Design, synthesis, and evaluation of substituted alkylindoles that activate G protein-coupled receptors distinct from the cannabinoid CB1 and CB2 receptors.

The alkylindole (AI), WIN55212-2, modulates the activity of several proteins, including cannabinoid receptors 1 and 2 (CB1R, CB2R), and at least additional G protein-coupled receptor (GPCR) that remains uncharacterized with respect to its molecular identity and pharmacological profile. Evidence suggests that such AI-sensitive GPCRs are expressed by the human kidney cell line HEK293. We synthesized fourteen novel AI analogues and evaluated their activities at AI-sensitive GPCRs using [35S]GTPγS and [3H]WIN55212-2 binding in HEK293 cell membranes, and performed in silico pharmacophore modeling to identify characteristics that favor binding to AI-sensitive GPCRs versus CB1R/CB2R. Compounds 10 and 12 stimulated [35S]GTPγS binding (EC50s = 3.5 and 1.1 nM, respectively), and this response was pertussis toxin-sensitive, indicating that AI-sensitive GPCRs couple to Gi/o proteins. Five AI analogues reliably distinguished two binding sites that correspond to the high and low affinity state of AI-sensitive GPCRs coupled or not to G proteins. In silico pharmacophore modeling suggest 3 characteristics that favor binding to AI-sensitive GPCRs versus CB1R/CB2R: 1) an s-cis orientation of the two aromatic rings in AI analogues, 2) a narrow dihedral angle between the carbonyl group and the indole ring plane [i.e., O-C(carbonyl)-C3-C2] and 3) the presence of a carbonyl oxygen. The substituted alkylindoles reported here represent novel chemical tools to study AI-sensitive GPCRs.

The Spicy Story of Cannabimimetic Indoles.

The Sterling Research Group identified pravadoline as an aminoalkylindole (AAI) non-steroidal anti-inflammatory pain reliever. As drug design progressed, the ability of AAI analogs to block prostaglandin synthesis diminished, and antinociceptive activity was found to result from action at the CB1 cannabinoid receptor, a G-protein-coupled receptor (GPCR) abundant in the brain. Several laboratories applied computational chemistry methods to ultimately conclude that AAI and cannabinoid ligands could overlap within a common binding pocket but that WIN55212-2 primarily utilized steric interactions via aromatic stacking, whereas cannabinoid ligands required some electrostatic interactions, particularly involving the CB1 helix-3 lysine. The Huffman laboratory identified strategies to establish CB2 receptor selectivity among cannabimimetic indoles to avoid their CB1-related adverse effects, thereby stimulating preclinical studies to explore their use as anti-hyperalgesic and anti-allodynic pharmacotherapies. Some AAI analogs activate novel GPCRs referred to as "Alkyl Indole" receptors, and some AAI analogs act at the colchicine-binding site on microtubules. The AAI compounds having the greatest potency to interact with the CB1 receptor have found their way into the market as "Spice" or "K2". The sale of these alleged "herbal products" evades FDA consumer protections for proper labeling and safety as a medicine, as well as DEA scheduling as compounds having no currently accepted medical use and a high potential for abuse. The distribution to the public of potent alkyl indole synthetic cannabimimetic chemicals without regard for consumer safety contrasts with the adherence to regulatory requirements for demonstration of safety that are routinely observed by ethical pharmaceutical companies that market medicines.

Evaluation of first generation synthetic cannabinoids on binding at non-cannabinoid receptors and in a battery of in vivo assays in mice.

Anecdotal reports suggest that abused synthetic cannabinoids produce cannabis-like "highs," but some of their effects may also differ from traditional cannabinoids such as Δ(9)-tetrahydrocannabinol (THC). This study examined the binding affinities of first-generation indole-derived synthetic cannabinoids at cannabinoid and noncannabinoid receptors and their effects in a functional observational battery (FOB) and drug discrimination in mice. All seven compounds, except JWH-391, had favorable affinity (≤159 nM) for both cannabinoid receptors. In contrast, binding at noncannabinoid receptors was absent or weak. In the FOB, THC and the six active compounds disrupted behaviors in CNS activation and muscle tone/equilibrium domains. Unlike THC, however, synthetic cannabinoids impaired behavior across a wider dose and domain range, producing autonomic effects and signs of CNS excitability and sensorimotor reactivity. In addition, mice acquired JWH-018 discrimination, and THC and JWH-073 produced full substitution whereas the 5-HT2B antagonist mianserin did not substitute in mice trained to discriminate JWH-018 or THC. Urinary metabolite analysis showed that the compounds were extensively metabolized, with metabolites that could contribute to their in vivo effects. Together, these results show that, while first-generation synthetic cannabinoids shared some effects that were similar to those of THC, they also possessed effects that differed from traditional cannabinoids. The high nanomolar (or absent) affinities of these compounds at receptors for most major neurotransmitters suggests that these divergent effects may be related to the greater potencies and/or efficacies at CB1 receptors; however, action(s) at noncannabinoid receptors yet to be assessed or via different signaling pathways cannot be ruled out.

Moving around the molecule: relationship between chemical structure and in vivo activity of synthetic cannabinoids.

Originally synthesized for research purposes, indole- and pyrrole-derived synthetic cannabinoids are the most common psychoactive compounds contained in abused products marketed as "spice" or "herbal incense." While CB1 and CB2 receptor affinities are available for most of these research chemicals, in vivo pharmacological data are sparse. In mice, cannabinoids produce a characteristic profile of dose-dependent effects: antinociception, hypothermia, catalepsy and suppression of locomotion. In combination with receptor binding data, this tetrad battery has been useful in evaluation of the relationship between the structural features of synthetic cannabinoids and their in vivo cannabimimetic activity. Here, published tetrad studies are reviewed and additional in vivo data on synthetic cannabinoids are presented. Overall, the best predictor of likely cannabimimetic effects in the tetrad tests was good CB1 receptor affinity. Further, retention of good CB1 affinity and in vivo activity was observed across a wide array of structural manipulations of substituents of the prototypic aminoalkylindole molecule WIN55,212-2, including substitution of an alkyl for the morpholino group, replacement of an indole core with a pyrrole or phenylpyrrole, substitution of a phenylacetyl or tetramethylcyclopropyl group for JWH-018's naphthoyl, and halogenation of the naphthoyl group. This flexibility of cannabinoid ligand-receptor interactions has been a particular challenge for forensic scientists who have struggled to identify and regulate each new compound as it has appeared on the drug market. One of the most pressing future research needs is determination of the extent to which the pharmacology of these synthetic cannabinoids may differ from those of classical cannabinoids.

The influence of beta-arrestin2 on cannabinoid CB1 receptor coupling to G-proteins and subcellular localization and relative levels of beta-arrestin1 and 2 in mouse brain.

CONTEXT: Beta-arrestins are known to couple to some G-protein-coupled receptors (GPCRs) to regulate receptor internalization, G-protein coupling and signal transduction, but have not been investigated for most receptors, and for very few receptors in vivo. Previous studies have shown that beta-arrestin2 deletion enhances the efficacy of specific cannabinoid agonists. OBJECTIVE: The present study hypothesized that brain cannabinoid CB1 receptors are regulated by beta-arrestin2. METHODS: Beta-arrestin2+/+ and -/- mice were used. Western blotting was used to determine the relative levels of each beta-arrestin subtype in mouse brain. Receptor binding was measured to determine whether deletion of beta-arrestin2 influences agonist binding to brain CB1 receptors, or the subcellular localization of CB1 in brain membranes subjected to differential centrifugation. A variety of cannabinoid agonists from different chemical classes were investigated for their ability to activate G-proteins in the presence and absence of beta-arrestin2 in cerebellum, hippocampus and cortex. RESULTS: No differences were found in the density of beta-arrestin1 or cannabinoid CB1 receptors in several brains of beta-arrestin2+/+ versus -/- mice. Differences between genotypes were found in the proportion of high- and low-affinity agonist binding sites in brain areas that naturally express higher levels of beta-arrestin2. Cortex from beta-arrestin2-/- mice contained less CB1 in the P1 fraction and more CB1 in the P2 fraction compared to beta-arrestin2+/+. Of the agonists assayed for activity, only Δ(9)-tetrahydrocannabinol (THC) exhibited a difference between genotypes, in that it was less efficacious in beta-arrestin2-/- than +/+ mouse membranes. CONCLUSION: Beta-arrestin2 regulates cannabinoid CB1 receptors in brain.

Use of SPME-HS-GC-MS for the analysis of herbal products containing synthetic cannabinoids.

The increasing prevalence and use of herbal mixtures containing synthetic cannabinoids presents a growing public health concern and legal challenge for society. In contrast to the plant-derived cannabinoids in medical marijuana and other cannabinoid-based therapeutics, the commonly encountered synthetic cannabinoids in these mendaciously labeled products constitute a structurally diverse set of compounds of relatively unknown pharmacology and toxicology. Indeed, the use of these substances has been associated with an alarming number of hospitalizations and emergency room visits. Moreover, there are already several hundred known cannabinoid agonist compounds that could potentially be used for illicit purposes, posing an additional challenge for public health professionals and law enforcement efforts, which often require the detection and identification of the active ingredients for effective treatment or prosecution. A solid-phase microextraction headspace gas chromatography-mass spectrometry method is shown here to allow for rapid and reliable detection and structural identification of many of the synthetic cannabinoid compounds that are currently or could potentially be used in herbal smoking mixtures. This approach provides accelerated analysis and results that distinguish between structural analogs within several classes of cannabinoid compounds, including positional isomers. The analytical results confirm the continued manufacture and distribution of herbal materials with synthetic cannabinoids and provide insight into the manipulation of these products to avoid legal constraints and prosecution.

Synthesis and pharmacology of 1-alkyl-3-(1-naphthoyl)indoles: steric and electronic effects of 4- and 8-halogenated naphthoyl substituents.

To develop SAR at both the cannabinoid CB(1) and CB(2) receptors for 3-(1-naphthoyl)indoles bearing moderately electron withdrawing substituents at C-4 of the naphthoyl moiety, 1-propyl and 1-pentyl-3-(4-fluoro, chloro, bromo and iodo-1-naphthoyl) derivatives were prepared. To study the steric and electronic effects of substituents at the 8-position of the naphthoyl group, the 3-(4-chloro, bromo and iodo-1-naphthoyl)indoles were also synthesized. The affinities of both groups of compounds for the CB(1) and CB(2) receptors were determined and several of them were evaluated in vivo in the mouse. The effects of these substituents on receptor affinities and in vivo activity are discussed and structure-activity relationships are presented. Although many of these compounds are selective for the CB(2) receptor, only three JWH-423, 1-propyl-3-(4-iodo-1-naphthoyl)indole, JWH-422, 2-methyl-1-propyl-3-(4-iodo-1-naphthoyl)indole, the 2-methyl analog of JWH-423 and JWH-417, 1-pentyl-3-(8-iodo-1-naphthoyl)indole, possess the desirable combination of low CB(1) affinity and good CB(2) affinity.

1-Pentyl-3-phenylacetylindoles and JWH-018 share in vivo cannabinoid profiles in mice.

BACKGROUND: Smoking of synthetic cannabinoid-enhanced "herbal incense" is an emerging substance abuse problem. The indole-derived cannabinoids identified in these products were originally developed as research tools and are structurally distinct from cannabinoids in the cannabis plant. Although abused by humans, most published research on this class of compounds has been performed in vitro. The purpose of this study was to evaluate a novel series of 1-pentyl-3-phenylacetylindoles in mice. METHODS: The potencies of these analogs to produce the cannabinoid agonist effects of antinociception, hypothermia and suppression of locomotion were evaluated in ICR mice. The major structural manipulations in the present series included the type of substituent (i.e., unsubstituted, methyl, methoxy, chloro, bromo, and fluoro) and the position of the substituent on the phenyl ring (i.e., 2-, 3- or 4-position). RESULTS: Potencies of this series of phenylacetylindoles for each cannabinoid effect were highly correlated with CB(1) receptor affinities reported previously. Active compounds produced a profile of effects that resembled that exhibited by Δ(9)-tetrahydrocannabinol (THC). The most critical factor affecting in vivo potency was the position of the substituent. Whereas compounds with 2- and 3-phenylacetyl substituents were efficacious with good potencies, 4-substituents resulted in compounds that had poor potency or were inactive. CONCLUSIONS: These results suggest that phenylacetylindoles with good CB(1) binding affinity share pharmacological properties with THC in mice; however, they also emphasize the complexity of molecular interactions of synthetic cannabinoids with CB(1) receptors and suggest that scheduling efforts based solely upon structural features should proceed with caution.

Hijacking of Basic Research: The Case of Synthetic Cannabinoids.

Gathering and communicating knowledge are important aspects of the scientific endeavor. Yet presentation of data in public forums such as scientific meetings and publications makes it available not only to scientists, but also to others who may have different ideas about how to use research findings. A recent example of this type of hijacking is the introduction of synthetic cannabinoids that are sprayed on herbal products and subsequently smoked for their marijuana-like intoxicating properties. Originally developed for the legitimate research purpose of furthering understanding of the cannabinoid system, these synthetic cannabinoids are being abused worldwide, creating issues for regulatory and law enforcement agencies that are struggling to keep up with the growing number of compounds of various structural motifs. Basic and clinical scientists need to provide advice now to facilitate decision-making about the health threats posed by this emerging problem.

Structural and pharmacological analysis of O-2050, a putative neutral cannabinoid CB(1) receptor antagonist.

Rimonabant, the prototypic antagonist of cannabinoid CB(1) receptors, has been reported to have inverse agonist properties at higher concentrations, which may complicate its use as a tool for mechanistic evaluation of cannabinoid pharmacology. Consequently, recent synthesis efforts have concentrated on discovery of a neutral antagonist using a variety of structural templates. The purpose of this study was to evaluate the pharmacological properties of the putative neutral cannabinoid CB(1) receptor antagonist O-2050, a sulfonamide side chain analog of Δ(8)-tetrahydrocannabinol. O-2050 and related sulfonamide cannabinoids exhibited good affinity for both cannabinoid CB(1) and CB(2) receptors. While the other sulfonamide analogs produced cannabinoid agonist effects in vivo (e.g., activity suppression, antinociception, and hypothermia), O-2050 stimulated activity and was inactive in the other two tests. O-2050 also decreased food intake in mice, an effect that was reminiscent of that produced by rimonabant. Unlike rimonabant, however, O-2050 did not block the effects of cannabinoid agonists in vivo, even when administered i.c.v. In contrast, O-2050 antagonized the in vitro effects of cannabinoid agonists in [(35)S]GTPγS and mouse vas deferens assays without having activity on its own in either assay. Further evaluation revealed that O-2050 fully and dose-dependently substituted for Δ(9)-tetrahydrocannabinol in a mouse drug discrimination procedure (a cannabinoid agonist effect) and that it inhibited forskolin-stimulated cyclic AMP signaling with a maximum efficacy of approximately half that of the full agonist CP55,940 [(-)-cis-3-[2-hydroxy-4(1,1-dimethyl-heptyl)phenyl]-trans-4-(3-hydroxy-propyl)cyclohexanol]. Together, these results suggest that O-2050 is not a viable candidate for classification as a neutral cannabinoid CB(1) receptor antagonist.

1-Bromo-3-(1',1'-dimethylalkyl)-1-deoxy-Δ(8)-tetrahydrocannabinols: New selective ligands for the cannabinoid CB(2) receptor.

Δ(8)-Tetrahydrocannabinol (26), 3-(1',1'-dimethylbutyl)- (12), 3-(1',1'-dimethylpentyl)- (13), 3-(1',1'-dimethylhexyl)- (14) and 3-(1',1'-dimethylheptyl)-Δ(8)-tetrahydrocannabinol (15) have been converted into the corresponding 1-bromo-1-deoxy-Δ(8)-tetrahydrocannabinols (25, 8-11). This was accomplished using a protocol developed in our laboratory in which the trifluoromethanesulfonate of a phenol undergoes palladium mediated coupling with pinacolborane. Reaction of this dioxaborolane with aqueous-methanolic copper(II) bromide provides the aryl bromide. The affinities of these bromo cannabinoids for the cannabinoid CB(1) and CB(2) receptors were determined. All of these compounds showed selectivity for the CB(2) receptor and one of them, 1-bromo-1-deoxy-3-(1',1'-dimethylhexyl)-Δ(8)-tetrahydrocannabinol (10), exhibits 52-fold selectivity for this receptor with good (28nM) affinity.

Synthesis and pharmacology of 1-methoxy analogs of CP-47,497.

Three 1-methoxy analogs of CP-47,497 (7, 8, and 19) have been synthesized and their affinities for the cannabinoid CB(1) and CB(2) receptors have been determined. Although these compounds exhibit selectivity for the CB(2) receptor none have significant affinity for either receptor. Modeling and receptor docking studies were carried out, which provide a rationalization for the weak affinities of these compounds for either receptor.

Discriminative stimulus properties of delta9-tetrahydrocannabinol (THC) in C57Bl/6J mice.

Primarily, rats have served as subjects in Delta(9)-tetrahydrocannabinol's (THC) discrimination studies although other species such as monkeys and pigeons have been used. While the introduction of the knockout and transgenic mice has vastly stimulated the study of the discriminative stimulus effects of drugs there is only a single published report of mice trained to discriminate THC. Thus, this study extended those results by providing a systematic replication that THC serves as an effective discriminative stimulus in mice and by further investigating the mechanisms of action involved in the THC discrimination model in the mouse. Male C57BL/6J mice were trained to discriminate 10 mg/kg THC from vehicle in 2-lever drug discrimination. THC fully and dose dependently substituted for itself. Cannabinoid indoles, except one with low cannabinoid CB(1) receptor affinity, substituted for THC. Anandamide failed to substitute for THC when administered alone but completely substituted when administered with the non-specific fatty acid amide hydrolase inhibitor, phenylmethylsulphonyl fluoride. As expected, nicotine failed to substitute for THC. Lastly, the cannabinoid CB(1) receptor antagonist rimonabant blocked THC's discriminative stimulus effects. Taken together these studies demonstrate THC's ability to produce discriminative stimulus effects as well as demonstrate its pharmacological specificity and mechanism of action in a two-lever drug discrimination mouse model.

Sensitivity to delta9-tetrahydrocannabinol is selectively enhanced in beta-arrestin2 -/- mice.

Little is known about the roles of beta-arrestins in the regulation of brain CB1 cannabinoid receptors. This study investigated the role of beta-arrestin2 in cannabinoid behavioral effects using beta-arrestin2 -/- mice and their wild-type counterparts. A variety of cannabinoid ligands from different chemical classes that exhibit a variety of efficacies for activation of CB1 receptors were investigated, including Delta-tetrahydrocannabinol, CP55940, methanandamide, JWH-073, and O-1812. Delta-tetrahydrocannabinol produced both greater antinociception and greater decreases in body temperature in beta-arrestin2 -/- compared with beta-arrestin2 +/+ mice. No significant differences were, however, present in either assay for the other CB1 agonists. Antagonist radioligand binding indicated no difference in the density of cannabinoid CB1 receptors in the cerebellum, cortex, or hippocampus of beta-arrestin2 +/+ and -/- mice. These data demonstrate that beta-arrestin2 may regulate cannabinoid CB1 receptor sensitivity in an agonist-specific manner.

Recent advances in the development of selective ligands for the cannabinoid CB(2) receptor.

Two subtypes of the mammalian cannabinoid receptor have been identified and successfully cloned since 1990. The CB(1) receptor is primarily located in the central nervous system and the CB(2) receptor is almost exclusively expressed in cells of the immune system. The CB(1) and CB(2) receptors are both G-protein coupled receptors and are involved in the inhibition of adenylate cyclase. The CB(2) receptor is of particular importance due to its involvement in signal transduction in the immune system, making it a potential target for therapeutic immune intervention. A number of these selective ligands are derivatives of traditional dibenzopyran based cannabinoids. These include the very recently synthesized (2'R)-1-methoxy-3-(2'-methylbutyl)- Delta (8)-THC (JWH-359) which has a 224 fold selectivity for the CB(2) receptor, readily comparable to the well known 1-deoxy-3-(1',1'-dimethylbutyl)- Delta (8)-THC (JWH-133) which has 200 fold selectivity for the CB(2) receptor. Several 9-hydroxyhexahydrocannabinols have also been synthesized and are found to be selective high affinity ligands for the CB(2) receptor. These are 1-deoxy-9beta-hydroxy-dimethylhexylhexahydrocannabinol (JWH-361, K(i) = 2.7 nM) and 1-deoxy-9beta-hydroxy-dimethylpentylhexahydrocannabinol (JWH-300, K(i) = 5.3 nM). CB(2) selective cannabi-mimetic indoles include 1-(2,3-dichlorobenzoyl)-2-methyl-3-(2-[1-morpholine]ethyl)-5-methoxyindole (L768242), (R)-3-(2-Iodo-5-nitrobenzoyl)-1-(1-methyl-2-piperidinylmethyl)-1H-indole (AM1241) and 1-propyl-2-methyl-3-(1-naphthoyl) indole (JWH-015), which exhibit significant selectivity for the CB(2) receptor coupled with weak affinity for the CB(1) receptor. Bristol-Meyer Squibb has produced a phenylalanine derived cannabimimetic indole which possesses high CB(2) affinity (K(i) = 8 nM) and very low affinity for the CB(1) receptor (K(i) = 4000 nM). This review will discuss the current advances and recent results in the structure-activity relationships (SAR) of selective ligands for the cannabinoid CB(2) receptor.

Acylation of N-p-Toluenesulfonylpyrrole Under Friedel-Crafts Conditions. Evidence for Organoaluminum Intermediates.

The Friedel-Crafts acylation of N-p-toluenesulfonylpyrrole under Friedel-Crafts conditions has been reinvestigated. Evidence is presented in support of the hypothesis that when AlCl(3) is used as the Lewis acid, acylation proceeds via reaction of an organoaluminum intermediate with the acyl halide. This leads to the production of the 3-acyl derivative as the major product. With weaker Lewis acids (EtAlCl(2), Et(2)AlCl) or less than one equivalent of AlCl(3) the relative amount of 2-acyl product is increased. A mechanistic rationalization is presented to explain these data.

Synthesis and pharmacology of 1-deoxy analogs of CP-47,497 and CP-55,940.

A series of 1-deoxy analogs of CP-47,497 (8 and 13, n=0-7) and 1-deoxy analogs of CP-55,940 (9, n=0-7) have been synthesized and their affinities for the cannabinoid CB(1) and CB(2) receptors have been determined. Although the majority of these compounds exhibit selectivity for the CB(2) receptor, none have greater than modest affinity for either receptor. The interactions of these 1-deoxy nontraditional cannabinoids with the CB(2) receptor are discussed.

CB2-receptor stimulation attenuates TNF-alpha-induced human endothelial cell activation, transendothelial migration of monocytes, and monocyte-endothelial adhesion.

Targeting cannabinoid-2 (CB(2)) receptors with selective agonists may represent a novel therapeutic avenue in various inflammatory diseases, but the mechanisms by which CB(2) activation exerts its anti-inflammatory effects and the cellular targets are elusive. Here, we investigated the effects of CB(2)-receptor activation on TNF-alpha-induced signal transduction in human coronary artery endothelial cells in vitro and on endotoxin-induced vascular inflammatory response in vivo. TNF-alpha induced NF-kappaB and RhoA activation and upregulation of adhesion molecules ICAM-1 and VCAM-1, increased expression of monocyte chemoattractant protein, enhanced transendothelial migration of monocytes, and augmented monocyte-endothelial adhesion. Remarkably, all of the above-mentioned effects of TNF-alpha were attenuated by CB(2) agonists. CB(2) agonists also decreased the TNF-alpha- and/or endotoxin-induced ICAM-1 and VCAM-1 expression in isolated aortas and the adhesion of monocytes to aortic vascular endothelium. CB(1) and CB(2) receptors were detectable in human coronary artery endothelial cells by Western blotting, RT-PCR, real-time PCR, and immunofluorescence staining. Because the above-mentioned TNF-alpha-induced phenotypic changes are critical in the initiation and progression of atherosclerosis and restenosis, our findings suggest that targeting CB(2) receptors on endothelial cells may offer a novel approach in the treatment of these pathologies.

Cannabinoid-2 receptor mediates protection against hepatic ischemia/reperfusion injury.

Hepatic ischemia-reperfusion (I/R) injury continues to be a fatal complication that can follow liver surgery or transplantation. We have investigated the involvement of the endocannabinoid system in hepatic I/R injury using an in vivo mouse model. Here we report that I/R triggers several-fold increases in the hepatic levels of the endocannabinoids anandamide and 2-arachidonoylglycerol, which originate from hepatocytes, Kupffer, and endothelial cells. The I/R-induced increased tissue endocannabinoid levels positively correlate with the degree of hepatic damage and serum TNF-alpha, MIP-1alpha, and MIP-2 levels. Furthermore, a brief exposure of hepatocytes to various oxidants (H2O2 and peroxynitrite) or inflammatory stimuli (endotoxin and TNF-alpha) also increases endocannabinoid levels. Activation of CB2 cannabinoid receptors by JWH133 protects against I/R damage by decreasing inflammatory cell infiltration, tissue and serum TNF-alpha, MIP-1alpha and MIP-2 levels, tissue lipid peroxidation, and expression of adhesion molecule ICAM-1 in vivo. JWH133 also attenuates the TNF-alpha-induced ICAM-1 and VCAM-1 expression in human liver sinusoidal endothelial cells (HLSECs) and the adhesion of human neutrophils to HLSECs in vitro. Consistent with the protective role of CB2 receptor activation, CB2-/- mice develop increased I/R-induced tissue damage and proinflammatory phenotype. These findings suggest that oxidative/nitrosative stress and inflammatory stimuli may trigger endocannabinoid production, and indicate that targeting CB2 cannabinoid receptors may represent a novel protective strategy against I/R injury. We also demonstrate that CB2-/- mice have a normal hemodynamic profile.