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

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  • 9-Tetrahydrocannabinol and Its Major Metabolite9-Tetrahydrocannabinol-11-oic Acid as15-Lipoxygenase Inhibitors

    SHUSO TAKEDA,1,2 RONGRONG JIANG,3 HIRONORI ARAMAKI,1 MASUMI IMOTO,4 AKIHISA TODA,4 REIKO EYANAGI,4

    TOSHIAKI AMAMOTO,5 IKUO YAMAMOTO,6 KAZUHITO WATANABE2,3

    1Department of Molecular Biology, Daiichi University of Pharmacy, 22-1 Tamagawa-cho, Minami-ku, Fukuoka 815-8511, Japan

    2Organization for Frontier Research in Preventive Pharmaceutical Sciences, Hokuriku University, Ho-3 Kanagawa-machi, Kanazawa920-1181, Japan

    3Department of Hygienic Chemistry, Faculty of Pharmaceutical Sciences, Hokuriku University, Ho-3 Kanagawa-machi, Kanazawa920-1181, Japan

    4Department of Hygienic Chemistry, Daiichi University of Pharmacy, 22-1 Tamagawa-cho, Minami-ku, Fukuoka 815-8511, Japan

    5NEUES Company Limited, Yaesu Center Building 3F, 1-6-6 Yaesu, Chuo-ku, Tokyo 103-0028, Japan

    6School of Pharmaceutical Sciences, Kyushu University of Health and Welfare, 1714-1 Yoshino-cho, Nobeoka 882-8508, Japan

    Received 18 May 2010; revised 26 August 2010; accepted 28 August 2010

    Published online 1 October 2010 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.22354

    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

    INTRODUCTION

    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

    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

    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

    9-Tetrahydrocannabinol (9-THC), a majorcannabinoid constituent of marijuana, has been

    1206 JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 3, MARCH 2011

  • 9- THC AND 9-THC-11-OIC ACID AS 15-LIPOXYGENASE INHIBITORS 1207

    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)

    R, a

    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.

    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

    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.

    MATERIALS AND METHODS

    Cannabinoids and Chemicals

    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.

    Enzyme Sources

    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

    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.

    Data Analysis

    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).

    RESULTS AND DISCUSSION

    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

    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.

    DOI 10.1002/jps JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 3, MARCH 2011

  • 1208 TAKEDA ET AL.

    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.

    (-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,

    15-LOX activity was completely and mostly inhibitedby 2 :M CBDD and NDGA, respectively, but not byCBE (Fig. 2, right panel).

    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, b