Sanaz Hashemian

Characterisation of (R)-2-(2-Fluorobiphenyl-4-yl)-N-(3-Methylpyridin-2-yl)Propanamide as a Dual Fatty Acid Amide Hydrolase: Cyclooxygenase Inhibitor

Background Increased endocannabinoid tonus by dual-action fatty acid amide hydrolase (FAAH) and substrate selective cyclooxygenase (COX-2) inhibitors is a promising approach for pain-relief. One such compound with this profile is 2-(2-fluorobiphenyl-4-yl)-N-(3-methylpyridin-2-yl)propanamide (Flu-AM1). These activities are shown by Flu-AM1 racemate, but it is not known whether its two single enantiomers behave differently, as is the case towards COX-2 for the parent flurbiprofen enantiomers. Further, the effects of the compound upon COX-2-derived lipids in intact cells are not known. Methodology/Principal Findings COX inhibition was determined using an oxygraphic method with arachidonic acid and 2-arachidonoylglycerol (2-AG) as substrates. FAAH was assayed in mouse brain homogenates using anandamide (AEA) as substrate. Lipidomic analysis was conducted in unstimulated and lipopolysaccharide + interferon γ- stimulated RAW 264.7 macrophage cells. Both enantiomers inhibited COX-2 in a substrate-selective and time-dependent manner, with IC50 values in the absence of a preincubation phase of: (R)-Flu-AM1, COX-1 (arachidonic acid) 6 μM; COX-2 (arachidonic acid) 20 μM; COX-2 (2-AG) 1 μM; (S)-Flu-AM1, COX-1 (arachidonic acid) 3 μM; COX-2 (arachidonic acid) 10 μM; COX-2 (2-AG) 0.7 μM. The compounds showed no enantiomeric selectivity in their FAAH inhibitory properties. (R)-Flu-AM1 (10 μM) greatly inhibited the production of prostaglandin D2 and E2 in both unstimulated and lipopolysaccharide + interferon γ- stimulated RAW 264.7 macrophage cells. Levels of 2-AG were not affected either by (R)-Flu-AM1 or by 10 μM flurbiprofen, either alone or in combination with the FAAH inhibitor URB597 (1 μM). Conclusions/Significance Both enantiomers of Flu-AM1 are more potent inhibitors of 2-AG compared to arachidonic acid oxygenation by COX-2. Inhibition of COX in lipopolysaccharide + interferon γ- stimulated RAW 264.7 cells is insufficient to affect 2-AG levels despite the large induction of COX-2 produced by this treatment.

Degradation of proteoglycans affects astrocytes and neurite formation in organotypic tissue cultures.

Chondroitin sulfate proteoglycans (CSPGs) promote nerve growth during development, and inhibit axonal growth in the adult CNS after injury. Chondroitinase ABC (ChABC) and methyl-umbelliferyl-β-d-xyloside (β-xyloside), two compounds that degrade CSPGs, promote regeneration after injury, however, they demonstrate opposing results in tissue culture. To elucidate the effect of the two compounds, organotypic tissue cultures, treated with ChABC or β-xyloside, were employed to monitor nerve fiber outgrowth and astrocytic migration. Rat ventral mesencephalon (VM) and spinal cord (SC) from embryonic day (E) 14 and E18 were treated early, from the plating day for 14 days in vitro, or late where treatment was initiated after being cultured for 14 days. In the early treatment of E14 VM and SC cultures, astrocytic migration and nerve fiber outgrowth were hampered using both compounds. Early treatment of E18 cultures reduced the astrocytic migration, while nerve growth was promoted by β-xyloside, but not by ChABC. In the late treated cultures of both E14 and E18 cultures, no differences in distances that astrocytes migrated or nerve fiber growth were observed. However, in β-xyloside-treated cultures, the confluency of astrocytic monolayer was disrupted. In E18 cultures both early and late treatments, neuronal migration was present in control cultures, which was preserved using ChABC but not β-xyloside. In conclusion, ChABC and β-xyloside had similar effects and hampered nerve fiber growth and astrocytic migration in E14 cultures. In E18 cultures nerve fiber growth was stimulated and neuronal migration was hampered after β-xyloside treatment while ChABC treatment did not exert these effects.

Plasma levels of the endocannabinoid anandamide, related N-acylethanolamines and linoleic acid-derived oxylipins in patients with migraine

There is evidence that patients with migraine have deficient levels of the endogenous cannabinoid receptor ligand anandamide (AEA). It is not known, however, if this is a localised or generalised phenomenon. In the present study, levels of AEA, related N-acylethanolamines (NAEs) and linoleic acid-derived oxylipins have been measured in the blood of 26 healthy women and 38 women with migraine (26 with aura, 12 without aura) who were matched for age and body-mass index. Blood samples were taken on two occasions: the first sample near the start of the menstrual cycle (when present) and the second approximately fourteen days later. For a subset of migraine patients, two additional blood samples were taken, one during a migraine attack and one approximately 1 month later (to be at the same stage in the menstrual cycle, when present). NAEs and oxylipins were measured by liquid chromatography coupled to mass spectrometry. Twenty-nine lipids were quantified, of which 16 were found to have a high reproducibility of measurement. There were no significant differences in the levels of AEA, the related NAEs stearoylethanolamide and oleoylethanolamide or any of the nine linoleic acid-derived oxylipins measured either between migraine patients with vs. without aura, or between controls and migraine patients (after stratification to take into account whether or not the individuals had regular menstruation cycles) in either of the first two samples. Levels of linoleoylethanolamide were lower in the patients with vs. without aura on the second sample but not in the first sample, but the biological importance of this finding is unclear. Due to time-dependent increases in their concentrations ex vivo prior to centrifugation, AEA and oleoylethanolamide levels in the samples collected during migraine attacks were not analysed, but for the other fourteen lipids, there were no significant differences in plasma concentrations during migraine vs. one month later. It is concluded that migraine is not associated with a generalised (as opposed to localised) deficiency in these lipids.

Novel propanamides as fatty acid amide hydrolase inhibitors

Fatty acid amide hydrolase (FAAH) has a key role in the control of the cannabinoid signaling, through the hydrolysis of the endocannabinoids anandamide and in some tissues 2-arachidonoylglycerol. FAAH inhibition represents a promising strategy to activate the cannabinoid system, since it does not result in the psychotropic and peripheral side effects characterizing the agonists of the cannabinoid receptors. Here we present the discovery of a novel class of profen derivatives, the N-(heteroaryl)-2-(4-((2-(trifluoromethyl)pyridin-4-yl)amino)phenyl)propanamides, as FAAH inhibitors. Enzymatic assays showed potencies toward FAAH ranging from nanomolar to micromolar range, and the most compounds lack activity toward the two isoforms of cyclooxygenase. Extensive structure-activity studies and the definition of the binding mode for the lead compound of the series are also presented. Kinetic assays in rat and mouse FAAH on selected compounds of the series demonstrated that slight modifications of the chemical structure could influence the binding mode and give rise to competitive (TPA1) or non-competitive (TPA14) inhibition modes.

Non-Serotonergic Neurotoxicity by MDMA (Ecstasy) in Neurons Derived from Mouse P19 Embryonal Carcinoma Cells

3,4-methylenedioxymethamphetamine (MDMA; ecstasy) is a commonly abused recreational drug that causes neurotoxic effects in both humans and animals. The mechanism behind MDMA-induced neurotoxicity is suggested to be species-dependent and needs to be further investigated on the cellular level. In this study, the effects of MDMA in neuronally differentiated P19 mouse embryonal carcinoma cells have been examined. MDMA produces a concentration-, time- and temperature-dependent toxicity in differentiated P19 neurons, as measured by intracellular MTT reduction and extracellular LDH activity assays. The P19-derived neurons express both the serotonin reuptake transporter (SERT), that is functionally active, and the serotonin metabolizing enzyme monoamine oxidase A (MAO-A). The involvement of these proteins in the MDMA-induced toxicity was investigated by a pharmacological approach. The MAO inhibitors clorgyline and deprenyl, and the SERT inhibitor fluoxetine, per se or in combination, were not able to mimic the toxic effects of MDMA in the P19-derived neurons or block the MDMA-induced cell toxicity. Oxidative stress has been implicated in MDMA-induced neurotoxicity, but pre-treatment with the antioxidants α-tocopherol or N-acetylcysteine did not reveal any protective effects in the P19 neurons. Involvement of mitochondria in the MDMA-induced cytotoxicity was also examined, but MDMA did not alter the mitochondrial membrane potential (ΔΨm) in the P19 neurons. We conclude that MDMA produce a concentration-, time- and temperature-dependent neurotoxicity and our results suggest that the mechanism behind MDMA-induced toxicity in mouse-derived neurons do not involve the serotonergic system, oxidative stress or mitochondrial dysfunction.

The Absence of CD47 Promotes Nerve Fiber Growth from Cultured Ventral Mesencephalic Dopamine Neurons

In ventral mesencephalic organotypic tissue cultures, two timely separated sequences of nerve fiber growth have been observed. The first appearing nerve fiber pattern is a long-distance outgrowth that occurs before astrocytes start to proliferate and migrate to form an astrocytic monolayer that finally surrounds the tissue slice. These long-distance growing nerve fibers are retracted as the astrocytes migrate, and are followed by a secondary outgrowth. The secondary outgrowth is persistent in time but reaches short distances, comparable with outgrowth seen from a dopaminergic graft implanted to the brain. The present study was focused on the interaction between the astrocytes and the long-distance growing non-glial associated nerve fibers. Cross talk between astroglia and neurite formation might occur through the integrin-associated protein CD47. CD47 serves as a ligand for signal regulatory protein (SIRP) α and as a receptor for the extracellular matrix protein thrombospondin-1 (TSP-1). Embryonic day 14 ventral mesencephalic tissue from CD47+/+ and CD47−/− mice was used to investigate astrocytic migration and the tyrosine hydroxylase (TH) –positive outgrowth that occurred remote from the astrocytes. TH-immunohistochemistry demonstrated that the non-glial-associated nerve fiber outgrowth in CD47−/− cultures reached significantly longer distances and higher density compared to nerve fibers formed in CD47+/+ cultures at 14 days in vitro. These nerve fibers often had a dotted appearance in CD47+/+ cultures. No difference in the astrocytic migration was observed. Further investigations revealed that the presence of CD47 in control culture did neither hamper non-glial-associated growth through SIRPα nor through TSP-1 since similar outgrowth was found in SIRPα mutant cultures and in CD47+/+ cultures treated with blocking antibodies against the TSP-1, respectively, as in the control cultures. In conclusion, long-distance growing nerve fiber formation is promoted by the absence of CD47, even though the presence of astrocytes is not inhibited.

TLR4 receptor expression and function in F11 dorsal root ganglion × neuroblastoma hybrid cells

TLR4 respond to bacterial LPS to produce inflammatory cytokines. TLR4 are expressed in dorsal root ganglia and play a role in pain. F11 dorsal root ganglia × mouse neuroblastoma cells possess many of the properties seen in nociceptive dorsal root ganglia neuronal cells. Here, we investigated the effect of 2 h and 6 h treatment with LPS upon the expression of inflammatory proteins in undifferentiated and differentiated F11 cells. The cells expressed mRNA for TRL4 (mouse, not rat) and proteins involved in TLR4 signaling. TLR4 expression was confirmed using immunohistochemistry. LPS produced modest increases in mouse and rat IL-6 and in mouse cyclooxygenase-2 levels in undifferentiated cells, but did not significantly affect mouse TNF-α expression. This contrasts with the robust effects of LPS upon cyclooxygenase-2 expression in cultured dorsal root ganglia neurons. F11 cells expressed the endocannabinoid metabolizing enzymes fatty acid amide hydrolase and N-acylethanolamine acid amidase (both murine), which were functionally active. These data suggest that F11 cells are not a useful model for the study of LPS-mediated effects but may be useful for the study of endocannabinoid catabolism.