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Publications (10 of 12) Show all publications
Deplano, A., Karlsson, J., Moraca, F., Svensson, M., Cristiano, C., Morgillo, C. M., . . . Onnis, V. (2021). Design, synthesis and in vitro and in vivo biological evaluation of flurbiprofen amides as new fatty acid amide hydrolase/cyclooxygenase-2 dual inhibitory potential analgesic agents. Journal of enzyme inhibition and medicinal chemistry (Print), 36(1), 940-953
Open this publication in new window or tab >>Design, synthesis and in vitro and in vivo biological evaluation of flurbiprofen amides as new fatty acid amide hydrolase/cyclooxygenase-2 dual inhibitory potential analgesic agents
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2021 (English)In: Journal of enzyme inhibition and medicinal chemistry (Print), ISSN 1475-6366, E-ISSN 1475-6374, Vol. 36, no 1, p. 940-953Article in journal (Refereed) Published
Abstract [en]

Compounds combining dual inhibitory action against FAAH and cyclooxygenase (COX) may be potentially useful analgesics. Here, we describe a novel flurbiprofen analogue, N-(3-bromopyridin-2-yl)-2-(2-fluoro-(1,1'-biphenyl)-4-yl)propanamide (Flu-AM4). The compound is a competitive, reversible inhibitor of FAAH with a Ki value of 13 nM and which inhibits COX activity in a substrate-selective manner. Molecular modelling suggested that Flu-AM4 optimally fits a hydrophobic pocket in the ACB region of FAAH, and binds to COX-2 similarly to flurbiprofen. In vivo studies indicated that at a dose of 10 mg/kg, Flu-AM4 was active in models of prolonged (formalin) and neuropathic (chronic constriction injury) pain and reduced the spinal expression of iNOS, COX-2, and NFκB in the neuropathic model. Thus, the present study identifies Flu-AM4 as a dual-action FAAH/substrate-selective COX inhibitor with anti-inflammatory and analgesic activity in animal pain models. These findings underscore the potential usefulness of such dual-action compounds.

Place, publisher, year, edition, pages
Taylor & Francis, 2021
Keywords
allodynia, cyclooxygenase, endocannabinoid, FAAH inhibition, fatty acid amide hydrolase, Flurbiprofen amides, hyperalgesia, non-steroidal anti-inflammatory drugs
National Category
Pharmacology and Toxicology Medicinal Chemistry
Identifiers
urn:nbn:se:umu:diva-183361 (URN)10.1080/14756366.2021.1875459 (DOI)000643799500001 ()33896320 (PubMedID)2-s2.0-85105117861 (Scopus ID)
Available from: 2021-05-25 Created: 2021-05-25 Last updated: 2023-09-05Bibliographically approved
Alhouayek, M., Stafberg, L., Karlsson, J., Halin Bergström, S. & Fowler, C. J. (2020). Effects of orthotopic implantation of rat prostate tumour cells upon components of the N-acylethanolamine and monoacylglycerol signalling systems: an mRNA study. Scientific Reports, 10(1), Article ID 6314.
Open this publication in new window or tab >>Effects of orthotopic implantation of rat prostate tumour cells upon components of the N-acylethanolamine and monoacylglycerol signalling systems: an mRNA study
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2020 (English)In: Scientific Reports, E-ISSN 2045-2322, Vol. 10, no 1, article id 6314Article in journal (Refereed) Published
Abstract [en]

There is good evidence that the N-acylethanolamine (NAE)/monoacylglycerol (MAG) signalling systems are involved in the pathogenesis of cancer. However, it is not known how prostate tumours affect these systems in the surrounding non-malignant tissue and vice versa. In the present study we have investigated at the mRNA level 11 components of these systems (three coding for anabolic enzymes, two for NAE/MAG targets and six coding for catabolic enzymes) in rat prostate tissue following orthotopic injection of low metastatic AT1 cells and high metastatic MLL cells. The MLL tumours expressed higher levels of Napepld, coding for a key enzyme in NAE synthesis, and lower levels of Naaa, coding for the NAE hydrolytic enzyme N-acylethanolamine acid amide hydrolase than the AT1 tumours. mRNA levels of the components of the NAE/MAG signalling systems studied in the tissue surrounding the tumours were not overtly affected by the tumours. AT1 cells in culture expressed Faah, coding for the NAE hydrolytic enzyme fatty acid amide hydrolase, at much lower levels than Naaa. However, the ability of the intact cells to hydrolyse the NAE arachidonoylethanolamide (anandamide) was inhibited by an inhibitor of FAAH, but not of NAAA. Treatment of the AT1 cells with interleukin-6, a cytokine known to be involved in the pathogenesis of prostate cancer, did not affect the expression of the components of the NAE/MAG system studied. It is thus concluded that in the model system studied, the tumours show different expressions of mRNA coding for key the components of the NAE/MAG system compared to the host tissue, but that these changes are not accompanied by alterations in the non-malignant tissue.

Place, publisher, year, edition, pages
Nature Publishing Group, 2020
National Category
Cancer and Oncology
Identifiers
urn:nbn:se:umu:diva-174861 (URN)10.1038/s41598-020-63198-y (DOI)000562162600022 ()32286386 (PubMedID)2-s2.0-85083523927 (Scopus ID)
Available from: 2020-09-14 Created: 2020-09-14 Last updated: 2023-03-24Bibliographically approved
Deplano, A., Karlsson, J., Svensson, M., Moraca, F., Catalanotti, B., Fowler, C. J. & Onnis, V. (2020). Exploring the fatty acid amide hydrolase and cyclooxygenase inhibitory properties of novel amide derivatives of ibuprofen. Journal of enzyme inhibition and medicinal chemistry (Print), 35(1), 815-823
Open this publication in new window or tab >>Exploring the fatty acid amide hydrolase and cyclooxygenase inhibitory properties of novel amide derivatives of ibuprofen
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2020 (English)In: Journal of enzyme inhibition and medicinal chemistry (Print), ISSN 1475-6366, E-ISSN 1475-6374, Vol. 35, no 1, p. 815-823Article in journal (Refereed) Published
Abstract [en]

Inhibition of fatty acid amide hydrolase (FAAH) reduces the gastrointestinal damage produced by non-steroidal anti-inflammatory agents such as sulindac and indomethacin in experimental animals, suggesting that a dual-action FAAH-cyclooxygenase (COX) inhibitor could have useful therapeutic properties. Here, we have investigated 12 novel amide analogues of ibuprofen as potential dual-action FAAH/COX inhibitors. N-(3-Bromopyridin-2-yl)−2-(4-isobutylphenyl)propanamide (Ibu-AM68) was found to inhibit the hydrolysis of [3H]anandamide by rat brain homogenates by a reversible, mixed-type mechanism of inhibition with a Ki value of 0.26 µM and an α value of 4.9. At a concentration of 10 µM, the compound did not inhibit the cyclooxygenation of arachidonic acid by either ovine COX-1 or human recombinant COX-2. However, this concentration of Ibu-AM68 greatly reduced the ability of the COX-2 to catalyse the cyclooxygenation of the endocannabinoid 2-arachidonoylglycerol. It is concluded that Ibu-AM68 is a dual-acting FAAH/substrate-selective COX inhibitor.

Place, publisher, year, edition, pages
Taylor & Francis, 2020
Keywords
Ibuprofen amides, FAAH inhibition, fatty acid amide hydrolase, endocannabinoid, cyclooxygenase
National Category
Pharmacology and Toxicology Medicinal Chemistry
Identifiers
urn:nbn:se:umu:diva-174734 (URN)10.1080/14756366.2020.1743283 (DOI)000560168200001 ()32200655 (PubMedID)2-s2.0-85082278965 (Scopus ID)
Available from: 2020-09-03 Created: 2020-09-03 Last updated: 2023-03-24Bibliographically approved
Deplano, A., Karlsson, J., Fowler, C. J. & Onnis, V. (2020). The fatty acid amide hydrolase and cyclooxygenase-inhibitory properties of novel amide derivatives of carprofen. Bioorganic chemistry (Print), 101, Article ID 104034.
Open this publication in new window or tab >>The fatty acid amide hydrolase and cyclooxygenase-inhibitory properties of novel amide derivatives of carprofen
2020 (English)In: Bioorganic chemistry (Print), ISSN 0045-2068, Vol. 101, article id 104034Article in journal (Refereed) Published
Abstract [en]

In experimental animals, inhibition of fatty acid amide hydrolase (FAAH) reduces the gastrointestinal damage produced by non-steroidal anti-inflammatory agents that act by inhibition of cyclooxygenase (COX). This suggests that compounds able to inhibit both enzymes may be potentially useful therapeutic agents. In the present study, we have investigated eight novel amide analogues of carprofen, ketoprofen and fenoprofen as potential FAAH/COX dual action inhibitors. Carpro-AM1 (2-(6-Chloro-9H-carbazol-2-yl)-N-(3-methylpyridin-2-yl)propenamide) and Carpro-AM6 (2-(6-Chloro-9H-carbazol-2-yl)-N-(3-chloropyridin-2-yl)propenamide) were found to be fully re-versible inhibitors of the hydrolysis of 0.5 mu M [H-3]anandamide in rat brain homogenates with IC50 values of 94 and 23 nM, respectively, i.e. 2-3 orders of magnitude more potent than carprofen in this respect. Both compounds inhibited the cyclooxygenation of arachidonic acid by ovine COX-1, and were more potent inhibitors of human recombinant COX-2 when 2-arachidonoylglycerol was used as substrate than when arachidonic acid was used. It is concluded that Carpro-AM1 and Carpro-AM6 are dual-acting FAAH/substrate-selective COX inhibitors.

Place, publisher, year, edition, pages
Elsevier, 2020
Keywords
Carprofen amides, FAAH inhibition, Fatty acid amide hydrolase, Endocannabinoid, Cyclooxygenase, Carprofen, Non-steroidal anti-inflammatory drugs
National Category
Pharmacology and Toxicology
Identifiers
urn:nbn:se:umu:diva-174031 (URN)10.1016/j.bioorg.2020.104034 (DOI)000552621700001 ()32599361 (PubMedID)2-s2.0-85086879503 (Scopus ID)
Note

Corrigendum to “The fatty acid amide hydrolase and cyclooxygenase-inhibitory properties of novel amide derivatives of carprofen” [Bioorg. Chem. 101 (2020) 104034]. DOI: 10.1016/j.bioorg.2020.104241

Available from: 2020-08-17 Created: 2020-08-17 Last updated: 2023-03-24Bibliographically approved
Szeremeta, J., Karlsson, J., Alhouayek, M. & Fowler, C. J. (2019). Low mRNA expression and activity of monoacylglycerol lipase in human SH-SY5Y neuroblastoma cells. Prostaglandins & other lipid mediators, 142, 59-67
Open this publication in new window or tab >>Low mRNA expression and activity of monoacylglycerol lipase in human SH-SY5Y neuroblastoma cells
2019 (English)In: Prostaglandins & other lipid mediators, ISSN 1098-8823, E-ISSN 2212-196X, Vol. 142, p. 59-67Article in journal (Refereed) Published
Abstract [en]

Relatively little is known about the endocannabinoid system in human neuroblastoma cell lines. In the present study, we have investigated the expression of the genes coding for the enzymes involved in the synthesis and catabolism of endocannabinoids in the SH-SY5Y cell line. The expression of MGLL, the gene coding for the 2-arachidonoylglycerol hydrolytic enzyme monoacylglycerol lipase (MAGL), was found to be 85 and 340 fold lower than the expression levels for the genes coding for alpha/beta-hydrolase domain containing 6 and 12 (ABHD6, ABHD12), which are alternative hydrolytic enzymes for this endocannabinoid. In comparison, mRNA levels of MGLL were 1.5 fold higher than ABHD6 and 2 fold lower than the levels of ABHD12 in DU-145 human prostate cells. In functional assays, the hydrolysis of the 2-arachidonoylglycerol homologue 2-oleoylglycerol by intact SH-SY5Y cells was partially inhibited by the ABHD6 inhibitor WWL70, but not by the MAGL inhibitor JZL184, whereas the reverse was true in DU-145 cells. The combination of JZL184 + WWL70 did, however produce a significantly greater inhibition of 2-OG hydrolysis than seen with WWL70 alone in the SH-SY5Y cells. The low MGLL expression in the SH-SY5Y cells was not due to epigenetic silencing, since levels were not affected by treatment with the methylation inhibitor 5-aza-2'-deoxycytidine and/or the histone acetylase inhibitor trichostatin A. The low MGLL expression in SH-SY5Y cells should be taken into account when using these cells in experiments investigating the involvement of the endocannabinoid system in models of physiological and pathological processes.

Place, publisher, year, edition, pages
Elsevier, 2019
Keywords
Monoacylglycerol Llipase, SH-SY5Y cells, Neuroblastoma, Endocannabinoid, DU-145 cells
National Category
Cancer and Oncology
Identifiers
urn:nbn:se:umu:diva-160287 (URN)10.1016/j.prostaglandins.2019.04.003 (DOI)000469159100007 ()30978461 (PubMedID)2-s2.0-85064523507 (Scopus ID)
Available from: 2019-06-17 Created: 2019-06-17 Last updated: 2023-03-24Bibliographically approved
Karlsson, J. (2018). Endocannabinoid metabolism: the impact of inflammatory factors and pharmacological inhibitors. (Doctoral dissertation). Umeå: Umeå universitet
Open this publication in new window or tab >>Endocannabinoid metabolism: the impact of inflammatory factors and pharmacological inhibitors
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Alternative title[sv]
Endokannabinoid metabolism : påverkan av inflammatoriska faktorer och farmakologiska inhibitorer
Abstract [en]

The endocannabinoid (eCB) system is an endogenous signaling system consisting of ligands (referred to as endocannabinoids, eCBs), receptors and metabolic enzymes. The eCB system is involved in homeostatic control of a variety of biological functions such as neuronal signaling, mood, appetite and pathological conditions such as pain, inflammation and tumour progression. The main eCBs N- arachidonoylethanolamine (AEA, anandamide) and 2-arachidonoylglycerol (2-AG) are synthesised upon stimuli when and where their action is demanded. The signaling is brief and the eCBs are quickly degraded. The enzyme primarily responsible for eCB degradation is fatty acid amide hydrolase (FAAH) for AEA and monoacylglycerol lipase (MAGL) for 2-AG. In addition, both substances are substrates for cyclooxygenase-2 (COX-2). COX-2 is upregulated in inflammation, pain and in several tumours including prostate cancers, but it is not known whether COX-2 contribute significantly to eCB metabolism under these conditions.

Increasing endogenous levels of eCBs by inhibiting their degradation is exploited as a future therapy for pain conditions. One suggested therapeutic strategy is dual inhibition of enzymes FAAH and COX-2 to raise AEA levels. Paper I and II of this thesis investigates FAAH and COX inhibitory effects of: the major metabolites and enantiomers of derivatives (flu-AM1 and ibu-AM5) of the current clinically used NSAIDs ibuprofen and flurbiprofen. The metabolites 3 ́hydroxyibuprofen and 4 ́hydroxyflurbiprofen retained the FAAH and COX- inhibitory effects seen by the parent compounds although at lower potencies. Both enantiomers of flu-AM1 were equally potent as FAAH inhibitors and displayed a useful substrate selective COX-2 inhibition profile, favoring eCBs as substrates rather than arachidonic acid.

Paper III explores the impact of COX-2 and the effect of (R)-flu-AM1 upon AEA levels and degradation in mouse leukemic macrophage RAW264.7 cells. Despite the high inhibitory potency in enzyme assays, neither (R)-flu-AM1 nor the combination of a FAAH inhibitor with flurbiprofen increased AEA levels in the intact cells to any great extent. This suggests that the eCB turnover in these cells is rather slow. Further, in paper IV, induction of COX-2 did not unmask an ability of this enzyme to “gate” the uptake of AEA analogous to that seen with FAAH.

Paper IV and V focus upon the role of the eCB system in prostate cancer. The eCB system is altered in cancer and is linked to the progression and prognosis of prostate cancer. How and whereby this change occurs is unknown. This thesis explores the impact of the inflammatory factors TNFα, IL-6 and lactic acid induced low pH upon the mRNA levels of eCB related enzymes and the functional impact upon AEA degradation in human DU145 and rat AT-1 prostate cancer cells. TNFα treatment of DU145 and IL-6 and lactic acid induced low pH exposure of AT-1 changed the mRNA levels of 2-AG related enzymes leaving AEA rather unaffected other than for a substantial induction of COX-2 mRNA in DU145 cells. Thus, AEA homeostasis was not shifted in prostate cancer cell lines exposed to inflammatory factors. The results suggest that COX-2 does not gate the uptake of AEA and is a minor contributor to AEA degradation in intact cells. 

Place, publisher, year, edition, pages
Umeå: Umeå universitet, 2018. p. 82
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1958
Keywords
endocannabinoid, anandamide, 2-AG, fatty acid amide hydrolase, cyclooxygenase-2, prostate cancer, catabolism, inflammation, inflammatory factors
National Category
Pharmacology and Toxicology
Identifiers
urn:nbn:se:umu:diva-147503 (URN)978-91-7601-870-5 (ISBN)
Public defence
2018-06-01, Major Groove, NUS byggnad 6L, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2018-05-09 Created: 2018-05-04 Last updated: 2018-06-09Bibliographically approved
Popova, D., Karlsson, J. & Jacobsson, S. O. P. (2017). Comparison of neurons derived from mouse P19, rat PC12 and human SH-SY5Y cells in the assessment of chemical- and toxin-induced neurotoxicity. BMC Pharmacology & Toxicology, 18, Article ID 42.
Open this publication in new window or tab >>Comparison of neurons derived from mouse P19, rat PC12 and human SH-SY5Y cells in the assessment of chemical- and toxin-induced neurotoxicity
2017 (English)In: BMC Pharmacology & Toxicology, E-ISSN 2050-6511, Vol. 18, article id 42Article in journal (Refereed) Published
Abstract [en]

Background: Exposure to chemicals might be toxic to the developing brain. There is a need for simple and robust in vitro cellular models for evaluation of chemical-induced neurotoxicity as a complement to traditional studies on animals. In this study, neuronally differentiated mouse embryonal carcinoma P19 cells (P19 neurons) were compared with human neuroblastoma SH-SY5Y cells and rat adrenal pheochromocytoma PC12 cells for their ability to detect toxicity of methylmercury (MeHg), okadaic acid and acrylamide. Methods: Retinoic acid-treated P19 and SH-SY5Y cells and nerve growth factor-stimulated PC12 cells, allowed to differentiate for 6 days, were exposed to MeHg, okadaic acid and acrylamide for 48 h. Cell survival and neurite outgrowth were assessed with the calcein-AM assay and fluorescence detection of antibodies against the cytoskeletal neuron-specific protein beta III-tubulin, respectively. The effects of glutathione (GSH) and the potent inhibitor of GSH synthesis buthionine sulfoximine (BSO) on the MeHg induced-toxicity were assessed using the PrestoBlue (TM) cell viability assay and the TMRE mitochondrial membrane potential assay. Results: Differentiated P19 cells developed the most extensive neuronal network among the three cell models and were the most sensitive neuronal model to detect neurotoxic effects of the test compounds. MeHg produced a concentration-dependent toxicity in differentiated P19 cells and SH-SY5Y cells, with statistically significant effects at concentrations from 0.1 mu M in the P19 neurons and 1 mu M in the SH-SY5Y cells. MeHg induced a decrease in the cellular metabolic activity and mitochondrial membrane potential (Delta Psi m) in the differentiated P19 cells and SH-SY5Y cells, that were attenuated by GSH. Okadaic acid and acrylamide also showed statistically significant toxicity in the P19 neurons, but not in the SH-SY5Y cells or the P12 cells. Conclusions: P19 neurons are more sensitive to detect cytotoxicity of MeHg, okadaic acid and acrylamide than retinoic acid-differentiated SH-SY5Y cells and nerve growth factor-treated PC12 cells. P19 neurons are at least as sensitive as differentiated SH-SY5Y cells to detect the loss of mitochondrial membrane potential produced by MeHg and the protective effects of extracellular GSH on MeHg toxicity. P19 neurons may be a useful model to study neurotoxic effects of chemicals.

Place, publisher, year, edition, pages
BIOMED CENTRAL LTD, 2017
Keywords
In vitro cytotoxicity, Neuronal cell cultures, Retinoic acid-treated P19 cells, Retinoic acid-treated SH-
National Category
Pharmacology and Toxicology
Identifiers
urn:nbn:se:umu:diva-137043 (URN)10.1186/s40360-017-0151-8 (DOI)000402970800002 ()28583171 (PubMedID)2-s2.0-85020229477 (Scopus ID)
Available from: 2017-06-28 Created: 2017-06-28 Last updated: 2023-03-23Bibliographically approved
Karlsson, J., Gouveia-Figueira, S., Alhouayek, M. & Fowler, C. J. (2017). Effects of tumour necrosis factor alpha upon the metabolism of the endocannabinoid anandamide in prostate cancer cells. PLOS ONE, 12(9), Article ID e0185011.
Open this publication in new window or tab >>Effects of tumour necrosis factor alpha upon the metabolism of the endocannabinoid anandamide in prostate cancer cells
2017 (English)In: PLOS ONE, E-ISSN 1932-6203, Vol. 12, no 9, article id e0185011Article in journal (Refereed) Published
Abstract [en]

Tumour necrosis factor a (TNF alpha) is involved in the pathogenesis of prostate cancer, a disease where disturbances in the endocannabinoid system are seen. In the present study we have investigated whether treatment of DU145 human prostate cancer cells affects anandamide (AEA) catabolic pathways. Additionally, we have investigated whether cyclooxygenase- 2 (COX-2) can regulate the uptake of AEA into cells. Levels of AEA synthetic and catabolic enzymes were determined by qPCR. AEA uptake and hydrolysis in DU145 and RAW264.7 macrophage cells were assayed using AEA labeled in the arachidonic and ethanolamine portions of the molecule, respectively. Levels of AEA, related N-acylethanolamines (NAEs), prostaglandins (PG) and PG-ethanolamines (PG-EA) in DU145 cells and medium were quantitated by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis. TNF alpha treatment of DU145 cells increased mRNA levels of PTSG2 (gene of COX-2) and decreased the mRNA of the AEA synthetic enzyme N-acylphosphatidylethanolamine selective phospholipase D. mRNA levels of the AEA hydrolytic enzymes fatty acid amide hydrolase (FAAH) and N-acylethanolamine-hydrolyzing acid amidase were not changed. AEA uptake in both DU145 and RAW264.7 cells was inhibited by FAAH inhibition, but not by COX-2 inhibition, even in RAW264.7 cells where the expression of this enzyme had greatly been induced by lipopolysaccharide + interferon. treatment. AEA and related NAEs were detected in DU145 cells, but PGs and PGE(2)-EA were only detected when the cells had been preincubated with 100 nM AEA. The data demonstrate that in DU145 cells, TNFa treatment changes the relative expression of the enzymes involved in the hydrolytic and oxygenation catabolic pathways for AEA. In RAW264.7 cells, COX-2, in contrast to FAAH, does not regulate the cellular accumulation of AEA. Further studies are necessary to determine the extent to which inflammatory mediators are involved in the abnormal endocannabinoid signalling system in prostate cancer.

Place, publisher, year, edition, pages
PUBLIC LIBRARY SCIENCE, 2017
National Category
Pharmacology and Toxicology
Identifiers
urn:nbn:se:umu:diva-140467 (URN)10.1371/journal.pone.0185011 (DOI)000410859200126 ()28910408 (PubMedID)2-s2.0-85029855578 (Scopus ID)
Available from: 2017-10-25 Created: 2017-10-25 Last updated: 2023-03-24Bibliographically approved
Gouveia-Figueira, S., Karlsson, J., Deplano, A., Hashemian, S., Svensson, M., Fredriksson Sundbom, M., . . . Fowler, C. J. (2015). Characterisation of (R)-2-(2-Fluorobiphenyl-4-yl)-N-(3-Methylpyridin-2-yl)Propanamide as a Dual Fatty Acid Amide Hydrolase: Cyclooxygenase Inhibitor. PLOS ONE, 10(9), Article ID e0139212.
Open this publication in new window or tab >>Characterisation of (R)-2-(2-Fluorobiphenyl-4-yl)-N-(3-Methylpyridin-2-yl)Propanamide as a Dual Fatty Acid Amide Hydrolase: Cyclooxygenase Inhibitor
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2015 (English)In: PLOS ONE, E-ISSN 1932-6203, Vol. 10, no 9, article id e0139212Article in journal (Refereed) Published
Abstract [en]

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 gamma-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 mu M; COX-2 (arachidonic acid) 20 mu M; COX-2 (2-AG) 1 mu M; (S)-Flu-AM1, COX-1 (arachidonic acid) 3 mu M; COX-2 (arachidonic acid) 10 mu M; COX-2 (2-AG) 0.7 mu M. The compounds showed no enantiomeric selectivity in their FAAH inhibitory properties. (R)-Flu-AM1 (10 mu M) greatly inhibited the production of prostaglandin D2 and E2 in both unstimulated and lipopolysaccharide + interferon.-stimulated RAW 264.7 macrophage cells. Levels of 2-AG were not affected either by (R)-Flu-AM1 or by 10 mu M flurbiprofen, either alone or in combination with the FAAH inhibitor URB597 (1 mu M). 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.-stimulated RAW 264.7 cells is insufficient to affect 2-AG levels despite the large induction of COX-2 produced by this treatment.

National Category
Pharmacology and Toxicology
Identifiers
urn:nbn:se:umu:diva-110569 (URN)10.1371/journal.pone.0139212 (DOI)000361800700192 ()26406890 (PubMedID)2-s2.0-84947261212 (Scopus ID)
Available from: 2015-11-06 Created: 2015-10-23 Last updated: 2023-03-23Bibliographically approved
Karlsson, J., Morgillo, C. M., Deplano, A., Smaldone, G., Pedone, E., Javier Luque, F., . . . Fowler, C. J. (2015). Interaction of the N-(3-Methylpyridin-2-yl) amide Derivatives of Flurbiprofen and Ibuprofen with FAAH: Enantiomeric Selectivity and Binding Mode. PLOS ONE, 10(11), Article ID e0142711.
Open this publication in new window or tab >>Interaction of the N-(3-Methylpyridin-2-yl) amide Derivatives of Flurbiprofen and Ibuprofen with FAAH: Enantiomeric Selectivity and Binding Mode
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2015 (English)In: PLOS ONE, E-ISSN 1932-6203, Vol. 10, no 11, article id e0142711Article in journal (Refereed) Published
Abstract [en]

Background Combined fatty acid amide hydrolase (FAAH) and cyclooxygenase (COX) inhibition is a promising approach for pain-relief. The Flu-AM1 and Ibu-AM5 derivatives of flurbiprofen and ibuprofen retain similar COX-inhibitory properties and are more potent inhibitors of FAAH than the parent compounds. However, little is known as to the nature of their interaction with FAAH, or to the importance of their chirality. This has been explored here. Methodology/Principal Findings FAAH inhibitory activity was measured in rat brain homogenates and in lysates expressing either wild-type or FAAH(T488A)-mutated enzyme. Molecular modelling was undertaken using both docking and molecular dynamics. The (R)-and (S)-enantiomers of Flu-AM1 inhibited rat FAAH with similar potencies (IC50 values of 0.74 and 0.99 mu M, respectively), whereas the (S)-enantiomer of Ibu-AM5 (IC50 0.59 mu M) was more potent than the (R)-enantiomer (IC50 5.7 mu M). Multiple inhibition experiments indicated that both (R)-Flu-AM1 and (S)-Ibu-AM5 inhibited FAAH in a manner mutually exclusive to carprofen. Computational studies indicated that the binding site for the Flu-AM1 and Ibu-AM5 enantiomers was located between the acyl chain binding channel and the membrane access channel, in a site overlapping the carprofen binding site, and showed a binding mode in line with that proposed for carprofen and other non-covalent ligands. The potency of (R)-Flu-AM1 was lower towards lysates expressing FAAH mutated at the proposed carprofen binding area than in lysates expressing wild-type FAAH. Conclusions/Significance The study provides kinetic and structural evidence that the enantiomers of Flu-AM1 and Ibu-AM5 bind in the substrate channel of FAAH. This information will be useful in aiding the design of novel dual-action FAAH: COX inhibitors.

National Category
Medicinal Chemistry Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-114918 (URN)10.1371/journal.pone.0142711 (DOI)000367628500049 ()2-s2.0-84958817302 (Scopus ID)
Available from: 2016-03-04 Created: 2016-01-29 Last updated: 2023-03-23Bibliographically approved
Organisations
Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0001-8572-5841

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