5-lipoxygenase (5-LO)
The primary target of Docebenone is the enzyme 5-lipoxygenase (5-LO). 5-LO is a non-heme iron-containing dioxygenase that catalyzes the first two steps in the leukotriene synthesis pathway: the oxidation of arachidonic acid to 5-HPETE and its subsequent dehydration to leukotriene A4 (LTA4). LTA4 is a critical intermediate that is further metabolized to LTB4 by LTA4 hydrolase or to the cysteinyl leukotrienes by LTC4 synthase. By selectively inhibiting 5-LO, Docebenone effectively shuts down the entire leukotriene cascade. This target is distinct from the cyclooxygenase (COX) enzymes targeted by NSAIDs, providing a different mechanism for modulating inflammation. The selectivity of Docebenone for 5-LO over other lipoxygenases and COX enzymes is a key feature of its activity.

When Trichomonas are treated with the 5-LO inhibitor doxylbenzoquinone, their capacity to secrete LTB4 is considerably reduced as compared to the outcomes of treating them with culture medium. TvSP's stimulatory effect on IL-8 production is significantly reduced by doxibenquinone [1]. A dependent increase in [Ca2+]i concentration is observed at 10-200 μM Doxibenquinone [2].

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Docebenone exhibits potent in vitro activity as a selective inhibitor of 5-lipoxygenase. In enzyme assays, it inhibits the production of leukotrienes from arachidonic acid. Its inhibitory effects have been demonstrated in various cell types, including neutrophils and mast cells. For instance, treatment of Trichomonas vaginalis with Docebenone significantly reduces their capacity to secrete LTB4. It also strongly inhibits the stimulatory effect of TvSP on IL-8 production in neutrophils. Furthermore, Docebenone has been shown to induce a concentration-dependent increase in intracellular calcium [Ca²⁺]i at concentrations of 10-200 μM. These in vitro studies confirm its efficacy at the enzyme and cellular levels, making it a reliable tool for investigating 5-LO-mediated processes.

Rats exposed to experimental acute necrotizing pancreatitis may benefit from doxibenone's protective effects [3]. Doxepinone, at 10-8-10-5 M, exhibits a dose-dependent inhibition of 55-97% on the release of SRS-A in monkeys. Doxobenone, at dosages of 10-8-10-5 M, reduced the antigen-induced release of SRS-A from these fragments by 25-93% in a dose-dependent manner [4].

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Docebenone is orally active and has demonstrated protective and anti-inflammatory effects in several in vivo models. In animal models of acute necrotizing pancreatitis, Docebenone has shown protective effects. It has also been shown to dose-dependently inhibit the release of slow-reacting substance of anaphylaxis (SRS-A) in monkeys, with inhibition ranging from 55-97% at concentrations of 10⁻⁸ to 10⁻⁵ M. Additionally, it reduced the antigen-induced release of SRS-A from tissue fragments by 25-93% in a dose-dependent manner. These results highlight the compound's ability to block the 5-LO pathway and reduce leukotriene-mediated inflammatory responses in a living organism.

T. vaginalis produced more than 714 pg/ml of LTB(4) per 1 × 10(7) trichomonads. The ability of trichomonads to secrete LTB(4) was inhibited by treatment of trichomonads with the 5-lipo-oxygenease inhibitor AA861, but not the cyclo-oxygenease I inhibitor FR122047. When neutrophils were incubated with TvSP obtained from 1 × 10(7) trichomonads, IL-8 protein secretion was significantly increased compared to results for cells incubated with medium alone. The stimulatory effect of TvSP on IL-8 production was strongly inhibited by pretreatment of TvSP with lipase, although pretreatment with heat or proteinase K showed little inhibitory effect. Moreover, TvSP-induced IL-8 production was efficiently inhibited when trichomonads were pretreated with AA861 or when neutrophils were pretreated with antagonists for BLT1 or BLT2. Conclusion: Our results suggest that LTB(4) receptors BLT1 and BLT2 are involved in IL-8 production in neutrophils induced by T. vaginalis[1].

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The in vitro enzyme assay for Docebenone typically involves measuring the activity of 5-lipoxygenase using a cell-free system. Purified 5-LO enzyme is incubated with arachidonic acid in the presence of varying concentrations of Docebenone. The reaction is initiated by the addition of the substrate, and after a defined incubation period, the reaction is stopped. The amount of leukotriene produced (such as LTB4 or LTC4) is then quantified using an Enzyme-Linked Immunosorbent Assay (ELISA) or High-Performance Liquid Chromatography (HPLC). By comparing the leukotriene levels in the presence of the inhibitor to a control without the inhibitor, the IC₅₀ value can be determined. This protocol is a standard biochemical approach for characterizing 5-LO inhibitors.

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[2].

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In vitro cell-based assays evaluate Docebenone's activity in living cells. Typically, cells that express 5-LO, such as neutrophils, mast cells, or macrophages, are used. The cells are first treated with Docebenone and then stimulated with a calcium ionophore like A23187 to activate the 5-LO pathway and trigger leukotriene synthesis. After a period of incubation, the cell culture supernatant is collected, and the concentration of leukotrienes (e.g., LTB4) is measured by ELISA. The reduction in leukotriene production in the presence of the compound is a measure of its cellular activity. This assay confirms that the compound can effectively inhibit 5-LO in a physiologically relevant cellular environment.

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.[3]

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The effects of 2,3,5-trimethyl-6-(12-hydroxy-5,10-dodecadiynyl)-1,4-benzoquinone (AA-861), a selective 5-lipoxygenase inhibitor, on immunological or non-immunological release of slow reacting substance of anaphylaxis (SRS-A) and histamine and its effects on experimental asthma were investigated. AA-861 showed a dose-dependent inhibition of SRS-A release, with no effect on histamine release from passively sensitized guinea pig, monkey (M. irus) and human lung fragments. An analysis of the anaphylactic diffusate from the human lung fragments, using the combined technique of high performance liquid chromatography and radioimmunoassay, revealed that AA-861 markedly suppresses biosynthesis of the leukotrienes. However, this drug inhibits the release of histamine as well as SRS-A from lung fragments of anaphylactic monkey (M. mulatta) and in the Ca ionophore-stimulated rat peritoneal cavity. AA-861 suppressed the anaphylactically-induced airway resistance in mepyramine- and cimetidine-treated guinea pigs. These results suggest that AA-861 may be clinically effective for treating allergy-related asthma by modulating the 5-lipoxygenase pathway and that an inhibitory mechanism of histamine release by AA-861 may be present in some species.[4]

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In vivo animal models are employed to assess the therapeutic potential of Docebenone. One common model is the carrageenan-induced paw edema model in rodents, which is used to study acute inflammation. In this model, inflammation is induced by injecting carrageenan into the paw of a rat or mouse. Docebenone is administered orally prior to or after the injection. The increase in paw volume is measured over time as an indicator of inflammation. A reduction in paw swelling in the treated group compared to the control group indicates an anti-inflammatory effect. Other models, such as asthma models where leukotrienes play a key role, can also be used to evaluate the compound's efficacy.

Docebenone has a molecular weight of 326.43 and is characterized as an orally bioactive compound. It is a lipophilic molecule due to its benzoquinone structure with a long hydrophobic side chain. This lipophilicity likely contributes to its good oral absorption and bioavailability. Detailed pharmacokinetic parameters such as half-life (t½), maximum plasma concentration (Cmax), and area under the curve (AUC) are not extensively reported in the public literature. However, its efficacy in various in vivo models after oral administration confirms that it reaches sufficient concentrations in the bloodstream and at the site of action to exert its pharmacological effects.

Comprehensive toxicity data for Docebenone is limited. As a 5-LO inhibitor, it is expected to have a different toxicity profile compared to non-selective COX inhibitors, as it does not affect prostaglandin synthesis, which is important for gastric mucosal protection and renal function. However, leukotrienes are involved in host defense, so there is a potential for increased susceptibility to infections. The compound is a benzoquinone, and some quinones can cause oxidative stress at high concentrations. It is classified as a research compound and is not intended for human use.

[1]. Leukotriene B(4) receptors BLT1 and BLT2 are involved in interleukin-8 production in human neutrophils induced by Trichomonas vaginalis-derived secretory products. Inflamm Res. 2012 Feb;61(2):97-102.

[2]. AA-861-induced Ca(2+) mobilization in Madin Darby canine kidney cells. Toxicol Appl Pharmacol. 1999 Dec 1;161(2):202-8.

[3]. Protective effect of AA-861 (5-lipoxygenase inhibitor) on experimental acute necrotizing pancreatitis in rats. Int J Pancreatol. 1993 Jun;13(3):201-8.

[4]. Effect of AA-861, a selective 5-lipoxygenase inhibitor, on models of allergy in several species. Jpn J Pharmacol. 1988 Jul;47(3):261-71.

Docebenone belongs to the benzoquinone class of compounds. Its structure is p-benzoquinone, where the hydrogen atom is replaced by three methyl groups and one 12-hydroxydodeca-5,10-diyn-1-yl group. It is an EC 1.13.11.34 (arachidonic acid 5-lipoxygenase) inhibitor and a ferroptosis inhibitor. It is a primary alcohol, alkyne compound, and also a 1,4-benzoquinone compound.

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Docebenone (AA-861) is a well-established and valuable research tool for studying the 5-lipoxygenase/leukotriene pathway. It has been extensively used in inflammation research, particularly in the context of asthma, allergic diseases, and other inflammatory conditions. Its antioxidant properties also make it useful for studying oxidative stress. The compound has not advanced to clinical trials and does not have FDA approval for any indication.

C21H26O3

326.44

326.188

C, 77.27; H, 8.03; O, 14.70

80809-81-0

1967

Light yellow to yellow solid powder

3.52

1

3

6

24

692

0

WDEABJKSGGRCQA-UHFFFAOYSA-N

InChI=1S/C21H26O3/c1-16-17(2)21(24)19(18(3)20(16)23)14-12-10-8-6-4-5-7-9-11-13-15-22/h22H,5,7-10,12,14-15H2,1-3H3

2-(12-hydroxydodeca-5,10-diynyl)-3,5,6-trimethylcyclohexa-2,5-diene-1,4-dione

A-61589; A61589; aa-861; 80809-81-0; Docebenonum; Docebenona; AA861; 2-(12-hydroxydodeca-5,10-diyn-1-yl)-3,5,6-trimethylcyclohexa-2,5-diene-1,4-dione; 2-(12-Hydroxy-5,10-dodecadiynyl)-3,5,6-trimethyl-p-benzoquinone; Docebenone

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~250 mg/mL (~765.86 mM)

Solubility in Formulation 1: ≥ 6.25 mg/mL (19.15 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 62.5 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 + to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)