Δ9-tetrahydrocannabinol and its major metabolite Δ9-tetrahydrocannabinol-11-oic acid as 15-lipoxygenase inhibitors

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<ul><li><p>9-Tetrahydrocannabinol and Its Major Metabolite9-Tetrahydrocannabinol-11-oic Acid as15-Lipoxygenase Inhibitors</p><p>SHUSO TAKEDA,1,2 RONGRONG JIANG,3 HIRONORI ARAMAKI,1 MASUMI IMOTO,4 AKIHISA TODA,4 REIKO EYANAGI,4</p><p>TOSHIAKI AMAMOTO,5 IKUO YAMAMOTO,6 KAZUHITO WATANABE2,3</p><p>1Department of Molecular Biology, Daiichi University of Pharmacy, 22-1 Tamagawa-cho, Minami-ku, Fukuoka 815-8511, Japan</p><p>2Organization for Frontier Research in Preventive Pharmaceutical Sciences, Hokuriku University, Ho-3 Kanagawa-machi, Kanazawa920-1181, Japan</p><p>3Department of Hygienic Chemistry, Faculty of Pharmaceutical Sciences, Hokuriku University, Ho-3 Kanagawa-machi, Kanazawa920-1181, Japan</p><p>4Department of Hygienic Chemistry, Daiichi University of Pharmacy, 22-1 Tamagawa-cho, Minami-ku, Fukuoka 815-8511, Japan</p><p>5NEUES Company Limited, Yaesu Center Building 3F, 1-6-6 Yaesu, Chuo-ku, Tokyo 103-0028, Japan</p><p>6School of Pharmaceutical Sciences, Kyushu University of Health and Welfare, 1714-1 Yoshino-cho, Nobeoka 882-8508, Japan</p><p>Received 18 May 2010; revised 26 August 2010; accepted 28 August 2010</p><p>Published online 1 October 2010 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.22354</p><p>ABSTRACT: 15-Lipoxygenase (15-LOX) is one of the key enzymes responsible for the for-mation of oxidized low-density lipoprotein, a major causal factor for atherosclerosis. 9-Tetrahydrocannabinol (9-THC), a major component of marijuana, has suggested to suppressatherosclerosis. Although 9-THC seems to be attractive for the prevention of atherosclerosis,there is no information about whether or not 15-LOX isoform can be inhibited by 9-THC. Inthe present study, 9-THC was found to be a direct inhibitor for 15-LOX with an IC50 (50%inhibition concentration) value of 2.42 :M. Furthermore, 9-THC-11-oic acid, a major andnonpsychoactive metabolite of 9-THC, but not another 9-THC metabolite 11-OH-9-THC(psychoactive), was revealed to inhibit 15-LOX. Taken together, it is suggested that 9-THCcan abrogate atherosclerosis via direct inhibition of 15-LOX, and that 9-THC-11-oic acid isshown to be an active metabolite of 9-THC in this case. 2010 Wiley-Liss, Inc. and theAmerican Pharmacists Association J Pharm Sci 100:12061211, 2011Keywords: 15-lipoxygenase; atherosclerosis; 9-Tetrahydrocannabinol; metabolism. oxida-tion; inhibition; 9-THC-11-oic acid; lipid/lipoprotein; low-density lipoprotein; structure-activity relationship</p><p>INTRODUCTION</p><p>15-Lipoxygenase (15-LOX) belongs to the structurallyand functionally related nonheme iron dioxygenasesfamily. Up to now, three major Lypoxygenase (LOX)isoforms have been discovered (i.e., 5-, 12-, and 15-LOX).13 Among them, 15-LOX has emerged as an at-tractive target for therapeutic intervention because15-LOX is suggested to play an important role in</p><p>Correspondence to: Kazuhito Watanabe (Telephone: +81-76-229-6220; Fax: +81-76-229-6221; E-mail: k-watanabe@hokuriku-u.ac.jp)Journal of Pharmaceutical Sciences, Vol. 100, 12061211 (2011) 2010 Wiley-Liss, Inc. and the American Pharmacists Association</p><p>atherosclerosis as well as in the progression of somecancers.46 Although atherosclerosis is a multifacto-rial disease that involves chronic inflammation atevery stage, from initiation to progression and even-tually plaque rupture,7 oxidized low-density lipopro-tein (oxLDL) is recognized as one of the key play-ers in the development of atherosclerosis, as clearlydemonstrated by Katagiris group.8 Enzymes knownin the formation of oxLDL are 15-LOX, myeloperox-idase, and NADPH oxidase.9 Among them, DNA mi-croarray, transgenic and knockout studies suggest animportant role of 15-LOX in atherosclerosis.911</p><p>9-Tetrahydrocannabinol (9-THC), a majorcannabinoid constituent of marijuana, has been</p><p>1206 JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 3, MARCH 2011</p></li><li><p>9- THC AND 9-THC-11-OIC ACID AS 15-LIPOXYGENASE INHIBITORS 1207</p><p>reported to suppress atherosclerosis in animalmodel.12 9-THC has been approved by the Foodand Drug Administration in the United States ofAmerica to reduce the nausea of cancer patients un-dergoing chemotherapy,13 and furthermore 9-THCis a component of Sativex (GW Pharm., UK)</p><p>R, a</p><p>cannabinoid extract used as an oral spray and hasbeen approved in Canada and Spain to treat pain andmuscle spasms of multiple sclerosis. Thus, 9-THCis being used as a medicine worldwide. It should benoted that the action of 9-THC described above isbasically based on the interaction of the cannabinoidwith cannabinoid receptor type 2 (CB2 receptor).Although 9-THC seems to have an attractive actionespecially on atherosclerosis, there are no reportsthat investigate the effect of 9-THC on 15-LOXenzyme itself.</p><p>In the current report, we attempted to investigatewhether or not 9-THC can directly inhibit 15-LOXactivity. Because 9-THC is known to be subjected toextensive metabolism after its administration, whichleads to its oxidative metabolites at the 11-position;that is, 11-OH-9-THC and 9-THC-11-oic acid,14,15</p><p>we also studied the effects of these metabolites. 11-OH-9-THC (or 11-OH-8-THC) is identified as ma-jor metabolite with psychoactive effect,16,17 whereasone of the dominating metabolites in urine 9-THC-11-oic acid (or 8-THC-11-oic acid) is psychologicallyinactive.18,199-THC was found to be a direct in-hibitor for 15-LOX, and the 11-oic metabolite was stillan inhibitor for 15-LOX in its physiological concentra-tion ranges after dosing of 9-THC.</p><p>MATERIALS AND METHODS</p><p>Cannabinoids and Chemicals</p><p>9-THC and cannabidiol (CBD) were isolated andpurified from the cannabis leaves according tothe methods described elsewhere.20 11-OH-9-THC,cannabidiol-2,6-dimethylether (CBDD) and can-nabielsoin (CBE) were prepared as describedpreviously.21,22 Purities of these cannabinoids werechecked to be at least above 95% by gas chromato-graphy.2325 9-THC-11-oic acid was provided by theNational Institute on Drug Abuse (NIDA, Bethesda,Maryland). 8-THC-11-oic acid was synthesized ac-cording to the methods described elsewhere.26 Nordi-hydroguaiaretic acid (NDGA) was purchased fromCayman Chemical Company (Ann Arbor, Michigan).All other reagents were of analytical grade.</p><p>Enzyme Sources</p><p>Measurements of the 5-Lipoxygenase (5-LOX) and 15-LOX activities were carried out using a commerciallyavailable LOX inhibitor screening assay kit (CaymanChemical Company). 5-LOX (lot nos. #0400028-1) and</p><p>15-LOX (lot nos. #193367-193368) screening enzymeswere purchased from Cayman Chemical Company.All inhibitors added to the reaction system were pre-pared just before use. After enzyme reactions, result-ing hydroperoxides were treated with chromogen todevelop the reaction, and then absorbance intensitieswere determined spectrophotometrically with a 96-well plate reader at 490 nm. No colorimetric changewas observed in control incubations that were per-formed by omitting enzymes or with heat denaturedenzymes and inhibitors in combination with chro-mogen. The concentrations of cannabinoids used inthis study were determined based on the solubilityand the concentration without interference with chro-mogen. Each assay was performed in triplicate.</p><p>Data Analysis</p><p>The concentration of the inhibitor that is requiredto produce 50% inhibition of the enzymatic activity(IC50) was determined from the curves plottingenzymatic activity versus inhibitor concentrationsusing Origin7.5J software (OriginLab Corpora-tion, Northampton, Massachusetts). The details ofthe calculations were described in our previousarticles.24,25,27 Differences were considered to be sig-nificant when the p value was calculated to be lessthan 0.05. All statistical analyses were performed byScheffes F test, which is a type of post-hoc test for an-alyzing results of analysis of variance testing. Thesecalculations were performed using Statview5.0J soft-ware (SAS Institute Inc., Cary, North Carolina).</p><p>RESULTS AND DISCUSSION</p><p>In the present study, to investigate the direct in-hibitory effects of 9-THC as well as its two metabo-lites 11-OH-9-THC and 9-THC-11-oic acid on15-LOX activity, we used a purified 15-LOX as an en-zyme source. Structural information of these cannabi-noids is shown in Figure 1. 9-THC is known tobe mainly subjected to oxidation of methyl group</p><p>Figure 1. Structures of 9-THC and its major metabo-lites, 11-OH-9-THC and 9-THC-11-oic acid. Numbers inthe structures correspond to the monoterpenoid nomencla-ture.</p><p>DOI 10.1002/jps JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 3, MARCH 2011</p></li><li><p>1208 TAKEDA ET AL.</p><p>Figure 2. Dose-dependent inhibition by 9-THC on 15-LOX activity. CBDD/NDGA and CBE were used as pos-itive and negative controls, respectively. Enzymatic reac-tions were initiated with linoleic acid, and then chromogenwas added to stop the reactions and to develop colorimet-ric reactions. The absorbance was monitored at 490 nm.The assay conditions are described under Materials andMethods. Each bar represents the mean S.D. (triplicatedeterminations) of the relative activity to the control.</p><p>(-CH3) at the 11-position by cytochrome P450-catalyzed reaction.14 We first determined the IC50value of 9-THC-mediated 15-LOX inhibition withpositive and negative controls, CBDD (a potent andselective 15-LOX inhibitor)/NDGA (a potent pan-LOXinhibitor) and CBE (an oxidized metabolite of CBD),respectively.25 The IC50 value of 9-THC was deter-mined to be 2.42 :M (Fig. 2). In the experiment,</p><p>15-LOX activity was completely and mostly inhibitedby 2 :M CBDD and NDGA, respectively, but not byCBE (Fig. 2, right panel).</p><p>As shown in Figure 2 (left panel), although 9-THChas revealed to inhibit 15-LOX activity directly, thereis no information about the inhibitory effect of itsmetabolites against 15-LOX activity so far. It is impor-tant to obtain the information whether or not 15-LOXcan be inhibited by the metabolites, because some-times metabolites but not their parent compoundshave much stronger activity in inhibition of enzymes,being known as metabolic activation. Some of 9-THC metabolites are known to be pharmacologicallymore active than the parent cannabinoid.28 Thus,the effects of 9-THC including its major metabo-lites, 11-OH-9-THC and 9-THC-11-oic acid, on 15-LOX were studied. The concentrations of cannabi-noids used here (2 :M) were determined based onthe IC50 value of 9-THC-mediated 15-LOX inhibi-tion (Fig. 2, left panel). 15-LOX activity was signifi-cantly inhibited by 9-THC and 9-THC-11-oic acidbut was not inhibited by 11-OH-9-THC, although aquite high concentration of 11-OH-9-THC (20 :M)inhibited the activity (Fig. 3b) to some extent, whichis comparable to 2 :M 9-THC-11-oic acid-mediatedinhibition. On the other hand, 5-LOX activity wasnot inhibited by the three cannabinoids with thesame concentrations (Fig. 3a). 5-LOX activity wasnot significantly inhibited by higher concentrationsof the cannabinoids (up to 5 :M) (data not shown).Thus, it is suggested that 15-LOX is susceptible to</p><p>Figure 3. Effects of 9-THC and its major metabolites, 11-OH-9-THC and 9-THC-11-oicacid on 5-LOX/15-LOX activities. (a) and (b), effects of 9-THC, 11-OH-9-THC, and 9-THC-11-oic acid on 5-LOX and 15-LOX activity, respectively, were examined in the presence of indicatedconcentrations of the cannabinoids. Enzymatic reactions were initiated with linoleic acid, andthen chromogen was added to stop the reactions and to develop colorimetric reactions. Theabsorbance was monitored at 490 nm. The assay conditions are described under Materialsand Methods. Each bar represents the mean S.D. (triplicate determinations) of the relativeactivity to the control. , significantly different (P </p></li><li><p>9- THC AND 9-THC-11-OIC ACID AS 15-LIPOXYGENASE INHIBITORS 1209</p><p>9-THC-mediated inhibition, and that its metabolite9-THC-11-oic acid is an active metabolite of 9-THC. The IC50 value of 9-THC-mediated 15-LOXinhibition seems to be high, even though 9-THC canbe accumulated up to 20-fold concentrations (i.e., ashigh as 1 :M) in some tissues compared with nor-mal ones after its taking.29,30 Then, we investigatedthe effect of 9-THC-11-oic acid on 15-LOX activityfocusing on the plasma concentration range (up to200 nM) after administration of 9-THC.31 15-LOXactivity was inhibited by 9-THC-11-oic acid in aconcentration-dependent manner (Fig. 4a), althoughthe activity was not significantly inhibited by 9-THC at the concentration ranging from 25 to 200 nM(data not shown). 9-THC including its metabolitesis known to be highly lipophilic.31 Of plasma 9-THCand its metabolites &gt;95% is bound to plasma pro-teins, mainly to lipoproteins. Although the avail-ability of 9-THC-11-oic acid in plasma seems tobe restricted, it should be emphasized that 15-LOXcan oxidize low-density lipoprotein itself directly toproduce oxLDL.3234 As evidenced by the results inFigure 2, CBE, which is the metabolite of CBD, hadno inhibitory effect on 15-LOX activity. CBD has beenshown to inhibit 15-LOX by our most recent report.25</p><p>However, different from the case of 9-THC, the in-hibition potential of CBD may be disappeared by itsmetabolism via hepatic drug-metabolizing enzymes(i.e., formation of CBE).27,3539 Furthermore, we havereported that CBD can be converted into 9-THCin acidic conditions that are mimicked by stomachacid.40 In acidic conditions, 9-THC can be isomer-ized to 8-THC.41 If this is the case with 9-THC-11-oic acid, the metabolic fate and action might bemodulated by its administration route (i.e., oral vs.intravenous administration). Based on this possibil-ity, we studied the effect of 8-THC-11-oic acid on 15-LOX activity. As shown in Figure 4b, different fromthe event of 9-THC-11-oic acid, 15-LOX activity wasnot significantly inhibited by 8-THC-11-oic acid upto 1 :M. Thus, it is suggested that the inhibition po-tency of 9-THC toward 15-LOX might be changedby the administration route of the cannabinoid espe-cially p.o. administration.</p><p>In general, 9-THC evokes its pharmacological ac-tion via interaction with cannabinoid receptor (CB)receptors.30,42 The anti-atherosclerotic effect of 9-THC in mice has been reported to be initiated bythe interaction with the receptors (especially CB2receptor).12 The binding affinities of 9-THC to CB2receptor are reported to be from 5.05 to 80.3 nM.42 Ac-cumulating evidence suggests that the expression ofCB receptors can be modulated by some diseases andphysiological conditions,30,43,44 suggesting that theaction of 9-THC would be modulated by the expres-sion status of CB receptors. Until now, there is no ex-perimental evidence demonstrating the relationship</p><p>Figure 4. Dose-dependent inhibition by 9-THC-11-oicacid on 15-LOX activity. Effects of 9-THC-11-oic acid (a)and 8-THC-11-oic acid (b) on 15-LOX activity was ex-amined in the presence of indicated concentrations of thecannabinoids. Based on the binding affinity for CB receptorof 9-THC-11-oic acid (1 :M),45 the maximum concen-tration was set at 1 :M. 15-LOX activity was significantlyinhibited by plasma concentrations (up to 200 nM) of 9-THC-11-oic acid but not by its isomer 8-THC-11-oic acid.Enzymatic reactions were initiated with linoleic acid, andthen chromogen was added to stop...</p></li></ul>

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