Background:The structurally identical monomers of cyclooxygenase-2 act as distinct catalytic and allosteric subunits. Results: 13-Methylarachidonic acid strongly potentiates cyclooxygenase-2-mediated endocannabinoid oxygenation without affecting arachidonic acid oxygenation. Conclusion: 13-Methylarachidonic acid is a substrate-selective allosteric potentiator of cyclooxygenase-2-mediated endocannabinoid oxygenation. Significance: 13-Methylarachidonic acid is a valuable tool for probing cyclooxygenase-dependent endocannabinoid oxygenation.

BACKGROUND AND PURPOSECannabinoid (CB) ligands have been demonstrated to have utility as novel therapeutic agents for the treatment of pain, metabolic conditions and gastrointestinal (GI) disorders. However, many of these ligands are centrally active, which limits their usefulness. Here, we examine a unique novel covalent CB receptor ligand, AM841, to assess its potential for use in physiological and pathophysiological in vivo studies. EXPERIMENTAL APPROACHThe covalent nature of AM841 was determined in vitro using electrophysiological and receptor internalization studies on isolated cultured hippocampal neurons. Mouse models were used for behavioural analysis of analgesia, hypothermia and hypolocomotion. The motility of the small and large intestine was assessed in vivo under normal conditions and after acute stress. The brain penetration of AM841 was also determined. KEY RESULTSAM841 behaved as an irreversible CB1 receptor agonist in vitro. AM841 potently reduced GI motility through an action on CB1 receptors in the small and large intestine under physiological conditions. AM841 was even more potent under conditions of acute stress and was shown to normalize accelerated GI motility under these conditions. This compound behaved as a peripherally restricted ligand, showing very little brain penetration and no characteristic centrally mediated CB1 receptor-mediated effects (analgesia, hypothermia or hypolocomotion). CONCLUSIONS AND IMPLICATIONSAM841, a novel peripherally restricted covalent CB1 receptor ligand that was shown to be remarkably potent, represents a new class of potential therapeutic agents for the treatment of functional GI disorders.

Lapatinib (LAP), an oral tyrosine kinase inhibitor for the treatment of metastatic breast cancer, has been associated with idiosyncractic hepatotoxicity. Recent investigations have implicated the importance of P450 3A4/5 enzymes in the formation of an electrophilic quinone imine (LAPQI) metabolite generated through further oxidation of O-dealkylated lapatinib (OD-LAP). In the current study, hepatic stress was observed via mitochondrial impairment. OD-LAP caused a time- and concentration-dependent decrease in oxygen consumption in HepG2 cells, whereas LAP did not alter the oxygen consumption rate. Interestingly, however, HepG2 cells transfected with human P450 3A4 did exhibit mitochondrial dysfunction via P450 3A4-mediated metabolism of LAP to OD-LAP. OD-LAP-induced mitochondrial toxicity was enhanced upon depletion of intracellular GSH levels, demonstrating that cellular GSH levels are important in the protection of mitochondrial function against LAPQI. Given the nature of LAPQI and the importance of GSH levels in LAP-induced mitochondrial stress, the activation of nuclear factor erythroid 2-related factor 2 (Nrf2) was evaluated, as this transcription factor induces the expression of NAD(P)H quinone oxidoreductase 1, glutathione S-transferase, UDP-glucuronosyltransferases, and glutathione synthetase, all of which might be expected to decrease the toxicity of LAP. Using a FRET-based target gene assay in HepG2 cells, OD-LAP was indeed found to activate Nrf2. Follow-up assays showed increased mRNA levels of Nrf2 target genes after a 4 h treatment with OD-LAP but not with LAP. LAP activation of Nrf2 was observed only when HepG2 cells were transduced with P450 3A4. The significance of Nrf2 protection was established in vivo in Nrf2-KO mice. Increased transaminase levels were found after a single LAP dose in both Nrf2-KO and control mice, indicating elevated hepatic necrosis, although transaminase levels reverted to baseline levels in the control mice upon repeat dosing. They continued to rise in Nrf2-KO mice, however, indicating the likelihood that Nrf-2 plays a significant role in combatting the hepatotoxicity triggered by LAP.

The synthesis of potent metabolically stable endocannabinoids is challenging. Here we report a chiral arachidonoyl ethanolamide (AEA) analogue, namely, (13 S,1' R)-dimethylanandamide (AMG315, 3a), a high affinity ligand for the CB1 receptor ( K of 7.8 ± 1.4 nM) that behaves as a potent CB1 agonist in vitro (EC = 0.6 ± 0.2 nM). (13 S,1' R)-dimethylanandamide is the first potent AEA analogue with significant stability for all endocannabinoid hydrolyzing enzymes as well as the oxidative enzymes COX-2. When tested in vivo using the CFA-induced inflammatory pain model, 3a behaved as a more potent analgesic when compared to endogenous AEA or its hydrolytically stable analogue AM356. This novel analogue will serve as a very useful endocannabinoid probe.

Oxanorbornadiene dicarboxylate (OND) reagents were explored for the purpose of binding and releasing chemical cargos from endogenous circulating serum albumins. ONDs bearing gadolinium chelates as model cargos exhibited variable conjugation efficiencies with albumin in rat subjects that are consistent with the observed reactivity of each linker and their observed stability toward serum hydrolases in vitro. The terminal elimination rate from circulation was dependent on the identity of the OND used, and increased circulation time of gadolinium cargo was achieved for linkers bearing electrophilic fragments designed to react with cysteine-34 of circulating serum albumin. This binding of and release from endogenous albumin highlights the potential of OND linkers in the context of optimizing the pharmacokinetic parameters of drugs or diagnostic agents.