intrathecal injection of RU38486 (C/RU, 2 μg), but not vehicle (C/N), on postoperative day 7 attenuated thermal hyperalgesia in CCI rats when examined at 30 min after the injection. *P< 0.05, as compared to sham rats. See Fig. 11.

RU38486 reverses allodynia

A single intrathecal injection of RU38486 (C/RU, 2 μg), but not vehicle (C/N), on postoperative day 7 attenuated mechanical allodynia in CCI rats when examined at 30 min after the injection. *P< 0.05, as compared to sham rats. See Fig. 12.

Results and Discussion We have identified methods of treating chronic pain through the use of a clinically available GR antagonist as well as the regulation of a cellular mechanism responsible for the development and maintenance of chronic pain, particularly pain from injury to the nervous system and inflammation. We have utilized clinically available GR antagonists, which are agents that possess the property of inhibiting GR activation and are suitable for clinical use.

RU486 is a known example of such agents. We have found that a GR antagonist may be used alone or in combination with other analgesics including opioids. We have also found that a GR antagonist may be used with an agent that modulates intracellular pathways leading to GR upregulation and activation under a chronic pain condition. Our results have demonstrated that a GR antagonist may be used with an agent that inhibits activation of glutamate receptors, such as Ν-methyl-D-aspartate receptors, and/or intracellular activation of protein kinases including protein kinase C. Examples of such agents are dextromethoφhan, ketamin, amentadine, mementin, methadone, certain tricyclic antidepressants such as nortriptyline and amitriptyline, etc. We have also discovered that a GR antagonist or its combination may be given through different methods including oral, intravenous, epidural, intrathecal, and other systemic delivery methods, and that a GR antagonist or its combinations may be given for a sustained period depending on the treatment requirement. Our studies have also shown that a GR antagonist or its combination may be given to patients with different pain conditions requiring pain control. The methods we have discovered are particularly effective for, but not limited to, the treatment of pain from injury to the nervous system and inflammation. The methods we have identified may be used in in-patient and out-patient settings in compliance with regulatory requirements.

EQUIVALENTS Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claim_s. All references, including patent documents, disclosed herein are incoφorated by reference in their entirety.

We claim:

Claims

Claims

1. A method for reducing or preventing opioid tolerance in a subject comprising administering to the subject in need of such treatment an amount of a compound that reduces activity of a glucocorticoid receptor effective to reduce or prevent opioid tolerance in the subject.

2. The method of claim 1, further comprising one or more additional administrations of the compound that reduces activity of a glucocorticoid receptor.

3. A method of claim 1, further comprising administering an opioid to the subject.

4. The method of claim 3, wherein the compound that reduces activity of a glucocorticoid receptor is administered prior to, substantially in conjunction with, or after administration of the opioid.

5. The method of claim 3, wherein the opioid is moφhine, meperidine, butoφhanol, oxymoφhone, methadone, propoxyphene, codeine, heroin, hydromoφhone, oxycodone, and hydrocodone, fentanyl, sufentanil, nalbuphrine, buprenoφhine, or tramadol.

6. The method of claim 1, wherein the subject is human.

7. The method of claim 1 , wherein the subject has a pain condition.

8. The method of claim 7, wherein the pain condition is injury pain, neuropathic pain, surgical pain, disease-associated pain, phantom pain, or inflammation-associated pain.

9. The method of claim 8, wherein the injury pain is nervous system injury pain.

10. The method of claim 1 , wherein the subj ect is undergoing pain treatment.

11. The method of claim 1 , wherein the compound that reduces activity of a glucocorticoid receptor is linked to a targeting molecule.

12. The method of claim 11 , wherein the targeting molecule's target is a neuronal cell.

13. The method of claim 1 , wherein the compound that reduces activity of a glucocorticoid receptor is a glucocorticoid receptor antagonist or an antibody or antigen- binding fragment thereof.

14. The method of claim 13, wherein the glucocorticoid receptor antagonist is RU38486, RU486, or RU39305.

15. The method of claim 1 , wherein the compound that reduces activity of a glucocorticoid receptor is an antisense molecule, an siRNA molecule, or an RNAi molecule.

16. The method of claim 1 , wherein the mode of administration of the compound that reduces activity of a glucocorticoid receptor is implantation, mucosal administration, intrathecal administration, epidural administration, intravenous administration, inhalation, or oral administration.

17. The method of claim 1 , wherein the compound that reduces activity of a glucocorticoid receptor is administered in combination with an opioid or non-opioid analgesic.

18. The method of claim 17, wherein the non-opioid analgesic is acetaminophen, ibuprophen, gabapentin, tramadol, a tricyclic antidepressant, a sodium channel blocker, or a muscle relaxant.

19. The method of claim 18, wherein the antidepressant is nortriptyline.

20. The method of claim 18, wherein the sodium channel blocker is lamotrigene.

21. The method of claim 18, wherein the muscle relaxant is cyclobenzaprine or tizanidine.

22. The method of claim 1, further comprising administering an agent that inhibits adenylyl cyclase (AC) activity or protein kinase A (PKA) activity.

23. The method of claim 22, wherein the agent that inhibits adenylate cyclase (AC) activity is 2 ' , 5 ' -dideoxyadenosine (ddA) .

24. The method of claim 22, wherein the agent that inhibits PKA activity is N-[2-(p- bromocinnamylamino) ethyl]-5-isoquinoline sulfonamide (H89).

25. The method of claim 1, further comprising administering an agent that inhibits activation of glutamate receptors, and/or intracellular activation of protein kinases.

26. The method of claim 25, wherein the agent that inhibits activation of glutamate receptors and/or intracellular activation of protein kinases is dextromethoφhan, ketamine, amentadine, mementine, methadone, or a tricyclic antidepressant.

27. The method of claim 26, wherein the tricyclic antidepressant is nortriptyline or amitriptyline.

28. The method of claim 1 , wherein the compound that reduces activity of a glucocorticoid receptor is formulated for sustained release.

29. An opioid composition comprising an opioid, and a compound that reduces activity of a glucocorticoid receptor, wherein administration of the opioid composition to a subject induces less opioid tolerance than the administration of the opioid to the subject in the absence of the compound that reduces activity of a glucocorticoid receptor.

30. The opioid composition of claim 29, wherein the opioid is moφhine, meperidine, butoφhanol, oxymoφhone, methadone, propoxyphene, codeine, heroin, hydromoφhone, oxycodone, hydrocodone, fentanyl, sufentanil, nalbuphrine, buprenoφhine, or tramadol.

31. The opioid composition of claim 29, wherein the compound that reduces activity of a glucocorticoid receptor is a glucocorticoid receptor antagonist or an antibody or antigen- binding fragment thereof.

32. The opoiod composition of claim 31 , wherein the glucocorticoid receptor antagonist is RU38486, RU486, or RU39305.

33. The opioid composition of claim 29, wherein the compound that reduces activity of a glucocorticoid receptor is an antisense molecule, an siRNA molecule, or an RNAi molecule.

34. The opioid composition of claim 29, wherein the compound that reduces activity of a glucocorticoid receptor is linked to a targeting molecule.

35. The opioid composition of claim 34, wherein the targeting molecule's target is a neuronal cell.

36. The opioid composition of claim 29, further comprising a non-opioid drug for treating pain.

37. The opioid composition of claim 36, wherein the non-opioid analgesic is acetaminophen, ibuprophen, gabapentin, tramadol, a tricyclic antidepressant, a sodium channel blocker, or a muscle relaxant.

38. The opioid composition of claim 37, wherein the antidepressant is nortriptyline.

39. The opioid composition of claim 37, wherein the sodium channel blocker is lamotrigene.

40. The opioid composition of claim 37, wherein the muscle relaxant is cyclobenzaprine or tizanidine.

41. The opioid composition of claim 29, further comprising an agent that inhibits adenylyl cyclase (AC) activity or protein kinase A (PKA) activity.

42. The opioid composition of claim 41, wherein the agent that inhibits adenylate cyclase (AC) activity is 2 ' , 5 ' -dideoxyadenosine (ddA) .

43. The opioid composition of claim 41, wherein the agent that inhibits PKA activity is N-[2-(p-bromocinnamylamino) ethyl]-5-isoquinoline sulfonamide (H89).

44. The opioid composition of claim 29, further comprising an agent that inhibits activation of glutamate receptors, and/or intracellular activation of protein kinases.

45. The opioid composition of claim 44 wherein the agent is dextromethoφhan, ketamine, amentadine, mementine, methadone, or a tricyclic antidepressant.

46. The opoiod composition of claim 45, wherein the tricyclic antidepressant is nortriptyline or amitriptyline.

47. The opioid composition of claim 29, wherein the compound that reduces activity of a glucocorticoid receptor is formulated for sustained release.

48. A method for reducing or preventing chronic pain in a subject comprising administering to the subject in need of such treatment an amount of compound that reduces activity of a glucocorticoid receptor effective to reduce or prevent the chronic pain in the subject.

49. The method of claim 48, further comprising one or more additional administrations of the compound that reduces activity of a glucocorticoid receptor.

50. The method of claim 48, wherein the compound that reduces activity of a glucocorticoid receptor is a glucocorticoid receptor antagonist or an antibody or antigen- binding fragment thereof.

51. The method of claim 50, wherein the glucocorticoid receptor antagonist is RU38486, RU486, or RU39305.

52. The method of claim 48, wherein the compound that reduces activity of a glucocorticoid receptor is an antisense molecule, an siRNA molecule, or an RNAi molecule.

53. The method of claim 48, wherein the compound that reduces activity of a glucocorticoid receptor is linked to a targeting molecule.

54. The method of claim 53, wherein the targeting molecule's target is a neuronal cell.

55. The method of claim 48 , wherein the compound that reduces activity of a glucocorticoid receptor is administered prior to, concurrently with, or subsequent to the onset of pain.

56. The method of claim 48, further comprising administering one or more opioid and/or non-opioid analgesics.

57. The method of claim 56, wherein the opioid analgesic is moφhine, meperidine, butoφhanol, oxymoφhone, methadone, propoxyphene, codeine, heroin, hydromoφhone, oxycodone, hydrocodone, fentanyl, sufentanil, nalbuphrine, buprenoφhine, or tramadol.

58. The method of claim 56, wherein the non-opioid analgesic is acetaminophen, ibuprophen, gabapentin, tramadol, a tricyclic antidepressant, a sodium channel blocker, or a muscle relaxant.

59. The method of claim 58, wherein the antidepressant is nortriptyline.

60. The method of claim 58, wherein the sodium channel blocker is lamotrigene.

61. The method of claim 58, wherein the muscle relaxant is cyclobenzaprine or tizanidine.

62. The method of claim 48, further comprising administering an agent that inhibits adenylyl cyclase (AC) activity or protein kinase A (PKA) activity.

63. The method of claim 62, wherein the agent that inhibits adenylate cyclase (AC) activity is 2', 5'-dideoxyadenosine (ddA).

64. The method of claim 62, wherein the agent that inhibits PKA activity is N-[2-(p- bromocinnamylamino) ethyl]-5-isoquinoline sulfonamide (H89).

65. The method of claim 48, wherein the subject is human.

66. The method of claim 48, wherein the chronic pain condition is injury pain, neuropathic pain, surgical pain, disease-associated pain, phantom pain, or inflammation- associated pain.

67. The method of claim 66, wherein the injury pain is nervous system injury pain.

68. The method of claim 48, wherein the subject is undergoing pain treatment.

69. The method of claim 48, wherein the mode of admimstration is implantation, mucosal administration, intrathecal administration, epidural administration, intravenous administration, inhalation, or oral administration.

70. The method of claim 48, further comprising administrating an agent that inhibits activation of glutamate receptors, and/or intracellular activation of protein kinases.

71. The method of claim 70, wherein the agent is dextromethoφhan, ketamine, amentadine, mementine, methadone, or a tricyclic antidepressant.

72. The method of claim 71 , wherein the tricyclic antidepressant is nortriptyline or amitriptyline.

73. The method of claim 48, wherein the compound that reduces activity of a glucocorticoid receptor is formulated for sustained release.

74. A kit for treating a subject in accordance with the method of claim 1 or 48, comprising a package housing at least one first container containing at least one dose of a compound that reduces activity of a glucocorticoid receptor, and instructions for using the compound that reduces activity of a glucocorticoid receptor for the prevention and/or treatment of chronic pain and/or opioid tolerance.

75. The kit of claim 74, wherein the compound that reduces activity of a glucocorticoid receptor is linked to a targeting molecule.

76. The kit of claim 75, wherein the targeting molecule's target is a neuronal cell.

77. The kit of claim 74, wherein the compound that reduces activity of a glucocorticoid receptor is a glucocorticoid receptor antagonist or an antibody or antigen-binding fragment thereof.

78. The kit of claim 77, wherein the glucocorticoid receptor antagonist is RU38486, RU486, or RU39305.

79. The kit of claim 74, wherein the compound that reduces activity of a glucocorticoid receptor is an antisense molecule, an siRNA molecule, or an RNAi molecule.

80. The kit of claim 74, further comprising an additional container containing at least one dose of an opioid or non-opioid analgesic.

81. The kit of claim 80, wherein the opioid analgesic is moφhine, meperidine, butoφhanol, oxymoφhone, methadone, propoxyphene, codeine, heroin, hydromoφhone, oxycodone, hydrocodone, fentanyl, sufentanil, nalbuphrine, buprenoφhine, or tramadol.

82. The kit of claim 80, wherein the non-opioid analgesic is acetaminophen, ibuprophen, gabapentin, tramadol, a tricyclic antidepressant, a sodium channel blocker, or a muscle relaxant.

83. The kit of claim 82, wherein the antidepressant is nortriptyline.

84. The kit of claim 82, wherein the sodium channel blocker is lamotrigene.

85. The kit of claim 82, wherein the muscle relaxant is cyclobenzaprine or tizanidine.

86. The kit of claim 74, further comprising a container containing an agent that inhibits adenylyl cyclase (AC) activity or protein kinase A (PKA) activity.

87. The kit of claim 86, wherein the agent that inhibits adenylate cyclase (AC) activity is 2', 5'-dideoxyadenosine (ddA).

88. The kit of claim 86, wherein the agent that inhibits PKA activity is N-[2-(p- bromocinnamylamino) ethyl]-5-isoquinoline sulfonamide (H89).

89. The kit of claim 74, wherein the chronic pain condition is injury pain, neuropathic pain, surgical pain, disease-associated pain, phantom pain, or inflammation-associated pain.

90. The kit of claim 89, wherein the injury pain is nervous system injury pain.

91. The kit of claim 74, further comprising a container containing an agent that inhibits activation of glutamate receptors, and/or intracellular activation of protein kinases.

92. The kit of claim 91, wherein the agent is dextromethoφhan, ketamine, amentadine, mementine, methadone, or a tricyclic antidepressant.

93. The kit of claim 92, wherein the tricyclic antidepressant is nortriptyline or amitriptyline.

94. The kit of claim 74, wherein the compound that reduces activity of a glucocorticoid receptor is formulated for sustained release.