The potency of the fatty acid amide hydrolase inhibitor URB597 is dependent upon the assay pH

The potency of the fatty acid amide hydrolase inhibitor URB597 is dependent upon the assay pH
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  See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/6796198 The potency of the fatty acid amide hydrolaseinhibitor URB597 is dependent upon the assay pH  Article   in  Pharmacological Research · January 2007 DOI: 10.1016/j.phrs.2006.07.006 · Source: PubMed CITATIONS 29 READS 29 3 authors , including:Ben PaylorUniversity of British Columbia - Vancouver 11   PUBLICATIONS   179   CITATIONS   SEE PROFILE All content following this page was uploaded by Ben Paylor on 07 August 2014. The user has requested enhancement of the downloaded file. All in-text references underlined in blue are added to the srcinal documentand are linked to publications on ResearchGate, letting you access and read them immediately.  Pharmacological Research 54 (2006) 481–485 The potency of the fatty acid amide hydrolase inhibitorURB597 is dependent upon the assay pH Ben Paylor, Sandra Holt, Christopher J. Fowler ∗  Department of Pharmacology and Clinical Neuroscience, Ume˚ a University, SE-901 87 Ume˚ a, Sweden Accepted 27 July 2006 Abstract Inhibitors of the enzyme fatty acid amide hydrolase (FAAH), the principal enzyme involved in the metabolism of the endogenous cannabinoidanandamide, have potential utility in the treatment of disorders including inflammation and inflammatory pain. The carbamate compound URB597(3  -carbamoyl-biphenyl-3-yl-cyclohexylcarbamate) potently and selectively inhibits FAAH by forming a covalent bond with a key serine residueof the enzyme. Little is known as to the pH dependency of this inhibition. Using a preincubation time of 10min, URB597 inhibited rat brainanandamide hydrolysis with p  I  50  values of 7.19 ± 0.02 and 7.75 ± 0.06 at pH 6 and 8, respectively. The inhibition was time-dependent, and secondorder rate constants of  ∼ 0.15 × 10 6 M − 1 min − 1 (pH 6) and ∼ 1.2 × 10 6 M − 1 min − 1 (pH 8) could be estimated. In intact C6 glioma cells and usinga preincubation time of 10min, URB597 inhibited the hydrolysis of 250nM [ 3 H]AEA hydrolysis with p  I  50  values of 5.58 ± 0.07 and 6.45 ± 0.07at extracellular pH values of 6 and 8, respectively. Since tissue pH is affected by inflammation, these data would suggest that the pH selectivity of the inhibition can contribute to the potency of the compound  in vivo .© 2006 Elsevier Ltd. All rights reserved. Keywords:  Anandamide; Fatty acid amide hydrolase; URB597; pH dependency; Carbamylation 1. Introduction Fatty acid amide hydrolase (FAAH) is the principal enzymeresponsible for the metabolism of the endogenous cannabinoidanandamide(arachidonoylethanolamide,AEA)[1].Theenzyme has been cloned, and its physiological role in the body hasbeen well characterized using both knockout mice and selectiveinhibitors [2–4]. Perhaps the most well-studied selective FAAH inhibitor is URB597 [5] (structure see Fig. 1), which produces potentially beneficial effects in models of inflammatory pain,inflammation, anxiety and depression [5–10]. Relatively little is known about the mechanism of actionof the compound. In their srcinal study, Kathuria et al. [5]reportedthattheinhibitionwastime-dependent,andcouldnotberemoved by dialysis, but the observed inhibition of brain FAAHmeasured ex vivo was lost 24h after administration [11], which is shorter than seen with other irreversible compounds [12]. It was hypothesized that the compound interacts covalently witha key serine residue (Ser 241 ) of FAAH on the basis of interac- ∗ Corresponding author. Fax: +46 90 7852752.  E-mail address:  cf@pharm.umu.se (C.J. Fowler). tions of carbamates with other enzymes [13], and recent data has supported this contention [14].In a recent study using a limited number of data points, wefound that both rat and mouse brain FAAH appeared to be moresensitive to inhibition by URB597 at pH 9 than at pH 7 fol-lowing a 60min preincubation [7]. This finding requires further investigation, since is not only of mechanistic interest, but alsoof pharmacological relevance in view of the fact that inflam-mation can affect both tissue and intracellular pH [15,16] and thereby potentially the potency of carbamate FAAH inhibitorslike URB597. In consequence, in the present study, we haveinvestigatedthepHdependencyofFAAHinhibitionbyURB597using both homogenates and intact cells. 2. Materials and methods 2.1. Materials [ 3 H-1,2-ethanolamine]AEA (specific activity 60Ci/mmol)was obtained from American Radiolabeled Chemicals, Inc, St.Louis, USA. URB597 and non-radioactive anandamide wereobtained from the Cayman Chemical Co., Ann Arbor, USA.Fatty acid free bovine serum albumin, active charcoal, and 1043-6618/$ – see front matter © 2006 Elsevier Ltd. All rights reserved.doi:10.1016/j.phrs.2006.07.006  482  B. Paylor et al. / Pharmacological Research 54 (2006) 481–485 Fig. 1. Structure of URB597. The arrow indicates the part of the moleculeinvolved in the carbamylation of the FAAH Ser 241 residue [13,14]. indomethacin were obtained from Sigma Aldrich Inc, St. Louis,USA. C6 glioma cells were obtained from the European Collec-tion of Cell Cultures, Porton Down, UK. 2.2. Preparation of rat brain homogenates Adult male Wistar and Sprague Dawley rats were used inthe study. Whole brains (minus cerebellum) were homogenized(20 strokes, hand held glass homogeniser) in 20mL of buffer(20mM HEPES, 1mM MgCl 2 , pH 7.0). Samples were thencentrifuged at  ∼ 35000 × g  for 20min (4 ◦ C), resuspended in20mL of buffer and centrifuged again. After the second cen-trifugation, the pellets were resuspended in 10mL of bufferand incubated at 37 ◦ C for 15min in order to hydrolyse allendogenous FAAH substrates. They were then centrifuged afinal time at ∼ 35000 × g  for 20min (4 ◦ C). Supernatants werediscarded and samples were resuspended in 1mL. 200mg − 1 (srcinal tissue weight) of Tris–HCl buffer (50mM Tris–HCl,1mM EDTA, 3mM MgCl 2 , pH 7.4) and frozen at  − 80 ◦ Cin 250  L aliquots. A modified Lowry procedure [17] wasused to determine protein content in each sample with bovineserum albumin as standard. Ethical permission for the ani-mal experiments was obtained from the local animal ethicscommittee. 2.3. Assay of FAAH activity in brain homogenates FAAH was assayed as described by Boldrup et al. [18].Briefly, membrane homogenates were diluted with buffer(10mM Tris–HCl, 1mM EDTA, pH either 6 or 8) to give pro-tein contents of 2.5  g (pH 6) or 0.8  g (pH 8) per assay. Thelower protein content at the higher pH reflects the pH profile of FAAH[19].Testcompoundsweredilutedwithethanolandcom- pared against controls which used the same amount of ethanol(10  Lassay − 1 ). Tubes were preincubated at 37 ◦ C for 10min(unless otherwise stated). Aliquots (25  L) of [ 3 H]AEA (in10mM Tris–HCl, 1mM EDTA, 1% (w/v) fatty acid free bovineserum albumin, pH 6 or 8 as appropriate) were added to give anassaysubstrateconcentrationof2  M.Thetubes(assayvolumes200  L) were then incubated for 10min at 37 ◦ C before beingput on ice and having 400  L of active charcoal mixture (80  Lcharcoal+300  L 0.5M HCl) added. Each tube was corked,vortexed three times and left at room temperature for 30min.Samples were then centrifuged to sediment the charcoal, andaliquots (200  L) of the supernatants were taken to determinetritium content by liquid scintillation spectroscopy with quenchcorrection. Blanks were in all cases tubes with buffer in place of membranes. 2.4. FAAH activity in C6 glioma cells The assay used was based on the method of Jonsson et al.[20].Briefly,C6gliomacells(passagerange16–25)wereseededinto24wellcultureplates(2 × 10 5 cellswell − 1 ,culturemediumF-10 Ham with 10% foetal bovine serum, 100unitsmL − 1 peni-cillin and 100  gmL − 1 streptomycin) and incubated for 18hat 37 ◦ C, 5% CO 2  in humidified atmospheric pressure. On theday of assay, the wells were washed twice with 500  L assaybuffer (116mM NaCl, 5.4mM KCl, 1.8mM CaCl 2 , 25mMHEPES, 1mM NaH 2 PO 4 , 0.8mM MgSO 4  in milliQ deionizedwater, pH 6 or 8) containing 1% (w/v) bovine serum albu-min. After removal of the buffer by aspiration, 345  L buffercontaining 0.1% (w/v) fatty acid-free bovine serum albuminwas added followed by 5  L of URB597 or ethanol vehicle.The wells were incubated for 10min at 37 ◦ C after which50  L [ 3 H]AEA was added, to give a final assay substrate con-centration of 0.25  M. After incubation for 5min at 37 ◦ C,the culture plates were placed on ice, and methanol (400  L)was added. The cells were collected by scraping the wells.Aliquots (400  L) were transferred to glass tubes, chloroform(200  L) was added, and the samples were vortex mixed twice.After centrifugation (1400 × g , 5min) to separate the phases,aliquots (200  L) of the aqueous phase were taken to deter-mine tritium content by liquid scintillation spectroscopy withquenchcorrection.Blankswereinallcaseswellsnotcontainingcells. 2.5. Analysis of data p  I  C 50  values [ − log 10 (IC 50  values)] were analyzed for dataexpressed as % of control using the built-in equation ‘sigmoiddose-response’(variableslope)oftheGraphPadPrismcomputerprogramme (GraphPad Software Inc., San Diego, CA, USA)with ‘top’ (uninhibited) values fixed at 100 and ‘bottom’ (min-imum activity) fixed at 0. Confidence intervals of the slopesfor the time-dependency data were also determined using theGraphPad Prism computer programme. 3. Results 3.1. pH-dependent inhibition of rat brain FAAH by URB597  Concentration–response curves for the inhibition of rat brain[ 3 H]AEA hydrolysis by URB597 following a preincubationtime of 10min are shown in Fig. 2A. The compound was less potent at pH 6 than at pH 8, with p  I  50  values of 7.19 ± 0.02and 7.75 ± 0.06, respectively, being found. These values cor-respond to IC 50  values of 64 and 18nM, respectively. Underthe same conditions, the acidic non-steroidal anti-inflammatorydrugindomethacinwasfoundtoexhibitthereversepattern,withp  I  50  values of 4.43 ± 0.03 and 4.05 ± 0.03 (corresponding toIC 50  values of 37 and 89  M, respectively) (data not shown), aresult in line with previous data [21].   B. Paylor et al. / Pharmacological Research 54 (2006) 481–485  483Fig. 2. Inhibition of 2  M [ 3 H]AEA hydrolysis by URB597 in rat brain homogenates at assay pH values of either 6 (unfilled symbols) or 8 (filled symbols). InPanel A, the homogenates were pre-incubated with URB597 for 10min whereas in Panel B, the compound was preincubated for the time shown on the abscissawith either 20nM (  ,  ) or 50nM (  ,  ) URB597 prior to addition of [ 3 H]AEA and incubation for a further 10min at 37 ◦ C. Shown are means ± S.E.M.,  n =3.The slopes ( × 10 − 3 log 10  unitsmin − 1 ) of the regression lines in Panel B were: 20nM URB597 at pH 6, − 1.3 ± 0.4; 50nM URB597 at pH 6,  − 3.4 ± 0.5; 20nMURB597 at pH 8,  − 7.1 ± 1.4; 50nM URB597 at pH 8,  − 26 ± 4.9. The 95% confidence intervals of the slopes for 20 and 50nM URB597 at pH 6 (( − 2.1 to − 0.4) × 10 − 3 log 10  unitsmin − 1 and ( − 4.4 to − 2.3) × 10 − 3 log 10  unitsmin − 1 , respectively) did not overlap with the corresponding 95% confidence intervals of theslopes for at pH 8 (( − 10 to − 4.0) × 10 − 3 log 10  unitsmin − 1 and ( − 37 to − 16) × 10 − 3 log 10  unitsmin − 1 , respectively). Theeffectofpreincubationtimeupontheobservedinhibitionat the two assay pH values are shown in Fig. 2B. Consistent with an irreversible process, the data fitted well to straight lineson a semilog plot, and from the slopes of the regression lines(given in the figure legend), rate constants of 7.8 × 10 − 3 and60 × 10 − 3 min − 1 couldbecalculatedfor50nMURB597atpH6and8respectively.Thecorrespondingvaluesfor20nMURB597were2.9 × 10 − 3 and16 × 10 − 3 min − 1 ,respectively.Fromthesevalues, second order rate constants of  ∼ 0.15 × 10 6 M − 1 min − 1 (pH 6) and ∼ 1.2 × 10 6 M − 1 min − 1 (pH 8) could be estimated.In order to demonstrate that the observed pH dependency didnot reflect a lack of stability of URB597 at high concentrations,experiments were undertaken where URB597 was first incu-bated for 20min at 37 ◦ C with 40  L buffer at pH 6, after whichan equal amount of buffer at pH 8 was added. Homogenateswerethenadded,andtheassaywasthenrunasdescribedabove,usinga10minpreincubationphase,andtheinhibitioncomparedwith parallel assays where the order of the first two buffers wasreversed. A lack of stability at pH 6 would give a lower potencyof URB597 for the former conditions [pH 6 then pH 8 priorto addition of enzyme] compared to the latter. This was not Fig.3. Inhibitionof250nM[ 3 H]AEAhydrolysisbyURB597inintactC6cells.Assays were conducted at either pH 6 (  ) or pH 8 (  ). The cells were prein-cubated with URB597 for 10min prior to addition of [ 3 H]AEA and incubationfor a further 5min at 37 ◦ C. Shown are means ± S.E.M.,  n =3–7. seen regardless of the inhibitor concentrations used (20, 50 and100nM) or the final assay pH used (6 or 8) (data not shown). 3.2. pH-dependent inhibition of AEA hydrolysis byURB597 in intact C6 glioma cells Onthebasisofinitialtimecourseexperimentsusinganincu-bation time range of 2–15min at pH 6 and 8, an incubation timeof 5min was chosen for the present experiments. The hydrol-ysis of AEA by C6 cells was inhibited by URB597, and thecompound was less potent at the lower extracellular pH value(Fig. 3). The p  I  50  values were 5.58 ± 0.07 and 6.45 ± 0.07 (cor-responding to IC 50  values of 2.6  M and 350nM, respectively)at pH e  values of 6 and 8, respectively. 4. Discussion The present study has demonstrated that URB597 is a moreeffective inhibitor of FAAH at pH 8 than at pH 6 in rat brainhomogenates,andthatthisisnotduetoaninstabilityofthecom-pound at the lower pH value. The second order rate constant forthetime-dependentinhibitionisabouteighttimeshigheratpH8thanatpH6.Ithasbeenknownformanyyearsthattherateofcar-bamylation of amino acid residues is highly dependent upon thep K  A  valuesoftheaminogroups[22],butinthecaseofURB597, the carbamylation is at the free hydroxyl group of Ser 241 [14].In this model, the cyclohexyl–NH–(CO)–O– group (arrowed inFig.1)becomescovalentlyboundtotheSer 241 ,anditispossiblethat the ionization status of either the molecule, and/or the pro-cess of interaction with other key residues (Lys 142 and Ser 217 )[13,14] determines the rate of carbamylation. In this respect, it can be noted that the srcinal model was based upon the inter-action of carbamate compounds with acetylcholinesterase, andthe second order rate constants seen here, albeit estimates, showthe same pH dependency as seen for inhibition of purified horseacetylcholinesterase by such compounds [23].  484  B. Paylor et al. / Pharmacological Research 54 (2006) 481–485 FAAH is intracellularly localized, and so URB597 will needto penetrate the cell to produce its effects. In the present study,we have used C6 glioma cells to study the pH dependency of URB597 in intact cells. These cells were chosen because theyhave been well characterized with respect to variations in extra-cellular and intracellular pH [24,25]. In our hands, C6 cells can buffer reasonably well a change in the extracellular pH from7.6 to 6.0, the intracellular pH values decreasing by about 0.8units over this range [26]. At pH 8, the potency of URB597 was about 20-fold lower than seen for the homogenates, presumablya reflection both of the need for the compound to penetrate thecell, and of the time course of this process. Interestingly, thepH sensitivity of the inhibition was retained, and the compoundwas about seven-fold less effective an inhibitor at pH 6 thanat pH 8. This differs from the observed pH dependency of theacidic non-steroidal anti-inflammatory agent ibuprofen, whichbecomes more potent as the pH is reduced both for intact C6glioma cells and for rat brain homogenates [26,27]. If this data is generalisable to all cells, it would suggest that the potency of URB597 is dependent upon the pH of its environment.While providing novel information on the interaction of FAAH with URB597, the data may have in vivo correlates.Studies in genetically modified mice have suggested that lossof activity of the peripheral enzyme is involved in regulatingthe oedema response to carrageenan, whilst the central FAAHis involved in the regulation of pain responses [28]. Inflamma- tion causes a marked decrease in tissue pH [15] and can also affect intracellular pH [16], and so it might be predicted that the potency of URB597 in preventing the oedema response tocarrageenan will be lower than for centrally mediated effectsof the compound. This appears to be the case (see [5,7,9,10]), although other factors, such as the influence of the vehicle used,the distribution of the compound in the body, and the degree of FAAH inhibition required to produce the given effect, will of course contribute. Nonetheless, the pH dependence of URB597maycontributetotheobservedpotencyofthecompoundinvivo. Acknowledgements The research was supported by grants from the SwedishResearch Council (Grant No. 12158, medicine), Gun and BertilStohne’s Foundation, Konung Gustaf V’s and Drottning Vic-torias Foundation, Stiftelsen f ¨or Gamla Tj¨anarinnor and theResearch Funds of the Medical Faculty, Ume˚a University. References [1] Deutsch DG, Chin SA. Enzymatic synthesis and degradation of anan-damide, a cannabinoid receptor agonist. Biochem Pharmacol 1993;46:791–6.[2] Fowler CJ. Metabolism of the endocannabinoids anandamide and 2-arachidonoyl glycerol, a review, with emphasis on the pharmacology of fatty acid amide hydrolase, a possible target for the treatment of neurode-generative diseases and pain. Curr Med Chem - Central Nervous SystemAgents 2004;4:161–74.[3] McKinney MK, Cravatt BF. Structure and function of fatty acid amidehydrolase. Annu Rev Biochem 2005;74:411–32.[4] Maccarrone M. Fatty acid amide hydrolase: a potential target for next gen-eration therapeutics. Curr Pharm Des 2006;12:759–72.[5] Kathuria S, Gaetani S, Fegley D, Vali˜no F, Duranti A, Tontini A, et al.Modulation of anxiety through blockade of anandamide hydrolysis. 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Pharmacological evaluation of cannabinoid recep-tor ligands in a mouse model of anxiety: further evidence for an anxi-olytic role for endogenous cannabinoid signaling. J Pharmacol Exp Ther2006;318:304–11.[11] Fegley D, Gaetani S, Duranti A, Tontini A, Mor M, Tarzia G, et al. Charac-terization of the fatty-acid amide hydrolase inhibitor URB597: effects onanandamide and oleoylethanolamide deactivation. J Pharmacol Exp Ther2005;313:352–8.[12] Quistad GB, Sparks SE, Segall Y, Nomura DK, Casida JE. Selectiveinhibitors of fatty acid amide hydrolase relative to neuropathy targetesteraseandacetylcholinesterase:toxicologicalimplications.ToxicolApplPharmacol 2002;179:57–63.[13] Basso E, Duranti A, Mor M, Piomelli D, Tontini A, Tarzia G, et al. Tandemmass spectrometric data-FAAH inhibitory activity relationships of somecarbamic acid  O -aryl esters. 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