Inhibitor Licofelone Research Articles

Immobilized Metal Affinity Chromatography as a Drug Discovery Platform for Metalloenzyme Inhibitors.

Immobilized metal-ion affinity chromatography (IMAC) used to purify recombinant proteins features a resin-bound 1:1 Ni(II)-iminodiacetic acid (IDA) complex. This hemi-saturated Ni(II)-IDA system containing exchangeable sites at the metal ion is re-cast as a surrogate of a coordinatively-unsaturated metalloenzyme active site, with utility for selecting compounds with metal-binding groups from mixtures as potential metalloenzyme inhibitors. Exchanging Ni(II) for other metal ions could broaden the scope of metalloenzyme target. This work examined the performance of Cu(II)-, Fe(III)-, Ga(III)-, Ni(II)-, or Zn(II)-IMAC resins to reversibly bind experimental or clinical metalloenzyme inhibitors of Zn(II)-ACE1, Zn(II)-HDAC, Fe(II)/(III)-5-LO or Cu(II)-tyrosinase from a curated mixture (1-17). Each IMAC system gave a distinct selection profile. The Zn(II)-IMAC system selectively bound the thiol-containing Zn(II)-ACE1 inhibitors captopril and omapatrilat, and the Fe(III)-IMAC system selectively bound the Fe(II)/(III)-5-LO inhibitor licofelone, demonstrating a remarkable IMAC-metalloenzyme metal ion match. IMAC provides a simple, water-compatible platform, which could accelerate metalloenzyme inhibitor discovery.

Licofelone, a dual cyclooxygenase/5-lipoxygenase inhibitor, reverses endotoxin-induced impaired atrial chronotropic responsiveness to cholinergic stimulation in rats

Evidence show that endotoxemia is linked with tachycardia. The exact mechanism of tachycardia is not well-understood, but it seems that impaired cardiac chronotropic responsiveness to cholinergic stimulation plays a role in this phenomenon. The aim of this experiment was to study the effect of licofelone as a dual cyclooxygenase (COX)/5-lipoxygenase (5-LOX) inhibitor in modulation of atrial chronotropic hyporesponsiveness to cholinergic stimulation in endotoxemic rats, compared with hydrocortisone and indomethacin in in vitro and in vivo studies. Rats were injected by either of lipopolysaccharide (LPS) or saline. The isolated atria were incubated with licofelone, hydrocortisone, or indomethacin in an organ bath set up. In a separate experiment, rats were injected with licofelone, hydrocortisone, or indomethacin prior to isolation of the atria. Then, in both experiments, chronotropic responsiveness to cumulative concentrations of carbacholine in organ bath was recorded. LPS injection decreased the chronotropic responsiveness to cholinergic stimulation in both in vitro and in vivo experiments, significantly (P < 0.0001), while either incubation of isolated atria with licofelone (a dual COX/5-LOX inhibitor) or injection of licofelone to animals could reverse it, completely (P < 0.01). Hydrocortisone (phospholipase A2 and COX-2 inhibitor) in vitro and in vivo (P < 0.001, P < 0.05, respectively) as well as indomethacin (COX inhibitor) in vitro and in vivo (P < 0.05, P < 0.01, respectively) exerted some lesser effects.Our data revealed that in endotoxemic rats, chronotropic hyporesponsiveness to cholinergic stimulation was modulated by the dual COX/5-LOX inhibitor licofelone, and this effect is comparable with hydrocortisone and indomethacin.

Licofelone Enhances the Efficacy of Paclitaxel in Ovarian Cancer by Reversing Drug Resistance and Tumor Stem-like Properties.

Drug development for first-line treatment of epithelial ovarian cancer (EOC) has been stagnant for almost three decades. Traditional cell culture methods for primary drug screening do not always accurately reflect clinical disease. To overcome this barrier, we grew a panel of EOC cell lines in three-dimensional (3D) cell cultures to form multicellular tumor spheroids (MCTS). We characterized these MCTS for molecular and cellular features of EOC and performed a comparative screen with cells grown using two-dimensional (2D) cell culture to identify previously unappreciated anticancer drugs. MCTS exhibited greater resistance to chemotherapeutic agents, showed signs of senescence and hypoxia, and expressed a number of stem cell-associated transcripts including ALDH1A and CD133, also known as PROM1 Using a library of clinically repurposed drugs, we identified candidates with preferential activity in MCTS over 2D cultured cells. One of the lead compounds, the dual COX/LOX inhibitor licofelone, reversed the stem-like properties of ovarian MCTS. Licofelone also synergized with paclitaxel in ovarian MCTS models and in a patient-derived tumor xenograft model. Importantly, the combination of licofelone with paclitaxel prolonged the median survival of mice (>141 days) relative to paclitaxel (115 days), licofelone (37 days), or vehicle (30 days). Increased efficacy was confirmed by Mantel-Haenszel HR compared with vehicle (HR = 0.037) and paclitaxel (HR = 0.017). These results identify for the first time an unappreciated, anti-inflammatory drug that can reverse chemotherapeutic resistance in ovarian cancer, highlighting the need to clinically evaluate licofelone in combination with first-line chemotherapy in primary and chemotherapy-refractory EOC.Significance: This study highlights the use of an in vitro spheroid 3D drug screening model to identify new therapeutic approaches to reverse chemotherapy resistance in ovarian cancer. Cancer Res; 78(15); 4370-85. ©2018 AACR.

Improved Antitumor Activity of a Therapeutic Melanoma Vaccine through the Use of the Dual COX-2/5-LO Inhibitor Licofelone

Immune-suppressive cell populations impair antitumor immunity and can contribute to the failure of immune therapeutic approaches. We hypothesized that the non-steroidal anti-inflammatory drug licofelone, a dual cyclooxygenase-2/5-LO inhibitor, would improve therapeutic melanoma vaccination by reducing immune-suppressive cell populations. Therefore, licofelone was administered after tumor implantation, either alone or in combination with a peptide vaccine containing a long tyrosinase-related protein 2-peptide and the adjuvant α-galactosylceramide, all formulated into cationic liposomes. Mice immunized with the long-peptide vaccine and licofelone showed delayed tumor growth compared to mice given the vaccine alone. This protection was associated with a lower frequency of immature myeloid cells (IMCs) in the bone marrow (BM) and spleen of tumor-inoculated mice. When investigating the effect of licofelone on IMCs in vitro, we found that the prostaglandin E2-induced generation of IMCs was decreased in the presence of licofelone. Furthermore, pre-incubation of BM cells differentiated under IMC-inducing conditions with licofelone reduced the secretion of cytokines interleukin (IL)-10 and -6 upon lipopolysaccharides (LPS) stimulation as compared to untreated cells. Interestingly, licofelone increased IL-6 and IL-10 secretion when administered after the LPS stimulus, demonstrating an environment-dependent effect of licofelone. Our findings support the use of licofelone to reduce tumor-promoting cell populations.

Abstract 2819: Targeting inflammation and pancreatitis blocks tumor-initiating stem cells and pancreatic cancer progression

Abstract Pancreatic cancer (PC) is a deadly disease with the lowest survival of all cancers. Recent development of genetically engineered mouse models (GEMs) for PC that recapitulate human disease progression has enabled development of new strategies to delay or inhibit pancreatic cancer and testing of experimental interventions in preclinical trials. Pancreatic tumor-initiating or cancer stem cell (CSC) populations contribute to tumor growth, metastasis, and resistance to therapy. We first found that expression of the CSC marker DclK1 occurs at an early stage of PC in both early and late pancreatic intraepithelial neoplasia (PanINs) and increases as the disease progresses. Genome-wide next generation sequencing of pancreatic ductal adenocarcinoma (PDAC) from GEMs revealed significantly increased DclK1 along with inflammatory genes. Genetic ablation of cyclo-oxygenase (COX)-2 decreased the DclK1 in GEMs. Induction of inflammation with cerulein induced pancreatitis in GEMs increased DclK1, and Dclk1 was reduced by the novel anti-inflammatory dual COX/5-lipoxygenase (5-LOX) inhibitor licofelone. We investigated the long term pharmacologic inhibitory efficacy of licofelone on PDAC in vivo using a GEM model. GEM (n = 86) and wild type mice (n = 24) were fed a diet containing different doses of licofelone for 300 days and evaluated for formation of PanINs and for their progression to PDAC. Dietary licofelone at tested doses significantly inhibited the incidence of PDAC (60-90%; p&amp;lt;0.0001) with a profound suppression of carcinoma in situ (35-60%; p&amp;lt;0.001) in male and female GEMs. Licofelone caused a dose-dependent suppression of pancreatic tumor COX-2 and 5-LOX activities and modulated miRNAs for inflammation markers and CSCs, including DclK1, CD133, CD44, and Lgr5 (p&amp;lt;0.001) in correlation with the PDAC inhibition. Licofelone also inhibited inflammation-induced CSCs in vitro. These studies provide the first evidence that modulation of inflammation with a dual COX-LOX inhibitor effectively blocks CSCs and inhibits pancreatic tumorigenesis. In summary, our preclinical data indicate that licofelone has potential for chemoprevention of PC and should be evaluated in other PDAC models in anticipation of future clinical trials. {Supported by NCI-CN-53300} Citation Format: Altaf Mohammed, Naveena B. Janakiram, Venkateshwar Madka, Misty Brewer, Rebekah L. Ritchie, Stan Lightfoot, Gaurav Kumar, Michael Sadeghi, Jagan Mohan R Patlolla, Hiroshi Yamada, Zobeida Cruz-Monserrate, Courtney W. Houchen, Vernon E. Steele, Chinthalapally V. Rao. Targeting inflammation and pancreatitis blocks tumor-initiating stem cells and pancreatic cancer progression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2819. doi:10.1158/1538-7445.AM2015-2819

Targeting pancreatitis blocks tumor-initiating stem cells and pancreatic cancer progression.

Recent development of genetically engineered mouse models (GEMs) for pancreatic cancer (PC) that recapitulates human disease progression has helped to identify new strategies to delay/inhibit PC development. We first found that expression of the pancreatic tumor-initiating/cancer stem cells (CSC) marker DclK1 occurs in early stage PC and in both early and late pancreatic intraepithelial neoplasia (PanIN) and that it increases as disease progresses in GEM and also in human PC. Genome-wide next generation sequencing of pancreatic ductal adenocarcinoma (PDAC) from GEM mice revealed significantly increased DclK1 along with inflammatory genes. Genetic ablation of cyclo-oxygenase-2 (COX-2) decreased DclK1 in GEM. Induction of inflammation/pancreatitis with cerulein in GEM mice increased DclK1, and the novel dual COX/5-lipoxygenase (5-LOX) inhibitor licofelone reduced it. Dietary licofelone significantly inhibited the incidence of PDAC and carcinoma in situ with significant inhibition of pancreatic CSCs. Licofelone suppressed pancreatic tumor COX-2 and 5-LOX activities and modulated miRNAs characteristic of CSC and inflammation in correlation with PDAC inhibition. These results offer a preclinical proof of concept to target the inflammation initiation to inhibit cancer stem cells early for improving the treatment of pancreatic cancers, with immediate clinical implications for repositioning dual COX/5-LOX inhibitors in human trials for high risk patients.

The Dual Cyclooxygenase/5-Lipoxygenase Inhibitor Licofelone Attenuates P-Glycoprotein-Mediated Drug Resistance in the Injured Spinal Cord

There are currently no proven effective treatments that can improve recovery of function in spinal cord injury (SCI) patients. Many therapeutic compounds have shown promise in pre-clinical studies, but clinical trials have been largely unsuccessful. P-glycoprotein (Pgp, Abcb1b) is a drug efflux transporter of the blood-spinal cord barrier that limits spinal cord penetration of blood-borne xenobiotics. Pathological Pgp upregulation in diseases such as cancer causes heightened resistance to a broad variety of therapeutic drugs. Importantly, several drugs that have been evaluated for the treatment of SCI, such as riluzole, are known substrates of Pgp. We therefore examined whether Pgp-mediated pharmacoresistance diminishes delivery of riluzole to the injured spinal cord. Following moderate contusion injury at T10 in male Sprague-Dawley rats, we observed a progressive, spatial spread of increased Pgp expression from 3 days to 10 months post-SCI. Spinal cord uptake of i.p.-delivered riluzole was significantly reduced following SCI in wild type but not Abcb1a-knockout rats, highlighting a critical role for Pgp in mediating drug resistance following SCI. Because inflammation can drive Pgp upregulation, we evaluated the ability of the new generation dual anti-inflammatory drug licofelone to promote spinal cord delivery of riluzole following SCI. We found that licofelone both reduced Pgp expression and enhanced riluzole bioavailability within the lesion site at 72 h post-SCI. This work highlights Pgp-mediated drug resistance as an important obstacle to therapeutic drug delivery for SCI, and suggests licofelone as a novel combinatorial treatment strategy to enhance therapeutic drug delivery to the injured spinal cord.

Licofelone Modulates Neuroinflammation and Attenuates Mechanical Hypersensitivity in the Chronic Phase of Spinal Cord Injury

Inflammation is a major factor shaping outcome during the early, acute phase of traumatic spinal cord injury (SCI). It is known that pro-inflammatory signaling within the injured spinal cord drives pathological alterations in neurosensory processing and shapes functional outcome early after injury. However, it is unclear whether inflammation persists into the chronic phase of injury or shapes sensory processing long after injury. To investigate these possibilities, we have performed biochemical and behavioral assessments 9 months after moderate thoracic spinal contusion injury in the rat. We have found that levels of the pro-inflammatory lipid mediators leukotriene B4 and prostaglandin E2 are elevated in the chronic spinal cord lesion site. Additionally, using metabolomic profiling, we have detected elevated levels of pro-oxidative and inflammatory metabolites, along with alterations in multiple biological pathways within the chronic lesion site. We found that 28 d treatment of chronically injured rats with the dual COX/5-LOX inhibitor licofelone elevated levels of endogenous anti-oxidant and anti-inflammatory metabolites within the lesion site. Furthermore, licofelone treatment reduced hypersensitivity of hindpaws to mechanical, but not thermal, stimulation, indicating that mechanical sensitivity is modulated by pro-inflammatory signaling in the chronic phase of injury. Together, these findings provide novel evidence of inflammation and oxidative stress within spinal cord tissue far into the chronic phase of SCI, and demonstrate a role for inflammatory modulation of mechanical sensitivity in the chronic phase of injury.

Inhibition of Prostaglandin E2 (PGE2) Signaling by Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) or EP4 Receptor Antagonism Expands Hematopoietic Stem and Progenitor Cells (HSPC) and Enhances Their Mobilization to Peripheral Blood in Mice and Baboons.

Abstract 84PGE2 is a physiological regulator of HSPC and exhibits both stimulatory and inhibitory activities. We recently reported that PGE2 increases CXCR4 expression and homing of HSPC to bone marrow (Hoggatt et al, Blood 2009). Since PGE2 is produced within the marrow microenvironment, we hypothesized that blocking PGE2 synthesis in vivo via inhibition of cyclooxygenases (COX) by NSAIDs would facilitate HSPC mobilization and serve as an adjunct to current mobilization regimens. Treatment of mice with the NSAID indomethacin b.i.d. along with G-CSF for 4 days resulted in an increase in peripheral blood (PB) CFU-GM over G-CSF alone (1.96±0.19 fold, p<0.001). Indomethacin treatment for 4 days without G-CSF resulted in marginal, but significant, HSPC mobilization. In addition, NSAID significantly increased mobilization of Sca-1+, c-kit+, Lineageneg (SKL) cells (G-CSF:1565±25 vs G-CSF + NSAID:6447±1324) and the primitive stem cell population of CD150+CD48− SKL cells (G-CSF:57±1 vs G-CSF + NSAID:217±53) per ml blood. Indomethacin synergistically enhanced mobilization by a single administration of AMD3100 alone or in combination with GROβ, indicating G-CSF independent mechanisms. To confirm enhanced mobilization in a non-human primate model, 4 baboons were mobilized with G-CSF + NSAID, or G-CSF alone in a crossover protocol. NSAID co-treatment increased CFU-GM (2845 vs 5975, p=0.03) and CD34+ cells (1061 vs 2201, p=0.03) per ml blood compared to G-CSF alone. Total CFC analysis indicated that NSAID enhanced mobilization of CFU-GM, BFU-E and CFU-GEMM. To determine if long-term repopulating cells were mobilized, lethally irradiated mice were transplanted with 2×106 murine PB mononuclear cells mobilized by G-CSF or G-CSF + NSAID. In mice receiving the G-CSF + NSAID mobilized graft, faster recovery of both neutrophils and platelets was observed (Days to 50%/100% recovery; Neutrophils: G-CSF 19/26 vs G-CSF + NSAID 14/22; Platelets: G-CSF 20/28 vs G-CSF + NSAID 16/24). In murine competitive repopulation assays, increased competitive repopulating units were quantitated in G-CSF + NSAID mobilized grafts compared to G-CSF. Since PGE2 can be produced by COX 1 and COX 2 enzymes, mice were treated with COX 1 specific (SC-560, Valeryl Salicylate), COX 2 specific (Valdecoxib, NS-398) or dual specific (Aspirin, Ibuprofen) NSAIDs with G-CSF. COX 1 specific NSAIDs had no effect on HSPC mobilization, while COX 2 specific and dual inhibitors significantly enhanced mobilization over G-CSF alone. The lipoxygenase (LOX) inhibitor baicalein had no effect on HSPC mobilization and the COX/LOX inhibitor Licofelone was no more effective than NSAIDs, indicating that the mobilization enhancing effect is COX specific and not due to general eicosanoid inhibition. Intriguingly, the dual COX inhibitors produced the greatest activity and a dose-dependent response (max response: 3.92±0.15 fold over G-CSF), indicating inhibition of both COX 1 and COX 2 is required for optimal enhancement. Since PGE2 signals through 4 G-protein coupled receptors (EP1-4), mice were administered EP specific antagonists + G-CSF, or EP specific agonists + NSAID + G-CSF. EP4 antagonists significantly enhanced mobilization over G-CSF alone (2.02±0.21 fold, p<0.005), while an EP1-3 antagonist had no effect. Similarly, a selective EP4 agonist (L-902,688) abrogated NSAID enhancement, while Butaprost (EP2) or Sulprostone (EP1/3) had no effect, indicating that a lack of EP4 receptor signaling is responsible for enhanced HSPC mobilization. Treatment of bone marrow cells with EP specific agonists/antagonists in vitro demonstrates inhibition of colonies by EP4 agonists and a block of inhibition by EP4 antagonists, suggesting a model in which a lack of EP4 signaling drives HSPC expansion. In vivo administration of NSAIDs for 4 days resulted in expansion of CFU-GM and SKL cells per femur, indicating that the mechanism of enhanced mobilization may be in part due to expansion of marrow HSPC. Our results define a novel role for NSAIDs and suggest that the addition of NSAIDs, particularly dual COX inhibitors (i.e. Aspirin or Ibuprofen), to current mobilization regimens may increase HSPC yield. In addition, we have identified novel mechanisms mediated by EP receptors and show that HSPC expansion and mobilization can be enhanced in vivo through antagonism of the EP4 receptor, defining a new pharmaceutical target for hematopoietic mobilization and transplantation.Disclosures: No relevant conflicts of interest to declare.

Licofelone, a dual COX/5-LOX inhibitor, induces apoptosis in HCA-7 colon cancer cells through the mitochondrial pathway independently from its ability to affect the arachidonic acid cascade

Nowadays, no data are available concerning the potential use of dual cyclooxygenase (COX)/5-lipoxygenase (LOX) inhibitors as anticancer agents in colon cancer treatment. Here, we report, for the first time, that the dual COX/5-LOX inhibitor licofelone triggers apoptosis in a dose- and time-dependent manner in HCA-7 colon cancer cells. Induction of apoptosis was related to the recruitment of the intrinsic mitochondrial apoptotic pathway, as shown by loss in mitochondrial membrane potential, cytochrome c release, caspase-9 and 3 activation and poly-(ADP-ribose)polymerase-1 cleavage. Moreover, licofelone induced the cleavage of the full-length p21(Bax) into p18(Bax), a more potent inducer of the apoptotic process than the uncleaved form. Pre-treatment of HCA-7 cells with the pan-caspase inhibitor z-VAD-fmk significantly blocked licofelone-induced apoptosis, confirming that this process occurred primarily in a caspase-dependent pathway. We also present evidences that licofelone was able to affect the arachidonic acid (AA) cascade, as it blocked the activity of 5-LOX and COX enzymes, and it induced, through the phosphorylation of cytoplasmic phospholipase A(2) (cPLA(2)), the release of unesterified AA from HCA-7 membrane phospholipids. However, apoptosis induction was not related to the ability of licofelone to affect the AA cascade, since neither exogenous prostaglandin E(2) and leukotriene B(4) addition, nor pharmacological inhibition of cPLA(2), was able to rescue HCA-7 cells from apoptosis. Even if further studies are needed to clarify the mechanism of licofelone-induced apoptosis, this study suggests that this drug, as well as similar dual COX/5-LOX inhibitors, may represent a novel and promising approach in colon cancer treatment.

Comparative Protection against Liver Inflammation and Fibrosis by a Selective Cyclooxygenase-2 Inhibitor and a Nonredox-Type 5-Lipoxygenase Inhibitor

In this study, we examined the relative contribution of cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LO), two major proinflammatory pathways up-regulated in liver disease, to the progression of hepatic inflammation and fibrosis. Separate administration of 4-[5-(4-chlorophenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide (SC-236), a selective COX-2 inhibitor, and CJ-13,610, a 5-LO inhibitor, to carbon tetrachloride-treated mice significantly reduced fibrosis as revealed by the analysis of Sirius Red-stained liver sections without affecting necroinflammation. Conversely, combined administration of SC-236 and 4-[3-[4-(2-methylimidazol-1-yl)-phenylthio]]phenyl-3,4,5,6-tetrahydro-2H-pyran-4-carboxamide (CJ-13,610) reduced both necroinflammation and fibrosis. These findings were confirmed in 5-LO-deficient mice receiving SC-236, which also showed reduced hepatic monocyte chemoattractant protein 1 expression. Interestingly, SC-236 and CJ-13,610 significantly increased the number of nonparenchymal liver cells with apoptotic nuclei (terminal deoxynucleotidyl transferase dUTP nick-end labeling-positive). Additional pharmacological profiling of SC-236 and CJ-13,610 was performed in macrophages, the primary hepatic inflammatory cell type. In these cells, SC-236 inhibited prostaglandin (PG) E2 formation in a concentration-dependent manner, whereas CJ-13,610 blocked leukotriene B4 biosynthesis. Of note, the simultaneous addition of SC-236 and CJ-13,610 resulted in a higher inhibitory profile on PGE2 biosynthesis than the dual COX/5-LO inhibitor licofelone. These drugs differentially regulated interleukin-6 mRNA expression in macrophages. Taken together, these findings indicate that both COX-2 and 5-LO pathways are contributing factors to hepatic inflammation and fibrosis and that these two pathways of the arachidonic acid cascade represent potential targets for therapy.

The lipoxygenase–cyclooxygenase inhibitor licofelone prevents thromboxane A2-mediated cardiovascular derangement triggered by the inflammatory peptide fMLP in the rabbit

Licofelone is an analogue of arachidonic acid that inhibits 5-lipoxygenase (LOX), cyclooxygenase (COX)-1 and COX-2. We investigated the effects of licofelone on cardiovascular derangements and production of thromboxane (Tx)A 2 induced by the inflammatory agonist n-formyl-methionyl-leucyl-phenylalanine (fMLP) in the rabbit, in comparison with those of aspirin or rofecoxib, inhibitors of COX-1 and COX-2, respectively. In control rabbits, injection of fMLP (30 nmol/kg) in the jugular vein evokes ischemic electrocardiographic (ECG) changes in the first 1–5 min, i.e. a profound depression of the ST segment and inversion of the T wave. Simultaneously, fMLP induces bradycardia and hypotension and increases TxB 2 blood levels. All changes are transient. Licofelone (60 mg/kg/5 days, p.os) prevented fMLP-induced ECG ischemic changes in all treated animals, reverted bradycardia and hypotension, and significantly reduced TxB 2. Aspirin (10 mg/kg/5 days, p.os) prevented ischemic ECG alterations in 2 out of 5 treated animals and did not modify either bradycardia or hypotension. One rabbit died two min after fMLP. In 2 rabbits, aspirin reduced TxB 2 levels by more than 80% respect to mean control values; the remaining two rabbits produced an amount of TxB 2 similar to controls. These two rabbits also showed ischemic ECG changes. Rofecoxib (10 mg/kg/5 days, p.os) did not prevent fMLP-induced ischemic ECG alteration, bradycardia and hypotension, and did not significantly modify the increase of TxB 2. These results indicate that the capacity of licofelone to efficiently suppress TxA 2 production, is responsible for the protection from the cardiovascular derangement triggered by an inflammatory stimulus.

Licofelone, a novel 5-LOX/COX-inhibitor, attenuates leukocyte rolling and adhesion on endothelium under flow

The main mechanism of action of non-steroidal anti-inflammatory drugs (NSAIDs) is the inhibition of cycloxygenases COX-1 and COX-2. During recent years, combined 5-LOX/COX-inhibition, interfering with the biosynthesis of both prostaglandins and leukotrienes (LTs), has emerged as a possibility to avoid side effects related to COX-inhibition. The aim of the present study was to investigate if there is a contribution of mechanisms other than the reduction of inflammatory prostaglandins and leukotrienes to the anti-inflammatory effect of the LOX/COX inhibitor licofelone. In a flow chamber assay, licofelone (10–30 μM) dose-dependently decreased both the rolling and adhesion of leukocytes on endothelial cells (EC). In contrast, no effects were found after treatment of EC with the unselective COX-1/COX-2 inhibitor indomethacin (30 μM), the potent and selective 5-LOX inhibitor, ZD-2138 (30 μM), the mainly COX-2 inhibitor aceclofenac (30 μM), the selective COX-2 inhibitor celecoxib (30 μM) and the combination of ZD-2138 with the selective COX-2 inhibitor celecoxib (30 μM). In the presence of licofelone (30 μM) the expression of E-selectin mRNA in cytokine-stimulated EC was attenuated, whereas no NSAID (30 μM) tested showed any effect on E-selectin expression. Moreover, licofelone treatment (30 μM) attenuated expression of VCAM-1 and ICAM-1 on inflammatory EC. The effect of licofelone on leukocyte recruitment was also evaluated in vivo. Using a mouse peritonitis model it was found that leukocyte accumulation was markedly reduced in licofelone treated animals (100 mg/kg) compared to untreated mice. Thus, the novel 5-LOX/COX inhibitor licofelone possesses anti-inflammatory activity that, in addition to COX/LOX inhibition, involves effects on leukocyte-endothelial interactions.

Eicosanoids, osteoarthritis, and crystal deposition diseases

Eicosanoids are produced by chondrocytes, synoviocytes, and subchondral osteoblasts within the osteoarthritic joint and are involved in normal joint physiology as well as in the pathogenesis of joint disorders such as osteoarthritis. Calcium-containing crystals are found in most osteoarthritic joints and have been implicated in osteoarthritis. Recent advances in the understanding of the potential role of eicosanoids in the pathogenesis of osteoarthritis and in potential therapeutic targeting of eicosanoid pathways are reviewed. The ability of interleukin-1beta to upregulate microsomal prostaglandin E2 synthase-1 in synovial fibroblasts and chondrocytes of patients with osteoarthritis has been demonstrated. A potential role for prostaglandin E2 in downregulating interleukin-1beta-induced inflammatory responses has also been described. Basic calcium phosphate crystals can upregulate cyclooxygenase-1 and cocylooxygenase-2 expression, both of which contributed to the observed increase in prostaglandin E2 production in human fibroblasts. Novel potential mechanisms of inhibition of eicosanoid synthesis are also discussed. Last, further evidence of amelioration of osteoarthritis in animal models by the dual 5-lipoxygenase/cyclooxygenase inhibitor licofelone has been reported. The inhibition of prostaglandin synthesis has long been a ornerstone of the pharmacologic treatment of osteoarthritis. Nevertheless, prostaglandins may have potentially beneficial as well as deleterious effects in osteoarthritis. In addition, other eicosanoids such as leukotrienes have also been implicated in the pathogenesis of osteoarthritis. Therefore, more selective inhibition of prostaglandin pathways and/or inhibition of leukotriene activity may prove to be effective therapeutic strategies in osteoarthritis.

Combined Lipoxygenase/Cyclo-oxygenase Inhibition in the Elderly

One of the categories of drugs most frequently used by the elderly, and probably the most commonly self-prescribed class of drug in this age group, is NSAIDs. However, NSAIDs are one of the primary causes of adverse drug reactions and are notorious for their gastric toxicity. They also inhibit renal function and reduce the efficacy of diuretics and ACE inhibitors, drugs that are commonly used by elderly patients. Recent studies have shown that cyclo-oxygenase (COX)-2 is important in renal physiology. This means that selective COX-2 inhibitors, while undoubtedly safer than NSAIDs in terms of gastric toxicity, are not devoid of renal toxicity (in addition to their now clearly established adverse effects on coronary heart disease risk). Both the gastric and renal toxicities induced by traditional NSAIDs and selective COX-2 inhibitors seem to be related to inhibition of prostaglandin, but not leukotriene, synthesis. Maintaining the correct balance between prostaglandins and leukotrienes is essential for continuing good health, but both classes of mediators also play an important role in the pathogenesis of several diseases.Recently, a new class of anti-inflammatory drugs, the lipoxygenase (LOX)/COX inhibitors, has been developed as a means of simultaneously inhibiting the synthesis of prostaglandins, thromboxanes and leukotrienes. Inhibition of leukotriene synthesis increases anti-inflammatory efficacy, particularly in rheumatic diseases, while reducing the risk of gastric damage. The LOX/COX inhibitor licofelone, which is currently in phase III trials, is the first of this new class and in the most advanced stage of development. Preliminary data with this drug seem promising, but further well designed clinical trials of this agent in the elderly will be necessary before a final evaluation is possible.

Pain and osteoarthritis: new drugs and mechanisms

This review discusses the recent literature on drugs used for symptomatic pain relief in patients with osteoarthritis (OA) as well as potential mechanisms underlying their pharmacologic action. Recent research has shed light on the molecular mechanisms underlying the contribution of prostaglandins to pain sensation. Moreover, the role of the enzymes cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX) in inflammation and subsequent structural changes of joints has been clarified. Based on the COX-1/COX-2 hypothesis, various selective COX-2 inhibitors with improved gastrointestinal tolerability as compared with conventional nonsteroidal anti-inflammatory drugs (NSAIDs) have been established for the symptomatic treatment of OA in recent years. Rational therapy with these compounds should be based on their diverse pharmacokinetic characteristics. Among the traditional NSAIDs, the mode of action of aceclofenac has been recently clarified in that the compound was shown to elicit preferential inhibition of COX-2 as a result of limited but sustained biotransformation to diclofenac. Novel mechanisms have also been proposed to account for the action of acetaminophen. Finally, there is evidence from animal models to suggest that the dual LOX/COX inhibitor licofelone may stop disease progression in OA. Clinical studies are under way to establish this compound for treatment of OA. It is anticipated that new insights into the pathophysiology of OA as well as novel therapeutics will improve the pharmacologic options in OA.

Licofelone, an inhibitor of cyclooxygenase and 5-lipoxygenase, specifically inhibits cyclooxygenase-1-dependent platelet activation

5-Lipoxygenase/cyclooxygenase inhibitors, possessing anti-inflammatory action and gastric safety due to cyclooxygenase-2 and 5-lipoxygenase inhibition and antiplatelet activity due to cyclooxygenase-1 blockade, would be beneficial in the treatment of ischemic disease because they may reduce, at the same time, inflammation, underlying the atherosclerotic process, and platelet activation, responsible for acute thrombotic events. In this study, we characterized the antiplatelet effects of the new 5-lipoxygenase/cyclooxygenase inhibitor licofelone ([2,2-dimethyl-6-(4-chlorophenyl)-7-phenyl-2,3,dihydro-1 H-pyrrolizine-5-yl]-acetic acid. Licofelone completely prevented platelet aggregation induced in platelet-rich plasma by threshold aggregating concentrations of arachidonic acid (0.87±0.14 mM) at threshold inhibitory concentrations of 0.75±0.35 μM ( n=5). Platelet-rich plasma aggregation induced by threshold aggregating concentrations of collagen/adrenalin (0.3±0.05 μg/ml and 0.4±0.1 μM, respectively) was reduced to 3.2±2% of control at licofelone 100 μM, ( P<0.05, n=6). Washed platelet aggregation induced by threshold aggregating concentrations of thrombin (0.07±0.01 U/ml) was only partially affected by licofelone at concentrations one or two order of magnitude higher than those fully preventing arachidonic acid-induced aggregation (44±11% of control at 100 μM, P<0.05, n=7). Failure to prevent aggregation triggered by high concentrations of collagen/adrenalin in aspirin-treated platelets supports cyclooxygenase-1 as a specific target of licofelone. In fact, licofelone inhibited thromboxane B 2 (TxB 2) production by all the agonists tested at concentrations between 0.5 and 50 μM. At this concentration, TxB 2 production was reduced at values similar to those of unstimulated platelets. These results indicate that, at clinically relevant concentrations, licofelone exerts a potent antiplatelet effect mediated by the inhibition of cyclooxygenase-1 activity.

Osteoarthritis therapy--are there still unmet needs?

Non-steroidal anti-inflammatory drugs (NSAIDs) and selective cyclooxygenase (COX)-2 inhibitors are commonly used to control pain and inflammation in osteoarthritis. However, these agents have been associated with gastrointestinal, renal and cardiovascular adverse effects. Together, these complications indicate a clear unmet need in the safety of current treatment options for the management of osteoarthritis. NSAIDs are known to have adverse gastrointestinal effects, and more recently it has been suggested that some selective COX-2 inhibitors are also associated with serious gastrointestinal complications. Selective COX-2 inhibitors have a similar capacity to NSAIDs to delay ulcer healing, and may not significantly decrease the incidences of perforation, ulceration and bleeding (the most clinically relevant gastrointestinal endpoints) compared with NSAIDs. These effects may be due to overlapping roles of COX-1 and COX-2 in physiological and pathophysiological processes. Furthermore, as COX-2 is integrally involved in renal homeostasis, selective COX-2 inhibitors are associated with negative effects on kidney function similar to those seen with NSAIDs. Electrolyte disturbances, oedema and hypertension have been correlated with the use of both drug classes. Additionally, selective COX-2 inhibitors have the potential to increase cardiovascular events, although further research is required to clearly determine such a risk. With the current unmet needs in the treatment of osteoarthritis, the opportunity exists for the development of new therapies. Novel agents include the COX-inhibiting nitric oxide donors and the lipoxygenase (LOX)/COX inhibitor licofelone. Initial results suggest that these therapies may have tolerability advantages over the NSAIDs and selective COX-2 inhibitors.

Licofelone, a dual lipoxygenase–cyclooxygenase inhibitor, downregulates polymorphonuclear leukocyte and platelet function

Polymorphonuclear leukocytes are strongly implicated in the pathogenesis of inflammatory disease. Polymorphonuclear leukocyte recruitment at sites of inflammation, mainly sustained by the β2-integrins, is followed by the synthesis and release of inflammatory mediators, such as leukotrienes, proteolytic enzymes and reactive oxygen species. Functional and metabolic interactions between polymorphonuclear leukocytes and platelets can contribute to and exacerbate the process. The effects of the dual 5-lipoxygenase and cyclooxygenase inhibitor licofelone ([2,2-dimethyl-6-(4-chlorophenyl)-7-phenyl-2,3-dihydro-1 H-pyrrolizine-5-yl]-acetic acid) were studied on arachidonic acid transcellular metabolism occurring between polymorphonuclear leukocytes and platelets. The formation of leukotriene C 4, a leukotriene A 4-derived metabolite, by mixed polymorphonuclear leukocyte/platelet suspensions stimulated with 10 μM A23187 was inhibited by licofelone with an IC 50 of 3.8±0.07 μM. The formation of 5,12-di-hydroxy-eicosatetraenoic acid (HETE) was abolished at concentrations ≥10 μM. Licofelone also inhibited the generation of reactive oxygen species by polymorphonuclear leukocytes stimulated with 1 μM n-formyl-methionyl-leucyl-phenylalanine (fMLP), 10 nM complement fraction 5a (C5a) and 1 μM platelet activating factor (PAF) with IC 50s of 24.4±0.6, 11.0±1.5 and 11.7±1.2 μM; elastase release induced by the three agonists was inhibited with IC 50s of 12.2±2.2, 23.5±8 and 2.6±1 μM, respectively. Homotypic polymorphonuclear leukocyte aggregation induced by fMLP, C5A and PAF was inhibited by licofelone with IC 50s of 23.7±4.8, 15.6±3.4 and 15.4±4 μM, respectively. The present study extends the anti-lipoxygenase and anti-cyclooxygenase activities of licofelone to the production of arachidonic acid metabolites generated as a consequence of polymorphonuclear leukocyte-platelet transcellular metabolism and to polymorphonuclear leukocyte responses relevant to the pathogenesis of inflammation. The coexistence within the same molecule of a wide spectrum of anti-inflammatory properties is of interest.