Cox-2 gene expression in chemically induced skin papillomas cannot predict subsequent tumor fate.

Elevated cyclooxygenase-2 (COX-2) expression is observed in a variety of premalignant neoplastic tissues, suggesting COX-2 expression might serve as a potential indicator of subsequent tumor development. However, it has not been possible to compare the relationship between Cox-2 gene expression in premalignant lesions and their subsequent fate, because conventional studies require tissue destruction for analysis of gene expression. To monitor COX-2 expression non-invasively during tumor development, we created a Cox-2 luciferase knock-in mouse, Cox-2(luc), in which the firefly luciferase coding region replaces the Cox-2 coding region. Luciferase activity was non-invasively, quantitatively and repeatedly monitored in Cox-2(luc/+) mice subjected to DMBA/TPA multistage skin tumor induction. Luciferase activity is significantly higher in all papillomas than in surrounding skin. However, the magnitude of Cox-2 promoter-driven luciferase activity in small papillomas cannot predict subsequent papilloma regression or growth. Elevated Cox-2 promoter-driven luciferase signal can be detected when papillomas first become visible, but not before this time.

COX-2 expression and function in the hyperalgesic response to paw inflammation in mice.

Peripheral inflammation and edema are often accompanied by primary and secondary hyperalgesia which are mediated by both peripheral and central mechanisms. The role of cyclooxygenase-2 (COX-2)-mediated prostanoid production in hyperalgesia is a topic of substantial current interest. We have established a murine foot-pad inflammation model in which both pharmacologic and genetic tools can be used to characterize the role of COX-2 in hyperalgesia. Zymosan, an extract from yeast, injected into the plantar surface of the hindpaw induces an edema response and an increase in COX-2 expression in the hindpaw, spinal cord and brain. Zymosan-induced primary hyperalgesia, measured as a decrease in hindpaw withdrawal latency in response to a thermal stimulus, is long-lasting and is not inhibited by pre-treatment with the systemic COX-2 selective inhibitor, parecoxib (20 mg/kg). In contrast, the central component of hyperalgesia, measured as a reduction in tail flick latency in response to heat, is reduced by parecoxib. Zymosan-induced primary hyperalgesia in Cox-2-/- mice is similar to that of their Cox-2+/+ littermate controls. However, the central component of hyperalgesia is substantially reduced in Cox-2-/- versus Cox-2+/+ mice, and returns to baseline values much more rapidly. Thus pharmacological data suggest, and genetic experiments confirm, (i) that primary hyperalgesia in response to zymosan inflammation in the mouse paw is not mediated by COX-2 function and (ii) that COX-2 function plays a major role in the central component of hyperalgesia in this model of inflammation.

Imaging cyclooxygenase-2 (Cox-2) gene expression in living animals with a luciferase knock-in reporter gene.

The cyclooxygenase-2 (Cox-2) gene plays a role in a variety of normal and pathophysiological conditions. Expression of the Cox-2 gene is induced in a broad range of cells, in response to many distinct stimuli. The ability to monitor and quantify Cox-2 expression noninvasively in vivo may facilitate a better understanding of the role of Cox-2, both in normal physiology and in different diseases. We generated a "knock-in" mouse in which the firefly luciferase reporter enzyme is expressed at the start site of translation of the endogenous Cox-2 gene. Correlation of luciferase and Cox-2 expression was confirmed in heterozygous Cox-2luc/+ mouse embryonic fibroblasts isolated from the knock-in mouse. In an acute sepsis model, following injection of interferon gamma and endotoxin, ex vivo imaging and Western blotting demonstrated coordinate Cox-2 and luciferase induction in multiple organs. Using both paw and air pouch inflammation models, we can monitor repeatedly localized luciferase expression in the same living mouse. Cox-2luc/+ knock-in mice should provide a valuable tool to analyze Cox-2 expression in many disease models.

Inhibitory effect of sildenafil on gastrointestinal smooth muscle: role of NO-cGMP transduction pathway.

Nitric oxide (NO) is an important neurotransmitter in the gut and has been demonstrated to be a key physiological mediator of non-adrenergic non-cholinergic (NANC) relaxation of gastrointestinal smooth muscle. In the present study the effect of PDE 5 inhibitor sildenafil on the gastrointestinal function (gastric emptying and intestinal transit) has been demonstrated in mice. Sildenafil (0.5-2 mg/kg, po) did not alter the percent gastric emptying however, in higher doses (5, 10 and 30 mg/kg, po) it inhibited the gastric emptying. On acute administration (0.5-5 mg/kg, po) it did not alter the intestinal transit but in higher doses (10 and 30 mg/kg, p.o.) delayed the intestinal transit. Further, the inhibitory effect of sildenafil was significantly blocked by L-NAME (10 mg/kg, ip), a non-selective NOS inhibitor and methylene blue (1 mg/kg, ip), a guanylate cyclase inhibitor. These findings suggest the participation of NO-cGMP transduction pathway in the inhibitory effect of sildenafil (higher doses) on the gastrointestinal smooth muscles and its potential application in patients with nutcracker oesophagus, hypertensive lower oesophageal sphincter (LOS), achalsia and diabetic gastroparesis or colitis where there is a loss of nNOS.

Aggravation of inflammatory bowel disease by cyclooxygenase-2 inhibitors in rats.

The objective of the present study was to determine the effect of a selective cyclooxygenase-2 (COX-2) inhibitor in in-vivo dextran sodium sulfate (DSS)-stimulated distal colon tissues of the rat. Longitudinal colon tissue sections from DSS-treated rats exhibited noticeable inflammation, altered contraction, increased myleoperoxidase activity, and oxidative stress. When the animals were pretreated with celecoxib, a selective COX-2 inhibitor, the flare of the colon was further worsened in terms of all the parameters studied. There was a reduction in PGE2 levels on chronic administration of celecoxib in DSS-treated animals. The results of the present study suggest that COX-2 enzyme and prostaglandins derived from COX-2 might play a defensive role in protecting ulceration of the colon akin to that seen in the upper gastrointestinal tract.

Isobolographic analysis of interaction between cyclooxygenase inhibitors and tramadol in acetic acid-induced writhing in mice.

Non-steroidal anti-inflammatory drugs (NSAIDs) and opioids are the most commonly used analgesics in the management of acute and chronic pain. Combined use of NSAIDs and opioids has been indicated for achieving better analgesia with reduced side effects. The present study was aimed at evaluating the combination of different NSAIDs, which inhibit cyclooxygenase (COX) enzymes and tramadol against acetic acid-induced writhing in mice. The expected beneficial effect of combination regimen was analyzed by isobolographic analysis. The oral and intrathecally administered tramadol, a mu-opioid and naproxen, a nonselective COX inhibitor produced dose-dependent antinociception, however, rofecoxib, a selective COX-2 inhibitor lacked analgesic efficacy in writhing test. Isobolographic analysis showed synergistic or supra-additive interactions for the combinations of naproxen and tramadol after oral and intrathecal administration. However, similar interaction was not observed when tramadol was combined with rofecoxib. Pretreatment with naloxone partially reversed the antinociceptive effect of tramadol per se and its combination with naproxen without modifying the per se effect of NSAID. The results demonstrated marked synergistic interaction between naproxen and tramadol and such interaction involved opioid as well as non-opioid mechanisms of tramadol and inhibition of COX-1 but not COX-2 by naproxen.

Modulatory effect of diclofenac on antispasmodic effect of pitofenone in cholinergic spasm.

Biliary, ureteric and intestinal colic are extremely common clinical conditions associated with smooth muscle spasm. In the present study, antispasmodic activity was carried out against acetylcholine (10-640 ng/ml)-induced contractions on guinea pig ileum. Acetylcholine (10-640 ng/ml) induced concentration-dependent contraction of smooth muscle. Diclofenac, in varying concentration (9.4 x 10(-5) mol/l and 14.1 x 10(-5) mol/l) shifted the concentration response curve of acetylcholine to the right without suppressing the maximal response. However, in higher concentration diclofenac (18.9 x 10(-5) mol/l) blocked the response in an unsurmountable fashion. Further, analgin (11.09 x 10(-5), 16.63 x 10(-5) and 22.18 x 10(-5) mol/l) in equimolar concentrations did not alter the concentration response curve of acetylcholine, but in higher concentration analgin (44.36 x 10(-5) mol/l) also blocked the response in an unsurmountable fashion. Pitofenone (2.5 x 10(-6) mol/l) also, shifted the concentration response curve of acetylcholine to right in a parallel fashion with no change in maximal response. The present study confirms the potent antispasmodic activity of diclofenac-pitofenone combination in comparison to analgin-pitofenone in molar equivalent concentration (in comparison to diclofenac) against acetylcholine-induced contractions of guinea pig ileum.

Pharmacological profile of parecoxib: a novel, potent injectable selective cyclooxygenase-2 inhibitor.

The antinociceptive, anti-inflammatory, antipyretic effects along with gastric safety profile of parecoxib, a novel, potent selective cyclooxygenase-2 inhibiting prodrug, and those of ketorolac, a nonselective cyclooxygenase inhibitor, were evaluated in various animal models. Parecoxib (up to 20 mg/kg, i.v.) had no effect in two acute pain models, namely, the acetic acid-induced writhing (visceral pain) and the formalin test (tonic pain). However, ketorolac (up to 10 mg/kg, i.v.) showed marked antinociceptive effects in these models. In the models of carrageenan-provoked inflammatory hyperalgesia and inflammation, and in lipopolysaccharide-induced pyrexia, parecoxib significantly reversed all the behavioral changes and it was found to be more potent than ketorolac. Further, ketorolac (10 mg/kg, i.v.) produced visible gastric lesions with prominent petechiae and hemorrhagic streaks. However, parecoxib was without any effect on gastric mucosa. The present results showed that the cyclooxygenase-2 inhibitor, parecoxib, when administered parenterally, has potent antihyperalgesic, anti-inflammatory, antipyretic effects and has a better safety profile than with ketorolac, with sparing of cyclooxygenase-1 in the stomach in these animal models.

Cholinergic-NO-cGMP mediation of sildenafil-induced antinociception.

Acetylcholine and cholinomimetic agents with predominant muscarinic action are known to increase the concentration of cGMP by activation of nitric oxide signaling pathway in the nociceptive conditions. The present study was aimed to investigate the NO-cGMP-PDE5 pathway in nociceptive conditions in the experimental animals. Nociceptive threshold was assessed by acetic acid-induced writhing assay (chemonociception) or carrageenan-induced hyperalgesia. Sildenafil [1-5 mg/kg, ip, 50-200 microg/paw, intraplantar (ipl)] produced dose dependent antinociception in both the tested models. Coadministration of acetylcholine (50 mcg/paw, ipl) or cholinomimetic agent, neostigmine (0.1 mcg/kg, ip and 25 ng/paw, ipl) augmented the peripheral antinociceptive effect of sildenafil. This effect was sensitive to blockade by L-NAME (20 mg/kg, ip, 100 microg/paw, ipl), a non-selective NOS inhibitor and methylene blue (1 mg/kg, ip), a guanylate cyclase inhibitor, which per se had little or no effect in both the models of nociception. Further, the per se analgesic effect of acetylcholine and neostigmine was blocked by both L-NAME and methylene blue in the models of nociception, suggesting the activation of NO-cGMP pathway. Also, both L-NAME and methylene blue blocked the per se analgesic effect of sildenafil. These results indicate the peripheral accumulation of cGMP may be responsible for antinociceptive effect, and a possible interaction between cholinergic agents and PDE5 system in models of nociception.

Effect of selective inhibition of cyclooxygenase-2 on lipopolysaccharide-induced hyperalgesia.

Lipopolysaccharide (LPS) is known to increase the expression and release of various pro-inflammatory mediators, including cyclooxygenase-2 (COX-2) and produce hyperalgesia. It is also well known that prostaglandins (PGs), synthesised both in the periphery and centrally by COX isoforms, play a key role in sensitisation of nociceptors and nociceptive processing. To investigate the role of COX-2 in LPS-induced hyperalgesia, parecoxib, a selective COX-2-inhibiting pro-drug, was injected intravenously 30 min before assessing hyperalgesia induced by intraperitoneal or subcutaneous administration of LPS (50 microg/mouse or 25 microg/paw of rat, respectively). Acetic acid-induced writhing and tail immersion assay in mice and paw withdrawal response to thermal and mechanical stimuli in rats were used to assess the effect of inhibition of COX-2 on LPSinduced hyperalgesia. Animals showed significant hyperalgesic behavior 8 h after LPS injection. Parecoxib (up to 20 mg/kg, i.v.) had no effect in the two acute nociceptive assays but showed marked antinociceptive activity in writhing and tail immersion assay in LPS-pretreated mice. Similarly, parecoxib reversed the hyperalgesia in the LPS-injected paw but not in the contralateral paw of rats. Pre-treatment with dexamethasone, an inhibitor of COX-2 expression before LPS injection significantly affected the development of hyperalgesia in both mice and rats. These findings suggest that inducible COX-2 derived PGs are involved in central nociceptive processing, which resulted in hyperalgesic behavior following LPS administration and inhibition of COX-2 or its expression attenuated LPS-induced hyperalgesia.

Modulatory effect of cyclooxygenase inhibitors on sildenafil-induced antinociception.

Peripheral activation of the NO-cGMP pathway has been implicated in various nociceptive conditions. The antinociceptive effect of the PDE-5 inhibitor, sildenafil, alone or in combination with cyclooxygenase inhibitor diclofenac and nimesulide, was assessed in the different animal models of peripheral nociception. In the present study we investigated the possible interaction between cyclooxygenase and NO-cGMP pathway in writhing assay and carrageenan-induced hyperalgesia in mice and rats, respectively. Sildenafil [1-2 mg/kg, i.p. or 50-100 microg/paw, intraplantar (i.pl.)], nimesulide (1-2 mg/kg, i.p. or 25-50 microg/paw, i.pl.) and diclofenac (1-2 mg/kg, i.p. or 25-50 microg/paw, i.pl.) exhibited an antinociceptive effect in both the models. When ineffective doses of sildenafil (0.5 mg/kg, i.p and 25 microg/paw, i.pl.) were co-administered with ineffective doses of nimesulide (0.5 mg/kg, i.p. and 10 microg/paw, i.pl.) and diclofenac (0.5 mg/kg, i.p. and 10 microg/paw, i.pl.), there was a significant increase in the antinociceptive effect in both the models of peripheral nociception. Further, the potentiation of the effect was blocked by L-NAME (20 mg/kg, i.p., 100 microg/paw, i.pl.), a non-selective NOS inhibitor and methylene blue (1 mg/kg, i.p.), a guanylate cyclase inhibitor. L-NAME or methylene blue itself had little or no effect on both the models of hyperalgesia. These results suggest that cyclooxygenase, NO and cGMP are relevant in the combination-induced antinociception. In conclusion, sildenafil induced antinociception, and its potentiation of the effect of the cyclooxygenase inhibitors nimesulide and diclofenac was probably mediated through the activation of the NO-cGMP pathway and inhibition of cyclic GMP degradation.

Effect of nimesulide on acetic acid- and leukotriene-induced inflammatory bowel disease in rats.

Inflammatory bowel disease (IBD) is a relapsing inflammation of intestine, which is mediated by release of inflammatory mediators. Both cyclo-oxygenase product prostaglandin (PGE2) and lipo-oxygenase product leukotriene (LTB4), may contribute to the pathogenesis of the inflammatory response. Nimesulide, a preferential COX-2 inhibitor was evaluated for its efficacy against experimental colitis in two different models (acetic acid- and LTB4-induced IBD) in rats. Inflammatory response was induced by intrarectal single administration of acetic acid or LTB4. Nimesulide (9 and 18 mg/kg, p.o.) significantly prevented development of inflammatory changes, decreased myeloperoxidase (MPO) activity, and also restored the altered contractility response of the isolated colon segment to KCl. The results suggested the involvement of both cyclo-oxygenase (COX) and lipo-oxygenase-mediated proinflammatory agents in colonic inflammatory process associated with IBD. Further, this study suggests that such therapeutic interventions may be of value in the treatment of IBD.

Paradoxical effects of opioid antagonist naloxone on SSRI-induced analgesia and tolerance in mice.

Selective serotonin reuptake inhibitors (SSRIs) have been used clinically as co-analgesics in various devastating painful conditions. Upon chronic treatment tolerance develops to their analgesic effect, which is often refractory to increasing dose. Although modulation of serotonergic pathways considerably explains their clinical efficacy, numerous reports nevertheless indicate the direct/indirect role of the opioidergic pathway in SSRI-induced analgesia. The present study was designed to investigate the effect, if any, of the opioid antagonist naloxone on SSRIs-induced analgesia and tolerance employing acetic acid-induced writhing assay. Two SSRIs, fluoxetine (FLX), and citalopram (CTP) were used in the study. Acute systemic (5-40 mg kg(-1) i.p.), or intrathecal (5-40 microg per mouse, i.t.) administration of fluoxetine or citalopram exhibited a dose-dependent and significant (p < 0.05) antinociceptive effect. Single systemic (2-5 mg kg(-1) i.p.) or intrathecal (1 microg per mouse, i.t.) administration of opioid antagonist naloxone blocked where as systemic ultra-low dose (10 ng/kg) or intrathecal (0.05 ng) naloxone potentiated the acute antinociceptive effect of both SSRIs (10 mg kg(-1) i.p. and 10 microg i.t.). Animals treated chronically over a 7-day period with SSRIs developed tolerance to their antinociceptive effect. Further, chronic administration of ultra-low dose of naloxone intrathecal (0.05 ng per mouse, i.t.) or systemic (10 ng kg(-1) i.p.) with fluoxetine or citalopram (10 microg i.t.; 5 mg kg(-1) i.p.) over a 7-day period reversed the tolerance to the antinociceptive effect of SSRIs. Thus, in ultra-low doses, naloxone paradoxically enhances SSRIs-induced analgesia and reverse tolerance through spinal and peripheral action. These effects of opioid antagonist naloxone on SSRIs-induced antinociception may have an implication in refractory cases upon chronic use of SSRIs as co-analgesics.

Protective effect of flavonoids against aging- and lipopolysaccharide-induced cognitive impairment in mice.

Flavonoids, naturally occurring polyphenolic compounds, are known to inhibit both lipopolysaccharide (LPS) stimulated tumor necrosis factor alpha and interleukin 6 release which modulate the proinflammatory molecules that have been reported in many progressive neurodegenerative disorders, including Alzheimer's disease (AD), viral and bacterial meningitis, AIDS dementia complex, and stroke. The present experiments were performed to study the possible effects of exogenously administered flavonoids (apigenin-7-glucoside and quercetin) on the cognitive performance in aged and LPS-treated mice (an animal model for AD) using passive avoidance and elevated plus-maze tasks. Aged and LPS-treated mice showed poor retention of memory in step-through passive avoidance and in plus-maze tasks. Chronic administration of the flavonoids apigenin-7-glucoside (5-20 mg/kg i.p.) and quercetin (25-100 mg/kg i.p.) dose dependently reversed the age-induced and LPS-induced retention deficits in both test paradigms. However, flavonoids after chronic administration in young mice did not show any improvement of memory retention in both paradigms. Apigenin-7-glucoside showed more efficacy as compared with quercetin in both models that may be probably due to its greater efficacy to inhibit cyclooxygenase-2 and inducible nitric oxide synthase. Chronic treatment with flavonoids did not alter the locomotor activity in both young and aged mice; however, aged mice showed improvement of performance on Rota-Rod test. The results showed that chronic treatment with flavonoids reverses cognitive deficits in aged and LPS-intoxicated mice which suggests that modulation of cyclooxygenase-2 and inducible nitric synthase by flavonoids may be important in the prevention of memory deficits, one of the symptoms related to AD.

Sildenafil, a phosphodiesterase-5 inhibitor, enhances the antinociceptive effect of morphine.

Various evidence has demonstrated a role of the nitric oxide (NO)/cGMP signaling pathway in the processing of nociception. The exact role of phosphodiesterase-5 (PDE-5) via the NO/cGMP pathway is not fully understood in pain response. The aim of the present study was to investigate the possible peripheral interaction between a PDE-5 inhibitor (sildenafil) and morphine. Carrageenan-induced hyperalgesia in rats and the acetic-acid-induced writhing test in mice were used as animal models. Local administration of sildenafil (50-200 microg/paw, i.pl.) exhibited a dose-dependent antinociceptive effect against the paw pressure test. Sildenafil also demonstrated an antinociceptive effect (1-10 mg/kg, i.p.) against in the writhing test. Co-administration of sildenafil (100 microg/paw, i.pl. and 2 mg/kg, i.p.) significantly enhanced the antinociceptive effect of morphine (2 microg/ paw, i.pl. and 2 mg/kg, i.p respectively). The antinociception produced by the drugs alone or combined was due to a local action, as its administration in the contralateral paws was ineffective. Pretreatment with N(G)-nitro-L-arginine methyl ester (an NO synthesis inhibitor), methylene blue (gunalyl cyclase inhibitor) or naloxone (opioid receptor antagonist) blocked the effect of a sildenafil-morphine combination in both tests. The results suggest that opioid receptor (NO and cGMP) mechanisms are involved in the combined antinociceptive effect. Further, sildenafil produced antinociception per se and increased the response of morphine, probably through the inhibition of cGMP degradation.

Modulation of NSAID-induced antinociceptive and anti-inflammatory effects by alpha2-adrenoceptor agonists with gastroprotective effects.

Tizanidine, an alpha2-adrenergic receptor agonist with myospasmolytic action, is indicated for the treatment of back pain either as monotherapy or in combination with nonsteridal anti-inflammatory drugs (NSAIDs). Tizanidine (0.25-1.0 mg/kg) significantly produced analgesic and anti-inflammatory effect in acetic acid induced writhing in mice and carrageenan-induced paw edema in rats, respectively. The effects were comparable with clonidine (0.25 and 0.50 mg/kg), another alpha2-agonist. Yohimbine (1 mg/kg), alpha2-adrenergic antagonist reversed the effect of tizanidine. Tizanidine (0.25 mg/kg) and clonidine (0.25 mg/kg) significantly potentiated the antinociceptive and anti-inflammatory effect of NSAIDs (nimesulide, meloxicam and naproxen). Tizanidine (1 mg/kg) did not alter basal pH, acidity (free and total) of gastric content and did not produce any mucosal injury in fasted rats. Tizanidine (1 mg/kg) significantly reduced meloxicam (UD50 3.21 mg/kg), nimesulide (UD50 24.52 mg/kg) and naproxen (UD50 14.10 mg/kg)-induced ulcerogenic effect (ulcer index, pH and free/total acidity). It is expected that tizanidine exerted gastrotprotection through stimulation of gastric and central alpha2-adrenergic receptors. Present investigation suggested that tizanidine not only enhance the analgesic and anti-inflammatory effect of NSAIDs but also improved gatstrointestinal tolerability of NSAIDs through modulation of central alpha-2-receptors. From this study, it can be speculated that tizanidine and NSAID combination therapy would prove to be a novel approach to treat nociceptive/inflammatory conditions with improved gastric tolerability of NSAIDs.

Modulatory role of cyclooxygenase inhibitors in aging- and scopolamine or lipopolysaccharide-induced cognitive dysfunction in mice.

Inflammation processes may play a critical role in the pathogenesis of the degenerative changes and cognitive impairments associated with Alzheimer's disease (AD). Non-steroidal anti-inflammatory drugs are reported to be effective in reducing the risk of developing AD or cognitive impairments. Present experiments were performed to study the possible effect of various NSAIDs on cognitive performance of young, aged and scopolamine or lipopolysaccharide (LPS) treated mice (an animal model of AD) using one trial step through type of passive avoidance and in elevated plus maze task. Chronic administration of NSAIDs at the ED(50) doses (nimesulide, rofecoxib and naproxen for 15 days) significantly reversed the age or scopolamine-induced retention deficits in both test paradigms. However, in both the memory paradigms chronic administration of NSAIDs failed to modulate the retention performance of young mice. Acute administration of LPS (50 mcg/mouse, i.p.) significantly exhibited retention deficits after 24 h and seventh day of its administration in both test paradigms. Chronic administration (7 days) of rofecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor (1.92 mg/kg, p.o.) significantly reversed the LPS-induced retention deficits in both tests. The results of this study showed chronic treatment of NSAIDs reverses the cognitive deficits in age and scopolamine or LPS treated mice. These findings establish a link between the central nervous system expression of various pro-inflammatory cytokines and learning impairment in mice.