Nonspecific Cyclooxygenase Inhibitor Research Articles

An Observational Study on Use of Intravenous Paracetamol for Closure of Patent Ductus Arteriosus in Newborns

Introduction: Failure or delay in spontaneous closure of ductus arteriosus is commonly seen among very low birth weight and very preterm neonates (<32 weeks), results in patent ductus arteriosus (PDA). Hemodynamically significant PDA (hs-PDA) is associated with significant morbidity and mortality if not timely intervened. At present, treatment modalities for hs- PDA remain pharmacological with nonspecific cyclo-oxygenase inhibitors such as ibuprofen/indomethacin or surgical ligation whenever pharmacological management is contraindicated. Recently, trials with prostaglandin synthase inhibitor, paracetamol are emerging as an effective treatment modality for PDA closure. In this retrospective observational cohort study, we evaluated the effectiveness of intravenous paracetamol as a first line therapy in very low birth weight infants with hemodynamically significant PDA.
 Methods: Twenty four preterm infants with hemodynamically significant PDA (hs-PDA) were treated with intravenous paracetamol 15mg/kg every 6 hourly and subsequent closure was evaluated clinically and by follow-up 2D-Echocardiography. The dosage of 15 mg/kg for IV paracetamol was chosen based on previously reported data for paracetamol in the treatment of PDA in preterm newborns.
 Results: PDA closure following intravenous paracetamol was evident in 22 preterm neonates (91.67%). There were no significant side effects noted with paracetamol therapy.
 Conclusions: This study concludes that intravenous paracetamol is an effective alternative for the pharmacological closure of hemodynamically significant patent ductus arteriosus in preterm infants.

Cyclooxygenase inhibition attenuates brain angiogenesis and independently decreases mouse survival under hypoxia.

Although cyclooxygenase (COX) role in cancer angiogenesis has been studied, little is known about its role in brain angioplasticity. In the present study, we chronically infused mice with ketorolac, a non‐specific COX inhibitor that does not cross the blood–brain barrier (BBB), under normoxia or 50% isobaric hypoxia (10% O2 by volume). Ketorolac increased mortality rate under hypoxia in a dose‐dependent manner. Using in vivo multiphoton microscopy, we demonstrated that chronic COX inhibition completely attenuated brain angiogenic response to hypoxia. Alterations in a number of angiogenic factors that were reported to be COX‐dependent in other models were assayed at 24‐hr and 10‐day hypoxia. Intriguingly, hypoxia‐inducible factor 1 was unaffected under COX inhibition, and vascular endothelial growth factor receptor type 2 (VEGFR2) and C‐X‐C chemokine receptor type 4 (CXCR4) were significantly but slightly decreased. However, a number of mitogen‐activated protein kinases (MAPKs) were significantly reduced upon COX inhibition. We conclude that additional, angiogenic factor‐independent mechanism might contribute to COX role in brain angioplasticity, probably including mitogenic COX effect on endothelium. Our data indicate that COX activity is critical for systemic adaptation to chronic hypoxia, and BBB COX is essential for hypoxia‐induced brain angioplasticity. These data also indicate a potential risk for using COX inhibitors under hypoxia conditions in clinics. Further studies are required to elucidate a complete mechanism for brain long‐term angiogenesis regulation through COX activity.

Cyclooxygenase and nitric oxide synthase pathways mediate the respiratory effects of TNF-α in rats

TNF-α is the key inflammatory cytokine. TNF-α receptors are expressed in brain stem regions involved in respiratory control and also in the carotid bodies, which are the sensory organs monitoring arterial blood O2. We hypothesised that the circulating tumour necrosis factor (TNF)-α may affect the lung ventilation and modulate the hypoxic ventilatory response via activation of cyclooxygenase (COX) and nitric oxide synthase (NOS) pathways. The aim of the current study was to compare the respiratory effects of TNF-α before and after pretreatment with diclofenac or L-NG-nitro arginine methyl ester (L-NAME) nonspecific inhibitors of COX and NOS, respectively. The hypoxic ventilatory response was measured in anaesthetised rats using rebreathing techniques. We found that TNF-α increased the lung ventilation in normoxia but decreased the ventilatory response to hypoxia. Pretreatment with each of these inhibitors reduced respiratory effects of TNF-α. We believe that activation of COX and NOS-related pathways and also “cross-talk” between them mediates the TNF-α respiratory effects and underlies the impact of inflammation on the respiratory function.

Female Rats Artificially Selected for Low and High Intrinsic Aerobic Capacity Swap Inflammatory Cascade in Resistance Arteries: Mechanisms of Cyclooxygenase‐Derived Prostanoids

Low‐capacity runner (LCR) rats and high‐capacity runner (HCR) rats are translational models of human physiology as they present a genetic predisposition towards disease and the ultra‐healthy state, respectively. Selected for their low (LCR) and high (HCR) intrinsic aerobic capacity, LCR rats display increased risk of metabolic syndrome and vascular inflammation, whereas HCR rats have decreased risk factors. Cyclooxygenase (COX) is an enzyme that plays a key role in inflammation as well as normal vascular physiology; however, it is unknown if rats with low or high intrinsic capacity present differences in the COX‐induced inflammatory cascade. As obese females display an increased risk to cardiovascular disease compared to males, female LCR rats were used as a model for metabolic syndrome. Therefore, we hypothesized that resistance arteries from female LCR rats would show increased COX activity, while arteries from female HCR rats would show decreased COX activity.Our rat model (female, 18–25 weeks old) study used LCR, HCR, and high‐response trained (HRT) rats as control (n=4/group). HRT rats have a trained aerobic capacity that is higher than LCR but lower than HCR. We quantified cardiac hypertrophy by dividing the left ventricle weight by 26.73 + tibia length (TL)3. Statistics: t‐test: p<0.05: * vs control (HRT); # vs. HCR; & vs. absence of indomethacin (Indo). No differences in fat pad weight between HRT and HCR were observed. However, LCR rats showed increased fat pad weight when compared to HCR [HRT: 1.05±0.2 vs. LCR: 1.57±0.2#; HCR: 0.92±0.1 (g)]. As expected, HCR presented an increased left ventricle weight, indicating physiologic hypertrophy, when compared to LCR. No differences were observed between LCR and control [HRT: 6.84±0.9 vs. LCR: 6.55±0.1#; HCR: 7.73±0.3; 26.73+TL3]. Mesenteric resistance arteries (MRA) were extracted and mounted onto a wire myograph. No significant differences were observed in the acetylcholine and phenylephrine (PE) concentration‐response curves between groups. As expected, arteries from HRT and LCR rats incubated with a nonspecific COX inhibitor [indomethacin (Indo), 10 μM] presented a significant decrease in contraction. Interestingly, indomethacin did not alter or decrease phenylephrine‐induced contraction in arteries from HCR [PE: Emax%: HRT: control 93±3 vs. Indo 3±0.6&; LCR: control 62±17 vs. Indo 2±0.7&; HCR: control 94±6 vs. Indo 80±9] (Fig 1). Overall, these data reveal a new, dichotomous mechanism for vascular inflammation and contractility in LCR and HCR rats. Specifically, we observed that arteries from female rats born with high aerobic capacity are unresponsive to COX inhibition during phenylephrine contraction, perhaps to maintain the high vascular flow needed for their higher exercise metabolism. This may be caused by a swap mechanism from a normal inflammatory state to a higher inflammatory‐resolution state. Our study constitutes the basis for the discovery of new therapeutic targets for vascular inflammation in people with high aerobic capacity.Support or Funding InformationThis work was supported by National Institutes of Health (NIH) (NIH: R00GM118885)Concentration‐response curves to phenylephrine, with and without indomethacin (10 uM), in mesenteric resistance arteries (MRA) from (A) HRT, (B) LCR, and (C) HCR rats. (D) The average maximum response (Emax) of MRA to phenylephrine, with and without indomethacin (10 uM). T‐test: & vs. absence of Indo p<0.05.Figure 1

CYCLOOXYGENASE MECHANISMS IN THE REGULATION OF RESPIRATORY EFFECTS OF TUMOR NECROSIS FACTOR

Introduction. It is known that the systemic level of the major pro-inflammatory cytokine increases in many respiratory diseases such as asthma, COPD and sleep apnea [1, 2]. The lung ventilation changes and the pathological types of breathing are typical in these diseases. By the reason, the research of the respiratory effects of cytokine is actual. The aim of this study was to compare the respiratory effects of tumor necrosis factor - α (TNF-α) before and after pretreatment with diclofenac, a nonspecific cyclooxygenase (COX) inhibitor. Materials and methods. Тhe experiments were performed in tracheostomized anaesthetized with urethane rats. A respiratory flow head connected to a pneumotachometer (AD Instruments ML141 Spirometer, Dunedin, New Zealand) was used to measure peak airflow and respiratory rate. The hypoxic ventilatory response was measured by using rebreathing with hypoxic gas mixture before and after the tail vein injection of TNF-α (10 μg/rat). In order to determine the role of the cyclooxygenase pathway in the ventilatory effects of TNF-α, introperitoneal administration of diclofenac, a nonspecific COX inhibitor, was used (0.5 mg/rat). Results and discussion. We have shown that the increase in level of TNF-α in blood increased the parameters of respiration such as minute ventilation (by 40%), tidal volume (by 18%), and the mean inspiratory flow (by 33%). The slope of the hypoxic ventilatory response decreased from 6.06 ± 0.91 to 3.48 ± 0.38 ml/min-1 mmHg-1 (by 40%) 40 min after administration of TNF-α (p < 0.05), the slope of tidal volume and mean inspiratory flow also decreased (by 27%) (p < 0.05). After pretreatment with diclofenac, the influence of TNF-α on breathing was dampened, as no significant changes were observed. Conclusion. We concluded that the elevation of inflammatory cytokine level in blood intensifies ventilation during the resting breathing that may be associated with increased central inspiratory activity. At the same time TNF-α reduces the chemoreflex sensitivity to hypoxia, thereby worsening the compensatory capabilities of the respiratory system. Thus, the results of our study suggest participation of inflammatory cytokines in mechanisms of central breathing control and chemoreception. Diclofenac pretreatment eliminated the respiratory effects of TNF-α. The data indicate that the ability of TNF-α to enhance basal ventilation and to reduce the ventilatory hypoxic response is mediated by the COX pathway.

Blood-Brain Barrier Is the Major Site for a Rapid and Dramatic Prostanoid Increase upon Brain Global Ischemia.

We and others have demonstrated a rapid and dramatic increase in brain prostanoids upon decapitation-induced brain global ischemia and injury. However, the mechanism for this induction, including the cell types involved, are unknown. In the present study, we have validated and applied a pharmacological approach to inhibit prostanoid synthesis in the blood-brain barrier including endothelial cells. Our results indicate that a nonspecific cyclooxygenase (COX) inhibitor, ketorolac, does not pass the blood-brain barrier and does not enter red blood cells but penetrates endothelial cells. Ketorolac treatment did not affect basal prostanoid levels but completely prevented prostanoid induction upon global ischemia. These data indicate that basal prostanoids are synthesized in brain parenchyma cells, while inducible prostanoids are synthesized in the blood-brain barrier, most likely in endothelial cells. However, future studies with cell and COX isoform-specific gene ablation are needed to further validate this conclusion. These findings identify endothelial cells as a possible target for the development of pharmacological approaches to selectively attenuate inducible prostanoid pools without affecting basal levels under brain ischemia, trauma, surgery, and other related conditions.

Cyclooxygenase-1 and -2 Play Contrasting Roles in Listeria-Stimulated Immunity.

Nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit cyclooxygenase (COX) activity and are commonly used for pain relief and fever reduction. NSAIDs are used following childhood vaccinations and cancer immunotherapies; however, how NSAIDs influence the development of immunity following these therapies is unknown. We hypothesized that NSAIDs would modulate the development of an immune response to Listeria monocytogenes-based immunotherapy. Treatment of mice with the nonspecific COX inhibitor indomethacin impaired the generation of cell-mediated immunity. This phenotype was due to inhibition of the inducible COX-2 enzyme, as treatment with the COX-2-selective inhibitor celecoxib similarly inhibited the development of immunity. In contrast, loss of COX-1 activity improved immunity to L. monocytogenes Impairments in immunity were independent of bacterial burden, dendritic cell costimulation, or innate immune cell infiltrate. Instead, we observed that PGE2 production following L. monocytogenes is critical for the formation of an Ag-specific CD8+ T cell response. Use of the alternative analgesic acetaminophen did not impair immunity. Taken together, our results suggest that COX-2 is necessary for optimal CD8+ T cell responses to L. monocytogenes, whereas COX-1 is detrimental. Use of pharmacotherapies that spare COX-2 activity and the production of PGE2 like acetaminophen will be critical for the generation of optimal antitumor responses using L. monocytogenes.

Nitric oxide synthase and cyclooxygenase modulate β-adrenergic cutaneous vasodilatation and sweating in young men.

β-Adrenergic receptor agonists such as isoproterenol induce cutaneous vasodilatation and sweating in humans, but the mechanisms underpinning this response remain unresolved. Using intradermal microdialysis, we evaluated the roles of nitric oxide synthase (NOS) and cyclooxygenase (COX) in β-adrenergic cutaneous vasodilatation and sweating elicited by administration of isoproterenol. We show that while NOS contributes to β-adrenergic cutaneous vasodilatation, COX restricts cutaneous vasodilatation. We also show that combined inhibition of NOS and COX augments β-adrenergic sweating These new findings advance our basic knowledge regarding the physiological control of cutaneous blood flow and sweating, and provide important and new information to better understand the physiological significance of β-adrenergic receptors in the skin. β-Adrenergic receptor agonists such as isoproterenol can induce cutaneous vasodilatation and sweating in humans, but the mechanisms underpinning this response remain unresolved. We evaluated the hypotheses that (1) nitric oxide synthase (NOS) contributes to β-adrenergic cutaneous vasodilatation, whereas cyclooxygenase (COX) limits the vasodilatation, and (2) COX contributes to β-adrenergic sweating. In 10 young males (25±5years), cutaneous vascular conductance (CVC) and sweat rate were evaluated at four intradermal forearm skin sites infused with (1) lactated Ringer solution (control), (2) 10mm Nω -nitro-l-arginine (l-NNA), a non-specific NOS inhibitor, (3) 10mm ketorolac, a non-specific COX inhibitor, or (4) a combination of l-NNA and ketorolac. All sites were co-administered with a high dose of isoproterenol (100μm) for 3min to maximally induce β-adrenergic sweating (β-adrenergic sweating is significantly blunted by subsequent activations). Approximately 60min after the washout period, three incremental doses of isoproterenol were co-administered (1, 10 and 100μm each for 25min). Increases in CVC induced by the first and second 100μm isoproterenol were attenuated by l-NNA alone, and those in response to all doses of isoproterenol were reduced by l-NNA with co-infusion of ketorolac (all P≤0.05). Ketorolac alone augmented increases in CVC induced by 10μm and by the second 100μm isoproterenol (both P≤0.05). While isoproterenol-induced sweating was not affected by the separate administration of l-NNA or ketorolac (all P>0.05), their combined administration augmented sweating elicited by the first 3min of 100μm isoproterenol (P=0.05). We show that while NOS contributes to β-adrenergic cutaneous vasodilatation, COX restrains the vasodilatation. Finally, combined inhibition of NOS and COX augments β-adrenergic sweating.

Lipopolysaccharide Cross-Tolerance Delays Platelet-Activating Factor-Induced Sudden Death in Swiss Albino Mice: Involvement of Cyclooxygenase in Cross-Tolerance.

Lipopolysaccharide (LPS) signaling through Toll-like receptor-4 (TLR-4) has been implicated in the pathogenesis of many infectious diseases. Some believe that TLR-mediated pathogenicity is due, in part, to the lipid pro-inflammatory mediator platelet-activating factor (PAF), but this has been questioned. To test the direct contribution of PAF in endotoxemia in murine models, we injected PAF intraperitoneally into Swiss albino mice in the presence and absence of LPS. PAF alone (5 μg/mouse) caused death within 15–20 min, but this could be prevented by pretreating mice with PAF-receptor (PAF-R) antagonists or PAF-acetylhydrolase (PAF-AH). A low dose of LPS (5 mg/kg body wt) did not impair PAF-induced death, whereas higher doses (10 or 20 mg/kg body wt) delayed death, probably via LPS cross-tolerance. Cross-tolerance occurred only when PAF was injected simultaneously with LPS or within 30 min of LPS injection. Tolerance does not appear to be due to an abundant soluble mediator. Histologic examination of lungs and liver and measurement of circulating TNF-α and IL-10 levels suggested that the inflammatory response is not diminished during cross-tolerance. Interestingly, aspirin, a non-specific cyclooxygenase (COX) inhibitor, partially blocked PAF-induced sudden death, whereas NS-398, a specific COX-2 inhibitor, completely protected mice from the lethal effects of PAF. Both COX inhibitors (at 20 mg/kg body wt) independently amplified the cross-tolerance exerted by higher dose of LPS, suggesting that COX-derived eicosanoids may be involved in these events. Thus, PAF does not seem to have a protective role in endotoxemia, but its effects are delayed by LPS in a COX-sensitive way. These findings are likely to shed light on basic aspects of the endotoxin cross-tolerance occurring in many disease conditions and may offer new opportunities for clinical intervention.

Cutaneous blood flow during intradermal NO administration in young and older adults: roles for calcium-activated potassium channels and cyclooxygenase?

Nitric oxide (NO) increases cutaneous blood flow; however, the underpinning mechanism(s) remains to be elucidated. We hypothesized that the cutaneous blood flow response during intradermal administration of sodium nitroprusside (SNP, a NO donor) is regulated by calcium-activated potassium (KCa) channels and cyclooxygenase (COX) in young adults. We also hypothesized that these contributions are diminished in older adults given that aging can downregulate KCa channels and reduce COX-derived vasodilator prostanoids. In 10 young (23 ± 5 yr) and 10 older (54 ± 4 yr) adults, cutaneous vascular conductance (CVC) was measured at four forearm skin sites infused with 1) Ringer (Control), 2) 50 mM tetraethylammonium (TEA), a nonspecific KCa channel blocker, 3) 10 mM ketorolac, a nonspecific COX inhibitor, or 4) 50 mM TEA + 10 mM ketorolac via intradermal microdialysis. All skin sites were coinfused with incremental doses of SNP (0.005, 0.05, 0.5, 5, and 50 mM each for 25 min). During SNP administration, CVC was similar at the ketorolac site (0.005-50 mM, all P > 0.05) relative to Control, but lower at the TEA and TEA + ketorolac sites (0.005-0.05 mM, all P < 0.05) in young adults. In older adults, ketorolac increased CVC relative to Control during 0.005-0.05 mM SNP administration (all P < 0.05), but this increase was not observed when TEA was coadministered (all P > 0.05). Furthermore, TEA alone did not modulate CVC during any concentration of SNP administration in older adults (all P > 0.05). We show that during low-dose NO administration (e.g., 0.005-0.05 mM), KCa channels contribute to cutaneous blood flow regulation in young adults; however, in older adults, COX inhibition increases cutaneous blood flow through a KCa channel-dependent mechanism.

Paracetamol for the Treatment of Patent Ductus Arteriosus in Very Low Birth Weight Infants

Persistent patent ductus arteriosus (PDA) is associated with significant co-morbidities and increased mortality in preterm infants, especially very low birth weight (VLBW) infants. A large number of studies on the management of PDA have been published. Despite PDA being such a common condition in preterm infants, there is no consensus on which PDAs to treat, when to treat and how best to treat. Nonspecific cyclo-oxygenase inhibitors such as indomethacin and ibuprofen have been the mainstay of medical treatment of PDA for decades. Ibuprofen has similar efficacy and higher safety profile when compared to indomethacin, as it is associated with fewer gastrointestinal and renal side effects, and is considered the drug of choice for PDA closure. Recently, there is a growing interest in paracetamol for PDA closure and it has been suggested as an alternative drug to treat PDA. Finding the optimal pharmacological treatment for PDA closure in VLBW continues to remain challenging. In this review article, we assessed the evidence of paracetamol for PDA closure VLBW infants.

Nicotine in high concentration causes contraction of isolated strips of rabbit corpus cavernosum.

It is well known that cigarette smoke can cause erectile dysfunction by affecting the penile vascular system. However, the exact effects of nicotine on the corpus cavernosum remains poorly understood. Nicotine has been reported to cause relaxation of the corpus cavernosum; it has also been reported to cause both contraction and relaxation. Therefore, high concentrations of nicotine were studied in strips from the rabbit corpus cavernosum to better understand its effects. The proximal penile corpus cavernosal strips from male rabbits weighing approximately 4 kg were used in organ bath studies. Nicotine in high concentrations (10-5~10-4 M) produced dose-dependent contractions of the corpus cavernosal strips. The incubation with 10-5 M hexamethonium (nicotinic receptor antagonist) significantly inhibited the magnitude of the nicotine associated contractions. The nicotine-induced contractions were not only significantly inhibited by pretreatment with 10-5 M indomethacin (nonspecific cyclooxygenase inhibitor) and with 10-6 M NS-398 (selective cyclooxygenase inhibitor), but also with 10-6 M Y-27632 (Rho kinase inhibitor). Ozagrel (thromboxane A2 synthase inhibitor) and SQ-29548 (highly selective TP receptor antagonist) pretreatments significantly reduced the nicotine-induced contractile amplitude of the strips. High concentrations of nicotine caused contraction of isolated rabbit corpus cavernosal strips. This contraction appeared to be mediated by activation of nicotinic receptors. Rho-kinase and cyclooxygenase pathways, especially cyclooxygenase-2 and thromboxane A2, might play a pivotal role in the mechanism associated with nicotine-induced contraction of the rabbit corpus cavernosum.

Cyclooxygenase pathway in modulation of the ventilatory response to hypercapnia by interleukin-1β in rats

At the present time very little is known about interactions between the systemic inflammatory and ventilatory control. Our previous study has demonstrated that the elevation of the major inflammatory cytokines interleukin-1β (IL-1β) in the cerebrospinal fluid (CSF) may affect the control of ventilation. The aim of the current study was to compare the respiratory effects of IL-1β before and after pretreatment with diclofenac, a nonspecific cyclooxygenase (COX) inhibitor. Using the method of rebreathing with hyperoxic gas mixture we demonstrate that the slope of the ventilatory response to carbon dioxide decreased almost twofold from 5.6±0.52 to 2.5±0.28mlmin−1mmHg−1 (p<0.01) 40min after cerebroventricular administration of IL-1β. In contrast, the basal level of lung ventilation increased after the elevation of IL-1β in CSF. Diclofenac pretreatment eliminated these respiratory effects of IL-1β. The data indicate that the ability of IL-1β to enhance basal ventilation and to reduce the ventilatory hypercapnic response is mediated by the COX pathway.

Dexmedetomidine-induced contraction involves c-Jun NH2 -terminal kinase phosphorylation through activation of the 5-lipoxygenase pathway in the isolated endothelium-denuded rat aorta.

Vasoconstriction induced by dexmedetomidine, a highly selective alpha-2 adrenoceptor agonist, mainly involves c-Jun NH2 -terminal kinase (JNK) phosphorylation in the isolated endothelium-denuded aorta. We carried out an in vitro study to determine the main arachidonic acid metabolic pathway that is involved in dexmedetomidine-induced JNK activation. Cumulative dexmedetomidine concentration-contractile response curves were generated in the endothelium-denuded rat aorta in the presence or absence of the following inhibitors: the JNK inhibitor SP600125, the phospholipase A2 inhibitor quinacrine dihydrochloride, the non-specific lipoxygenase (LOX) inhibitor nordihydroguaiaretic acid, the 5-LOX inhibitor AA-861, the dual 5-LOX and cyclooxygenase (COX) inhibitor phenidone, the non-specific COX inhibitor indomethacin, the cytochrome p450 epoxygenase inhibitor fluconazole, the COX-1 inhibitor SC-560, and the COX-2 inhibitor NS-398. The effect of the alpha-2 adrenoceptor inhibitor rauwolscine and other inhibitors, such as quinacrine dihydrochloride, nordihydroguaiaretic acid, AA-861, phenidone, indomethacin and the protein kinase C inhibitor GF 109203X, on dexmedetomidine-induced JNK phosphorylation was investigated in rat aortic vascular smooth muscle cells with western blotting. The effect of dexmedetomidine on 5-LOX and COX-2 expression was investigated in vascular smooth muscle cells. SP600125, quinacrine dihydrochloride, nordihydroguaiaretic acid, AA-861, phenidone, rauwolscine and chelerythrine attenuated dexmedetomidine-induced contraction. Indomethacin slightly attenuated dexmedetomidine-induced contraction. Fluconazole and SC-560 had no effect on dexmedetomidine-induced contraction, whereas NS-398 attenuated contraction. SP600125, rauwolscine, quinacrine dihydrochloride, nordihydroguaiaretic acid, AA-861, phenidone and GF 109203X attenuated dexmedetomidine-induced JNK phosphorylation. 5-LOX and COX-2 were upregulated by dexmedetomidine. Thus, dexmedetomidine-induced alpha-2 adrenoceptor-mediated contraction is mediated mainly by 5-LOX and partially by COX-2, which leads to JNK phosphorylation.

In vitro Effects of the Organophosphorus Pesticide Malathion on the Reactivity of Rat Aorta

Background and Purpose: There is a remarkable paucity of studies analyzing the role of the endothelium-derived relaxing factors on the vascular effects of organophosphates. This study was carried out to evaluate the vascular effects of malathion and the role of nitric oxide (NO) and prostacyclin (PGI₂). Methods: Vascular reactivity measuring isometric forces in vitro (‘organ chambers') and flow cytometry (cells loaded with DAF-FM DA) were used. Results: In rat thoracic aorta segments contracted with phenylephrine (Phe) (10^-7 mol/l), malathion (10^-10 to 10^-5 mol/l) induced concentration-dependent relaxation in arteries with intact endothelium (n = 7; p < 0.05). Malathion-mediated relaxation was blocked by N-nitro-<smlcap>L</smlcap>-arginine methyl ester (<smlcap>L</smlcap>-NAME; 10^-4 mol/l), a nonspecific NO synthase inhibitor, and/or indomethacin (10^-5 mol/l), a nonspecific cyclooxygenase inhibitor (n = 10, p < 0.05). In thoracic aorta rings, with and without endothelium, Phe (10^-10 to 10^-5 mol/l) evoked concentration-dependent contraction, which was reduced in the presence of malathion. In rings with or without endothelium, incubated with malathion, <smlcap>L</smlcap>-NAME and indomethacin, the Phe-induced contraction was restored. The role of NO was confirmed using flow cytometry. Malathion evokes endothelium-dependent relaxation through the M₁ muscarinic receptor, since this relaxation was clearly blocked by atropine (M₁ and M₂ blocker) and pirenzepine (M₁ blocker), but was less blocked by gallamine (M₂ blocker) or 4-DAMP (M₃ blocker). Conclusions: These findings suggest that the organophosphate compound effects on vascular reactivity depend of NO and PGI₂.

Hydrogen peroxide elicits constriction of skeletal muscle arterioles by activating the arachidonic acid pathway.

AimsThe molecular mechanisms of the vasoconstrictor responses evoked by hydrogen peroxide (H2O2) have not been clearly elucidated in skeletal muscle arterioles.Methods and ResultsChanges in diameter of isolated, cannulated and pressurized gracilis muscle arterioles (GAs) of Wistar-Kyoto rats were determined under various test conditions. H2O2 (10–100 µM) evoked concentration-dependent constrictions in the GAs, which were inhibited by endothelium removal, or by antagonists of phospholipase A (PLA; 100 µM 7,7-dimethyl-(5Z,8Z)-eicosadienoic acid), protein kinase C (PKC; 10 µM chelerythrine), phospholipase C (PLC; 10 µM U-73122), or Src family tyrosine kinase (Src kinase; 1 µM Src Inhibitor-1). Antagonists of thromboxane A2 (TXA2; 1 µM SQ-29548) or the non-specific cyclooxygenase (COX) inhibitor indomethacin (10 µM) converted constrictions to dilations. The COX-1 inhibitor (SC-560, 1 µM) demonstrated a greater reduction in constriction and conversion to dilation than that of COX-2 (celecoxib, 3 µM). H2O2 did not elicit significant changes in arteriolar Ca2+ levels measured with Fura-2.ConclusionsThese data suggest that H2O2 activates the endothelial Src kinase/PLC/PKC/PLA pathway, ultimately leading to the synthesis and release of TXA2 by COX-1, thereby increasing the Ca2+ sensitivity of the vascular smooth muscle cells and eliciting constriction in rat skeletal muscle arterioles.

Role of prostaglandins in spinal transmission of the exercise pressor reflex in decerebrated rats

Previous studies found that prostaglandins in skeletal muscle play a role in evoking the exercise pressor reflex; however the role played by prostaglandins in the spinal transmission of the reflex is not known. We determined, therefore, whether or not spinal blockade of cyclooxygenase (COX) activity and/or spinal blockade of endoperoxide (EP) 2 or 4 receptors attenuated the exercise pressor reflex in decerebrated rats. We first established that intrathecal doses of a non-specific COX inhibitor Ketorolac (100μg in 10μl), a COX-2-specific inhibitor Celecoxib (100μg in 10μl), an EP2 antagonist PF-04418948 (10μg in 10μl), and an EP4 antagonist L-161,982 (4μg in 10μl) effectively attenuated the pressor responses to intrathecal injections of arachidonic acid (100μg in 10μl), EP2 agonist Butaprost (4ng in 10μl), and EP4 agonist TCS 2510 (6.25μg in 2.5μl), respectively. Once effective doses were established, we statically contracted the hind limb before and after intrathecal injections of Ketorolac, Celecoxib, the EP2 antagonist and the EP4 antagonist. We found that Ketorolac significantly attenuated the pressor response to static contraction (before Ketorolac: 23±5mmHg, after Ketorolac 14±5mmHg; p<0.05) whereas Celecoxib had no effect. We also found that 8μg of L-161,982, but not 4μg of L-161,982, significantly attenuated the pressor response to static contraction (before L-161,982: 21±4mmHg, after L-161,982 12±3mmHg; p<0.05), whereas PF-04418948 (10μg) had no effect. We conclude that spinal COX-1, but not COX-2, plays a role in evoking the exercise pressor reflex, and that the spinal prostaglandins produced by this enzyme are most likely activating spinal EP4 receptors, but not EP2 receptors.

Optimization of Naltrexone Diclofenac Codrugs for Sustained Drug Delivery Across Microneedle-Treated Skin

The purpose of this work was to optimize the structure of codrugs for extended delivery across microneedle treated skin. Naltrexone, the model compound was linked with diclofenac, a nonspecific cyclooxygenase inhibitor to enhance the pore lifetime following microneedle treatment and develop a 7day transdermal system for naltrexone. Four different codrugs of naltrexone and diclofenac were compared in terms of stability and solubility. Transdermal flux, permeability and skin concentration of both parent drugs and codrugs were quantified to form a structure permeability relationship. The results indicated that all codrugs bioconverted in the skin. The degree of conversion was dependent on the structure, phenol linked codrugs were less stable compared to the secondary alcohol linked structures. The flux of naltrexone across microneedle treated skin and the skin concentration of diclofenac were higher for the phenol linked codrugs. The polyethylene glycol link enhanced solubility of the codrugs, which translated into flux enhancement. The current studies indicated that formulation stability of codrugs and the flux of naltrexone can be enhanced via structure design optimization. The polyethylene glycol linked naltrexone diclofenac codrug is better suited for a 7day drug delivery system both in terms of stability and drug delivery.

Cyclooxygenase-2 Regulated by the Nuclear Factor-κB Pathway Plays an Important Role in Endometrial Breakdown in a Female Mouse Menstrual-like Model

The role of prostaglandins (PGs) in menstruation has long been proposed. Although evidence from studies on human and nonhuman primates supports the involvement of PGs in menstruation, whether PGs play an obligatory role in the process remains unclear. Although cyclooxygenase (COX) inhibitors have been used in the treatment of irregular uterine bleeding, the mechanism involved has not been elucidated. In this study, we used a recently established mouse menstrual-like model for investigating the role of COX in endometrial breakdown and its regulation. Administration of the nonspecific COX inhibitor indomethacin and the COX-2 selective inhibitor DuP-697 led to inhibition of the menstrual-like process. Furthermore, immunostaining analysis showed that the nuclear factor (NF)κB proteins P50, P65, and COX-2 colocalized in the outer decidual stroma at 12 to 16 hours after progesterone withdrawal. Chromatin immunoprecipitation analysis showed that NFκB binding to the Cox-2 promoter increased at 12 hours after progesterone withdrawal in vivo, and real-time PCR analysis showed that the NFκB inhibitors pyrrolidine dithiocarbamate and MG-132 inhibited Cox-2 mRNA expression in vivo and in vitro, respectively. Furthermore, COX-2 and NFκB inhibitors similarly reduced endometrial breakdown, suggesting that NFκB/COX-2-derived PGs play a critical role in this process. In addition, the CD45(+) leukocyte numbers were sharply reduced following indomethacin (COX-1 and COX-2 inhibitor), DuP-697 (COX-2 inhibitor), and pyrrolidine dithiocarbamate (NFκB inhibitor) treatment. Collectively, these data indicate that NFκB/COX-2-induced PGs regulate leukocyte influx, leading to endometrial breakdown.

Diclofenac delays micropore closure following microneedle treatment in human subjects

Drugs absorbed poorly through the skin are commonly delivered via injection with a hypodermic needle, which is painful and increases the risk of transmitting infectious diseases. Microneedles (MNs) selectively and painlessly permeabilize the outermost skin layer, allowing otherwise skin-impermeable drugs to cross the skin through micron-sized pores and reach therapeutic concentrations. However, rapid healing of the micropores prevents further drug delivery, blunting the clinical utility of this unique transdermal technique. We present the first human study demonstrating that micropore lifetime can be extended following MN treatment. Subjects received one-time MN treatment and daily topical application of diclofenac sodium. Micropore closure was measured with impedance spectroscopy, and area under the admittance–time curve (AUC) was calculated. AUC was significantly higher at MN+diclofenac sodium sites vs. placebo, suggesting slower rates of micropore healing. Colorimetry measurements confirmed the absence of local erythema and irritation. This mechanistic human proof-of-concept study demonstrates that micropore lifetime can be prolonged with simple topical administration of a non-specific cyclooxygenase inhibitor, suggesting the involvement of subclinical inflammation in micropore healing. These results will allow for longer patch wear time with MN-enhanced delivery, thus increasing patient compliance and expanding the transdermal field to a wider variety of clinical conditions.