Docebenone (AA 861)

Effect of AA-861, a 5-lipoxygenase inhibitor, on leukotriene synthesis in human polymorphonuclear leukocytes and on cyclooxygenase and 12-lipoxygenase activities in human platelets

AA-861, a selective inhibitor of 5-lipoxygenase of arachidonic acid, was tested for ability to inhibit leukotriene C4 and leukotriene B4 synthesis in human polymorphonuclear leukocytes after calcium ionophore stimulation. AA-861 dose-dependently inhibited leukotriene B4 and leukotriene C4 generation in human polymorphonuclear leukocytes; the concentration required to inhibit generation by 50% (IC50) was 3 X 10(-7) M for leukotriene B4 and 1 X 10(-8) M for leukotriene C4. BW-755C inhibited the generation of leukotriene C4 with an IC50 of about 10(-5) M, indicating that AA-861 is about 1,000 times more potent than BW-755C. AA-861 did not affect the activity of either cyclooxygenase or 12-lipoxygenase at a concentration up to 10(-5) M in human platelets. AA-861 did not inhibit histamine release from human basophils. These results indicate that AA-861 selectively inhibits 5-lipoxygenase but not cyclooxygenase or 12-lipoxygenase in human specimens.

Protective effect of AA-861 (5-lipoxygenase inhibitor) on experimental acute necrotizing pancreatitis in rats

The effect of the 5-lipoxygenase inhibitor AA-861 on acute necrotizing pancreatitis was studied in an experimental model. Pancreatitis was induced in rats by retrograde injection of 0.4 mL/kg body wt of 6% taurocholic acid into the pancreatic duct. The animals were divided into three groups: control group; administered AA-861 in a single dose of 30 mg/kg; and administered AA-861 in a single dose of 60 mg/kg. The following parameters were examined: serum amylase, lipase, trypsin, blood sugar, and survival rate. Histology of the pancreas was also studied. The serum amylase and lipase activities in groups 2 and 3 were lower than those in group 1 in the early phase after induction of pancreatitis. The elevation of serum trypsin was not suppressed by AA-861. Blood sugar was more efficiently controlled in groups 2 and 3 than in group 1. The survival rates in groups 2 and 3 were better than that in group 1, but there were no significant differences among the three groups. Histologically, massive tissue necrosis with hemorrhage, edema, and inflammatory cell infiltration was prominent in group 1, whereas such changes were obviously suppressed in groups 2 and 3. The results suggest that AA-861 may prove useful in the treatment of acute pancreatitis.

Inhibitors of Human 5-Lipoxygenase Potently Interfere With Prostaglandin Transport

5-Lipoxygenase (5-LO) is the key enzyme in the formation of pro-inflammatory leukotrienes (LT) which play an important role in a number of inflammatory diseases. Accordingly, 5-LO inhibitors are frequently used to study the role of 5-LO and LT in models of inflammation and cancer. Interestingly, the therapeutic efficacy of these inhibitors is highly variable. Here we show that the frequently used 5-LO inhibitors AA-861, BWA4C, C06, CJ-13,610 and the FDA approved compound zileuton as well as the pan-LO inhibitor nordihydroguaiaretic acid interfere with prostaglandin E₂ (PGE₂) release into the supernatants of cytokine-stimulated (TNFα/IL-1β) HeLa cervix carcinoma, A549 lung cancer as well as HCA-7 colon carcinoma cells with similar potencies compared to their LT inhibitory activities (IC₅₀ values ranging from 0.1-9.1 ?M). In addition, AA-861, BWA4C, CJ-13,610 and zileuton concentration-dependently inhibited bacterial lipopolysaccharide triggered prostaglandin (PG) release into human whole blood. Western Blot analysis revealed that inhibition of expression of enzymes involved in PG synthesis was not part of the underlying mechanism. Also, liberation of arachidonic acid which is the substrate for PG synthesis as well as PGH₂ and PGE₂ formation were not impaired by the compounds. However, accumulation of intracellular PGE₂ was found in the inhibitor treated HeLa cells suggesting inhibition of PG export as major mechanism. Further, experiments showed that the PG exporter ATP-binding cassette transporter multidrug resistance protein 4 (MRP-4) is targeted by the inhibitors and may be involved in the 5-LO inhibitor-mediated PGE₂ inhibition. In conclusion, the pharmacological effects of a number of 5-LO inhibitors are compound-specific and involve the potent inhibition of PGE₂ export. Results from experimental models on the role of 5-LO in inflammation and pain using 5-LO inhibitors may be misleading and their use as pharmacological tools in experimental models has to be revisited. In addition, 5-LO inhibitors may serve as new scaffolds for the development of potent prostaglandin export inhibitors.

AA-861 appears to suppress leukocyte infiltration induced by traumatic brain injury in rats

Objective: To study the effect of 2,3,5-Trimethyl-6-(12-hydroxy-5,10-dodecadiynyl)-1,4-benzoquinone (AA-861) on intercellular adhesion molecule 1 (ICAM-1) and P-selectin expression, leukotriene B4 (LTB4) level, and myeloperoxidase (MPO) activity 24 hours after traumatic brain injury (TBI).
Methods: This study was carried out in the laboratory of the Department of Clinical Pharmacology, Osmangazi University, Eskisehir, Turkey in 2006. Traumatic brain injury was induced in 2 sets of animals using Feeney`s weight-drop method. The first set was used to study the expression of ICAM-1, P-selectin, CD11a, and mouse anti-rat granulocyte monoclonal antibody (HIS48). The second was used to study tissue changes in LTB4 level, and MPO activity. The rats were sacrificed at 0.5, 4, 24, 48, and 72 hours post-injury.
Results: Intercellular adhesion molecule (p=0.000001) and P-selectin expression (p=0.00002) peaked at 24 hours, remained high at 48 hours (p=0.00012 for ICAM-1, and p=0.00002 for P-selectin), and 72 hours (p=0.000008 for ICAM-1, p=0.0011 for P-selectin). The HIS48 intensity was significantly increased at 24-72 hours (p=0.022), while the intensity of CD11a became significant only at 72 hours (p=0.040). Myeloperoxidase activity increased notably at 24 hours (p=0.00077), and peaked at 48 hours (p=0.00001). The LTB4 increased markedly at 4 hours (p=0.000004), and peaked at 24 hours (p=0.000001). Pretreatment with AA-861 considerably suppressed the expression of ICAM-1 (p=0.0053), and P-selectin (p=0.0018) on microvascular endothelium, and lowered MPO activity (p=0.0007), and LTB4 level (p=0.008) at 24 hours.
Conclusion: The present results suggest that AA-861 might be a potential mediator in the treatment of brain inflammation in TBI.

AA-861-induced Ca(2+) mobilization in Madin Darby canine kidney cells

The effect of 2,3,5-trimethyl-6-(12-hydroxy-5,10-dodecadiynyl)-1, 4-benzoquinone (AA-861), a 5-lipoxygenase inhibitor, on Ca(2+) mobilization in Madin Darby canine kidney (MDCK) cells has been examined by fluorimetry using fura-2 as a Ca(2+) indicator. AA-861 at 10-200 microM increased [Ca(2+)](i) concentration dependently. The signal comprised an initial rise and a sustained phase. Ca(2+) removal inhibited the Ca(2+) signals by reducing both the initial rise and the sustained phase. In Ca(2+)-free medium, pretreatment with 50 microM AA-861 abolished the Ca(2+) release induced by thapsigargin (1 microM), an endoplasmic reticulum Ca(2+) pump inhibitor, and carbonylcyanide m-chlorophenylhydrazone (CCCP; 2 microM), a mitochondrial uncoupler. Pretreatment with CCCP, thapsigargin and gly-phe-beta-naphthylamide to deplete the Ca(2+) stores in mitochondria, the endoplasmic reticulum, and lysosomes, respectively, only partly inhibited AA-861-induced Ca(2+) release. This suggests AA-861 released Ca(2+) from multiple internal pools. Addition of 3 mM Ca(2+) induced a [Ca(2+)](i) rise after pretreatment with 50 microM AA-861 in Ca(2+)-free medium. AA-861 (50 microM)-induced internal Ca(2+) release was not altered by inhibition of phospholipase C with U73122 (2 microM) but was inhibited by 40% by inhibition of phospholipase A(2) with aristolochic acid (40 microM). Collectively, we found that AA-861 increased [Ca(2+)](i) in MDCK cells by releasing Ca(2+) from multiple internal stores in a manner independent of the formation of inositol-1,4,5-trisphosphate, followed by Ca(2+) entry from external medium.