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Effect of spinal cyclooxygenase inhibitors in rat using the formalin test and in vitro prostaglandin E2 release
Abstract
Spinally delivery of the non-specific cyclooxygenase inhibitor, S(+)-ibuprofen, reduces the second phase of the formalin test and the evoked release of prostaglandin E2 (prostaglandin E2) from rat spinal cord in vitro. Using two selective cyclooxygenase-2 inhibitors, SC58125 (1-[(4-methysufonyl)phenyl]-3-tri-fluoromethyl-5-(4-fluorophenyl)p yrazole) and SC-236 (4-[5-(4-chlorophenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfon amide), we observed that neither agent at the highest dose/concentration employed altered the second phase of the formalin test after intrathecal delivery or K+-evoked prostaglandin E2 release from spinal cord in vitro, although ibuprofen was effective in both models. These observations suggest that cyclooxygenase-2 may not be associated with spinal prostanoid synthesis acutely or with facilitated nociception which occurs within the limited time frame of the formalin test.
... It is of interest that Ca v 2.2±/± mice showed a reduced level of responses only in the early half of phase 2 of the formalin test (Figure 5B). This early half of phase 2 seems to correspond to phase 2A reported by Yaksh and colleagues (Malmberg and Yaksh, 1992; Dirig et al., 1997). They proposed that phase 2 could be separated further into two qualitatively different subphases: phase 2A, the ®rst half of phase 2 in which pain responses are sensitive to anti-in¯ammatory drugs such as cyclooxygenase inhibitors; and phase 2B, the latter half of phase 2 in which pain responses are resistant to these drugs (Malmberg and Yaksh, 1992; Dirig et al., 1997). ...
... This early half of phase 2 seems to correspond to phase 2A reported by Yaksh and colleagues (Malmberg and Yaksh, 1992; Dirig et al., 1997). They proposed that phase 2 could be separated further into two qualitatively different subphases: phase 2A, the ®rst half of phase 2 in which pain responses are sensitive to anti-in¯ammatory drugs such as cyclooxygenase inhibitors; and phase 2B, the latter half of phase 2 in which pain responses are resistant to these drugs (Malmberg and Yaksh, 1992; Dirig et al., 1997). Our result further con®rmed the presence of two distinguishable subphases in phase 2 and indicates that phase 2A is completely dependent upon the N-type VDCC function. ...
The importance of voltage-dependent Ca2+ channels (VDCCs) in pain transmission has been noticed gradually, as several VDCC blockers have been shown to be effective in inhibiting this process. In particular, the N-type VDCC has attracted attention, because inhibitors of this channel are effective in various aspects of pain-related phenomena. To understand the genuine contribution of the N-type VDCC to the pain transmission system, we generated mice deficient in this channel by gene targeting. We report here that mice lacking N-type VDCCs show suppressed responses to a painful stimulus that induces inflammation and show markedly reduced symptoms of neuropathic pain, which is caused by nerve injury and is known to be difficult to treat by currently available therapeutic methods. This finding clearly demonstrates that the N-type VDCC is essential for development of neuropathic pain and, therefore, controlling the activity of this channel can be of great importance for the management of neuropathic pain.
... COX inhibitors, when delivered intrathecally, have an analgesic action on acute nociceptive and inflammatory pain (11,25,(45)(46)(47). However, the extent to which this is mediated by inhibition of constitutively expressed COX1 or COX2 is disputed, as is whether COX2 induced in microglia or endothelial cells contributes (36,45,(48)(49)(50). ...
... COX inhibitors, when delivered intrathecally, have an analgesic action on acute nociceptive and inflammatory pain (11,25,(45)(46)(47). However, the extent to which this is mediated by inhibition of constitutively expressed COX1 or COX2 is disputed, as is whether COX2 induced in microglia or endothelial cells contributes (36,45,(48)(49)(50). We found no change in mechanical, thermal, or acute chemical (formalin phase 1) pain sensitivity in the neural COX2-null mice relative to littermate control mice, indicating minimal contribution of COX2 expressed in neurons or glia to basal nociception. ...
A cardinal feature of peripheral inflammation is pain. The most common way of managing inflammatory pain is to use nonsteroidal antiinflammatory agents (NSAIDs) that reduce prostanoid production, for example, selective inhibitors of COX2. Prostaglandins produced after induction of COX2 in immune cells in inflamed tissue contribute both to the inflammation itself and to pain hypersensitivity, acting on peripheral terminals of nociceptors. COX2 is also induced after peripheral inflammation in neurons in the CNS, where it aids in developing a central component of inflammatory pain hypersensitivity by increasing neuronal excitation and reducing inhibition. We engineered mice with conditional deletion of Cox2 in neurons and glial cells to determine the relative contribution of peripheral and central COX2 to inflammatory pain hypersensitivity. In these mice, basal nociceptive pain was unchanged, as was the extent of peripheral inflammation, inflammatory thermal pain hypersensitivity, and fever induced by lipopolysaccharide. By contrast, peripheral inflammation-induced COX2 expression in the spinal cord was reduced, and mechanical hypersensitivity after both peripheral soft tissue and periarticular inflammation was abolished. Mechanical pain is a major symptom of most inflammatory conditions, such as postoperative pain and arthritis, and induction of COX2 in neural cells in the CNS seems to contribute to this.
... However, growing evidence suggests that NSAIDs exert a central action in addition to a peripheral action. [3][4][5][6][7] The NSAIDs used clinically include a diversity of structural classes including salicylates, pyrazoles, oxicams, fenamates, arylacetic acids, and arylpropionic acids. A subclass ofNSAIDs, arylpropionic acid derivatives (AP As), is characterized by the presence of an asymmetric carbon atom which creates a chiral center at the a-carbon to the carboxylate function. ...
... It is well known that the inhibitory effect of ketoprofen on PG synthesis in inflammatory cells is due to its S( + )-enantiomer, 16 • 17 and it has also been demonstrated that S( + )-ketoprofen inhibits inducible COX (COX-2). 18 Although the pharmacokinetics of ketoprofen and its enantiomers have been studied in a 79.3 ± 4.9 (9) 69.6 ± 4.2 (6) [ (Tables I and ll). The analgesic potency that we found for rac(±)-ketoprofen is equivalent to that described elsewhere. ...
Many studies indicate that the S-enantiomers of arylpropionic (APA) nonsteroidal antiinflammatory drugs (NSAIDs) are the pharmacologically active enantiomers. S(+)-ketoprofen (dexketoprofen) stereoselectively inhibits cyclooxygenase (COX) in vitro but very little is known about the differential activity of ketoprofen enantiomers in vivo. We examined the analgesic, antiinflammatory, and antipyretic activities of S(+)-ketoprofen in rats and mice. First, we measured the antinociceptive action of S(+)-ketoprofen in abdominal pain models. After intravenous administration. 0.5 mg/kg S(+)-ketoprofen inhibited 92.1+/-2.2% of writhing in mice. Stereoselectivity in the activity was detected; intravenous administration of the R(-)-enantiomer resulted in no statistically significant activity in a dose range of 0.15-1 mg/kg. Similar results were obtained after oral administration in mice. In the rat, S(+)-ketoprofen was a more potent analgesic than diclofenac by both intravenous and oral administration. There was no significant difference between the analgesic effect of S(+)-ketoprofen treatment and the twofold dose of the racemic form in both the mouse and rat models. Second, we measured the antiinflammatory activity of S(+)-ketoprofen using a carrageenan-induced paw edema model in the rat. Intravenous administration of 5 mg/kg of S(+)-ketoprofen almost completely inhibited edema formation. After oral administration, S(+)-ketoprofen is both more potent and effective than diclofenac. Third, we measured antipyretic activity. S(+)-ketoprofen showed a marked antipyretic action (ED50 = 1.6 mg/kg) and was the most potent of the NSAIDs tested. S(+)-ketoprofen is a potent antiinflammatory, analgesic, and antipyretic agent in vivo, consistent with its potent anti-COX activity.
... It has been reported that systemic or spinal administration of the non-selective COX inhibitor ibuprofen, but not selective COX-2 inhibitors, such as NS 398 or SC58125, produced antinociception in the formalin test [19,20]. This indicates an important participation of COX-1, but not COX-2, in this test. ...
... In contrast, there is evidence that NS 398 was able to reduce formalin-induced flinches [21]. Our results confirm previous observations [19,20] about the lack of efficacy of selective COX-2 inhibitors in the formalin test. However, to our knowledge, this is the first report on the lack of efficacy of a clinically available selective COX-2 inhibitor in acute pain. ...
... Moreover, other mechanisms such as peripheral activation of the nitric oxide-cGMP pathway could also contribute to their actions [13,14,17]. There is evidence that systemic or spinal administration of the non-selective COX inhibitor ibuprofen, but not the selective COX-2 inhibitors NS-398 or SC58125, produced antinociception in 5% formalin test in the rat [18][19][20], indicating an important participation of COX-1, but not COX-2 in this particular test. In general, our results in the rat 1% or 5% formalin tests confirm previous observations [18][19][20] about the lack of efficacy of selective COX-2 inhibitors in the rat 5% formalin test and in other models [21,22]. ...
... There is evidence that systemic or spinal administration of the non-selective COX inhibitor ibuprofen, but not the selective COX-2 inhibitors NS-398 or SC58125, produced antinociception in 5% formalin test in the rat [18][19][20], indicating an important participation of COX-1, but not COX-2 in this particular test. In general, our results in the rat 1% or 5% formalin tests confirm previous observations [18][19][20] about the lack of efficacy of selective COX-2 inhibitors in the rat 5% formalin test and in other models [21,22]. ...
The peripheral antinociceptive effect of the selective COX-2 inhibitor celecoxib in the formalin-induced inflammatory pain was compared with that of resveratrol (COX-1 inhibitor) and diclofenac (non-selective COX inhibitor). Rats received local pretreatment with saline, celecoxib, diclofenac or resveratrol followed by 50 microl of either 1% or 5% formalin. Peripheral administration of celecoxib did not produce antinociception at either formalin concentration. In contrast, diclofenac and resveratrol produced a dose-dependent antinociceptive effect in the second phase of both 1% and 5% formalin test. The peripheral antinociception produced by diclofenac or resveratrol was due to a local action, as drug administration in the contralateral paw was ineffective. Results indicate that the selective COX-2 inhibitor celecoxib does not produce peripheral antinociception in formalin-induced inflammatory pain. In contrast, selective COX-1 and non-selective COX inhibitors (resveratrol and diclofenac, respectively) are effective drugs in this model of pain.
... We also studied herein the consequences of reducing the effect of PGs during the induction of inflammation, because in inflammation, PG synthesis and release in the spinal cord are upregulated (see introductory remarks). Local transmitters and modulators may contribute to this upregulation, because NMDA (Sorkin, 1993), kainic acid (Yang et al., 1996b), substance P (Hua et al., 1999), depolarization by K ϩ (Dirig et al., 1997), and capsaicin (Dirig and Yaksh, 1999) have been shown to stimulate PG synthesis and release in the spinal cord . During inflammation, interleukin-1-mediated induction of COX-2 may be particularly important (Samad et al., 2001). ...
Prostaglandins (PGs) are local mediators of several functions in the CNS. Both primary afferent neurons and intrinsic cells in the spinal cord produce PGs, with a marked upregulation during peripheral inflammation. Therefore, the significance of spinal PGs in the neuronal processing of mechanosensory information was herein investigated. In anesthetized rats, the discharges of spinal nociceptive neurons with input from the knee joint were extracellularly recorded. Topical administration of prostaglandin E(2) (PGE(2)) to the spinal cord facilitated the discharges and expanded the receptive field of dorsal horn neurons to innocuous and noxious pressure applied to the knee joint, the ankle, and the paw, thus mimicking inflammation-induced central sensitization. Conversely, topical administration of the PG synthesis inhibitor indomethacin to the spinal cord before and during development of knee joint inflammation attenuated the generation of inflammation-induced spinal neuronal hyperexcitability. However, after development of inflammation, the responses of spinal neurons to mechanical stimuli were only reduced by systemic indomethacin but not by indomethacin applied to the spinal cord. Thus, spinal PG synthesis is important for the induction and initial expression but not for the maintenance of spinal cord hyperexcitability. Spinal PGE(2) application facilitated dorsal horn neuronal firing elicited by ionophoretic delivery of NMDA, suggesting that an interaction of PGs and NMDA receptors may contribute to inflammation-induced central sensitization. However, after development of inflammation, spinal indomethacin failed to reduce responses to ionophoretic delivery of NMDA or AMPA, suggesting that such an interaction is not required for the maintenance of central sensitization.
... The central levels of PGE 2 that, according to our calculations, would be reached in preclinical headache studies would exceed the levels attained in these hyperalgesic states induced by IL-1b and LPS. The finding that central administration of COX inhibitors reduces or blocks pain behavior or pain hypersensitivity (34,43,44) further supports a role for central elaboration of PGE 2 in pain hypersensitivity. Hence, a central action of PGE 2 could also explain the development of the behavior that is commonly observed in preclinical headache studies (17,20,31). ...
Background:
Application of inflammatory mediators to the cranial dura has been used as a method to activate and sensitize neurons in the meningeal sensory pathway in preclinical behavioral studies of headache mechanisms. However, the relatively high concentrations and volumes used in these studies raise the question of whether the applied agents might pass through the dura to act directly on central neurons, thus bypassing the dural afferent pathway.
Methods:
We used a radiolabeling approach to quantify the meningeal permeability of two of the inflammatory mediators, 5-HT and PGE2, when applied to the cranial dura as part of an inflammatory mixture used in preclinical headache models.
Results:
Both agents could be detected in samples taken four hours after dural application in the cerebrospinal fluid (CSF) and, in measurements made only for PGE2, in the central nervous system (CNS) as well. Based on our measurements, we made estimates of the CSF and CNS levels that would be attained with the higher concentrations and volumes of 5HT and PGE2 that were exogenously applied in previous pre-clinical headache studies. These estimated levels were comparable to or larger than normal endogenous levels, potentially large enough to have physiological effects.
Conclusions:
The finding that the cranial meninges are permeable to the two tested inflammatory mediators PGE2 and 5-HT raises some uncertainty about whether the behavioral changes observed in prior pre-clinical headache studies with these as well as other agents can be attributed entirely to the activation of dural nociceptors, particularly when the agents are applied at concentrations several orders of magnitude above physiological levels.
... An increased release of PGE 2 into the splenic vein of an anaesthetized dog was detected after a nociceptive injection of bradykinin into the spleen (Ferreira et al., 1973). Subsequently, PGE 2 release from spinal cord slices in vitro was reported after stimulation with capsaicin or K + (60 Mm) (Malmberg and Yaksh, 1994;Dirig et al., 1997). PGE 2 was also collected by microdialysis with a probe implanted in the spinal cord of rats in vivo. ...
... Cyclo-oxygenase (COX) inhibitors are known to be effective in suppressing central nervous system activity, as demonstrated in the thermal hyperalgesia 12 and formalininduced pain models in rats. 13 Despite being effective analgesics, opioids have several adverse side effects, including acute respiratory and hemodynamic suppression, postoperative nausea and vomiting, rigidity, urinary retention, and cough. We tested whether coadministration of a NSAID with the synthetic opioid, fentanyl, could decrease the opioid concentration necessary for effectively inhibiting movement caused by a skin incision and thus potentially decrease the incidence of opioid-induced side effects and complications. ...
We hypothesized that nonsteroidal anti-inflammatory drugs decrease the plasma fentanyl concentration required to produce immobility in 50% of patients in response to skin incision (Cp50incision) compared with placebo under target-controlled infusion (TCI) propofol anesthesia.
Sixty-two unpremedicated patients scheduled to undergo gynecologic laparoscopy were randomly assigned to receive placebo (control group) or flurbiprofen axetil 1 mg·kg(-1) (flurbiprofen group) preoperatively. General anesthesia was induced with fentanyl and propofol, and intubation was performed after succinylcholine 1 mg·kg(-1). Propofol was administered via a target-controlled infusion (TCI) system (Diprifusor™) set at an effect-site concentration of 5 μg·mL(-1). Fentanyl was given by a TCI system using the STANPUMP software (Schafer model). The concentration for the first patient was set at 3 ng·mL(-1) and modified in each group according to the up-down method. Skin incision was performed after more than ten minutes equilibration time. Serum fentanyl concentration, bispectral index (BIS), and hemodynamic parameters were measured two minutes before and after skin incision. The Cp50incision of fentanyl was derived from the mean of the crossovers (i.e., the serum fentanyl concentrations of successive participants who responded and those who did not or vice versa).
Ten and 11 independent crossover pairs were collected in the control and flurbiprofen groups, respectively, representing 42 of 62 enrolled patients. The mean (SD) fentanyl Cp50incision was less in the flurbiprofen group [0.84 (0.63) ng·mL(-1)] than in the control group [1.65 (1.15) ng·mL(-1)]; P = 0.007; however, there were no differences in BIS, blood pressure, or heart rate, between groups.
Preoperative flurbiprofen axetil decreased the Cp50incision of fentanyl by 49% during propofol anesthesia without changing the BIS or hemodynamic variables.
... CNS PGs have been shown to be important in both thermal and mechanical hyperalgesia (41). Furthermore, COX-2 mRNA can be induced in the spinal cord after peripheral inflammation (39,40,42), and selective inhibitors of COX-2 applied directly to the lumbar spinal cord prevented inflammatory hyperalgesia (43). These observations point to a role for COX-2 expressed in the spinal cord in hyperalgesia. ...
The enzymes cyclooxygenase-1 and cyclooxygenase-2 (COX-1 and COX-2) catalyze the conversion of arachidonic acid to prostaglandin (PG) H2, the precursor of PGs and thromboxane. These lipid mediators play important roles in inflammation and pain and in normal physiological functions. While there are abundant data indicating that the inducible isoform, COX-2, is important in inflammation and pain, the constitutively expressed isoform, COX-1, has also been suggested to play a role in inflammatory processes. To address the latter question pharmacologically, we used a highly selective COX-1 inhibitor, SC-560 (COX-1 IC50 = 0.009 mu M; COX-2 IC50 = 6.3 mu M). SC-560 inhibited COX-1-derived platelet thromboxane B2, gastric PGE2, and dermal PGE2 production, indicating that it was orally active, but did not inhibit COX-2-derived PGs in the lipopolysaccharide-induced rat air pouch. Therapeutic or prophylactic administration of SC-560 in the rat carrageenan footpad model did not affect acute inflammation or hyperalgesia at doses that markedly inhibited in vivo COX-1 activity. By contrast, celecoxib, a selective COX-2 inhibitor, was anti-inflammatory and analgesic in this model. Paradoxically, both SC-560 and celecoxib reduced paw PGs to equivalent levels. Increased levels of PGs were found in the cerebrospinal fluid after carrageenan injection and were markedly reduced by celecoxib, but were not affected by SC-560. These results suggest that, in addition to the role of peripherally produced PGs, there is a critical, centrally mediated neurological component to inflammatory pain that is mediated at least in part by COX-2.
... fonamide NS 398 , produced antinociception in the forma-Ž . lin test Dirig et al., 1997;Euchenhofer et al., 1998 , indicating an important participation of cyclooxygenase-1, but not cyclooxygenase-2, in this test. On the other hand, there is evidence to support the participation of the cyclooxygenase-2 enzyme in formalin-induced acute nociception, as NS 398 was able to reduce the formalin-induced Ž . ...
The involvement of the nitric oxide–cyclic GMP pathway in the antinociceptive action of the cyclooxygenase-2 preferential inhibitor meloxicam was assessed in the rat formalin test. Rats received local pretreatment with saline or meloxicam and then 50 μl of dilute formalin (1%). Local administration of meloxicam produced a dose-dependent antinociception in the second phase of the formalin test. The antinociception produced by meloxicam was due to a local action as its administration in the contralateral paw was ineffective. Local pretreatment of the paws with saline or NG-d-nitro-arginine methyl ester (d-NAME) did not affect the antinociception produced by meloxicam. However, NG-l-nitro-arginine methyl ester (l-NAME, a NO synthesis inhibitor) or 1H-(1,2,4)-oxadiazolo(4,2-a)quinoxalin-1-one (ODQ, a soluble guanylyl cyclase inhibitor) blocked in a dose-dependent way the effect of meloxicam. It is concluded that the peripheral antinociceptive effect of meloxicam involves a local NO–cyclic GMP pathway.
... A more likely scenario is that several linoleic acid metabolites contribute to TRPV1 activation. It is also possible that other factors capable of sensitizing TRPV1 (15)(16) were coreleased under these conditions. Thus, the resultant TRPV1 agonist activity could be an entourage effect of several lipids acting in concert. ...
Transient receptor potential vanilloid 1 (TRPV1) plays a major role in hyperalgesia and allodynia and is expressed both in the peripheral and central nervous systems (CNS). However, few studies have evaluated mechanisms by which CNS TRPV1 mediates hyperalgesia and allodynia after injury. We hypothesized that activation of spinal cord systems releases endogenous TRPV1 agonists that evoke the development of mechanical allodynia by this receptor. Using in vitro superfusion, the depolarization of spinal cord triggered the release of oxidized linoleic acid metabolites, such as 9-hydroxyoctadecadienoic acid (9-HODE) that potently activated spinal TRPV1, leading to the development of mechanical allodynia. Subsequent calcium imaging and electrophysiology studies demonstrated that synthetic oxidized linoleic acid metabolites, including 9-HODE, 13-HODE, and 9 and 13-oxoODE, comprise a family of endogenous TRPV1 agonists. In vivo studies demonstrated that intrathecal application of these oxidized linoleic acid metabolites rapidly evokes mechanical allodynia. Finally, intrathecal neutralization of 9- and 13-HODE by antibodies blocks CFA-evoked mechanical allodynia. These data collectively reveal a mechanism by which an endogenous family of lipids activates TRPV1 in the spinal cord, leading to the development of inflammatory hyperalgesia. These findings may integrate many pain disorders and provide an approach for developing analgesic drugs.
... tiinflammatory capacity of the different NSAIDs is now proposed to be associated with the capacity of inhibiting cyclooxy- genase-2 activity. A central analgesic effect of NSAIDs Ž has also been proposed McCormak, 1994; Cashman, 1996; . Yaksh et al., 1998 , which is probably mediated by regula- Ž tion of cyclooxygenase-2 activity in the spinal cord Dirig . et al., 1997; Smith et al., 1998 . Regulation of cyclooxy- genase-1 activity, on the other hand, is thought to be responsible for the gastric and renal side effects of NSAIDs, Ž as well as for their antithrombotic activity for a review, . see Pairet and Engelhardt, 1996; Vane and Botting, 1998 . Cyclooxygenase-2 is presently regarded as an important ...
The ability of metamizol to inhibit cyclooxygenase-1 and cyclooxygenase-2 activities has been evaluated using different cyclooxygenase sources. Metamizol inhibited purified cyclooxygenase-1 and cyclooxygenase-2 with an IC50 of about 150 microg/ml. A similar IC50 value for cyclooxygenase-2 was obtained in lipopolysaccharide-activated broken murine macrophages. Consistent with these findings, molecular models of the complexes between cyclooxygenase-1 or cyclooxygenase-2 with 4-methylaminoantipyrine, the major active derivative of metamizol, suggested a common binding mode to both isoforms. In intact cells, however, the inhibition profiles were markedly different. The IC50 values of metamizol for cyclooxygenase-1 in intact bovine aortic endothelial cells (BAEC) cells and human platelets were 1730 +/- 150 microg/ml and 486 +/- 56 microg/ml, respectively. Inhibition of cyclooxygenase-2 activity in murine macrophages and primary human leukocytes activated by lipopolysaccharide yielded IC50 values of 12 +/- 1.8 microg/ml and 21 +/- 2.9 microg/ml, respectively. These data indicate that the IC50 values obtained with purified enzymes or disrupted cells cannot always be extrapolated to the cyclooxygenase inhibitory activity of nonsteroidal antiinflammatory drugs (NSAIDs) in intact cells. The data presented here also indicate that cyclooxygenase-2 inhibition could play an important role in the pharmacological effects of metamizol.
... Although COX-2 production of prostaglandin, particularly prostaglandin E 2 , which regulates many physiological processes including vasomotility, platelet aggregation, and immunomodulation, 34 has been proposed to perpetuate the ischemic cascade, 12 administration of COX-2 inhibitors after an acute noxious stimulus did not appear to alter prostaglandin synthesis in the rat spinal cord. 35 Thus, further studies are required to understand COX-2 inhibitor effects in the spinal cord after ischemia and to determine whether the neurodegenerative effects of COX-2 are mediated through the synthesis of prostaglandin E 2 or other mediators. The present study focused on the behavioral effects of COX-2 inhibition after ischemia. ...
Cyclooxygenase-2 (COX-2), an enzyme that is induced in the central nervous system after various insults, has been localized to neurons and in cells associated with the cerebral vasculature, where it may be involved in the inflammatory component of the ischemic cascade. COX-2 is part of the initial reaction that involves the arachidonic acid cascade, which produces molecules that support an inflammatory response. The present study evaluated the pharmacological effects of a specific long-acting COX-2 inhibitor, SC-236, in a reversible rabbit spinal cord ischemia model using clinical rating scores (behavioral analysis) as the primary end point.
SC-236 was administered (10 to 100 mg/kg SC) 5 minutes after the start of occlusion to groups of rabbits exposed to ischemia induced by temporary (10 to 40 minutes) occlusion of the infrarenal aorta. Behavioral analysis, which allowed for the calculation of an ET(50) value representing the duration of ischemia (minutes) associated with a 50% probability of resultant permanent paraplegia, was conducted 18 and 48 hours later. A drug was determined to be neuroprotective if it prolonged the ET(50) significantly compared with the appropriate control group.
Since SC-236 is not readily soluble in aqueous solutions, it was dissolved in 100% dimethyl sulfoxide (DMSO) for subcutaneous administration. Therefore, the vehicle-treated control group consisted of rabbits given an equal volume of DMSO without drug. In the DMSO-treated control group, the ET(50) assessed 18 hours after initiation of aortal occlusion was 18.84+/-3.19 minutes. In contrast, treatment with 100 mg/kg of SC-236 given 5 minutes after the start of occlusion prolonged the ET(50) of the group significantly to 30.04+/-3.55, an effect that was still evident 48 hours later. In addition, lower doses of the drug (10 and 50 mg/kg) also showed a trend for an increase in ET(50). SC-236 (100 mg/kg) did not significantly alter body temperature after a subcutaneous injection.
The present study suggests that COX-2 plays an important role in the ischemic cascade of events that translate into ischemia-induced behavioral deficits and furthermore that selective COX-2 inhibitors may be useful in the treatment of ischemic stroke to improve behavioral functions.
... We also studied herein the consequences of reducing the effect of PGs during the induction of inflammation, because in inflammation , PG synthesis and release in the spinal cord are upregulated (see introductory remarks). Local transmitters and modulators may contribute to this upregulation, because NMDA (Sorkin, 1993), kainic acid (Yang et al., 1996b), substance P (Hua et al., 1999), depolarization by K (Dirig et al., 1997), and capsaicin (Dirig and Yaksh, 1999) have been shown to stimulate PG synthesis and release in the spinal cord (Vanegas and Schaible, 2001). During inflammation, interleukin-1-mediated induction of COX-2 may be particularly important (Samad et al., 2001 ). ...
Prostaglandins (PGs) are local mediators of several functions in the CNS. Both primary afferent neurons and intrinsic cells in the spinal cord produce PGs, with a marked upregulation during peripheral inflammation. Therefore, the significance of spinal PGs in the neuronal processing of mechanosensory information was herein investigated. In anesthetized rats, the discharges of spinal nociceptive neurons with input from the knee joint were extracellularly recorded. Topical administration of prostaglandin E(2) (PGE(2)) to the spinal cord facilitated the discharges and expanded the receptive field of dorsal horn neurons to innocuous and noxious pressure applied to the knee joint, the ankle, and the paw, thus mimicking inflammation-induced central sensitization. Conversely, topical administration of the PG synthesis inhibitor indomethacin to the spinal cord before and during development of knee joint inflammation attenuated the generation of inflammation-induced spinal neuronal hyperexcitability. However, after development of inflammation, the responses of spinal neurons to mechanical stimuli were only reduced by systemic indomethacin but not by indomethacin applied to the spinal cord. Thus, spinal PG synthesis is important for the induction and initial expression but not for the maintenance of spinal cord hyperexcitability. Spinal PGE(2) application facilitated dorsal horn neuronal firing elicited by ionophoretic delivery of NMDA, suggesting that an interaction of PGs and NMDA receptors may contribute to inflammation-induced central sensitization. However, after development of inflammation, spinal indomethacin failed to reduce responses to ionophoretic delivery of NMDA or AMPA, suggesting that such an interaction is not required for the maintenance of central sensitization.
Voltage-dependent Ca2+ channels (VDCCs) play essential roles in various aspects of cellular activities including muscle contraction, neurotransmitter release, control of neuronal excitability, and gene expression (Catterall, 1998 Hofmann et al., 1999). To date, several kinds of VDCCs have been identified by electrophysiological and pharmacological means, and molecular biological studies have identified ten mammalian genes coding for the pore-forming α1 subunit (Ertel et al., 2000). Although many studies using pharmacological tools have revealed the physiological functions of VDCC subtypes, such studies have some limitations. The pharmacological specificity of the tools used to distinguish among different channels is not absolute, and the administration of these drugs and toxins to all the cells of interest is sometimes difficult. Genetic manipulation of VDCCs is an important alternative, since it can be used to eliminate completely a specific VDCC in all the cells of an animal. An increasing number of knockout animals carrying mutations in VOCC genes have been reported, to study the in vivo functions of different VDCCs. This chapter reviews the abnormalities found in VDCC knockout mice and the deduced in vivo functions of the VDCC.
After tissue or nerve injury, animals develop an increased sensitivity to noxious and non-noxious cutaneous stimuli, paralleling the development of hyperalgesia and allodynia in humans. While these changes depend partly on sensitization in peripheral tissues, there is also evidence that the increased sensitivity depends on sensitization within the central nervous system (CNS). Central sensitization after tissue or neural injury has been defined as a change in excitability of CNS neurons which is triggered by and outlasts nociceptive afferent inputs (Woolf and Chong, 1993). There is evidence indicating that persistent nociceptive responses to chemical stimulation with formalin depend on a sensitization of spinal cord neurons, which occurs in the early period after injury. Indeed, the formalin test has been used now for 10 years as an animal model of noxious stimulus-induced plasticity. In this paper, I will debate the evidence supporting the view that the formalin test reflects a sensitization of spinal cord neurons. Additionally, I will discuss some of the potential mediators that lead to the development of formalin injury-induced plasticity in spinal cord.
The discovery of an inducible form of cyclooxygenase (COX-2) requires a refinement of the theory that inhibition of cyclooxygenase activity explains both therapeutic effects and side-effects of non-steroidal anti-inflammatory drugs (NSAIDs). Selective COX-2 inhibitors have demonstrated in clinical trials a significantly better gastrointestinal tolerability than classical NSAIDs, for the same anti-inflammatory activity. Their tolerability in patients with active ulcer or with a recent history of ulcer as well as in patients suffering from cardiovascular or renal diseased has still to be investigated in detail. Their therapeutic potential in several new indications, including pre-term labour, colorectal cancer and Alzheimer's disease, is currently being investigated.
The pharmacological and toxicological properties of nimesulide have been previously reviewed (see [1-8]). The major part of these reviews has been concerned with the preclinical actions of the drug. A key issue concerning the interpretation of the in vitro effects of nimesulide has been the relationship of these to the plasma or synovial fluid concentrations of the drug, which are found during therapy. The review by Bennett and Villa [4] is noteworthy for having discriminated the in vitro effects, which are known to occur at therapeutic drug concentrations with those which are above this range. Thus, generally speaking although nimesulide has preferential COX-2 selectivity it is also an inhibitor of histamine release and actions, leukotriene B4 and C4, and platelet activating factor (PAF) production, the adherence and activation of neutrophils, collagenase and other metalloproteinases, glucocorticoid receptor phosphorylation, interleukin-6 production, calcium channel activation and is an antioxidant within the range of drug concentrations encountered therapeutically [4]. Thus, nimesulide can be regarded as having multifactorial actions in relation to its anti-inflammatory activity.
In vivo microdialysis has aided preclinical pain research for more than a decade. This valuable tool has allowed correlations between behavioral signs of pain and CNS neurotransmitter release. Here, we review studies that describe the effects of noxious stimulation on neurotransmitter concentrations in microdialysate obtained from either spinal cord or pain modulatory centers in the brain. We conclude that such studies are currently evolving from models of acute pain to a more modern approach that assesses release in animal models of lasting painful conditions.
In this review article we undertake an assessment of the comparative performance of cyclooxygenase-2 (COX-2) selective agents in managin a nonarthritic conditions. Clinical and in-vivo reports were identified through the use of the computer databases MEDLINE and EMBASE. Our findings reveal that, generally, clinical evidence for the effectiveness of COX-2 selective agents in the treatment of nonarthritic conditions is either equivocal or lacking. In the management of postoperative dental pain, rofecoxib 50 mg exhibits analgesic efficacy comparable with that of ibuprofen 400 mg. For celecoxib 200 mg, analgesic efficacy appears comparable with that of aspirin 650 mg, but less than that of ibuprofen 400 mg or rofecoxib 50 mg. In addition to the probability of a type II statistical error in some of these studies, we also present novel arguments for previously unconsidered sources of confounding. In-vivo outcomes using established animal models of clinical pain are consistent with limited clinical utility SC-58125 and SC-236 at the highest dose employed failed to alter the second phase of the formalin test, and NS-398, nimesulide, and L-745337 had no effect upon writhings induced by intraperitoneal acetic acid. Overall, we conclude that at present there are insufficient grounds for assuming analgesic equivalence between COX-2 selective and dual inhibitors across a wide range of acute tonic pains. For many pains, in order to achieve maximal analgesia, inhibition of both COX isoenzymes appears to be requisite.
Neurogenic inflammation occurs in many tissues, but has been principally studied in skin. Evidence accumulated over the last two decades supports the hypothesis that neurogenic inflammation is of prime importance in arthritis. Joints are richly innervated by neuropeptide-containing primary afferent and autonomic fibers, peptides are released into the synovial cavity of arthritic joints, and exogenous and endogenous neuropeptides exert proinflammatory actions on articular tissues. Acute and chronic interruption of the articular nerve supply renders joints less susceptible to arthritis.Evidence also indicates that neuronal activation and neurogenic inflammation may underlie the symmetrical nature of arthritis, in both humans and experimental animals. The evidence for neurogenic inflammation in the maintenance of arthritis is reviewed, and a hypothesis is suggested for mechanisms by which the spread of arthritis may occur.
The purpose of this study was to investigate the allodynic effect of bicuculline (BIC) given topically to the dorsal surface of the rat spinal cord, and to determine if spinal prostaglandins (PGs) mediate the allodynic state arising from spinal GABAA-receptor blockade. Male Sprague–Dawley rats (325–400 g) were anaesthetized with halothane and maintained with urethane for the continuous monitoring of blood pressure (MAP), heart rate (HR) and cortical electroencephalogram (EEG). A laminectomy was performed to expose the dorsal surface of the spinal cord. Unilateral application of BIC (0.1 μg in 0.1 μl) to the L5 or L6 spinal segment induced a highly localized allodynia (e.g. one or two digits) on the ipsilateral hind paw. Thus, hair deflection (brushing the hair with a cotton-tipped applicator) in the presence, but not absence of BIC, evoked an increase in MAP and HR, abrupt motor responses (MR; e.g. withdrawal of the hind leg, kicking, and/or scratching) on the affected side, and desynchrony of the EEG. BIC-allodynia was dose-dependent, yielding ED50’s (95% CI's) of 45 ng (31–65) for MAP; 68 ng (46–101) for HR and 76 ng (60–97) for MR. Allodynia was sustained for up to 2 h with repeated BIC application without any detectable change in the location or area of peripheral sensitization. Pretreatment with either the EP1- receptor antagonist, SC-51322, the cyclooxygenase (COX)-2 selective inhibitor, NS-398, or the NMDA-receptor antagonist, AP-7, inhibited BIC-allodynia in a dose-dependent manner. The results demonstrate: (a) BIC, applied to the dorsal surface of the spinal cord, induces highly localized allodynia; (b) this effect can be sustained with repeated BIC application; (c) it is evoked by NMDA-dependent afferent input; (d) spinal PGs are synthesized by constitutive COX-2 during BIC-allodynia; and (e) spinal PGs contribute to the abnormal processing of tactile input via spinal EP1-receptors.
The spinal cord is one of the sites where non-steroidal anti-inflammatory drugs (NSAIDs) act to produce analgesia and antinociception. Expression of cyclooxygenase(COX)-1 and COX-2 in the spinal cord and primary afferents suggests that NSAIDs act here by inhibiting the synthesis of prostaglandins (PGs). Basal release of PGD2, PGE2, PGF2α and PGI2 occurs in the spinal cord and dorsal root ganglia. Prostaglandins then bind to G-protein-coupled receptors located in intrinsic spinal neurons (receptor types DP and EP2) and primary afferent neurons (EP1, EP3, EP4 and IP). Acute and chronic peripheral inflammation, interleukins and spinal cord injury increase the expression of COX-2 and release of PGE2 and PGI2. By activating the cAMP and protein kinase A pathway, PGs enhance tetrodotoxin-resistant sodium currents, inhibit voltage-dependent potassium currents and increase voltage-dependent calcium inflow in nociceptive afferents. This decreases firing threshold, increases firing rate and induces release of excitatory amino acids, substance P, calcitonin gene-related peptide (CGRP) and nitric oxide. Conversely, glutamate, substance P and CGRP increase PG release. Prostaglandins also facilitate membrane currents and release of substance P and CGRP induced by low pH, bradykinin and capsaicin. All this should enhance elicitation and synaptic transfer of pain signals in the spinal cord. Direct administration of PGs to the spinal cord causes hyperalgesia and allodynia, and some studies have shown an association between induction of COX-2, increased PG release and enhanced nociception. NSAIDs diminish both basal and enhanced PG release in the spinal cord. Correspondingly, spinal application of NSAIDs generally diminishes neuronal and behavioral responses to acute nociceptive stimulation, and always attenuates behavioral responses to persistent nociception. Spinal application of specific COX-2 inhibitors sometimes diminishes behavioral responses to persistent nociception.
Prostaglandins (PGs) are known to be involved in inflammatory and nociceptive processing. Since the discovery of at least two isozymes of cyclooxygenase (COX), inhibition of COX-2 has been suggested to be responsible for the therapeutic effects of non-steroidal anti-inflammatory drugs (NSAIDs). In the present study, the effects of a rather selective COX-2 inhibitor, NS-398 (0.3–27 mg/kg i.p.), were studied using the rat formalin test as a model of acute nociception. Diclofenac (non-selective COX inhibitor; 0.3–27 mg/kg i.p.) was used as a control. NS-398 revealed antinociceptive activity only at a dose (27 mg/kg) which results in plasma concentrations which most likely do not selectively inhibit COX-2. By contrast, diclofenac inhibited formalin-induced flinching behaviour over the whole dose range tested. Our results suggest that PGs mediating nociception in the formalin test of the rat are most likely produced via the COX-1 as well as COX-2 pathways. Thus, in an acute model of nociception a non-selective COX inhibitor may offer advantages as compared to a selective COX-2 inhibitor.
Das nicht steroidale Analgetikum Metamizol ist ein populäres, potentes nicht-opioid-Analgetikum und Antipyretikum, dessen analgetische Potenz vergleichbar ist mit Opiaten. Abgesehen von spasmolytischen Eigenschaften unterscheidet es sich von den klassischen NSAR durch schwache antiinflammatorische Wirkungen sowie fehlende gastro-renale Nebenwirkungen. Der molekulare Wirkungsmechanismus ist noch nicht vollständig bekannt. Meine Dissertation deutet darauf hin, dass ein Teil der pharmakologischen Eigenschaften dadurch erklärt werden kann, dass es als Prodrug nach Abspalten der Sulfonsäuregruppe exklusiv im ZNS durch die Fettsäure-Amid-Hydrolase an Arachidonsäure gekoppelt wird. Es ensteht Methyl-Amino-Antipyrin-Arachidonat, ein Arachidonoyl-Amid, ein potenter Cyclooxygenase-Inhibitor und Agonist an Cannabis-Rezeptoren. The non-steroidal antiinflammatory drug dipyrone is a popular, potent non-opioid analgesic and antipyretic with analgesic potency comparable to that of opioids. Apart from its spasmolytic effect, the weak antiinflammatory effects and the absence of an gastro-renal side effects are features that distinguish it from the common non-steroidal antiinflammatory drugs. The molecular mechanism of action of dipyrone is yet not known. My data support the idea,that the pharmacological effects of the prodrug dipyrone are at least partly due to a arachidonoyl amide of methyl-amino-antipyrine, which is a part of the dipyrone-metabolic pathway. This drug is formed by fetty acid amide hydrolase in the CNS and ist a potent cyclooxygenase inhibitor and cannabinoid receptor ligand.
Myrica rubra Sieb. et Zucc. leaves are commonly used as an astringent, antidiarrheic, and analgesics in folk medicine in China. In the present study, the analgesic activity of myricetin, a major compound in Myrica rubra Sieb. et Zucc. leaves was evaluated in vivo. The analgesic effect of myricetin was tested by a serial of models, such as acetic acid-induced writhing response, formalin-induced paw licking and hot plate test. The sedative activity was evaluated by pentobarbital-induced sleep time. Platelet aggregation induced by collagen and arachidonic acid was also performed in vitro. Myricetin showed a significant inhibition on chemical nociceptive models such as the acetic acid-induced writhing response and the licking time on the late phase in the formalin test in a dose-dependent manner, but did not manifest a signicant effect in hot plate test. Myricetin was also not able to increase the sleeping time induced by pentobarbital, which further indicated that the analgesic effect of myricetin was unrelated to sedation. In addition, myricetin inhibited the content of PGE2 in the peritoneal fluid and platelet aggregation induced by collagen and arachidonic acid in vitro. These results collectively demonstrated that myricetin possessed potent analgesic activity, which was related with peripheral analgesia, but, not with the opioid system. Myricetin may be a potent COX-1 inhibitor with anti-platelet activity.
Hyperalgesia and allodynia are frequent symptoms of disease and may be useful adaptations to protect vulnerable tissues. Both may, however, also emerge as diseases in their own right. Considerable progress has been made in developing clinically relevant animal models for identifying the most significant underlying mechanisms. This review deals with experimental models that are currently used to measure (sect. II) or to induce (sect. III) hyperalgesia and allodynia in animals. Induction and expression of hyperalgesia and allodynia are context sensitive. This is discussed in section IV. Neuronal and nonneuronal cell populations have been identified that are indispensable for the induction and/or the expression of hyperalgesia and allodynia as summarized in section V. This review focuses on highly topical spinal mechanisms of hyperalgesia and allodynia including intrinsic and synaptic plasticity, the modulation of inhibitory control (sect. VI), and neuroimmune interactions (sect. VII). The scientific use of language improves also in the field of pain research. Refined definitions of some technical terms including the new definitions of hyperalgesia and allodynia by the International Association for the Study of Pain are illustrated and annotated in section I.
Senescence accelerated prone mouse 6 (SAMP6) mice have been known to be a model for accelerated aging. Compared with the normal control senescence accelerated resistant mouse 1 (SAMR1) mice, although the SAMP6 mice have normal bone mass at 4 months, they exhibit a significantly lower bone mass at 8 months. It was recently reported that SAMP6 has memory deficit at 4 months of age, indicating that the change of nervous function might be already detected at 4 months of age. To assess whether SAMP6 mice exhibit an age-related abnormality of nociceptive transmission, we examined a battery of tests using the von Frey test for mechanically induced response, the hot plate test for thermally induced response, and the formalin paw test for chemically induced response. SAMP6 and SAMR1 showed similar response patterns in the von Frey test and the hot plate test. In the formalin paw test, 1-month-old SAMP6 and SAMR1 had similar responses, while 4-month-old SAMP6 exhibited attenuated phase 2 response, but normal phase 1 response. These findings indicate that onset of age-related phenotypes in SAMP6 differs in different tissues. SAMP6 could be useful to delineate the involvement of age-related nociceptive mechanisms.
The highly disagreeable sensation of pain results from an extraordinarily complex and interactive series of mechanisms integrated at all levels of the neuroaxis, from the periphery, via the dorsal horn to higher cerebral structures. Pain is usually elicited by the activation of specific nociceptors ('nociceptive pain'). However, it may also result from injury to sensory fibres, or from damage to the CNS itself ('neuropathic pain'). Although acute and subchronic, nociceptive pain fulfils a warning role, chronic and/or severe nociceptive and neuropathic pain is maladaptive. Recent years have seen a progressive unravelling of the neuroanatomical circuits and cellular mechanisms underlying the induction of pain. In addition to familiar inflammatory mediators, such as prostaglandins and bradykinin, potentially-important, pronociceptive roles have been proposed for a variety of 'exotic' species, including protons, ATP, cytokines, neurotrophins (growth factors) and nitric oxide. Further, both in the periphery and in the CNS, non-neuronal glial and immunecompetent cells have been shown to play a modulatory role in the response to inflammation and injury, and in processes modifying nociception. In the dorsal horn of the spinal cord, wherein the primary processing of nociceptive information occurs, N-methyl-D-aspartate receptors are activated by glutamate released from nocisponsive afferent fibres. Their activation plays a key role in the induction of neuronal sensitization, a process underlying prolonged painful states. In addition, upon peripheral nerve injury, a reduction of inhibitory interneurone tone in the dorsal horn exacerbates sensitized states and further enhance nociception. As concerns the transfer of nociceptive information to the brain, several pathways other than the classical spinothalamic tract are of importance: for example, the postsynaptic dorsal column pathway. In discussing the roles of supraspinal structures in pain sensation, differences between its 'discriminative-sensory' and 'affective-cognitive' dimensions should be emphasized. The purpose of the present article is to provide a global account of mechanisms involved in the induction of pain. Particular attention is focused on cellular aspects and on the consequences of peripheral nerve injury. In the first part of the review, neuronal pathways for the transmission of nociceptive information from peripheral nerve terminals to the dorsal horn, and therefrom to higher centres, are outlined. This neuronal framework is then exploited for a consideration of peripheral, spinal and supraspinal mechanisms involved in the induction of pain by stimulation of peripheral nociceptors, by peripheral nerve injury and by damage to the CNS itself. Finally, a hypothesis is forwarded that neurotrophins may play an important role in central, adaptive mechanisms modulating nociception. An improved understanding of the origins of pain should facilitate the development of novel strategies for its more effective treatment.
We have established that mu-opioid receptor activation causes a presynaptic inhibition of neurotransmitter release that is mediated by 12-lipoxygenase metabolites of arachidonic acid in midbrain neurons [1]. We further demonstrated that the efficacy of opioids was enhanced synergistically by treatment of brain neurons with inhibitors of the other major enzymes responsible for arachidonic acid metabolism; cyclooxygenase (COX-1) and 5-lipoxygenase. These findings explain a mechanism of analgesic action of NSAIDs in the central nervous system that is both independent of prostanoid release and inhibited by opioid antagonists, as well as the synergistic interaction of opioids with NSAIDs. These findings also suggest new avenues for development of centrally active medications involving combinations of lowered doses of opioids and specific 5-lipoxygenase inhibitors.
In the nervous system, prostanoids are well recognized as mediators in a variety of processes, including fever generation, modulation of the stress response, sleep/wake cycle, control of cerebral blood flow, and hyperalgesia. Two isoforms of cyclooxygenase (COX), the enzyme that catalyzes the conversion of arachidonic acid to prostanoids, are now recognized: a constitutively expressed COX-1 and a highly regulated COX-2. New molecular and pharmacologic tools have provided a better understanding of the roles of COX-generated prostanoids in the nervous system. Other studies reveal that COX may represent an important target for new therapeutic approaches to neurologic disorders. This review summarizes our current understanding of cyclooxygenase expression and prostanoid actions in the nervous system, with special reference to COX-2 and studies demonstrating its expression in different cell types responding to a variety of stimuli. A brief review of the molecular biology, pharmacology, and primary actions of COX-2 outside of the nervous system provides a context for understanding potential neurobiological roles for COX-2 and prostanoid production. Information about the role of COX in human neurological disorders, including cerebrovascular disease, Alzheimer' s disease, and hyperalgesia, is covered in the last section.
The role of nonsteroidal anti-inflammatory drugs (NSAIDs) is examined in the control of cancer pain with a particular focus on their use as adjuvants to opioids in advanced cancer pain. These agents have both a peripheral effect on inflammation and a role in attenuating central pain pathways. The possibility of obtaining the benefits of NSAIDs with fewer side-effects by using COX-2-specific agents is discussed. The gastrointestinal, renal, haemostatic, cognitive and hypersensitivity side-effects of NSAIDs are reviewed and their potential impact assessed. The evidence for the efficacy of NSAIDs as single agents for cancer pain is reviewed together with the nine papers which have reported the effects of NSAIDs as adjuvants to opioids in cancer pain. All of these papers reported positive results of NSAIDs, but, in the absence of any randomized, double-blind controlled trials, where NSAIDs were used as adjuvants on a long-term basis alongside optimal opioid use, definite conclusions cannot be reached. Guidelines for the safe use of NSAIDs are suggested. Finally, suggestions for future research are made.
After peripheral injury and inflammation, the close relationship between stimulus intensity and response to acute injurious stimuli is altered. Peripheral injury and inflammation increase a subject’s responsiveness to noxious stimuli (i.e. pain reports, paw withdrawal latency, or electrophysiological recordings) relative to the un-injured state (Raja et al., 1988). This hypersensitivity can be expressed as a decreased response latency to a noxious stimulus (hyperalgesia) or a nocifensive response (i.e. pain report or escape attempts) to an innocuous stimulus (allodynia). These altered stimulus-response relationships may result from peripheral as well as central mechanisms; this review will focus on spinal prostanoid synthesis and hyperalgesia after peripheral injury and inflammation.
The pharmacological profile of the analgesic agent, 1-[(4, 5-bis(4-methoxyphenyl)-2-thiazoyl)carbonyl]-4-methylpiperazine hydrochloride (FR122047), was investigated. In recombinant human cyclooxygenase enzyme assays, the inhibition of prostaglandin E(2) formation by FR122047 was 2300 times more selective for cyclooxygenase-1 than cyclooxygenase-2. Oral administration of FR122047 (3.2-100 mg/kg) dose dependently reduced the phase 2 response (10-60 min) of the formalin test in rats. This effect was 3 times less potent than that of indomethacin. FR122047 (1-32 mg/kg; p. o.) showed a dose-dependent analgesic effect against the acetic acid-induced writhing response in mice. Furthermore, FR122047 (0. 01-10 mg/kg, p.o.) inhibited the increase in 6-keto prostaglandin F(1alpha) level in acetic acid-injected mouse peritoneal cavity. However, a selective cyclooxygenase-2 inhibitor, NS-398, had no effect in these cyclooxygenase-1 sensitive pain models. These results suggest that FR122047, a selective cyclooxygenase-1 inhibitor, shows an analgesic effect in chemical nociceptive models and may be a useful analgesic agent.
Increasing amounts of experimental and clinical data support the role of selective cyclooxygenase (COX)-2 inhibition in anti-inflammatory processes and the involvement of COX-1 inhibition in the side effects associated with non steroidal anti-inflammatory drug use. This review will focus on the differences in the structure of the COX-1 and COX-2 molecules, particularly the active site and how they are bound by various NSAIDs to achieve COX-2 selectivity. This COX-2 selectivity will then be characterized in pharmacological assays in vitro and in animal models in vivo. Finally, clinical information available for this new class of selective inhibitors will be discussed.
Prostaglandins are important mediators in spinal nociceptive processing. They are produced by cyclo-oxygenase isoforms, cyclo-oxygenase-1 and -2, which are both constitutively expressed in the central nervous system. The present immunohistochemical study details localization and regulation of cyclo-oxygenase-1 and -2 and neuronal nitric oxide synthase in lumbar spinal cord before and after induction of a painful paw inflammation in mice. Cyclo-oxygenase-1 immunoreactivity was found in glial cells of the dorsal and ventral horns, but not in neurons. In unstimulated mice, cyclo-oxygenase-2 immunoreactivity was found in motoneurons of the ventral horns and in lamina X, but not in dorsal horn neurons. After induction of a paw inflammation with zymosan, cyclo-oxygenase-2 immunoreactivity increased dramatically in dorsal horn neurons of laminae I–VI and X, paralleled by a significant increase in prostaglandin E2 release from lumbar spinal cord. Cyclo-oxygenase-2 was co-localized with neuronal nitric oxide synthase immunoreactivity in several neurons in superficial laminae of the dorsal horns and in the area surrounding the central canal. Nitric oxide synthase was distributed in the cytoplasm and extended to processes of some neurons. In contrast, electron microscopy revealed that cyclo-oxygenase-2 immunoreactivity was restricted to the nuclear membrane and rough endoplasmic reticulum.
Recognition of two isoforms of cyclooxygenase and reports that nonsteroidal anti-inflammatory drugs may be beneficial in devastating neurological conditions such as Alzheimer's disease have led to increased interest in cyclooxygenase function in the nervous system. In the present paper we review current data on the multiplicity of cyclooxygenase and prostaglandin mediated effects in the central nervous system (CNS). We discuss CNS cells types, including neurons, glia, and cerebrovascular elements, where cyclooxygenases-1 and -2 are expressed under normal conditions or can be induced by physiological or pathological stimuli. We also address physiological processes such as pain sensitization, CNS inflammation and fever induction that are regulated or modified by cyclooxygenase activity. Finally, we describe potential roles of cyclooxygenase in neurological diseases and rationales for nonsteroidal anti-inflammatory drug use in the treatment of neurodegenerative disorders, stroke and CNS injury.
Twisted gastrulation (TSG) is involved in specifying the dorsal-most cell fate in Drosophila embryos, but its mechanism of action is poorly understood. TSG has been proposed to modify the action of Short gastrulation (SOG), thereby increasing signalling by the bone morphogenetic protein (BMP) Decapentaplegic. SOG, an inhibitor of BMP signalling, is in turn inactivated by the protease Tolloid. Here we identify Tsg gene products from human, mouse, Xenopus, zebrafish and chick. Expression patterns in mouse and Xenopus embryos are consistent with in vivo interactions between Tsg, BMPs and the vertebrate SOG orthologue, chordin. We show that Tsg binds both the vertebrate Decapentaplegic orthologue BMP4 and chordin, and that these interactions have multiple effects. Tsg increases chordin's binding of BMP4, potentiates chordin's ability to induce secondary axes in Xenopus embryos, and enhances chordin cleavage by vertebrate tolloid-related proteases at a site poorly used in Tsg's absence; also, the presence of Tsg enhances the secondary axis-inducing activity of two products of chordin cleavage. We conclude that Tsg acts as a cofactor in chordin's antagonism of BMP signalling.
Inflammation causes the induction of cyclooxygenase-2 (Cox-2), leading to the release of prostanoids, which sensitize peripheral nociceptor terminals and produce localized pain hypersensitivity. Peripheral inflammation also generates pain hypersensitivity in neighbouring uninjured tissue (secondary hyperalgesia), because of increased neuronal excitability in the spinal cord (central sensitization), and a syndrome comprising diffuse muscle and joint pain, fever, lethargy and anorexia. Here we show that Cox-2 may be involved in these central nervous system (CNS) responses, by finding a widespread induction of Cox-2 expression in spinal cord neurons and in other regions of the CNS, elevating prostaglandin E2 (PGE2) levels in the cerebrospinal fluid. The major inducer of central Cox-2 upregulation is interleukin-1beta in the CNS, and as basal phospholipase A2 activity in the CNS does not change with peripheral inflammation, Cox-2 levels must regulate central prostanoid production. Intraspinal administration of an interleukin-converting enzyme or Cox-2 inhibitor decreases inflammation-induced central PGE2 levels and mechanical hyperalgesia. Thus, preventing central prostanoid production by inhibiting the interleukin-1beta-mediated induction of Cox-2 in neurons or by inhibiting central Cox-2 activity reduces centrally generated inflammatory pain hypersensitivity.
The purpose of this study was to determine whether ES-242-1, a novel N-methyl-D-aspartate (NMDA) receptor antagonist of microbial origin, has anti-nociception at the spinal level and to evaluate how its anti-nociceptive effect differs from that of MK-801, a non-competitive NMDA receptor antagonist. Agents were injected intrathecally (0.1, 1.0 and 10 microg) through a previously implanted PE tube in rats. Formalin (2%, 100 microl) was injected subcutaneously into the left hindpaw 15 min after each antagonist administration. Licking time as a nociceptive behavior was measured in three stages after formalin-injection, such as early phase (0-9 min), late first phase (10-29 min) and late second phase (30-60 min). In the early phase, the largest dose of ES-242-1 significantly decreased total licking time, although MK-801 did not show any significant reduction. With the treatment of 1.0 and 10 microg MK-801, total licking time in both late first and second phases was significantly suppressed, although the smallest dose (0.1 microg) of ES-242-1 showed a significant reduction in the late second phase. These results indicate that ES-242-1 is highly effective against tonic pain, such as inflammatory pain.
While acute pain has a fundamental role to operate a protective system, chronic pain associated with inflammation and nerve injury often outlasts its biological usefulness. Therefore, there has recently been great interest in the neurochemical mechanisms of hyperalgesia to noxious stimuli and tactile pain (allodynia) to innocuous stimuli with a hope to relieve persistent, intractable pain. Over several decades non-steroidal anti-inflammatory drugs and opioids have been employed for clinical management of pain. The introduction of molecular biology to pain research has enabled us to describe the mechanism of pain at the molecular level and to develop analgesics with selectivity for targets and with less adverse effects. This review focuses on current knowledge concerning mechanisms and pathways for pain induced by prostaglandins and their interactions with novel neuropeptides nociceptin/orphanin FQ and nocistatin derived from the same opioid precursor protein.
Intrathecal phospholipase A2 (PLA2) and cyclooxygenase-2 (COX-2), but not COX-1, inhibitors attenuate facilitated pain states generated by peripheral injury/inflammation and by direct activation of spinal glutamate and substance P receptors. These results are consistent with the constitutive expression of PLA2 and COX-2 in spinal cord, the spinal release of prostaglandins by persistent afferent input, and the effects of prostaglandins on spinal excitability. Whereas the acute actions of COX-2 inhibitors are clearly mediated by constitutively expressed spinal COX-2, studies of spinal COX-2 expression indicate that it is upregulated by neural input and circulating cytokines. Given the intrathecal potency of COX-2 inhibitors, the comparable efficacy of intrathecal versus systemic COX-2 inhibitors in hyperalgesic states not associated with inflammation, and the onset of antihyperalgesic activity prior to COX-2 upregulation, it is argued that a principal antihyperalgesic mechanism of COX-2 inhibitors lies with modulation of constitutive COX-2 present at the spinal level.
Non-steroidal anti-inflammatory drugs (NSAIDs) are frequently used as analgesics. Although the results of clinical studies indicate considerable disparity in the analgesic efficacy of NSAIDs, the pre-clinical models generally used for the study of nociception do not allow a clear distinction to be made between the analgesic properties of agents belonging to this family. As clinical pain is characterized by hyperalgesia, we evaluated the effects of NSAIDs with similar chemical structures but different selectivities for cyclo-oxygenase (COX)-1 and COX-2 in a new behavioural model of central hyperalgesia in rats. We assessed the effects of lornoxicam, piroxicam, and meloxicam on the reduction of hindpaw nociceptive thresholds to thermal stimulation produced by a 10% formaldehyde (formalin) injection into rat tail. Each drug was administered intraperitoneally (i.p.) at its ED(50)for the anti-inflammatory effect (namely the inhibition of carrageenan-induced hindpaw oedema). At this dose (1.3 mg kg(-1), 1.0 mg kg(-1), and 5.8 mg kg(-1), respectively), lornoxicam, piroxicam, and meloxicam produced the same anti-inflammatory effect, did not modify thermal nociceptive thresholds, and significantly reduced the hyperalgesia. However, only lornoxicam was fully effective for prevention of hyperalgesia. Our results indicate a dissociation between the anti-inflammatory and the anti-hyperalgesic activity of NSAIDs, where the latter seems to be more evident after the block of both COX-1 and COX-2. Finally, they suggest that our experimental model of thermal hindpaw hyperalgesia can be effectively utilized to assess the ability of different drugs to reduce central sensitization, and thus hyperalgesia.
Nonsteroidal anti-inflammatory drugs (NSAIDs) are frequently used as analgesics. Despite the fact that clinical experience indicates a considerable disparity in the analgesic efficacy of NSAIDs, the animal models of nociception do not allow a clear distinction to be made between the analgesic properties of these agents. In contrast to nociceptive pain, clinical pain is characterised by hyperalgesia. Therefore, we evaluated the anti-hyperalgesic effects of the four NSAIDs nimesulide, diclofenac, celecoxib and rofecoxib which are widely used to treat inflammatory pain. We performed two animal studies in which each drug was administered intraperitoneally (i.p.) at its previously defined ED50 for the anti-inflammatory effect in the rat (i.e. the inhibition of carrageenan-induced hindpaw oedema measured by plethysmometry). In the first study, nimesulide (2.9 mg/kg) completely inhibited the development of thermal hindpaw hyperalgesia induced by the injection of formalin in the tail, whereas diclofenac (3.0 mg/kg) or celecoxib (12.7 mg/kg) partly reduced the hyperalgesia, and rofecoxib (3.0 mg/kg) was ineffective. In the second study, nimesulide and diclofenac were significantly more effective than celecoxib and rofecoxib in reducing the mechanical hindpaw hyperalgesia induced by the intraplantar injection of Freund's complete adjuvant (FCA). The anti-hyperalgesic activity of the drugs was also investigated in patients with rheumatoid arthritis. After a single oral dose, all drugs reduced the inflammatory hyperalgesia. However, only nimesulide was effective 15 minutes after treatment. Moreover, nimesulide (100 mg) was significantly more effective than rofecoxib (25 mg). Overall, our data demonstrate that NSAIDs may show different anti-hyperalgesic properties. Nimesulide seems to be particularly effective and fast-acting against inflammatory pain.
We have prepared polyclonal antiserum against sheep seminal vesicle prostaglandin H synthase (also termed cyclooxygenase) which cross-reacted with human cyclooxygenase, thereby enabling us to directly determine the synthetic rate of cyclooxygenase protein and its modulation by the monokine interleukin-1 (IL-1). Cultured human dermal fibroblast cells were labeled with [35S]methionine, and the membrane-bound cyclooxygenase was solubilized and immunoprecipitated 35S-labeled fibroblast cyclooxygenase migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular size of approximately 73,000 daltons, similar to that of native sheep cyclooxygenase and of cyclooxygenase covalently labeled by [3H]aspirin, i.e. [3H]acetylcyclooxygenase. Additional validation of the immunoprecipitated 35S-labeled cyclooxygenase band indicated that it was specifically displaced by unlabeled sheep cyclooxygenase. N-terminal amino acid radiosequence analysis of [3H]proline-labeled cyclooxygenase revealed [3H]proline residues in positions 3, 6, and 8, consistent with the previously reported N-terminal sequence of sheep cyclooxygenase. Endoglycosidase H treatment of 35S-labeled fibroblast cyclooxygenase caused a decline in apparent molecular size (due to removal of mannose residues) which was similar to that seen with the native sheep cyclooxygenase. [35S]Methionine pulse-chase experiments indicated a half-life of 1 h for fibroblast cyclooxygenase. The monokine interleukin-1 stimulated fibroblast cyclooxygenase synthesis in a time- and dose-dependent fashion; as little as 0.03 unit/ml of IL-1 produced significant stimulation of 35S-labeled cyclooxygenase synthesis. Maximum stimulation was 3-10-fold after preincubation of the cells with 0.3 unit/ml of IL-1 for 12-16 h. IL-1 treatment of cells yielded parallel dose-response curves for stimulation of prostaglandin E2 formation, increased cellular cyclooxygenase activity, and increased synthetic rate of newly formed cyclooxygenase, suggesting that the IL-1 effect is mediated mainly, if not solely, via induction of cyclooxygenase synthesis.
Nonsteroidal anti-inflammatory drugs (NSAIDs) currently available for clinical use inhibit both COX-1 and COX-2. This suggests
that clinically useful NSAIDs inhibit pro-inflammatory prostaglandins (PGs) derived from the activity of COX-2, as well as
PGs in tissues like the stomach and kidney (via COX-1). A new class of compounds has recently been developed (SC-58125) that
have a high degree of selectivity for the inducible form of cyxlooxygenase (COX-2) over the constitutive form (COX-1). This
unique class of compounds exhibit a time-dependent irreversible inhibition of COX-2, while reversibly inhibiting COX-1. The
molecular basis of this selectivity was probed by site-directed mutagenesis of the active site of COX-2. The sequence differences
in the active site were determined by amino acid replacement of the COX-2 sequences based on the known crystal structure of
COX-1, which revealed a single amino acid difference in the active site (valine 509 to isoleucine) and a series of differences
at the mouth of the active site. Mutants with the single amino acid substitution in the active site and a combination of three
changes in the mouth of the active site were made in human COX-2, expressed in insect cells and purified. The single amino
acid change of valine 509 to isoleucine confers selectivity of COX-2 inhibitors in the class of SC-58125 and others of the
same class (SC-236, NS-398), while commonly used NSAIDs such as indomethacin showed no change in selectivity. Substitutions
of COX-1 sequences in COX-2 at the mouth of the active site of COX-2 did not change the selectivity of SC-58125. This indicates
that the single amino acid substitution of isoleucine at position 509 for a valine is sufficient to confer COX-2 selectivity
in this example of a diaryl-heterocycle COX inhibitor.
Subcutaneous injection of formalin into the dorsal surface of the hindpaw evoked a two-phased flinching (phase 1:0-9 min; phase 2: 10-60 min) of the injected paw. Intrathecal administration of the nonsteroidal anti-inflammatory drugs (NSAID) produced minimal effects upon phase 1, but showed a significant, though submaximal, dose-dependent suppression of the phase 2 response. Ordering of i.t. potency was (ID50 in nmol): indomethacin (1.9) > or = flurbiprofen (2.1) > ketorolac (5.2) > or = zomepirac (5.9) > S(+)ibuprofen (16) > or = ibuprofen(racemic) (19) > acetylsalicylic acid (27) > acetaminophen (250) > R(-)ibuprofen (> 270) = 0. Intraperitoneal administration also produced a dose-dependent inhibition of phase 2, but only at doses which were 100 to 1000 times higher than those required to produce similar effects after i.t. injection. Intrathecal and i.p. dose-response curves showed similar distinct plateaus of maximum achievable inhibition (intrinsic activity) of the phase 2 behavior, ranging from 20 to 50% of the control response. Varying the time of drug injection reveals that injection 9 min after formalin yielded effects the same as those observed when the agent was given 2 min before formalin. Pretreatment at longer intervals indicated that the duration of the antinociceptive effect was between 3 to 6 hr after the i.t. injection. The i.t. injection of the highest doses of the several NSAID were without significant effect upon the 52.5 degrees C hot plate test. These studies indicate that NSAID have a powerful effect upon spinal nociceptive processing evoked by the s.c. injection of formalin.
To purify and characterize the isoform of prostaglandin endoperoxide synthase (rPGSi) induced by human chorionic gonadotropin in granulosa cells of rat preovulatory follicles, solubilized cell extracts were subjected to anionic exchange chromatography, column fractions were resolved by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and rPGSi was visualized by immunoblotting and silver staining techniques. Immunoreactive rPGSi and peroxidase activity co-eluted at pH 6.5 and 6.0. Amino-terminal amino acid sequence of three immunoreactive rPGSi bands (Mr = 72,000, 70,000, and 59,000) were identical, Mr = 59,000 being a proteolytic fragment. Alignment of the amino-terminal sequences of rPGSi with those of ovine PGS (oPGS) indicated that only 15 of 26 residues were identical (58% identity). In contrast, rPGSi was closely related to the deduced amino acid sequence of PGS-related cDNA clones isolated from chicken and mouse cell lines, with 77% (20/26 residues) and 96% (25/26 residues) identity, respectively. Whereas tryptic digests of oPGS generated fragments of Mr = 38,000 and Mr = 33,000, only a small peptide appeared cleaved from the carboxyl terminus of rPGSi. Peroxidase activity of partially purified rPGSi exhibited lower apparent Km and maximal velocity than that of oPGS. Collectively, these results document the existence of a novel rat PGS isoform (based on purification, enzymatic activity, and amino-terminal amino acid sequence) which is hormonally induced and obligatory for a known biological process, ovulation.
Prostaglandins sensitize sensory neurons to activation by mechanical, thermal and chemical stimuli. This sensitization also results in an increase in the stimulus-evoked release of the neuroactive peptides, substance P and calcitonin gene-related peptide from sensory neurons. The cellular transduction cascade underlying the prostaglandin-induced augmentation of peptide release is not known. Therefore, we examined whether the sensitizing action of prostaglandins on peptide release from sensory neurons grown in culture is mediated by the second messenger, adenosine 3', 5' cyclic monophosphate (cAMP). Prostaglandin E2 and carba prostacyclin (a stable analog of prostaglandin I2) significantly increase the content of cAMP-like immunoreactive substance (icAMP) in the sensory neuron cultures at concentrations that also augment the bradykinin- or capsaicin-evoked release of peptides. Furthermore, pretreating sensory neurons with agents that increase intracellular cAMP mimics the sensitizing action of prostaglandins. Exposing cultures to either forskolin (0.1-10 microM), cholera toxin (1.5 micrograms), or 8-bromo-cAMP (100 microM) results in a significant enhancement of the bradykinin- or capsaicin-stimulated release of both substance P-like and calcitonin gene-related peptide-like immunoreactive substances. Pretreating sensory neurons with the adenylyl cyclase inhibitor, 9-tetrahydro-2-furyl adenine (5 mM), abolishes the prostaglandin-induced increases in icAMP content and attenuates the prostaglandin E2 or carba prostacyclin enhancement of the evoked release of calcitonin gene-related peptide-like immunoreactive substance. These results demonstrate that the cAMP transduction cascade mediates the sensitizing actions of prostaglandins on peptide release from sensory neurons.
Injection of formalin into the hind paw evokes a biphasic flinching of the injured paw. Pharmacological characterization of this behavior has implicated the spinal release of excitatory amino acids (EAAs) and cyclooxygenase (COX) products. To address this hypothesis, we examined the effect of paw formalin injection on release of EAAs and prostaglandin E2-like immunoreactivity (PGE2-LI) from the spinal cord in unanesthetized rats using a dialysis probe placed in the lumbar subarachnoid space. To assess the contribution of spinal COX products, the effects of S(+)- and R(-)-ibuprofen (active and inactive COX inhibitors) were examined. Paw formalin injection evoked a biphasic spinal release of PGE2-LI with an increase above resting concentrations of 110% in the 0-10 min sample, and of 83% in the 20-30 min sample. Significantly increased release of glutamate (Glu; 110%) and aspartate (Asp; 112%) was only observed in the 0-10 min sample. Saline injection into the paw had no effect on behavior, PGE2-LI, or EAA release. Intraperitoneal administration of 10 mg/kg, but not 1 mg/kg, S(+)-ibuprofen reduced paw flinching, blocked the elevated levels of PGE2-LI, and suppressed Glu and Asp release to 50% of control. Intrathecal delivery of 10 micrograms, but not 1 microgram, S(+)-ibuprofen also suppressed formalin-induced behavior, PGE2-LI, Glu, and Asp release. R(-)-ibuprofen showed no effect on formalin-induced behaviors or spinal release. These data demonstrate that paw formalin injection produces spinal release of PGE2-LI corresponding to the biphasic behavioral response and that the evoked release is blocked by antinociceptive doses of COX inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)
The subcellular locations of prostaglandin endoperoxide synthase-1 and -2 (PGHS-1 and -2) were determined by quantitative confocal fluorescence imaging microscopy in murine 3T3 cells and human and bovine endothelial cells using immunocytofluorescence with isozyme-specific antibodies. In all of the cell types examined, PGHS-1 immunoreactivity was found equally distributed in the endoplasmic reticulum (ER) and nuclear envelope (NE). PGHS-2 immunoreactivity was also present in the ER and NE. However, PGHS-2 staining was twice as concentrated in the NE as in the ER. A histofluorescence staining method was developed to localize cyclooxygenase/peroxidase activity. In quiescent 3T3 cells, which express only PGHS-1, histofluorescent staining was most concentrated in the perinuclear cytoplasmic region. In contrast, histochemical staining for PGHS-2 activity was about equally intense in the nucleus and in the cytoplasm, a pattern of activity staining distinct from that observed with PGHS-1. Our results indicate that there are significant differences in the subcellular locations of PGHS-1 and PGHS-2. It appears that PGHS-1 functions predominantly in the ER whereas PGHS-2 may function in the ER and the NE. We speculate that PGHS-1 and PGHS-2 acting in the ER and PGHS-2 functioning in the NE represent independent prostanoid biosynthetic systems.
A comparison was made of the spontaneous nociceptive behaviors elicited by s.c. injection into the rat hind paw of the following 8 irritants: acetic acid, carrageenan, formalin, kaolin, platelet-activating factor, mustard oil (given topically), serotonin, and yeast. Two distinct quantifiable behaviors indicative of pain were identified: flinching/shaking of the paw and hindquarters and licking/biting of the injected paw. These behaviors were prolonged and intense after formalin and acetic acid. Formalin-induced flinching was biphasic across time, a finding potentially useful for the study of both acute and tonic pain. Of the remaining test agents, only yeast caused significant spontaneous behavioral activity, which was of low intensity but long duration. Different time-courses for nociceptive behavior and development of edema were demonstrated for formalin, acetic acid and yeast. It is therefore unlikely that these endpoints are causally related. Overall, the present data strongly support the use of formalin as a noxious stimulus in tonic pain research.
The substituted glucopyranose ring structure 2-hydroxypropyl-β-cyclodextrin (CDEX) increases the solubility of molecules by inclusion of the agent in the lipophilic interior of the ring. This property is of particular use for the administration of molecules by the intracerebral (ICV) or intrathecal (IT) routes. In concentrations up to 40% w/v (isotonic), this agent (10 μl) has no effect upon nociceptive or motor function after IT injection or on EEG and general behavior after ICV injection in rats. Using 20% CDEX, there is no change in the ED50 as compared to saline on the hot plate (HP) after IT injection of morphine, D-Ala2-D-Leu5 enkephalin or Tyr-Aib-Gly-gPhe-mAib-NH2, (Aib: α-aminoisobutyric acid) although there is an increase in their respective durations of effect. Cyclic peptide opioids: Tyr-c[D-A2bu-Gly-D-βNal(1)-D-Leu] (A2bu: α, γ-diaminobutyric acid; β-Na(1): β-naphthylalanine(1) or Tyr-c[DA2bu-Gly-βNal(1)-D-Leu] are insoluble in saline but are readily dissolved in CDEX, and display a naloxone-sensitive antinociception following spinal administration. In other studies, saline insoluble capsaicin is administered in 25% dimethylsulfoxide (DMSO) or 20% CDEX (15 μl; 5 mg/ml) which result in a significant reduction in the spinal levels of substance P and calcitonin gene related peptide and an increase in the HP latency. DMSO alone, but not CDEX alone, reduces the levels of the two peptides. These data emphasize the utility of complexation with CDEX for intracerebral drug delivery and compatibility with brain and spinal tissue.
1. Decide on what organ or other biological material to study and select the proper compound for this study from the knowledge of precursor acid metabolism. Develop the RIA for this compound or a stable derivative thereof. 2. Whichever sample type is assayed (extracted or unextracted), the possibility of the presence of nonspecific interfering factors must be constantly kept in mind. Make dilution tests and assay for parallelism; however, do not accept parallelism as an absolute criterion of absence of nonspecific influence. 3. Use two or more completely different separation techniques for comparison when validating the RIA. 4. When evaluating the RIA, do not limit the investigation to the traditional reliability criteria: sensitivity, specificity, precision, and accuracy. The RIA may seem reliable at this stage and still give completely unrealistic values when applied to biological material. The method must be evaluated by other studies as well. 5. In the experimental design, aim at following the changes in prostaglandin levels in a series of samples instead of measuring absolute levels in single samples. 6. Make comparative studies employing different quantitative methods.
Current thinking emphasizes that protracted small afferent input can evoke mechanisms that mediate a significant potentiation of spinal nociceptive processing and that this facilitory component has a unique pharmacology. To investigate the behavioral parallels of this spinal facilitation, we evaluated the effects of pre- and post-treatment of intrathecal morphine (mu agonist) and MK801 (N-methyl-D-aspartate [NMDA] antagonist) on the formalin test. Intraplantar formalin resulted in a biphasic appearance of flinching behavior (phase 1 = 0-5 min; phase 2 = 10-60 min). Morphine and MK801 were administered intrathecally 15 min before formalin injection in the pretreatment study and 9 min after formalin injection in the posttreatment study. Pretreatment with intrathecal morphine produced comparable dose-dependent suppressions of the phase 1 and phase 2 behaviors (ED50 = 0.5 micrograms [95% CI = 0.3-0.9] and 0.3 micrograms [95% CI = 0.1-0.7], respectively). Posttreatment with morphine also resulted in comparable suppression of the phase 2 response (ED50 = 0.2 micrograms [95% CI = 0.1-0.3]). At the highest dose of intrathecal morphine (10 micrograms), an almost complete suppression of formalin-evoked behavior was observed. Pretreatment with MK801 inhibited the second-phase response more strongly than the first-phase response (ED50 = 1.6 micrograms [95% CI = 0.5-5.7] vs. 0.1 microgram [95% CI = 0.3 - 0.4], respectively). In contrast, posttreatment with the highest dose of MK801 had no effect on the phase 2 response.(ABSTRACT TRUNCATED AT 250 WORDS)
CP-96,345 has been identified as being a highly selective, nonpeptidic agent with subnanomolar affinity for the NK1 receptor. In the present study, we observed that pre but not posttreatment with this agent will produce depression in the second, but not the first phase of the agitation behavior induced by the injection of formalin into a rat's hindpaw. This effect is monotonically dose dependent after intrathecal (10-200 micrograms/10 microliters) or systemic (1-15 mg/kg, ip) administration. Even at the highest dose examined (400 micrograms/10 microliters), there was only a transient motor weakness of the hindpaw. The stereoisomer CP-96,344 has no binding affinity, and has no effect on the formalin response, but shows the same dose profile for motor dysfunction at the highest dose. In contrast, Spantide, a peptidic sP ligand, had only a modest effect upon the formalin response at 1 microgram/10 microliters and produced a prominent, long-lasting motor dysfunction at 4 micrograms/10 microliters. These results provide the first suggestion of sP antagonists having prominent analgesic activity with a significant therapeutic index (analgesic to motor), and emphasizes the probable role of the NK1 class of receptors in the spinal cord in mediating at least one class of nociceptive afferent input.
We have studied the effect of glucocorticoids administered in vivo on the activity and synthesis of the cyclooxygenase enzyme (COX) in mice treated with or without concurrent intravenous administration of LPS. Mouse peritoneal macrophages from LPS-treated animals showed a two to three fold increase in COX activity determined by the production of PGE2 and PGI2 after stimulation of the cells with exogenous arachidonate. Dexamethasone injected simultaneously with LPS, 12 h before killing of the animal and removal of the macrophages, completely blocked the LPS-induced increase COX activity in peritoneal macrophages. The regulation observed in COX activity by LPS and dexamethasone are due primarily to changes in COX mass as determined by immunoprecipitation of [35S]methionine endogenously labeled enzyme. In contrast, the COX present in the nonadherent cells and in renal medullary microsomes obtained from the same animals, showed no significant changes between treatments. These results indicate that LPS in vivo stimulates COX synthesis in the peritoneal macrophages but not in the kidney. The effect of dexamethasone to inhibit COX synthesis is selective to the LPS-induced enzyme.
The effects of intrathecal administration of prostaglandins on pain responses in conscious mice were evaluated by using hot plate and acetic acid writhing tests. Prostaglandin D2 (0.5-3 ng/mouse) had a hyperalgesic action on the response to a hot plate during a 3-60 min period after injection. Prostaglandin E2 showed a hyperalgesic effect at doses of 1 pg to 10 ng/mouse, but the effect lasted shorter (3-30 min) than that of prostaglandin D2. Similar results were obtained by acetic acid writhing tests. The hyperalgesic effect of prostaglandin D2 was blocked by simultaneous injection of a substance P antagonist (greater than or equal to 100 ng) but not by AH6809, a prostanoid EP1-receptor antagonist. Conversely, prostaglandin E2-induced hyperalgesia was blocked by AH6809 (greater than or equal to 500 ng) but not by the substance P antagonist. Prostaglandin F2 alpha had little effect on pain responses. These results demonstrate that both prostaglandin D2 and prostaglandin E2 exert hyperalgesia in the spinal cord, but in different ways.
We have prepared polyclonal antiserum against sheep seminal vesicle prostaglandin H synthase (also termed cyclooxygenase) which cross-reacted with human cyclooxygenase, thereby enabling us to directly determine the synthetic rate of cyclooxygenase protein and its modulation by the monokine interleukin-1 (IL-1). Cultured human dermal fibroblast cells were labeled with [35S]methionine, and the membrane-bound cyclooxygenase was solubilized and immunoprecipitated 35S-labeled fibroblast cyclooxygenase migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular size of approximately 73,000 daltons, similar to that of native sheep cyclooxygenase and of cyclooxygenase covalently labeled by [3H]aspirin, i.e. [3H]acetylcyclooxygenase. Additional validation of the immunoprecipitated 35S-labeled cyclooxygenase band indicated that it was specifically displaced by unlabeled sheep cyclooxygenase. N-terminal amino acid radiosequence analysis of [3H]proline-labeled cyclooxygenase revealed [3H]proline residues in positions 3, 6, and 8, consistent with the previously reported N-terminal sequence of sheep cyclooxygenase. Endoglycosidase H treatment of 35S-labeled fibroblast cyclooxygenase caused a decline in apparent molecular size (due to removal of mannose residues) which was similar to that seen with the native sheep cyclooxygenase. [35S]Methionine pulse-chase experiments indicated a half-life of 1 h for fibroblast cyclooxygenase. The monokine interleukin-1 stimulated fibroblast cyclooxygenase synthesis in a time- and dose-dependent fashion; as little as 0.03 unit/ml of IL-1 produced significant stimulation of 35S-labeled cyclooxygenase synthesis. Maximum stimulation was 3-10-fold after preincubation of the cells with 0.3 unit/ml of IL-1 for 12-16 h. IL-1 treatment of cells yielded parallel dose-response curves for stimulation of prostaglandin E2 formation, increased cellular cyclooxygenase activity, and increased synthetic rate of newly formed cyclooxygenase, suggesting that the IL-1 effect is mediated mainly, if not solely, via induction of cyclooxygenase synthesis.
Many studies of pain and nociception use short-lasting acute stimuli which may have limited relevance to prolonged or chronic pain states. Using extracellular single-unit recording in the dorsal horn of the rat lumbar spinal cord the present study examines the response of neurones to a long-lasting nociceptive stimulus, i.e., 50 microliter 5% formalin injected into the corresponding receptive field in the ipsilateral hind paw, and modulation of this response by an opioid. Formalin produced a distinct biphasic excitatory response in all convergent neurones tested; an immediate acute or phasic peak of neuronal firing (mean maximum 22 spikes/sec) 0-10 min post injection, and a second more prolonged tonic excitatory response (mean maximum 12 spikes/sec) over a period 20-65 min after formalin. Cells only activated by innocuous stimuli were not excited by formalin indicating the involvement of C fibre afferents in the excitatory response of convergent neurones to formalin. Both the biphasic nature and the time course of the neuronal response are similar to those observed in behavioural studies. Intrathecal DAGO (Tyr-D-AlaGlyMePheGly-ol), a potent and selective mu opioid receptor agonist, applied 20 min prior to formalin completely inhibited both peaks of excitation. Co-administration of intrathecal naloxone with the agonist restored the biphasic response. By contrast, when the administration of naloxone was delayed to 2 min post formalin so that inhibition of the first peak by DAGO pretreatment occurred, there was no subsequent second peak of activity although antagonism of the opioid would have occurred. When DAGO was applied 2 min post formalin so the initial acute response occurred, the inhibitory effect of the agonist on the second peak was far less. Thus the relative ability of DAGO to modulate the biphasic excitatory response of cells to formalin depends on whether the agonist is administered prior to or after the formalin and the appearance of the second peak may depend on the presence of the first. These results are discussed in light of the role of these neurones in nociception, opioid effects and changes in neural systems following peripheral stimuli.
Experiments with guinea-pig lung suggest that some of the therapeutic effects of sodium salicylate and aspirin-like drugs are due to inhibition of the synthesis of prostaglandins.
Catabolism of prostaglandin (PG) E2 was studied in homogenates of spinal cord and spinal nerve roots of the cat. Spinal roots enzymatically converted PGE2 to a product (metabolite I) with the chromatographic mobility of 15-keto-PGE2. Little metabolic degradation occurred in the spinal cord; however, incubation of PGE2 with combined spinal cord and nerve root tissue yielded a second metabolite (metabolite II) in addition to metabolite I. Metabolite II was identified as 15-keto-13, 14-dihydro-PGE2. These results prove that spinal nerve roots, unlike the spinal cord, contain 15-hydroxyprostaglandin dehydrogenase (15-PGDH) which is the major and rate-limiting enzyme in the inactivation of prostaglandins. The location and functional significance of 15-PGDH in peripheral nerves remain to be elucidated.
Prostacyclin (PGI2) is a potent prostanoid producing various symptoms of inflammation, including an increased sensitivity to noxious stimulation. One component of these PGI2-mediated actions may involve activation or sensitization of sensory neurons to enhance release of neuroactive peptides. We, therefore, examined whether PGI2 and carba prostacyclin (CPGI2), a stable analog of PGI2, could alter the resting and evoked release of the neuropeptides, substance P (SP) and calcitonin gene-related peptide (CGRP) from embryonic rat sensory neurons grown in culture. Treating isolated sensory neurons with CPGI2 (10-1000 nM) for 30 min caused a 3-fold increase in the resting release of both peptides. One nM CPGI2, a concentration that did not alter the resting release, significantly enhanced neuropeptide release evoked by capsaicin, 100 nM bradykinin, or 40 mM KCl. Similarly, 10 nM PGI2 did not alter resting release, but augmented capsaicin-stimulated release of SP and CGRP 2-3 fold. In contrast, prostaglandin F2 alpha was ineffective in altering either resting or capsaicin-evoked peptide release. Our results demonstrate that low concentrations of PGI2 sensitize sensory neurons to other stimuli, whereas higher concentrations evoke release directly. This PGI2-induced augmentation of neuropeptide release may be one mechanism contributing to neurogenic inflammation.
Both gamma-aminobutyric acid (GABA)A and GABAB receptor subtypes have been implicated in spinally mediated antinociception in acute pain models. In the current study, the formalin test was used as a model of protracted nociception to examine the effect of intrathecally (i.t.) administered baclofen (GABAB agonist), muscimol (GABAA agonist) or midazolam (a benzodiazepine) on antinociception. At doses that did not affect motor function, baclofen (0.3 and 1.0 micrograms, i.t.) decreased the flinch response in a dose-dependent manner during Phase 1 and Phase 2. This effect was reversible by the GABAB-specific antagonist, CGP35348 ([P-(3-aminopropyl)-P-diethoxymethyl-phosphinic acid]). Muscimol (0.3 and 1.0 microgram i.t.) evoked a dose-dependent, bicuculline-reversible decrease in flinching during Phase 1 and Phase 2, but midazolam had no effect on either phase. No attenuation of the quiescent period between Phase 1 and Phase 2 was seen upon administration of baclofen, muscimol or midazolam. Additionally, no increase in nocifensive behavior was observed upon administration of either GABAA or GABAB antagonists alone. Therefore, our conclusions are that both GABAA and GABAB agonists are antinociceptive at the spinal cord level and that endogenous spinal GABA levels are insufficient for a GABA potentiator to act alone in an antinociceptive manner.
Considerable debate exists regarding the cellular source of prostaglandins in the mammalian central nervous system (CNS). At least two forms of prostaglandin endoperoxide synthase, or cyclooxygenase (COX), the principal enzyme in the biosynthesis of these mediators, are known to exist. Both forms have been identified in the CNS, but only the distribution of COX 1 has been mapped in detail. In this study, we used Western blot analysis and immunohistochemistry to describe the biochemical characterization and anatomical distribution of the second, mitogen‐inducible form of this enzyme, COX 2 in the rat brain.
COX 2‐like immunoreactive (COX 2‐ir) staining occurred in dendrites and cell bodies of neurons, structures that are typically postsynaptic. It was noted in distinct portions of specific cortical laminae and subcortical nuclei. The distribution in the CNS was quite different from COX 1. COX 2‐ir neurons were primarily observed in the cortex and allocortical structures, such as the hippocampal formation and amygdala. Within the amygdala, neurons were primarily observed in the caudal and posterior part of the deep and cortical nuclei. In the diencephalon, COX 2–ir cells were also observed in the paraventricular nucleus of the hypothalamus and in the nuclei of the anteroventral region surrounding the third ventricle, including the vascular organ of the lamina terminalis. COX 2–ir neurons were also observed in the subparafascicular nucleus, the medial zona incerta, and pretectal area. In the brainstem, COX 2‐ir neurons were observed in the dorsal raphe nucleus, the nucleus of the brachium, of the inferior colliculus, and in the region of the subcoeruleus. The distribution of COX 2‐ir neurons in the CNS suggests that COX 2 may be involved in processing and integration of visceral and special sensory input and in elaboration of the autonomic, endocrine, and behavioral responses.
The enzyme cyclo-oxygenase catalyses the oxygenation of arachidonic acid, leading to the formation of prostaglandins. Recently two forms of cyclo-oxygenase have been described: a constitutive (COX-1) enzyme present in most cells and tissues, and an inducible (COX-2) isoenzyme observed in many cells in response to pro-inflammatory cytokines. Constitutive and inducible forms of human cyclo-oxygenase (hCOX-1 and hCOX-2) were cloned and expressed in insect cells, utilizing a baculovirus expression system. hCOX-1 had a specific activity of 18.8 mumol of O2/mg with a Km of 13.8 microM for arachidonate and Vmax. of 1500 nmol of O2/nmol of enzyme, whereas hCOX-2 had a specific activity of 12.2 mumol of O2/mg with a Km of 8.7 microM for arachidonate and a Vmax. of 1090 nmol of O2/nmol of enzyme. Indomethacin inhibited both hCOX-1 and hCOX-2, whereas NS-398 and Dup-697 selectively inhibited hCOX-2. Both NS-398 and Dup-697 exhibited time-dependent inactivation of hCOX-2, as did indomethacin on both enzymes. The competitive inhibitor of hCOX-1, mefenamic acid, also displayed competitive inhibition of hCOX-2. These results demonstrate the ability to generate selective non-steroidal anti-inflammatory drugs (NSAIDs), which could provide useful improvement therapeutically in the treatment of chronic inflammatory disease.
Nonsteroidal antiinflammatory drugs (NSAIDs) are widely used for the treatment of inflammatory diseases, but significant side effects such as gastrointestinal erosion and renal damage limit their use. NSAIDs inhibit the enzyme cyclooxygenase (COX), which catalyzes the conversion of arachidonic acid to prostaglandins (PGs) and thromboxane. Two forms of COX have been identified--COX-1, which is constitutively expressed in most tissues and organs, and the inducible enzyme, COX-2, which has been localized primarily to inflammatory cells and tissues. In an animal model of acute inflammation (injection of carrageenan into the footpad), edema was produced that was associated with marked accumulation of COX-2 mRNA and thromboxane. A selective inhibitor of COX-2 (SC-58125) inhibited edema at the inflammatory site and was analgesic but had no effect on PG production in the stomach and did not cause gastric toxicity. These data suggest that selective inhibition of COX-2 may produce superior antiinflammatory drugs with substantial safety advantages over existing NSAIDs.
The antinociceptive effect of spinally delivered S- and R-flurbiprofen, enantiomers of 2-arylpropionic acid, was studied in rats using the formalin test. Intrathecal injections of S- or R-flurbiprofen produced a significant reduction of the second phase of the formalin test, with no effect on the first phase. The maximal suppression of the second phase was similar for both agents (about 50% at the highest doses). While both agents were active, S-flurbiprofen was significantly more potent than R-flurbiprofen. The potency ratio between the dose-response curves was 10 (6-20; 95% confidence intervals). These results demonstrate that both S- and R-flurbiprofen produce antinociception after spinal administration. The potency difference is similar to that for inhibition of cyclooxygenase in brain tissue and supports the hypothesis that cyclooxygenase products are involved in prolonged spinal nociceptive transmission.
Repetitive C afferent input evokes a facilitated state of processing that results in increased receptive fields and exaggerated responses to afferent input ("wind-up"). These phenomena underlie the behavioral phenomena of secondary hyperalgesia and this in turn is an important component of postoperative pain. The initiation of this facilitated component is not well blocked by even higher concentrations of volatile anesthetics, but it can be prevented by pretreatment with agents known to block afferent input (local anesthetics) or C-fiber transmitter release (opiates) or to act at one of several links to block a complex spinal cascade involving the N-methyl-D-aspartate receptor, nitric oxide synthase, and cyclooxygenase. These fundamental mechanisms promise to have an impact on the management of postoperative pain.
Prostaglandins play important and diverse roles in the CNS. The first step in prostaglandin synthesis involves enzymatic oxidation of arachidonic acid, which is catalyzed by prostaglandin H(PGH) synthase, also referred to as cyclooxygenase. We have cloned an inducible form of this enzyme from rat brain that is nearly identical to a murine, mitogen-inducible cyclooxygenase identified from fibroblasts. Our studies indicate that this gene, here termed COX-2, is expressed throughout the forebrain in discrete populations of neurons and is enriched in the cortex and hippocampus. Neuronal expression is rapidly and transiently induced by seizures or NMDA-dependent synaptic activity. No expression is detected in glia or vascular endothelial cells. Basal expression of COX-2 appears to be regulated by natural synaptic activity in the developing and adult brain. Both basal and induced expression of COX-2 are inhibited by glucocorticoids, consistent with COX-2 regulation in peripheral tissues. Our studies indicate that COX-2 expression may be important in regulating prostaglandin signaling in brain. The marked inducibility in neurons by synaptic stimuli suggests a role in activity-dependent plasticity.
Nonsteroidal anti-inflammatory drugs (NSAIDs) currently available for clinical use inhibit both COX-1 and COX-2. This suggests that clinically useful NSAIDs inhibit pro-inflammatory prostaglandins (PGs) derived from the activity of COX-2, as well as PGs in tissues like the stomach and kidney (via COX-1). A new class of compounds has recently been developed (SC-58125) that have a high degree of selectivity for the inducible form of cyxlooxygenase (COX-2) over the constitutive form (COX-1). This unique class of compounds exhibit a time-dependent irreversible inhibition of COX-2, while reversibly inhibiting COX-1. The molecular basis of this selectivity was probed by site-directed mutagenesis of the active site of COX-2. The sequence differences in the active site were determined by amino acid replacement of the COX-2 sequences based on the known crystal structure of COX-1, which revealed a single amino acid difference in the active site (valine 509 to isoleucine) and a series of differences at the mouth of the active site. Mutants with the single amino acid substitution in the active site and a combination of three changes in the mouth of the active site were made in human COX-2, expressed in insect cells and purified. The single amino acid change of valine 509 to isoleucine confers selectivity of COX-2 inhibitors in the class of SC-58125 and others of the same class (SC-236, NS-398), while commonly used NSAIDs such as indomethacin showed no change in selectivity. Substitutions of COX-1 sequences in COX-2 at the mouth of the active site of COX-2 did not change the selectivity of SC-58125. This indicates that the single amino acid substitution of isoleucine at position 509 for a valine is sufficient to confer COX-2 selectivity in this example of a diaryl-heterocycle COX inhibitor.
Prostaglandins (PG) have been described as mediators in spinal nociceptive processing after peripheral inflammation. Enzymes essential for PG biosynthesis, cyclooxygenase isozymes COX-1 and COX-2, have not yet been investigated in the spinal cord. In two studies on rats with adjuvant-induced peripheral inflammation levels of mRNA expression of both COX isoforms were analyzed in the lumbar section of the spinal cord using reverse transcription-polymerase chain reaction (RT-PCR) technique. We could show that mRNA of both COX isoforms is expressed constitutively in the spinal cord with COX-2 as the predominant isoform. Six hours after induction of peripheral inflammation, levels of COX-2 mRNA expression were raised significantly in respect to untreated control rats and returned to baseline within 3 days after induction of inflammation. COX-2 might therefore be regarded as the COX isozyme responsible for spinal PG release in nociceptive processing under a peripheral inflammatory stimulus.
Formalin injected subcutaneously into the paw is a frequently used pain assay; it evokes an initial period of licking and flinching followed by a period of quiescence and last by a second period of intense and protracted licking and flinching. The prominent second phase is believed to reflect the development of a central (spinal cord) facilitation. This conclusion is based on the assumption that formalin evokes an initial burst of activity in fine afferent fibers, followed by prolonged low levels of activity in C fibers. Detailed reports substantiating this essential assumption have not been published. Thus, we recorded in situ from single sural nerve fibers, identified by their conduction velocity and modality, in the barbiturate anesthetized rat. Following formalin (2.5%, 50 microliters) injection into their receptive fields, phase-1 activity was prominent in A beta and A delta fibers as well as in high-threshold C nociceptive afferent fibers. Phase-2 activity was observed in A delta fibers with receptive fields in hairy skin and in all mechanically sensitive C fibers. Mean phase-2 activity in these fibers was 1/2-2/3 of the magnitude achieved in phase 1. Mechanically insensitive fibers and A delta and C fibers with receptive field centers outside of the injection zone began firing 15 min or more post-injection and would contribute to phase-2, but not phase-1, behavioral activity. Intravenous infusion of low doses of lidocaine yielding plasma levels of 3.6-7.9 micrograms/ml administered during phase 2 blocked formalin-evoked activity in primary afferent fibers in a dose-related fashion without blocking either electrically or mechanically evoked activity. Effective plasma doses were comparable to those found to relieve neuropathic pain. These data indicate that phase 2 in the formalin test is more closely related to ongoing afferent input than had previously been thought.
The chapter compares and contrasts the structural and kinetic properties of prostaglandin endoperoxide H synthase-1 (PGHS-1) and -2. It also discusses the description of the interactions of the two isozymes with nonsteroidal anti-inflammatory drugs (NSAIDs). There are three general areas of study important to understanding more about PGHS isozymes: mechanisms of catalysis, regulation of gene expression, and subcellular functional independence. The chapter also explain how is arachidonic acid specifically channeled to PGHS-2 following mobilization in cells in response to TPA and how is the localization of PGHS-2 in the nuclear envelope important for PGHS-2 functioning. The chapter also describes the structures and regulation of expression of the PGHS-1 and -2 genes. The concepts that PGHS-1 and PGHS-2 represent two separate prostaglandin biosynthetic pathways and two separate prostaglandin signaling pathways need to be tested. The chapter also discusses how the two isozymes may act independently in intact cells to mediate the formation of prostanoids destined to act on cell surface and/or nuclear targets to mediate different biological and pathobiological events.
Pharmacological studies have implicated the spinal activation of excitatory amino acids, nitric oxide, and prostaglandins systems in the development of tactile and thermal hypersensitivity and central sensitization after peripheral inflammation. In the present study, using a chronically placed loop dialysis catheter, we examined in the unanesthetized rat the effect of carrageenan/kaolin (C/K)-induced knee joint inflammation on the time course of spinal release of several active factors including excitatory amino acids (glutamate, aspartate), citrulline (a marker of nitric oxide formation), and prostaglandin E2 (PGE2) as well as the concomitant development of tactile and thermal hypersensitivity. Infection of C/K in the knee evoked a significant release of glutamate, with an initial peak seen immediately after knee C/K injection (179 +/- 22%) and with a progressive and consistent increase over a period of 24 h (153-186%). Comparable changes in the concentration of aspartate (123-179%) were observed. Citrulline was constantly above baseline for the 24-h period (121-158%). PGE2 was significantly increased at 10 min (146 +/- 11%) with no change observed between 3-5 h. At 24 h, PGE2 was again significantly (143 +/- 18%) increased. Behaviorally, a prominent thermal and tactile allodynia developed after injection with the peak seen by 1-3 h after induction of the inflammation. This hypersensitivity state, while diminished in its intensity, persisted for the entire observation period. These data suggest that increased spinal release of excitatory amino acids (EAA), nitric oxide and/or PGE2 is involved in the maintenance of the pain state initiated by acute peripheral inflammation.
A series of sulfonamide-containing 1,5-diarylpyrazole derivatives were prepared and evaluated for their ability to block cyclooxygenase-2 (COX-2) in vitro and in vivo. Extensive structure-activity relationship (SAR) work was carried out within this series, and a number of potent and selective inhibitors of COX-2 were identified. Since an early structural lead (1f, SC-236) exhibited an unacceptably long plasma half-life, a number of pyrazole analogs containing potential metabolic sites were evaluated further in vivo in an effort to identify compounds with acceptable pharmacokinetic profiles. This work led to the identification of 1i (4-[5-(4-methylphenyl)-3-(trifluoromethyl)- H-pyrazol-1-yl]benzenesulfonamide, SC-58635, celecoxib), which is currently in phase III clinical trials for the treatment of rheumatoid arthritis and osteoarthritis.
To administer drugs into the spinal subarachnoid space of unanesthetized and intact rats and rabbits, a procedure is described whereby a polyethylene catheter (PE-10) may be inserted through a puncture of the atlanto-occipital membrane and secured to the skull. Calibration experiments carried out with bromophenol blue dye, 3H-naloxone and 14C-urea revealed first, that there was little rostro-caudal diffusion of the injectate along the spinal axis and secondly, that even for compounds such as naloxone which can rapidly permeate neural tissues, the levels which do appear in the brain are small following the spinal subarachnoid administration of the drug. Control injections, administered either acutely or repeatedly over a prolonged period of time, had no detectable effect on the animal's behavior. These observations, as well as the lack of pathology in the spinal cords of rats having such catheters for periods of up to 4 months suggests that the implant is well tolerated.
Central and Peripheral Mechanism for the Antialgesic Action of Acetylsalicylic Acid
- Eds
Eds, Central and Peripheral Mechanism for the Antialgesic Action of Acetylsalicylic Acid. Raven Press, New York, NY, pp. 137–152.
Pre-clinical models of nociception
- T L Yaksh
Yaksh, T.L., 1997. Pre-clinical models of nociception. In: Biebuyck, J.F., Ž. Lynch, C., Maze, M., Saidman, L.J., Yaksh, T.L., Zapol, W. Eds, Pre-clinical Models of Nociception. Lippincott-Raven, Philadelphia, PA, in press.
Intrathecally administered cyclooxygenase-2 inhibitors attenuate carrageenan-induced thermal hyperalgesia in the rat
- D L Hammond
- S A Gregory
Hammond, D.L., Gregory, S.A., 1996. Intrathecally administered cyclo-oxygenase-2 inhibitors attenuate carrageenan-induced thermal hyper-algesia in the rat. Soc. Neurosci. Abs. 26th Annual Meeting, 712.17.
Central and peripheral mechanism for the antialgesic action of acetylsalicylic acid
- T L Yaksh