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Fatty acid amide hydrolase - A target for anti-inflammatory therapies?
Umeå University, Faculty of Medicine, Pharmacology and Clinical Neuroscience, Pharmacology.
2005 (English)Doctoral thesis, comprehensive summary (Other academic)Alternative title
Fettsyraamidohydrolas - Ett mål för antiinflammatoriska läkemedel? (Swedish)
Abstract [en]

Anti-inflammatory drugs are a widely used class of therapeutic agents, but the use of non-steroidal anti-inflammatory drugs (NSAID) is hampered by their gastrointestinal side-effects. Recent reports that cyclooxygenase-2 inhibitors may cause cardiovascular events underline the importance of identifying new therapeutic strategies for the treatment of inflammation. One such target could be agents modifying the endogenous cannabinoid (endocannabinoid) system, since there is evidence that this system plays a role in our natural defence against inflammation. The levels of the endocannabinoid anandamide (arachidonoyl ethanolamide, AEA) are low under normal conditions, and stand under strict regulatory control of synthesising and degrading enzymes. Fatty acid amide hydrolase (FAAH) is the main enzyme degrading AEA, hydrolysing it to ethanolamine and arachidonic acid. The focus of this thesis lies in exploring the pharmacology of FAAH to evaluate its possibilities as a target for new anti-inflammatory drugs.

In Papers I and II, the effects of the ambient pH on the properties of FAAH were investigated, since tissue pH is known to decrease under inflammatory conditions. In homogenates, it was found that the activity of FAAH decreased as the assay pH was decreased, consistent with the known pH profile of the enzyme. More importantly, the sensitivity of the enzyme to inhibition by FAAH inhibitors changed. In particular, the sensitivity of the enzyme to inhibition by the NSAID ibuprofen increased seventeen-fold as the assay pH decreased from 8.37 to 5.28. A similar pattern was found using intact C6 glioma cells when the extracellular, but not the intracellular pH was reduced. Thus, at an extracellular pH value of 6.2, (R)-ibuprofen, (S)-flurbiprofen and (R,S)-flurbiprofen inhibited the metabolism of AEA with IC50 values of 26, 14 and 15 µM, respectively. These values are in theory reachable upon normal dosing of the compounds. In Paper III, the effect of the selective FAAH inhibitor URB597 and the NSAID indomethacin were investigated in vivo upon the oedema response to carrageenan administration in the paw of anaesthetised mice. Both compounds reduced the oedema in a manner completely blocked by the CB2 receptor antagonist SR144528. In Paper IV, the effect of inflammation upon endocannabinoid synthesis was investigated in mice. Lipopolysaccharide-induced pulmonary inflammation was found not to affect the release of AEA to any obvious extent, and did not change the activities of the AEA synthesising enzymes N-acyl transferase or N-acyl phosphatidylethanolamine phospholipase D, or of FAAH in lung tissue.

The results of this thesis would suggest that FAAH inhibitors can produce anti-inflammatory effects, and that the endocannabinoid system contributes to the actions of the NSAID indomethacin in the carrageenan model of inflammation, but that an increased endocannabinoid synthesis (a prerequisite for FAAH inhibition as a therapeutic strategy) is not an obligatory response to an inflammatory stimulus.

Place, publisher, year, edition, pages
Umeå: Farmakologi och klinisk neurovetenskap , 2005. , 97 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 944
Keyword [en]
Pharmacology, FAAH, anandamide, endocannabinoid, pH, inflammation, NSAID
Keyword [sv]
Farmakologi
National Category
Pharmacology and Toxicology
Research subject
Medical Pharmacology
Identifiers
URN: urn:nbn:se:umu:diva-504ISBN: 91-7305-823-8 (print)OAI: oai:DiVA.org:umu-504DiVA: diva2:143644
Public defence
2005-05-13, E04, 6E, Norrlands universitetssjukhus, Umeå, 13:00 (English)
Opponent
Supervisors
Available from: 2005-04-19 Created: 2005-04-19 Last updated: 2009-11-16Bibliographically approved
List of papers
1. Effects of pH on the inhibition of fatty acid amidohydrolase by ibuprofen.
Open this publication in new window or tab >>Effects of pH on the inhibition of fatty acid amidohydrolase by ibuprofen.
Show others...
2001 (English)In: British Journal of Pharmacology, ISSN 0007-1188, Vol. 133, no 4, 513-520 p.Article in journal (Refereed) Published
Abstract [en]

The pharmacological properties of fatty acid amidohydrolase (FAAH) at different assay pH values were investigated using [(3)H]-anandamide ([(3)H]-AEA) as substrate in rat brain homogenates and in COS-1 [corrected] cells transfected with wild type and mutant FAAH. Rat brain hydrolysis of [(3)H]-AEA showed pH dependency with an optimum around pH 8-9. Between pH 6.3 and 8.2, the difference in activity was due to differences in the V(max), rather than the K(M) values. For inhibition of rat brain [(3)H]-AEA metabolism by a series of known FAAH inhibitors, the potencies of the enantiomers of ibuprofen and phenylmethylsulphonyl fluoride (PMSF) were higher at pH 5.28 than at pH 8.37, whereas the reverse was true for oleyl trifluoromethylketone (OTMK) and arachidonoylserotonin. At both pH values, (-)ibuprofen was a mixed-type inhibitor of FAAH. The K(i)((slope)) and K(i)((intercept)) values for (-)ibuprofen at pH 5.28 were 11 and 143 microM, respectively. At pH 8.37, the corresponding values were 185 and 3950 microM, respectively. The pH dependency for the inhibition by OTMK and (-)ibuprofen was also seen in COS-1 [corrected] cells transiently transfected with either wild type, S152A or C249A FAAH. No differences in potencies between the wild type and mutant enzymes were seen. It is concluded that the pharmacological properties of FAAH are highly pH-dependent. The higher potency of ibuprofen at lower pH values raises the possibility that in certain types of inflamed tissue, the concentration of this compound following oral administration may be sufficient to inhibit FAAH.

Keyword
Anandamide, fatty acid amidohydrolase, ibuprofen, pH dependency
Identifiers
urn:nbn:se:umu:diva-4508 (URN)10.1038/sj.bjp.0704113 (DOI)11399668 (PubMedID)
Available from: 2005-04-19 Created: 2005-04-19Bibliographically approved
2. Anandamide metabolism by fatty acid amide hydrolase in intact C6 glioma cells. Increased sensitivity to inhibition by ibuprofen and flurbiprofen upon reduction of extra- but not intracellular pH.
Open this publication in new window or tab >>Anandamide metabolism by fatty acid amide hydrolase in intact C6 glioma cells. Increased sensitivity to inhibition by ibuprofen and flurbiprofen upon reduction of extra- but not intracellular pH.
2003 (English)In: Archives of Pharmacology, ISSN 0028-1298, Vol. 367, no 3, 237-244 p.Article in journal (Refereed) Published
Abstract [en]

The metabolism of anandamide by fatty acid amidohydrolase (FAAH) at different intra- and extracellular pH values has been investigated in intact C6 rat glioma cells. The cellular uptake of anandamide at 37 degrees C was found to decrease by 28% when the extracellular pH (pH(e)) was reduced from pH 7.4 to pH 6.2. In contrast, a selective decrease in intracellular pH (pH(i)), accomplished by acidifying the cells followed by incubation in sodium-free buffer at pH 7.4, did not affect the uptake. Anandamide uptake was inhibited by (R)-ibuprofen, with pI(50) values of 3.05+/-0.57, 3.66+/-0.23 and 3.94+/-0.88 at pH(e) values of 7.4, 6.8 and 6.2, respectively. In the presence of phenylmethylsulfonyl fluoride, however, (R)-ibuprofen failed to inhibit the uptake of anandamide. A reduction in pH(e) from 7.4 to 6.2 produced a 17% reduction in the FAAH-catalyzed metabolism of anandamide in the intact C6 cells. However, an increased sensitivity of FAAH activity to inhibition by (R)-ibuprofen as well as (R,S)-flurbiprofen and (S)-flurbiprofen was seen at a lower pH(e). For (R)-ibuprofen, pI(50) values of 3.57+/-0.08, 4.04+/-0.05 and 4.59+/-0.04 were found at pH(e) values of 7.4, 6.8 and 6.2, respectively. For (R,S)- and (S)-flurbiprofen, the pI(50) values at pH(e) 7.4 were 4.02+/-0.05 and 4.13+/-0.18, respectively at a pH(e) of 7.4, and 4.81+/-0.11 and 4.84+/-0.10, respectively, at a pH(e) of 6.2. In contrast, intracellular acidification did not affect either the rate of anandamide metabolism or its inhibition by (R)-ibuprofen or (S)-flurbiprofen. It is concluded that a reduction of extracellular pH produces an enhanced accumulation of the acidic NSAIDs ibuprofen and flurbiprofen into C6 glioma cells and thereby an inhibition of anandamide metabolism.

Identifiers
urn:nbn:se:umu:diva-4509 (URN)10.1007/s00210-002-0686-z (DOI)12644895 (PubMedID)
Available from: 2005-04-19 Created: 2005-04-19Bibliographically approved
3. Inhibitors of fatty acid amide hydrolase reduce carrageenan-induced hind paw inflammation in the mouse: Comparison with indomethacin and possible involvement of cannabinoid receptors.
Open this publication in new window or tab >>Inhibitors of fatty acid amide hydrolase reduce carrageenan-induced hind paw inflammation in the mouse: Comparison with indomethacin and possible involvement of cannabinoid receptors.
Manuscript (Other academic)
Identifiers
urn:nbn:se:umu:diva-4510 (URN)
Available from: 2005-04-19 Created: 2005-04-19 Last updated: 2010-08-26Bibliographically approved
4. Lipopolysaccharide-induced pulmonary inflammation is not accompanied by a release of anandamide into the lavage fluid or a down-regulation of the activity of fatty acid amide hydrolase.
Open this publication in new window or tab >>Lipopolysaccharide-induced pulmonary inflammation is not accompanied by a release of anandamide into the lavage fluid or a down-regulation of the activity of fatty acid amide hydrolase.
Show others...
2004 (English)In: Life Sciences, ISSN 0024-3205, Vol. 76, no 4, 461-472 p.Article in journal (Refereed) Published
Abstract [en]

The effect of lipopolysaccharide inhalation upon lung anandamide levels, anandamide synthetic enzymes and fatty acid amide hydrolase has been investigated. Lipopolysaccharide exposure produced a dramatic extravasation of neutrophils and release of tumour necrosis factor alpha into the bronchoalveolar lavage (BAL) fluid, which was not accompanied by epithelial cell injury. The treatment, however, did not change significantly the levels of anandamide and the related compound palmitoylethanolamide in the cell-free fraction of the BAL fluid. The activities of the anandamide synthetic enzymes N-acyltransferase and N-acylphosphatidylethanolamine phospholipase D and the activity of fatty acid amide hydrolase in lung membrane fractions did not change significantly following the exposure to lipopolysaccharide. The non-selective fatty acid amide hydrolase inhibitor phenylmethylsulfonyl fluoride was a less potent inhibitor of lung fatty acid amide hydrolase than expected from the literature, and a dose of 30 mg/kg i.p. of this compound, which produced a complete inhibition of brain anandamide metabolism, only partially inhibited the lung metabolic activity.

Keyword
Acyltransferases/metabolism, Administration; Inhalation, Amidohydrolases/analysis/*metabolism, Animals, Arachidonic Acids/analysis/*metabolism, Brain/drug effects/metabolism, Bronchoalveolar Lavage Fluid/chemistry, Cell Membrane/drug effects/enzymology, Disease Models; Animal, Down-Regulation, Female, Lipopolysaccharides/administration & dosage/*pharmacology, Mice, Mice; Inbred C57BL, Neutrophils/drug effects/immunology, Phenylmethylsulfonyl Fluoride/pharmacology, Phospholipase D/metabolism, Pneumonia/chemically induced/*metabolism, Polyunsaturated Alkamides, Tumor Necrosis Factor-alpha/biosynthesis
Identifiers
urn:nbn:se:umu:diva-5780 (URN)10.1016/j.lfs.2004.09.005 (DOI)15530507 (PubMedID)
Available from: 2008-01-09 Created: 2008-01-09 Last updated: 2009-11-16Bibliographically approved

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