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Pharmacology of Palmitoylethanolamide and Related Compounds
Umeå University, Faculty of Medicine, Pharmacology and Clinical Neuroscience, Pharmacology.
2005 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Anandamide (AEA) is an endogenous fatty acid which activates the same cannabinoid receptors as ∆9-tetrahydrocannabinol, the psychoactive substance in marijuana. In vivo, anandamide exerts a number of actions including effects upon pain and inflammation. However, AEA has a short duration of action since it is rapidly metabolised, primarily by the intracellular enzyme fatty acid amide hydrolase (FAAH).

The general aim of this thesis has been to identify and characterize compounds capable of preventing the metabolism of anandamide. The chemical approach was based on the endogenous anti-inflammatory compound palmitoylethanolamide (PEA), a compound related to anandamide with the ability to inhibit anandamide degradation by substrate competition, but without the ability to directly activate cannabinoid receptors.

A number of compounds were identified as inhibitors of rat brain FAAH in the initial in vitro studies, without having major affinity for the cannabinoid receptors. In particular, palmitoylisopropylamide (PIA) was found to reduce the metabolism of AEA in intact C6 glioma cells with potency similar to the prototypical AEA reuptake inhibitor AM404. This compound was in addition found to exert less effect upon C6 glioma cell proliferation than either AM404 or the closely related uptake inhibitor VDM11. To evaluate if PIA was effective in vivo, a model of mast cell dependent inflammation, oedema of the ear following local injection of compound 48/80, was set up using anaesthetised mice. Initially, a CB2 cannabinoid receptor selective agonist was used to probe the model and demonstrated to produce an anti-oedema effect. In contrast, the compound was inactive in vitro in skin slice preparations. PIA showed a similar pattern, although there was a large variation in responses which affected the significance of the result obtained, as did the vehicle used to dissolve the compound.

Taken together, the present data would suggest that PIA can inhibit the degradation of AEA without having deleterious effects upon cell proliferation or affinity for the cannabinoid receptors. Further experimentation is necessary to elucidate the usefulness of this compound in vivo.

Place, publisher, year, edition, pages
Umeå: Farmakologi och klinisk neurovetenskap , 2005. , 82 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 942
Keyword [en]
Pharmacology, anandamide, cannabinoid, FAAH, palmitoylethanolamide, palmitoylisopropylamide, inflammation, mast cells
Keyword [sv]
Farmakologi
National Category
Pharmacology and Toxicology
Research subject
Medical Pharmacology
Identifiers
URN: urn:nbn:se:umu:diva-445ISBN: 91-7305-821-1 (print)OAI: oai:DiVA.org:umu-445DiVA: diva2:143476
Public defence
2005-03-04, E04, 6E, Norrlands universitetssjukhus, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2005-02-09 Created: 2005-02-09 Last updated: 2009-11-17Bibliographically approved
List of papers
1. Effects of homologues and analogues of palmitoylethanolamide upon the inactivation of the endocannabinoid anandamide.
Open this publication in new window or tab >>Effects of homologues and analogues of palmitoylethanolamide upon the inactivation of the endocannabinoid anandamide.
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2001 (English)In: British Journal of Pharmacology, ISSN 0007-1188, Vol. 133, no 8, 1263-1275 p.Article in journal (Refereed) Published
Abstract [en]

The ability of a series of homologues and analogues of palmitoylethanolamide to inhibit the uptake and fatty acid amidohydrolase (FAAH)-catalysed hydrolysis of [3H]-anandamide ([3H]-AEA) has been investigated.Palmitoylethanolamide and homologues with chain lengths from 12–18 carbon atoms inhibited rat brain [3H]-AEA metabolism with pI50 values of ∼5. Homologues with chain lengths eight carbon atoms gave <20% inhibition at 100 μM.R-palmitoyl-(2-methyl)ethanolamide, palmitoylisopropylamide and oleoylethanolamide inhibited [3H]-AEA metabolism with pI50 values of 5.39 (competitive inhibition), 4.89 (mixed type inhibition) and 5.33 (mixed type inhibition), respectively.With the exception of oleoylethanolamide, the compounds did not produce dramatic inhibition of [3H]-WIN 55,212-2 binding to human CB2 receptors expressed on CHO cells. Palmitoylethanolamide, palmitoylisopropylamide and R-palmitoyl-(2-methyl)ethanolamide had modest effects upon [3H]-CP 55,940 binding to human CB1 receptors expressed on CHO cells.Most of the compounds had little effect upon the uptake of [3H]-AEA into C6 and/or RBL-2H3 cells. However, palmitoylcyclohexamide (100 μM) and palmitoylisopropylamide (30 and 100 μM) produced more inhibition of [3H]-AEA uptake than expected to result from inhibition of [3H]-AEA metabolism alone.In intact C6 cells, palmitoylisopropylamide and oleoylethanolamide inhibited formation of [3H]-ethanolamine from [3H]-AEA to a similar extent as AM404, whereas palmitoylethanolamide, palmitoylcyclohexamide and R-palmitoyl-(2-methyl)ethanolamide were less effective.These data provide useful information upon the ability of palmitoylethanolamide analogues to act as 'entourage' compounds. Palmitoylisopropylamide may prove useful as a template for design of compounds that reduce the cellular accumulation and metabolism of AEA without affecting either CB1 or CB2 receptors.

Identifiers
urn:nbn:se:umu:diva-4392 (URN)10.1038/sj.bjp.0704199 (DOI)11498512 (PubMedID)
Available from: 2005-02-09 Created: 2005-02-09 Last updated: 2009-11-16Bibliographically approved
2. Modifications of the ethanolamine head in N-palmitoylethanolamine: synthesis and evaluation of new agents interfering with the metabolism of anandamide.
Open this publication in new window or tab >>Modifications of the ethanolamine head in N-palmitoylethanolamine: synthesis and evaluation of new agents interfering with the metabolism of anandamide.
2003 (English)In: Journal of Medicinal Chemistry, ISSN 0022-2623, Vol. 46, no 8, 1440-1448 p.Article in journal (Refereed) Published
Abstract [en]

The endogenous fatty acid amide anandamide (AEA) has, as a result of its actions on cannabinoid and vanilloid receptors, a number of important pharmacological properties including effects on nociception, memory processes, spasticity, and cell proliferation. Inhibition of the metabolism of AEA, catalyzed by fatty acid amide hydrolase (FAAH), potentiates the actions of AEA in vivo and therefore may be a useful target for drug development. In the present study, we have investigated whether substitution of the headgroup of the endogenous alternative FAAH substrate palmitoylethanolamide (PEA) can result in the identification of novel compounds preventing AEA metabolism. Thirty-seven derivatives of PEA were synthesized, with the C16 long chain of palmitic acid kept intact, and comprising 20 alkylated, 12 aromatic, and 4 halogenated amides. The ability of the PEA derivatives to inhibit FAAH-catalyzed hydrolysis of [(3)H]AEA was investigated using rat brain homogenates as a source of FAAH. Inhibition curves were analyzed to determine the potency of the inhibitable fraction (pI(50) values) and the maximal attained inhibition for the compound, given that solubility in an aqueous environment is a major issue for these compounds. In the alkylamide family, palmitoylethylamide and palmitoylallylamide were inhibitors of AEA metabolism with pI(50) values of 5.45 and 5.47, respectively. Halogenated derivatives (Cl and Br) exhibit pI(50) values of approximately 5.5 but rather low percentages of maximal inhibition. The -OH group of the ethyl head chain of N-palmitoylethanolamine was not necessary for interaction with FAAH. Amides containing aromatic moieties were less potent inhibitors of AEA metabolism. Compounds containing amide and ester bonds, 13 and 37, showed pI(50) values of 4.99 and 5.08, respectively. None of the compounds showed obvious affinity for CB(1) or CB(2) receptors expressed on Chinese hamster ovary (CHO) cells. It is concluded that although none of the compounds were dramatically more potent than PEA itself at reducing the metabolism of AEA, the lack of effect of the compounds at CB(1) and CB(2) receptors makes them useful templates for development of possible therapeutic FAAH inhibitors.

Identifiers
urn:nbn:se:umu:diva-4393 (URN)10.1021/jm0209679 (DOI)12672243 (PubMedID)
Available from: 2005-02-09 Created: 2005-02-09 Last updated: 2009-11-16Bibliographically approved
3. AM404 and VDM 11 non-specifically inhibit C6 glioma cell proliferation at concentrations used to block the cellular accumulation of the endocannabinoid anandamide.
Open this publication in new window or tab >>AM404 and VDM 11 non-specifically inhibit C6 glioma cell proliferation at concentrations used to block the cellular accumulation of the endocannabinoid anandamide.
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2003 (English)In: Archives of Toxicology, ISSN 0340-5761, Vol. 77, no 4, 201-207 p.Article in journal (Refereed) Published
Abstract [en]

AM404 [ N-(4-hydroxyphenyl)arachidonylamide] and VDM 11 [(5 Z,8 Z,11 Z,14 Z)- N-(4-hydroxy-2-methylphenyl)-5,8,11,14-eicosatetraenamide] are commonly used to prevent the cellular accumulation of the endocannabinoid anandamide, and thereby to potentiate its actions. However, it has been reported that AM404 can produce an influx of calcium into cells, which might be expected to have deleterious effects on cell proliferation. In the present study, AM404 and VDM 11 were found to reduce C6 glioma cell proliferation with IC(50) values of 4.9 and 2.7 microM, respectively. The inhibition of cell proliferation following a 96-h exposure was not accompanied by dramatic caspase activation, and was not prevented by either a combination of cannabinoid and vanilloid receptor antagonists, or by the antioxidant alpha-tocopherol, suggestive of a non-specific mode of action. Similar results were seen with palmitoylisopropylamide, although this compound only produced significant inhibition of cell proliferation at 30 microM concentrations. AM404 (1 microM), VDM 11 (1 microM) and palmitoylisopropylamide (3-30 microM), i.e. concentrations producing relatively modest effects on cell proliferation per se, reduced the vanilloid receptor-mediated antiproliferative effects of anandamide, as would be expected for compounds preventing the cellular accumulation of anandamide (and thereby access to its binding site on the vanilloid receptor). It is concluded that concentrations of AM404 and VDM 11 that are generally used to reduce the cellular accumulation of anandamide have deleterious effects upon cell proliferation, and that lower concentrations of these compounds may be more appropriate to use in vitro.

Identifiers
urn:nbn:se:umu:diva-4394 (URN)10.1007/s00204-002-0435-6 (DOI)12698235 (PubMedID)
Available from: 2005-02-09 Created: 2005-02-09 Last updated: 2009-11-17Bibliographically approved
4. The cannabinoid CB2 receptor selective agonist JWH133 reduces mast cell oedema in response to compound 48/80 in vivo but not the release of beta-hexosaminidase from skin slices in vitro.
Open this publication in new window or tab >>The cannabinoid CB2 receptor selective agonist JWH133 reduces mast cell oedema in response to compound 48/80 in vivo but not the release of beta-hexosaminidase from skin slices in vitro.
2006 (English)In: Life Sciences, ISSN 0024-3205, Vol. 78, no 6, 598-606 p.Article in journal (Refereed) Published
Abstract [en]

In a recent study so far published in abstract form, it was reported that the CB(2) receptor selective agonist AM1241 diminishes oedema produced as a result of mast cell degranulation in vivo. It is, however, not known whether other structurally different CB(2) agonists share this effect, and whether this is due to a direct effect on mast cell function. In the present study, we have investigated the effects of JWH133, a CB(2) receptor selective agonist, together with the anti-inflammatory agent palmitoylethanolamide and its analogue palmitoylisopropylamide, on compound 48/80-induced oedema and degranulation in vivo and in vitro. JWH133 (20 and 200 microg/mouse i.p.) significantly reduced the ability of compound 48/80 to induce oedema in vivo in the anaesthetised mouse following its injection into the ear pinna. Palmitoylethanolamide (200 microg/mouse i.p) also reduced the response to compound 48/80, whereas no firm conclusions could be drawn for palmitoylisopropylamide (20 and 200 microg/mouse i.p.). The CB(2) selective antagonist/inverse agonist SR144528 (60 microg/mouse i.p.) appeared to produce anti-inflammatory effects per se in this model, making it hard to interpret the effects of JWH133 in terms of CB(2) receptor mediated activation. In contrast to the situation in vivo, neither JWH133 (0.3 and 3 microM) nor palmitoylethanolamide (10 microM) affected mast cell degranulation, measured by following the release of the granular protein beta-hexosaminidase, produced by compound 48/80 in vitro in mouse skin slices. The two compounds were also ineffective in inhibiting the binding of [(3)H]pyrilamine to histamine H(1) receptors in vitro. It is concluded that the ability of JWH133 to affect mast cell dependent inflammation in vivo may be mediated by an indirect action upon the mast cells.

Keyword
Animals, Cannabinoids/*pharmacology, Capillary Permeability/drug effects, Edema/*prevention & control, Female, Mast Cells/*drug effects/physiology, Mice, Mice; Inbred BALB C, Palmitic Acids/pharmacology, Polymerase Chain Reaction, Receptor; Cannabinoid; CB2/*agonists, Receptors; Histamine H1/drug effects, Skin/*drug effects/secretion, beta-N-Acetylhexosaminidase/*secretion, p-Methoxy-N-methylphenethylamine/*pharmacology
Identifiers
urn:nbn:se:umu:diva-5883 (URN)10.1016/j.lfs.2005.05.059 (DOI)16111718 (PubMedID)
Available from: 2008-04-02 Created: 2008-04-02 Last updated: 2009-11-17Bibliographically approved
5. Characterisation of palmitoylisopropylamide in mast cell dependent systems in vitro and in vivo.
Open this publication in new window or tab >>Characterisation of palmitoylisopropylamide in mast cell dependent systems in vitro and in vivo.
Manuscript (Other academic)
Identifiers
urn:nbn:se:umu:diva-4396 (URN)
Available from: 2005-02-09 Created: 2005-02-09 Last updated: 2010-01-13Bibliographically approved

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