Cyclooxygenase-2 Inhibitor Nimesulide Research Articles

Acute heat trauma model in rats, gender-dependent thermoresistance, and screening of potential thermoprotectors

Heat trauma (HT) is an urgent medical and social problem. Heat damage is a widespread effect of the environment on humans, driven by global warming, military conflicts, technological disasters, work in hot environments, and engagement in extreme sports and tourism. The aim of the study: to propose a model of acute HT in rats that does not cause the death of animals, to determine the dependence of thermoresistance on gender, and to compare the effectiveness of the thermoprotective effects of a range of non-steroidal anti-inflammatory drugs (NSAIDs), paracetamol, and glucosamine hydrochloride in this model. Materials and Methods: The experiment was conducted on adult white rats of both genders. Acute HT was modelled by using a specially developed method involving heat exposure to animals at +55 °C for 30 minutes, followed by a recovery period of 60 minutes. Rectal temperature was measured every 15 minutes. The degree of hyperthermia in males and females was determined. The presence and intensity of the thermoprotective effect of glucosamine hydrochloride (G h/ch), diclofenac sodium, acetylsalicylic acid (ASA), nimesulide, etoricoxib, celecoxib, and paracetamol were evaluated through intragastric administration 60 minutes before heat exposure. The results were analyzed using the STATISTICA 12.0 program. Results: It was established that heat exposure at +55 °C for 30 minutes effectively replicates acute HT in rats without causing animal fatalities, adhering to bioethical requirements. Body temperature increases by 10-13 %, characterized as a heat stroke. Occasionally, thermoresistant animals are encountered, where the temperature increase during the first 15 minutes of exposure is less than 1 °C. These animals should not be used for further modelling of heat trauma. Male rats are more sensitive to the effect of high environmental temperatures than females, exhibiting greater hyperthermia (temperature increase of 5.03±0.39°C compared to 3.72±0.22 °C in females, p<0.01). The thermoprotective effect of glucosamine hydrochloride depends on gender, being more pronounced in males. Among the 6 tested COX inhibitors, the most significant thermoprotective effect was observed in the highly selective COX-2 inhibitor celecoxib and the weakly selective central inhibitor paracetamol, warranting in-depth research into their impact on organ and system states following heat trauma, as well as the mechanisms of their thermoprotective action. The thermoprotective effect is not associated with selectivity towards COX (cyclooxygenase): it is not observed in the highly selective COX-2 inhibitor etoricoxib and moderately selective COX-2 inhibitor nimesulide, as well as in non-selective COX inhibitors such as diclofenac sodium and aspirin, which also slows down the recovery of body temperature after heat exposure. Conclusions: A convenient and simple model of acute HT in rats is proposed, demonstrating higher thermosensitivity and a more pronounced thermoprotective effect of glucosamine hydrochloride in males. A significant thermoprotective effect was identified in celecoxib and paracetamol, surpassing other investigated NSAIDs. The mechanism and specific features of this effect require further clarification

Safety of partial selective COX-2 inhibitors in patients with cross-reactive NSAID hypersensitivity and factors affecting safety

Aims: Partial selective COX-2 inhibitors, such as nimesulide, or meloxicam are tolerated by the majority of the patients with cross-reactive NSAID hypersensitivity. This study aimed to obtain more information about the safety of partial selective COX-2 inhibitors; nimesulide and meloxicam in non-immunologic type, cross-reactive NSAID hypersensitivity and to detect risk factors for intolerance to these drugs.
 Methods: This is a retrospective study of patients with suggestive of cross-reactive NSAID hypersensitivity who admitted to our clinic over a period of 10 years. Patients who had a reliable history of immediate type NSAIDs hypersensitivity with at least 2 chemically unrelated class and/or positive ASA provocation test and who underwent alternative drug provocation test with partial selective COX-2 inhibitors (nimesulide and/or meloxicam) were included to study. Patients’ demographics, comorbidities, atopy status, duration of NSAID hypersensitivity, total number of reactions, reaction grades, clinical phenotypes, pulmonary function test parameters and results of alternative drug provocation test results are recorded. Patients with and without reactions during alternative provocation tests with nimesulide and/or meloxicam were compared in terms of these data. 
 Results: A total of 560 patients were included in the study, 378 (67.5%) of them were female. Allergic comorbidities were detected in 394 (72.6%) patients. Asthma, other drug allergies and nasal polyp were the most common comorbidities. Alternative drug provocation test positivity with nimesulide and/or meloxicam was detected in 50 of 560 (8.9%) patients. Provocation test positivity was 33/541 (6.1%) with nimesulide, 30/517 (5.8%) with meloxicam and 13/498 (2.3%) with both nimesulide and meloxicam. Duration of NSAID hypersensitivity was shorter and allergic comorbidities, asthma, nasal polyp and the coexistence of asthma and nasal polyp were more common in patients with a positive provocation test.
 Conclusion: The partial selective COX-2 inhibitors nimesulide and meloxicam are well tolerated alternatives in patients with cross-reactive NSAID hypersensitivity. Hypersensitivity to these drugs is significantly higher in patients with asthma and/or nasal polyp than other group of cross-reactive NSAID hypersensitive patients and also in patients with a shorter duration of NSAID hypersensitivity.

Depletion of tumor-associated macrophages inhibits lung cancer growth and enhances the antitumor effect of cisplatin.

In lung cancer, tumor-associated macrophages (TAMs), especially M2-like TAMs, represent the main tumor progression components in the tumor microenvironment (TME). Therefore, M2-like TAMs may serve as a therapeutic target. The purpose of this study was to investigate the effect of M2-like TAM depletion in the TME on tumor growth and chemotherapy response in lung cancer. The levels of secreted monocyte chemoattractant protein (MCP-1) and prostaglandin E2 (PGE2) in the supernatants of lung cancer cell lines A549 and LLC were evaluated via ELISA. Cell migration assays were performed to assess the recruitment ability of macrophage cell lines THP-1 and J774-1 cells. Differentiation of macrophages was assessed via flow cytometry. Immunohistochemical staining was performed to visualize M2-like TAMs in transplanted lung cancer in mouse. We used the COX-2 inhibitor nimesulide to inhibit the secretion of MCP-1 and PGE2, which promotes macrophage migration and M2-like differentiation. Nimesulide treatment decreased the secretion of MCP-1 and PGE2 from lung cancer cells. Nimesulide treatment suppressed the migration of macrophages by blocking MCP-1. Lung cancer supernatant induced the differentiation of macrophages toward the M2-like phenotype, and nimesulide treatment inhibited M2-like differentiation by blocking MCP-1 and PGE2. In the lung cancer mouse model, treatment with nimesulide depleted M2-like TAMs in the TME and enhanced the tumor inhibitory effect of cisplatin. Our results indicated that blocking the secretion of MCP-1 and PGE2 from tumor cells depleted M2-like TAMs in the TME and the combination therapy with cisplatin considerably suppressed tumor growth in the LLC mouse model.

Alcohol reinstatement after prolonged abstinence from alcohol drinking by female adolescent rats: Roles of cyclooxygenase-2 and the prostaglandin E2 receptor 1

BackgroundAdolescent alcohol misuse is a global problem that can significantly increase the reinstatement of alcohol drinking during re-exposure after abstinence, but the mechanism that causes this increase is unknown. Female adolescents are an understudied population but they are particularly vulnerable to adolescent-onset alcohol abuse. We aimed to determine how adolescent-onset alcohol drinking affects pro-inflammatory mediators endothelin-1 (ET-1), cyclooxygenase-2 (COX-2), and prostaglandin E2 (PGE2) in the brain and the role of COX-2 and PGE2 in EtOH reinstatement in adolescent females. MethodsAdolescent female rats were exposed to a 2-bottle choice paradigm of water vs 5% ethanol (EtOH) every other day over a 21 day period. ET-1 and COX-2 proteins were measured in the dorsal striatum (DS) after a 4 week abstinence from EtOH drinking. The COX-2 inhibitor nimesulide was then administered during abstinence prior to an EtOH reinstatement or sucrose preference or to measure PGE2 content. The PGE2 receptor 1 (EP1) antagonist SC-51089 was then administered prior to EtOH reinstatement during which EtOH intake was measured. ResultsEtOH drinking significantly increased ET-1 by 33.8 ± 8.9% and COX-2 by 71.4 ± 24.3% in the DS. Treatment with nimesulide during abstinence attenuated EtOH intake during reinstatement after prolonged abstinence by 40.3 ± 12.4% compared to saline controls. Adolescent EtOH drinking and abstinence increased PGE2 150.5 ± 30.9% in the DS and nimesulide attenuated this increase. SC-51089 treatment during abstinence attenuated EtOH reinstatement by 48.1 ± 8.4% compared to DMSO controls. ConclusionsThese experiments identified a prostaglandin-mediated mechanism that offers a putative pharmacological target to attenuate EtOH reinstatement after adolescent-onset EtOH drinking.

Identification of Gene Networks Associated with the Anti-Leukemic Effect of Anti-Inflammatory Drugs on Acute Myeloid Leukemia Cell Lines

IntroductionDespite recent advances in therapy, acute myeloid leukemia (AML) remain a medical challenge with high morbidity and mortality rates. For most patients, allogeneic hematopoietic stem cell transplantation remain the only curative option, but due to the advanced age at diagnosis, a significant proportion of patients are not elegible to this form of therapy. Nevertheless, novel therapies are warranted. There is preclinical evidence that anti-inflammatory compounds, such as COX-2 inhibitors and steroids, may have anti-neoplastic activity in different tumor types, including AML; nevertheles the mechanisms associated with its anti-neoplastic activity are not clear. Therefore, the aim of this work was to evaluate the anti-leukemic effect of the anti-inflammatory compounds nimesulide and prednisolone in AML cell lines and to identify genes and molecular pathways associated with cytotoxicity through transcriptome analysis.MethodsThe leukemic cell lines HL-60, THP-1, OCI-AML2 and OCI-AML3 were treated with nimesulide and prednisolone at 100 µM alone and in combination and with cytarabine at 2.5 µM. Twenty four hours after treatment , we measured the amount of cell death using Annexin V Apoptosis Detection Kit FITC (ThermoFisher) and the cell cycle was analized after fixing the cells with 70% alcohol and incubation with propidium iodide (1mg/ml) and RNAse (10mg/ml). In another experiment, we harvested the cells after 4 hours of treatment for transcriptome analysis. RNA was extracted from control (DMSO) and treatment groups (1 - nimesulide, 2 - prednisolone , 3 - nimesulide and prednisolone) with RNeasy Mini Kit (Qiagen). The Illumina® NEBNext® Ultra II Directional RNA Library Prep Kit was used for library preparation, following the manufacturer protocol using Poly(A) mRNA Magnetic Isolation Module. Equimolar amount of libraries was sequenced using an Illumina NextSeq 500, following the manufacturer's instructions, on the Oklahoma Medical Research Foundation Genomics Core (USA). The sequences obtained with the RNA-Seq technique were aligned in the human genome of reference GRCh37.75 by the software Spliced Transcripts Alignment to a Reference (STAR) v2.5 and to obtain normalized counts in FPKM, the software Expectation-Maximization (RSEM) v1.3.0 was used. To identify network of genes correlated with the treatment (modules), we used the Weighted Correlation Network Analysis (WGCNA). Functional enrichment analysis of the WGCNA differentially expressed modules was performed using the Integrated Annotation, Visualization and Discovery Database (DAVID) v6.8 in order to correlate with biological processes.ResultsIn the cell cycle analysis, we observed a significant increase (p < 0.05) in the sub-G0 phase (cell death) after treatment with nimesulide alone, and in combination with prednisolone (figure 1). No effect was observed in the prednisolone only group. The cell cycle effect induced by nimesulide on HL-60 and OCI-AML2 was similar to the induced by cytarabine, a standard chemotherapy agent for AML that in known to induce arrest in the S phase. In addition, the cell line arrest in THP-1 was greater with nimesulide than with cytarabine, while OCI-AML3 was less sensitive to both nimesulide and cytarabine. Regarding cell death mechanism, treatment with nimesulide induced predominantly an increase in late apoptosis that was potentiated after combined treatment with nimesulide and cytarabine (figure 2). After the demonstration of cell cycle arrest and apoptosis induction after treatment with nimesulide, we performed whole transcriptome sequencing followed by WGCNA analysis. We have identified gene modules that were significantly correlated with anti-inflammatory treatments, being 1 module down-regulated (lightyellow with p = 0.00052) and 2 modules up-regulated (lightcyan with p = 0 .00025 and tan with p = 0.000038). Analysis of functional enrichment using DAVID showed up-regulation of gene networks associated with apoptotic processes and autophagy and down- regulation of gene networks associated with cell cycle and RNA splicing pathwaysConclusionsThe COX-2 inhibitor nimesulide caused cell cycle arrest and apoptosis in AML cell lines and potentiated the cytotoxic effects of cytarabine. This treatment was associated with up- regulation of autophagy and apoptosis and down-regulation of cell cycle and RNA splicing gene networks. [Display omitted] DisclosuresSantos: Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees. Campregher: Astellas: Consultancy.

Chronic cyclooxygenase-2 inhibition prevents the worsening of hypertension and endothelial dysfunction induced by ouabain in resistance arteries of spontaneously hypertensive rats

AimPrevious studies raise cyclooxygenase (COX) activation as a possible mechanism involved in the pathophysiology of ouabain-induced hypertension. We hypothesized that inhibition of COX-2 activity might prevent ouabain-induced vascular dysfunction and worsening of hypertension in spontaneously hypertensive rats (SHR). MethodsSHR were exposed to ouabain or vehicle and treated or not with the selective COX-2 inhibitor nimesulide for 5 weeks. Systolic blood pressure was measured by plethysmography. Vascular reactivity by wire myograph and protein expression by Western-blot were assessed in mesenteric resistance arteries (MRA) of groups. Thromboxane A2 (TXA2) production by ELISA was evaluated in MRA supernatants of groups. ResultsNoradrenaline-induced maximal contraction (Emax) was greater in MRA from SHR receiving ouabain than those of vehicle group. In situ inhibition of COX-2, TXA2 synthase, or TP receptor reduced the Emax to noradrenaline in MRA of ouabain to vehicle levels. TXA2 production was higher in ouabain than in vehicle group. Ouabain enhanced expression of cytoplasmic tyrosine kinase Src (c-Src)/ERK1/2/COX-2/TXA2 synthase/TP receptor in SHR MRA, but did not change NFkB/iKB ratio. Anticontractile effect of nitric oxide (NO) was smaller in MRA from ouabain- than vehicle-treated SHR, as well as eNOS and nNOS expression. Nimesulide co-treatment prevented the ouabain-induced worsening of hypertension and noradrenaline MRA hypercontractility in SHR. ConclusionOuabain worsen hypertension and induce MRA hypercontractility in SHR associated with upregulated c-Src/ERK1/2/COX-2/TXA2 synthase/TXA2/TP receptor axis. These effects were prevented by COX-2 inhibition.

COX-2 Inhibition Antagonizes Intra-Accumbens 2-Arachidonoylglycerol-Mediated Reduction in Ethanol Self-Administration in Rats.

Ethanol (EtOH) self-administration is particularly sensitive to the modulation of CB1 signaling in the nucleus accumbens (NAc) shell, and EtOH consumption increases extracellular levels of the endogenous cannabinoid CB1 receptor agonist 2-arachidonoyl glycerol (2-AG) in this brain region. Stimulation of CB1 receptor with agonists increases EtOH consumption, suggesting that EtOH-induced increases in 2-AG might sustain motivation for EtOH intake. In order to further explore this hypothesis, we analyzed the alterations in operant EtOH self-administration induced by intra-NAc shell infusions of 2-AG itself, the CB1 inverse agonist SR141716A, the 2-AG clearance inhibitor URB602, anandamide, and the cyclooxygenase-2 (COX-2) inhibitor nimesulide. Surprisingly, self-administration of 10% EtOH was dose-dependently reduced by either intra-NAc shell SR141716A or 2-AG infusions. Similar effects were found by intra-NAc shell infusions of URB602, suggesting again a role for accumbal 2-AG on the modulation of EtOH intake. Intra-NAc shell anandamide did not alter EtOH self-administration, pointing to a specific role for 2-AG in the modulation of EtOH self-administration. Finally, the inhibitory effect of intra-NAc shell 2-AG on EtOH intake was significantly reversed by pretreatment with nimesulide, suggesting that oxidative metabolites of 2-AG might mediate these inhibitory effects on operant self-administration. We propose that 2-AG signaling in the NAc exerts an inhibitory influence on EtOH consumption through a non-CB1 receptor mechanism involving the COX-2 pathway.

Abstract 5811: Evaluation of antitumor efficacy of CA102N, H-Nim and hyaluronic acid (HA) conjugate, alone or in combination with 5-fluorouracil (5-FU) in in vivo human colorectal cancer models

Abstract CA102N is a conjugate of HA and H-Nim (N-(4-Amino-2-phenoxyphenyl, methanesulfonamide), a derivative of cyclooxygenase-2 inhibitor Nimesulide, and a CD44 receptor targeted system currently in preclinical development for metastatic colorectal cancer. The in vivo antitumor activities of CA102N was evaluated singly or combined with 5-FU in CR5038 (Patient Derived Xenograft (PDX) and HT29 Xenograft models. Inhibitory activity was assessed according to tumor-volume changes. CA102N (i.v. 200mg/kg, BIW) monotherapy did not show significant tumor growth inhibition at either the end of the treatment (21 days) or prolonged observational (45 days) period, in CR5038 PDX-SCID metastatic model in comparison to vehicle control or 5-FU (i.v. 50mg/kg, QW). Notably, the combination therapy displayed a marked reduction in tumor growth (&amp;gt;70%) compared to 5-FU alone (p=0.0003) or vehicle control (p&amp;lt;0.0001) in addition to prolonged median survival (p&amp;lt;0.05). Alternatively, HT29 xenografts nude mice were found to have significantly reduced tumor burden treated with either single agents CA102N (400 mg/kg, QW) or 5-FU (50 mg/kg, QW), (p&amp;lt;0.012) compared to control vehicle. The drug combination further displayed a synergistic antitumoral effect and greater median survival (p &amp;lt;0.05) in HT29 model. CA102N was well tolerated alone or in the combination therapy. Based on the results of this study, combined treatment of CA102N and 5-FU resulted in a complete response at the regimen given. The combination of CA102N and 5-FU represents a promising potential therapeutic strategy and a new approach to reverse drug resistant in the treatment of colorectal cancer. Studies presently are ongoing to further evaluate its efficacy in CT26 murine colorectal models. Citation Format: Eskouhie Tchaparian, Louis Lin. Evaluation of antitumor efficacy of CA102N, H-Nim and hyaluronic acid (HA) conjugate, alone or in combination with 5-fluorouracil (5-FU) in in vivo human colorectal cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5811.

Prostaglandin Signaling Governs Spike Timing-Dependent Plasticity at Sensory Synapses onto Mouse Spinal Projection Neurons.

Highly correlated presynaptic and postsynaptic activity evokes spike timing-dependent long-term potentiation (t-LTP) at primary afferent synapses onto spinal projection neurons. While prior evidence indicates that t-LTP depends upon an elevation in intracellular Ca2+ within projection neurons, the downstream signaling pathways that trigger the observed increase in glutamate release from sensory neurons remain poorly understood. Using in vitro patch-clamp recordings from female mouse lamina I spino-parabrachial neurons, the present study demonstrates a critical role for prostaglandin synthesis in the generation of t-LTP. Bath application of the selective phospholipase A2 (PLA2) inhibitor arachidonyl trifluoromethyl ketone (AACOCF3) or the cyclooxygenase 2 (Cox-2) inhibitor nimesulide prevented t-LTP at sensory synapses onto spino-parabrachial neurons. Similar results were observed following the block of the EP2 subtype of prostaglandin E2 (PGE2) receptor with PF 04418948. Meanwhile, perfusion with PGE2 or the EP2 agonist butaprost potentiated the amplitude of monosynaptic primary afferent-evoked EPSCs while decreasing the paired-pulse ratio, suggesting a presynaptic site of action. Cox-2 was constitutively expressed in both spinal microglia and lamina I projection neurons within the superficial dorsal horn (SDH). Suppression of microglial activation with minocycline had no effect on the production of t-LTP, suggesting the possibility that prostaglandins produced within projection neurons could contribute to an enhanced probability of glutamate release at primary afferent synapses. Collectively, the results suggest that the amplification of ascending nociceptive transmission by the spinal SDH network is governed by PLA2-Cox-2-PGE2 signaling.SIGNIFICANCE STATEMENT Long-term potentiation (LTP) of primary afferent synapses contributes to the sensitization of spinal nociceptive circuits and has been linked to greater pain sensation in humans. Prior work has implicated elevated glutamate release in the generation of spike timing-dependent LTP (t-LTP) at sensory synapses onto ascending spinal projection neurons, but the underlying mechanisms remain unknown. Here we provide evidence that the activation of EP2 prostaglandin receptors by prostaglandin E2, occurring downstream of phospholipase A2 and cyclooxygenase 2 activation, mediates t-LTP at these synapses via changes in presynaptic function. This suggests that prostaglandins can increase the flow of nociceptive information from the spinal cord to the brain independently of their known ability to suppress synaptic inhibition within the dorsal horn.

Abstract A75: Meloxicam and nimesulide modulate immunity in pancreatic cancer

Abstract Background: Meloxicam and nimesulide have been studied as potential cancer immunomodulatory agents. Those drugs have been shown to have immune stimulatory effects on the tumor stroma and the microenvironment of several tumors including pancreatic ductal adenocarcinoma. They have several immune effects in vitro and in animal models. We compared the immunomodulatory activity of two preferential Cox-2 inhibitors drugs, meloxicam and nimesulide in advanced pancreatic cancer patient’s peripheral blood mononuclear cells (PBMCs) with good and poor Karnofsky. Materials and Methods: Repository PBMCs from advanced pancreatic patients with Karnofsky &amp;lt; 50% (n=10) and &amp;gt; 80% (n=10) were stimulated with different concentrations of meloxicam and nimesulide in vitro. We performed dose-response curve with concentrations of both drugs ranging from 0.5, 1, 10, 50 and 100 μM and we performed the ELISPOT assays for Interferon-gamma using human anti-CD3 and CMV whole lysate as basal T cell stimulation. Th2 cytokines were measured as well by ELISPOT and Cytokine ELISA. Results: In patients with Karnofsky &amp;gt; 80% both meloxicam and nimesulide were effective in promoting IFN-gamma production after stimulation with both drugs starting at 0.5 μM. The use of meloxicam was associated with significant production of IFN-gamma at 0.5 μM (p&amp;lt;0.001) and 1μM (p&amp;lt;0.0001). Nimesulide significantly induced IFN-gamma secretion at 0.5 μM (p=0.005), 1 μM (p=0.0002) and at 10 μM (p&amp;lt;0.0001). Meloxicam and nimesulide treatment resulted in different responses in PBMCs IFG-gamma production from pancreatic cancer patients that had a Karnofsky &amp;lt;50%. Nimesulide was more effective than meloxicam at promoting IFN-gamma production at 1μM (p&amp;lt;0.005) and 10μM (p&amp;lt;0.0001). Meloxicam did not improve the IFN-gamma secretion at low doses such as 0.5, 1 and 10 μM (n.s.). However, meloxicam at 50 μM and 100 μM was able to induce IFN-gamma (p&amp;lt;0.05 and p&amp;lt;0.0001, respectively). Additionally PBMCs treated with nimesulide in both groups were able to decrease Th2 cytokines such as IL-6, IL-18, TNF-alpha and IL-8 (statistical analysis in progress). Discussion: This preliminary data suggests that the use of meloxicam and nimesulide as putative immunomodulatory drugs may be less effective when pancreatic patients have a poor Karnosky although at high concentration they are effective. Also, the potent induction of IFN-gamma in patients with a good Karnofsky suggest synergy of these drugs in combination with standard of care treatment, immunogenic chemotherapy, anti-stroma strategies and active specific immunotherapy as a combinatorial approach to move forward into animal models of pancreatic cancer and eventually into the clinic. Grant Support: Junior Physician Scientist Fellowship Grant from CICS. Citation Format: Juan Marquez, Erik Ramos, Pedro Alejandro Lucero-Diaz, Fernando Durazo-Bustamante, Gerardo Palacios Diaz De Leon, Rosana Soto-Soto, Sara Carrillo-Marquez, Arturo Suplee-Rivera, Alejandro Camacho-Hernandez, Daniel Camacho-Gil.{Authors}. Meloxicam and nimesulide modulate immunity in pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr A75.

COX-2 plays a role in angiogenic DBA+ uNK cell subsets activation and pregnancy protection in LPS-exposed mice

IntroductionAlthough uterine Natural Killer (uNK) cells have cytoplasmic granules rich in perforin and granzymes, these cells do not degranulate in normal pregnancy. DBA lectin+ uNK cells produce angiogenic factors which stimulate remodeling of uterine arterioles to increase blood flow within the growing feto-placental unit. We sought to investigate the importance of COX-2 on mouse pregnancy inoculated with Gram-negative bacteria Lipopolysaccharide (LPS) by treating with a selective COX-2 inhibitor (nimesulide). MethodsWe have combined histochemical, immunohistochemical, stereological, morphometric, behavioral, and litter analyses to investigate mouse pregnancy inoculated with LPS with or without pre-treatment with nimesulide 30 min before LPS injections, focusing on DBA+ uNK cell response and viability of the pregnancy. ResultsLPS caused sickness behavior, an immature DBA+ uNK influx, decreased mature DBA+ uNK cell numbers, and triggered a new DBAlow uNK appearance. These effects of LPS, except the sickness behavior, were prevented by nimesulide. COX-2 inhibition also prevented the down-regulation of uNK perforin and spiral arteriole α-actin expression stimulated by LPS. While the litter size from Nimesulide + LPS-treated mothers was significantly smaller compared to those from LPS-treated group, nimesulide alone showed no effect on the offspring. DiscussionCollectively, our data indicate that COX-2 changes angiogenic DBA+ uNK cells in order to protect mouse pregnancy after LPS injection.

Adenosine signalling mediates the anti-inflammatory effects of the COX-2 inhibitor nimesulide

Extracellular adenosine formation from ATP is controlled by ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase/CD39) and ecto-5′-nucleotidase (e-5NT/CD73); the latter converts AMP to adenosine and inorganic phosphate, representing the rate limiting step controlling the ratio between extracellular ATP and adenosine. Evidence that cellular expression and activity of CD39 and CD73 may be subject to changes under pathophysiological conditions has identified this pathway as an endogenous modulator in several diseases and was shown to be involved in the molecular mechanism of drugs, such as methotrexate, salicylates , interferon-β. We evaluated whether CD73/adenosine/A2A signalling pathway is involved in nimesulide anti-inflammatory effect, in vivo and in vitro. We found that the adenosine A2A agonist, 4-[2-[[6-amino-9-(N-ethyl-β-d-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzenepropanoic acid hydrochloride (CGS21680, 2mg/kg ip.), inhibited carrageenan-induced rat paw oedema and the effect was reversed by co-administration of the A2A antagonist -(2-[7-amino-2-[2-furyl][1,2,4]triazolo[2,3-a][1,3,5]triazin-5-yl-amino]ethyl)phenol (ZM241385; 3mg/kg i.p.). Nimesulide (5mg/kg i.p.) anti-inflammatory effect was inhibited by pre-treatment with ZM241385 (3mg/kg i.p.) and by local administration of the CD73 inhibitor, adenosine 5′-(α,β-methylene)diphosphate (APCP; 400μg/paw). Furthermore, we found increased activity of 5′-nucleotidase/CD73 in paws and plasma of nimesulide treated rats, 4h following oedema induction. In vitro, the inhibitory effect of nimesulide on nitrite and prostaglandin E2 production by lipopolysaccharide-activated J774 cell line was reversed by ZM241385 and APCP. Furthermore, nimesulide increased CD73 activity in J774 macrophages while it did not inhibit nitrite accumulation by lipopolysaccharide-activated SiRNA CD73 silenced J774 macrophages. Our data demonstrate that the anti-inflammatory effect of nimesulide in part is mediated by CD73-derived adenosine acting on A2A receptors.

Anti-platelet eff ects of nimesulide in isoproterenol-induced myocardial ischaemia and infarction in rabbits

Background The present study was designed to determine the eff ects of cyclooxygenase-2 (COX-2) inhibitor-nimesulide on platelet activating factor (PAF) and arachidonic acid (AA)-induced platelet aggregation during myocardial infarction (MI) and ischaemia in rabbits.Methods Rabbits were divided into three groups; group I served as control and contains normal rabbits that received saline, group II rabbits received saline before induction of MI, and group III rabbits received nimesulide (25 mg/kg) before MI induction. Myocardial infarction was induced by isoproterenol (ISP) (65 mg/kg) and confi rmed by biochemical, electrophysiological and histopathological methods. Platelet aggregation was induced with arachidonic acid (AA) and platelet activating factor (PAF), and monitored by using light transmission aggregometery.Results When platelets were challenged with two diff erent aggregating agents, nimesulide-treated infarcted rabbits showed resistance to platelet aggregation while non-treated infarcted rabbits were found prone to aggregation with both aggregating agents (P >0.05).Conclusion These results suggest platelet inhibitory eff ects of nimesulide during experimental ischaemia and infarction in rabbits.

Role of phosphorylated extracellular signal-regulated kinase, calcitonin gene-related peptide and cyclooxygenase-2 in experimental rat models of migraine.

Although migraine is a common neurological condition, the pathomechanism is not yet fully understood. Activation of the trigeminovascular system (TVS) has an important function in this disorder and neurogenic inflammation and central sensitization are important mechanisms underlying this condition. Nitroglycerin (NTG) infusion in rats closely mimics a universally accepted human model of migraine. Electrical stimulation of the trigeminal ganglion (ESTG) of rats can also activate TVS during a migraine attack. Numerous studies have revealed that phosphorylated extracellular signal-regulated kinase (p-ERK), calcitonin gene-related peptide (CGRP) and cyclooxygenase-2 (COX-2) are involved in pain and nociceptive pathways. However, few studies have examined whether p-ERK, CGRP and COX-2 are involved in neurogenic inflammation and central sensitization. In the present study, the expression of p-ERK, CGRP and COX-2 was detected in the dura mater, trigeminal ganglion (TG) and spinal trigeminal nucleus caudalis in NTG-induced rats and ESTG models by immunohistochemistry. The three areas considered were crucial components of the TVS. The selective COX-2 inhibitor nimesulide was used in ESTG rats to examine the association between p-ERK, CGRP and COX-2. The results demonstrated that p-ERK, CGRP and COX-2 mediated neurogenic inflammation and central sensitization in migraine. In addition, the expression of p-ERK and CGRP was attenuated by the COX-2 inhibitor.

Reduced thrombosis in Klkb1−/− mice is mediated by increased Mas receptor, prostacyclin, Sirt1, and KLF4 and decreased tissue factor

AbstractThe precise mechanism for reduced thrombosis in prekallikrein null mice (Klkb1−/−) is unknown. Klkb1−/− mice have delayed carotid artery occlusion times on the rose bengal and ferric chloride thrombosis models. Klkb1−/− plasmas have long-activated partial thromboplastin times and defective contact activation–induced thrombin generation that partially corrects upon prolonged incubation. However, in contact activation–induced pulmonary thromboembolism by collagen/epinephrine or long-chain polyphosphate, Klkb1−/− mice, unlike F12−/− mice, do not have survival advantage. Klkb1−/− mice have reduced plasma BK levels and renal B2R mRNA. They also have increased expression of the renal receptor Mas and plasma prostacyclin. Increased prostacyclin is associated with elevated aortic vasculoprotective transcription factors Sirt1 and KLF4. Treatment of Klkb1−/− mice with the Mas antagonist A-779, COX-2 inhibitor nimesulide, or Sirt1 inhibitor splitomicin lowers plasma prostacyclin and normalizes arterial thrombosis times. Treatment of normal mice with the Mas agonist AVE0991 reduces thrombosis. Klkb1−/− mice have reduced aortic tissue factor (TF) mRNA, antigen, and activity. In sum, Klkb1−/− mice have a novel mechanism for thrombosis protection in addition to reduced contact activation. This pathway arises when bradykinin delivery to vasculature is compromised and mediated by increased receptor Mas, prostacyclin, Sirt1, and KLF4, leading to reduced vascular TF.

Abstract 19092: Cyclooxygenase 2 Mediates Increased Endothelium-Dependent Vascular Contraction in Hyperhomocysteinemia and Hyperlipidemia

Objectives: We recently reported that elevation of plasma homocysteine level_hyperhomocysteinemia (HHcy) accelerates atherosclerosis and vascular inflammation in hyperlipidemia (HLP). In this study, we examined the role of cyclooxygenase 2 (COX2)[[Unable to Display Character: —]]an inducible isoform of COX in responsible to inflammation, in HHcy/HPL-induced endothelial dysfunction (ED). Methods: Endothelial function was assessed in the thoracic aorta of a novel severe HHcy (plasma Hcy=221 μM) mouse model: human cystathionine β-synthase (CBS) transgenic, mouse CBS and apolipoprotine E gene deficient mouse fed with a high fat diet for 8 weeks (HHcy/HLP Cbs-/-¬ mice). Results: Endothelium-dependent relaxation to acetylcholine (ACh) was significantly impaired in HHcy/HLP Cbs-/- mice compared to HHcy/HLP Cbs+/+ mice. In the presence of eNOS inhibitor N(G)-nitro-L-Arg-methyl ester (L-NAME), ACh induced endothelium-dependent contraction in HHcy/HLP Cbs-/- but not in HHcy/HLP Cbs+/+ mice. L-NAME plus COX1/2 inhibitor indomethacin (INDO) completely blocked the HHcy/HLP-induced endothelium-dependent contraction. Moreover, INDO, COX2 inhibitor nimesulide, and thromboxane A2/prostaglandin H2 receptor antagonist SQ29548, but not COX1 inhibitor SC560, improved vascular relaxation to ACh in HHcy/HLP Cbs-/-¬ mice, indicating that COX2 plays a major role. Preincubation the aorta with antioxidants Tempol, polyethylene glycol superoxide dismutase and apocynin also improved endothelial function in HHcy/HLP Cbs-/- mice. Expression of COX2 and 3-nitrotyrosin (3-NT), but not COX1, was markedly increased in the endothelium of aorta of HHcy/HLP Cbs-/- mice. ICAM-1, VCAM-1, and TNFα were markedly increased in the aorta of HHcy/HLP Cbs-/- mice. Moreover, COX2 and 3-NT, but not COX1, was significantly increased in cultured human aortic endothelial cells treated with a combination of DL-Hcy (500 μM) and oxidized-LDL (200 μg/ml) for 48 hrs. Furthermore, DL-Hcy/oxidized-LDL-induced COX2 was prevented by PEG-SOD. Finally, HHcy markedly accelerated urinary 2,3-dinor TXB2, TXB2, 8-isoprostan and 6-keto prostaglandin F1α levels in HPL. Conclusions: COX2 mediates HHcy-induced endothelium-dependent vascular contraction in HPL via oxidative stress-dependent pathway.

Cyclooxygenase-1 inhibition attenuates angiotensin II-salt hypertension and neurogenic pressor activity in the rat

Cyclooxygenase (COX)-derived prostanoids contribute to angiotensin II (ANG II) hypertension (HTN). However, the specific mechanisms by which prostanoids act are unclear. ANG II-induced HTN is caused partly by increased sympathetic nervous system activity, especially in a setting of high dietary salt intake. This study tested the hypothesis that COX-derived prostanoids cause ANG II-salt sympathoexcitation and HTN. Experiments were conducted in conscious rats. Infusion of ANG II (150 ng·kg(-1)·min(-1) sc) caused a marked HTN in rats on 2% salt diet, but a much smaller increase in blood pressure in rats on 0.4% salt diet. The nonselective COX inhibitor ketoprofen (2 mg/kg sc) given throughout the ANG-II infusion period attenuated HTN development in rats on 2% NaCl diet, but not in rats on 0.4% NaCl diet. The acute depressor response to ganglion blockade was used to assess neurogenic pressor activity in rats on 2% NaCl diet. Ketoprofen-treated rats showed a smaller fall in arterial pressure in response to ganglion blockade during ANG-II infusion than did nontreated controls. In additional experiments, ketoprofen-treated rats exhibited smaller increases in plasma norepinephrine levels and whole body norepinephrine spillover than we previously reported in ANG II-salt HTN. Finally, the effects of the selective COX-1 inhibitor SC560 (10 mg·kg(-1)·day(-1) ip) and the selective COX-2 inhibitor nimesulide (10 mg·kg(-1)·day(-1) ip) were investigated. Treatment with SC560 but not nimesulide significantly reduced blood pressure and the depressor response to ganglion blockade in ANG II-salt HTN rats. The results suggest that COX-1 products are critical for sympathoexcitation and the full development of ANG II-salt HTN in rats.

GW24-e3751 Oxidative stress-dependent cyclooxygenase 2-derived vasoconstrictive prostaglandins contribute to hyperhomocysteinemia-induced endothelial dysfunction in hyperlipidemia

ObjectivesWe recently reported that elevation of plasma homocysteine level—hyperhomocysteinemia (HHcy) accelerates atherosclerosis and vascular inflammation in hyperlipidemia. Prostanoids are local mediators of inflammation and are synthesised from arachidonic acid by...

Cyclooxygenase‐2 inhibition and thromboxane A2 receptor antagonism attenuate hypoxic pulmonary vasoconstriction in a porcine model

Hypoxic pulmonary vasoconstriction (HPV) causes pulmonary hypertension that may lead to right heart failure. We hypothesized that the COX-2 inhibitor nimesulide and the thromboxane A(2) receptor antagonist daltroban would attenuate HPV. Haemodynamic measurements and blood sampling were performed in 18 anaesthetized, mechanically ventilated pigs, with mean ± SEM weights of 31.3 ± 0.6 kg, in normoxia (F(i)O(2)~0.21) and hypoxia (F(i)O(2)~0.10), before and 5, 15 and 45 min after initiation of right atrial infusion of nimesulide (n = 6) or daltroban (n = 6), respectively, and in six control pigs. Compared with normoxia, hypoxia (n = 18) increased mean pulmonary artery pressure by 15.8 ± 0.8 mmHg (P < 0.001), pulmonary vascular resistance (PVR) by 2.7 ± 0.3 WU (P < 0.05) and mean right atrial pressure by 2.3 ± 0.3 mmHg (P < 0.001). In the control pigs, mean pulmonary artery pressure, PVR and mean right atrial pressure remained stable (P = ns) throughout 45 min hypoxia, compared with hypoxia baseline. Nimesulide decreased mean pulmonary artery pressure by 3.7 ± 1.3 mmHg after 45 min (P < 0.013), as well as PVR by 0.8 ± 0.2 WU (P < 0.05), levelling off after 15 min. Daltroban transiently increased (P < 0.001) mean pulmonary artery pressure and mean right atrial pressure by 7.2 ± 1.2 and 2.7 ± 0.4 mmHg, respectively, but they returned to hypoxia baseline (P = ns) within 5 min. Daltroban then decreased mean pulmonary artery pressure to after 45 min be 4.2 ± 1.6 mmHg lower (P < 0.005) than at hypoxia baseline. COX-2 inhibition and thromboxane A(2) receptor antagonism attenuate HPV by decreasing mean pulmonary artery pressure by approximately 10-11%, as measured 45 min after initiation of nimesulide or daltroban infusion respectively.

Identification of a Class of Novel Tubulin Inhibitors

We previously developed a series of anticancer agents based on cyclooxygenase-2 (COX-2) inhibitor nimesulide as a lead compound. However, the molecular targets of these agents still remain unclear. In this study, we synthesized a biotinylated probe based on a representative molecule of the compound library and performed protein pull-down assays to purify the anticancer targets of the compound. Via proteomic approaches, the major proteins bound to the probe were identified to be tubulin and heat shock protein 27 (Hsp27), and the compound inhibited tubulin polymerization by binding at the colchicine domain. However, the tubulin inhibitory effect of the compound activated the Hsp27 phosphorylation and possibly overrode the direct Hsp27 inhibitory effects, which made it difficult to solely validate the Hsp27 target. Taken together, the compound was a dual ligand of tubulin and Hsp27, inhibited tubulin polymerization, and had the potential to be a class of new chemotherapeutic agents.