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The endocannabinoid system: a translational study from Achilles tendinosis to cyclooxygenase
Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience, Pharmacology.
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The endogenous cannabinoids anandamide (arachidonoyl ethanolamide, AEA) and 2-arachidonoyl glycerol (2-AG) exert their effect by activating cannabinoid receptors (CB). These receptors mediate a broad range of physiological functions such as beneficial effects in pain and inflammation, although little is known about the expression of CB receptors in human pain conditions. AEA and 2-AG are short- lived molecules due to their rapid cellular accumulation and metabolism. The enzymes primarily responsible for their degradation are fatty acid amide hydrolase (FAAH) for AEA and monoacylglycerol lipase (MGL) for 2-AG. Inhibition of endocannabinoid metabolism is a potential approach for drug development, and there is a need for the identification of novel compounds with inhibitory effects upon FAAH and MGL.

In Paper I of this thesis, the expression of CB1 receptors in human Achilles tendon was examined. We found expression of CB1 receptors in tenocytes, blood vessel wall as well as in the perineurium of the nerve. A semi-quantitative analysis showed an increase of CB1 receptors in painful human Achilles tendinosis.

In papers II and III, termination of AEA signalling was investigated via inhibition of FAAH. In Paper II, Flu-AM1, an analogue of flurbiprofen, was investigated. The compound inhibited both FAAH and the oxygenation of 2-AG by cyclooxygenase-2. In Paper III the antifungal compound ketoconazole was shown to inhibit the cellular uptake of AEA in HepG2, CaCo-2 and C6 cell lines in a manner consistent with inhibition of FAAH.

The role of FAAH in gating the cellular accumulation of AEA was investigated in Paper IV. FAAH has been shown to control the concentration gradient of AEA across the plasmamembrane in RBL2H3 cells, whereas no such effect is seen in other FAAH-expressing cell lines. To determine whether this effect is assay dependent or due to intrinsic differences between the cell lines, we assayed four cell lines with different levels of FAAH expression using the same methodology. We found that the sensitivity of FAAH uptake inhibition was not dependent on the expression level of FAAH, suggesting that factors other than FAAH are important for uptake.

Paper V is focused on the inhibition of MGL. Prior to this study no selective inhibitors of the enzyme had been described. Thus, we screened a number of compounds for their inhibitory effect on MGL. Troglitazone was found to be an inhibitor of MGL, although its potency was dependent upon the enzyme assay used. 

Place, publisher, year, edition, pages
Umeå: Umeå universitet , 2014. , 72 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1663
National Category
Pharmacology and Toxicology
Identifiers
URN: urn:nbn:se:umu:diva-91573ISBN: 978-91-7601-089-1 (print)OAI: oai:DiVA.org:umu-91573DiVA: diva2:737043
Public defence
2014-09-05, Hörsal E04 Unod R1, Byggnad 6E, Norrlands universitetssjukhus, Umeå, 09:00 (Swedish)
Opponent
Supervisors
Available from: 2014-08-15 Created: 2014-08-11 Last updated: 2014-08-14Bibliographically approved
List of papers
1. Increased expression of cannabinoid CB(1) receptors in achilles tendinosis
Open this publication in new window or tab >>Increased expression of cannabinoid CB(1) receptors in achilles tendinosis
2011 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 6, no 9, e24731- p.Article in journal (Refereed) Published
Abstract [en]

Background: The endogenous cannabinoid system is involved in the control of pain. However, little is known as to the integrity of the cannabinoid system in human pain syndromes. Here we investigate the expression of the cannabinoid receptor 1 (CB(1)) in human Achilles tendons from healthy volunteers and from patients with Achilles tendinosis.

Methodology: Cannabinoid CB(1) receptor immunoreactivity (CB(1)IR) was evaluated in formalin-fixed biopsies from individuals suffering from painful Achilles tendinosis in comparison with healthy human Achilles tendons.

Principal Findings: CB(1)IR was seen as a granular pattern in the tenocytes. CB(1)IR was also observed in the blood vessel wall and in the perineurium of the nerve. Quantification of the immunoreactivity in tenocytes showed an increase of CB(1) receptor expression in tendinosis tissue compared to control tissue.

Conclusion: Expression of cannabinoid receptor 1 is increased in human Achilles tendinosis suggesting that the cannabinoid system may be dysregulated in this disorder.

Place, publisher, year, edition, pages
San Francisco, CA: Public Library of Science, 2011
National Category
Biological Sciences
Identifiers
urn:nbn:se:umu:diva-47905 (URN)10.1371/journal.pone.0024731 (DOI)000294802800087 ()
Available from: 2011-10-04 Created: 2011-10-03 Last updated: 2017-12-08Bibliographically approved
2. Inhibition of fatty acid amide hydrolase and cyclooxygenase by the N-(3-methylpyridin-2-yl)amide derivatives of flurbiprofen and naproxen
Open this publication in new window or tab >>Inhibition of fatty acid amide hydrolase and cyclooxygenase by the N-(3-methylpyridin-2-yl)amide derivatives of flurbiprofen and naproxen
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2013 (English)In: European Journal of Pharmacology, ISSN 0014-2999, E-ISSN 1879-0712, Vol. 720, no 1-3, 383-390 p.Article in journal (Refereed) Published
Abstract [en]

Inhibitors of the metabolism of the endogenous cannabinoid ligand anandamide by fatty acid amide hydrolase (FAAH) reduce the gastric damage produced by non-steroidal anti-inflammatory agents and synergise with them in experimental pain models. This motivates the design of compounds with joint FAAH/cyclooxygenase (COX) inhibitory activity. Here we present data on the N-(3-methylpyridin-2-yl) amide derivatives of flurbiprofen and naproxen (Flu-AM1 and Nap-AM1, respectively) with respect to their properties towards these two enzymes. Flu-AM1 and Nap-AM1 inhibited FAAH-catalysed hydrolysis of [H-3]anandamide by rat brain homogenates with IC50 values of 044 and 0.74 mu M. The corresponding values for flurbiprofen and naproxen were 29 and > 100 mu M, respectively. The inhibition by Flu-AM1 was reversible, mixed-type, with K-slope(i) and K-intercept(i) values of 0.21 and 1.4 mu M, respectively. Flurbiprofen and Flu-AM1 both inhibited COX in the same manner with the order of potencies COX-2 vs. 2-arachidonoylglycerol > COX-1 vs. arachidonic acid > COX-2 vs. arachidonic acid with flurbiprofen being approximately 2-3 fold more potent than Flu-AM 1 in the assays. Nap-AM1 was a less potent inhibitor of COX. Flu-AM1 at low micromolar concentrations inhibited the FAAH-driven uptake of [H-3]anandamide into RBL2H3 basophilic leukaemia cells in vitro, but did not penetrate the brain in vivo sufficiently to block the binding of [F-18]DOPP to brain FAAH. It is concluded that Flu-AM 1 is a dual-action inhibitor of FAAH and COX that may be useful in exploring the optimal balance of effects on these two enzyme systems in producing peripheral alleviation of pain and inflammation in experimental models.

Place, publisher, year, edition, pages
Elsevier, 2013
Keyword
Fatty acid amide hydrolase, Cyclooxygenase, Anandamide, 2-arachidonoylglycerol, Non-steroidal anti-inflammatory drug
National Category
Pharmacology and Toxicology
Identifiers
urn:nbn:se:umu:diva-84519 (URN)10.1016/j.ejphar.2013.09.065 (DOI)000327488000047 ()
Funder
Swedish Research Council, 12158NIH (National Institute of Health), 1R21MH094424
Available from: 2014-01-14 Created: 2014-01-08 Last updated: 2017-12-06Bibliographically approved
3. Ketoconazole Inhibits the Cellular Uptake of Anandamide via Inhibition of FAAH at Pharmacologically Relevant Concentrations
Open this publication in new window or tab >>Ketoconazole Inhibits the Cellular Uptake of Anandamide via Inhibition of FAAH at Pharmacologically Relevant Concentrations
2014 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, no 1, e87542- p.Article in journal (Refereed) Published
Abstract [en]

Background: The antifungal compound ketoconazole has, in addition to its ability to interfere with fungal ergosterol synthesis, effects upon other enzymes including human CYP3A4, CYP17, lipoxygenase and thromboxane synthetase. In the present study, we have investigated whether ketoconazole affects the cellular uptake and hydrolysis of the endogenous cannabinoid receptor ligand anandamide (AEA). Methodology/Principal Findings: The effects of ketoconazole upon endocannabinoid uptake were investigated using HepG2, CaCo2, PC-3 and C6 cell lines. Fatty acid amide hydrolase (FAAH) activity was measured in HepG2 cell lysates and in intact C6 cells. Ketoconazole inhibited the uptake of AEA by HepG2 cells and CaCo2 cells with IC50 values of 17 and 18 mu M, respectively. In contrast, it had modest effects upon AEA uptake in PC-3 cells, which have a low expression of FAAH. In cell-free HepG2 lysates, ketoconazole inhibited FAAH activity with an IC50 value (for the inhibitable component) of 34 mu M. Conclusions/Significance: The present study indicates that ketoconazole can inhibit the cellular uptake of AEA at pharmacologically relevant concentrations, primarily due to its effects upon FAAH. Ketoconazole may be useful as a template for the design of dual-action FAAH/CYP17 inhibitors as a novel strategy for the treatment of prostate cancer.

National Category
Pharmacology and Toxicology
Identifiers
urn:nbn:se:umu:diva-86616 (URN)10.1371/journal.pone.0087542 (DOI)000330288000212 ()
Available from: 2014-04-29 Created: 2014-03-03 Last updated: 2017-12-05Bibliographically approved
4. Involvement of Fatty Acid Amide Hydrolase and Fatty Acid Binding Protein 5 in the Uptake of Anandamide by Cell Lines with Different Levels of Fatty Acid Amide Hydrolase Expression: A Pharmacological Study
Open this publication in new window or tab >>Involvement of Fatty Acid Amide Hydrolase and Fatty Acid Binding Protein 5 in the Uptake of Anandamide by Cell Lines with Different Levels of Fatty Acid Amide Hydrolase Expression: A Pharmacological Study
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2014 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, no 7, e103479- p.Article in journal (Refereed) Published
Abstract [en]

Background:

The endocannabinoid ligand anandamide (AEA) is removed from the extracellular space by a process ofcellular uptake followed by metabolism. In many cells, such as the RBL-2H3 cell line, inhibition of FAAH activity reduces theobserved uptake, indicating that the enzyme regulates uptake by controlling the intra- : extracellular AEA concentrationgradient. However, in other FAAH-expressing cells, no such effect is seen. It is not clear, however, whether these differencesare methodological in nature or due to properties of the cells themselves. In consequence, we have reinvestigated the roleof FAAH in gating the uptake of AEA.Methodology/Principal Findings: The effects of FAAH inhibition upon AEA uptake were investigated in four cell lines: AT1rat prostate cancer, RBL-2H3 rat basophilic leukaemia, rat C6 glioma and mouse P19 embryonic carcinoma cells. SemiquantitativePCR for the cells and for a rat brain lysate confirmed the expression of FAAH. No obvious expression of atranscript with the expected molecular weight of FLAT was seen. FAAH expression differed between cells, but all four couldaccumulate AEA in a manner inhibitable by the selective FAAH inhibitor URB597. However, there was a difference in thesensitivities seen in the reduction of uptake for a given degree of FAAH inhibition produced by a reversible FAAH inhibitor,with C6 cells being more sensitive than RBL-2H3 cells, despite rather similar expression levels and activities of FAAH. Thefour cell lines all expressed FABP5, and AEA uptake was reduced in the presence of the FABP5 inhibitor SB-FI-26, suggestingthat the different sensitivities to FAAH inhibition for C6 and RBL2H3 cells is not due to differences at the level of FABP-5.Conclusions/Significance: When assayed using the same methodology, different FAAH-expressing cells display differentsensitivities of uptake to FAAH inhibition.

Place, publisher, year, edition, pages
PLoS ONE, 2014
National Category
Pharmacology and Toxicology
Identifiers
urn:nbn:se:umu:diva-91572 (URN)10.1371/journal.pone.0103479 (DOI)000339954800048 ()25078278 (PubMedID)
Available from: 2014-08-11 Created: 2014-08-11 Last updated: 2017-12-05Bibliographically approved
5. Inhibition of monoacylglycerol lipase by troglitazone, N-arachidonoyl dopamine and the irreversible inhibitor JZL184: comparison of two different assays
Open this publication in new window or tab >>Inhibition of monoacylglycerol lipase by troglitazone, N-arachidonoyl dopamine and the irreversible inhibitor JZL184: comparison of two different assays
2010 (English)In: British Journal of Pharmacology, ISSN 0007-1188, E-ISSN 1476-5381, Vol. 161, no 7, 1512-1526 p.Article in journal (Refereed) Published
Abstract [en]

BACKGROUND AND PURPOSE: Drugs used clinically usually have a primary mechanism of action, but additional effects on other biological targets can contribute to their effects. A potentially useful additional target is the endocannabinoid metabolizing enzyme monoacylglycerol lipase (MGL). We have screened a range of drugs for inhibition of MGL and compared the observed potencies using different MGL enzyme assays.

EXPERIMENTAL APPROACH: MGL activity was screened using recombinant human MGL (cell lysates and purified enzyme) with 4-nitrophenyl acetate (NPA) as substrate. 2-Oleolyglycerol metabolism by rat cerebellar cytosolic MGL and by recombinant MGL was also investigated.

KEY RESULTS: Among the 96 compounds screened in the NPA assay, troglitazone, CP55,940, N-arachidonoyl dopamine and AM404 inhibited NPA hydrolysis by the lysates with IC(50) values of 1.1, 4.9, 0.78 and 3.1µM, respectively. The potency for troglitazone is in the same range as its primary pharmacological activity, activation of peroxisome proliferator-activated receptor (PPAR) γ. Among PPARγ ligands, the potency order towards human MGL was troglitazone > ciglitazone > rosiglitazone > 15-deoxy-Δ(12,14) -prostaglandin J(2) ≈ CAY 10415 > CAY 10514. In contrast to the time-dependent inhibitor JZL184, the potency of troglitazone was dependent upon the enzyme assay system used. Thus, troglitazone inhibited rat cytosolic 2-oleoylglycerol hydrolysis less potently (IC(50) 41µM) than hydrolysis of NPA by the human MGL lysates.

CONCLUSIONS AND IMPLICATIONS: 'Hits' in screening programmes for MGL inhibitors should be assessed in different MGL assays. Troglitazone may be a useful lead for the design of novel, dual action MGL inhibitors/PPARγ activators.

Keyword
monoacylglycerol lipase; cannabinoid; troglitazone; rosiglitazone; peroxisome proliferator-activated receptor γ
Identifiers
urn:nbn:se:umu:diva-41591 (URN)10.1111/j.1476-5381.2010.00974.x (DOI)000283948800008 ()20735405 (PubMedID)
Available from: 2011-03-29 Created: 2011-03-29 Last updated: 2017-12-11Bibliographically approved

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