New natural noncannabinoid ligands for cannabinoid type-2 (CB2) receptors

Since the discovery that Delta 9-tetrahydrocannabinol and related cannabinoids from Cannabis sativa L. act on specific physiological receptors in the human body and the subsequent elucidation of the mammalian endogenous cannabinoid system, no other natural product class has been reported to mimic the effects of cannabinoids. We recently found that N-alkyl amides from purple coneflower (Echinacea spp.) constitute a new class of cannabinomimetics, which specifically engage and activate the cannabinoid type-2 (CB2) receptors. Cannabinoid type-1 (CB1) and CB2 receptors belong to the family of G protein-coupled receptors and are the primary targets of the endogenous cannabinoids N-arachidonoyl ethanolamine and 2-arachidonoyl glyerol. CB2 receptors are believed to play an important role in distinct pathophysiological processes, including metabolic dysregulation, inflammation, pain, and bone loss. CB2 receptors have, therefore, become of interest as new targets in drug discovery. This review focuses on N-alkyl amide secondary metabolites from plants and underscores that this group of compounds may provide novel lead structures for the development of CB2-directed drugs.     

Modulation of gastric acid secretion by cannabinoids in rats

The current study aimed to evaluate the role of cannabinoid receptors in the regulation of gastric acid secretion and oxidative stress in gastric mucosa. To fulfill this aim, gastric acid secretion stimulated with histamine (5 mg/kg, subcutaneous [SC]), 2‐deoxy‐ d ‐glucose (D‐G) (200 mg/kg, intravenous) or ‐carbachol (4 μg/kg, SC) in the 4‐hour pylorus‐ligated rats. The CB1R agonist ( N‐arachidonoyl dopamine, 1 mg/kg, SC) inhibited gastric acid secretion stimulated by D‐G and carbachol but not in histamine, reduced pepsin content, and increased mucin secretion. Furthermore, it decreased malondialdehyde (MDA) and nitric oxide (NO) contents with an increase in glutathione (GSH) and paraoxonase 1 (PON‐1). Meanwhile, CB2R antagonist (AM630, 1 mg/kg, SC) inhibited gastric acid secretion stimulated by D‐G and reduced MDA and NO contents with an increase in GSH and PON‐1. Meanwhile, CB1R antagonist rimonabant or CB2R agonist GW 405833 had no effect on stimulated gastric acid secretion. Therefore, both CB1R agonist and CB2R antagonist may exert antisecretory and antioxidant potential in the stomach.     

Novel 5-substituted 1H-tetrazoles as cyclooxygenase-2 (COX-2) inhibitors

A series of novel 5-substituted 1H-tetrazoles as cyclooxygenase-2 (COX-2) inhibitors was prepared via treatment of various diaryl amides with tetrachlorosilane/sodium azide. All compounds were tested in cyclooxygenase (COX) assays in vitro to determine COX-1 and COX-2 inhibitory potency and selectivity. Tetrazoles contained a methylsulfonyl or sulfonamide group as COX-2 pharmacophore displayed only low inhibitory potency towards COX-2. Most potent compounds showed IC(50) values of 6 and 7 μM for COX-2. All compounds showed IC(50) values greater 100 μM for COX-1 inhibition.     

Evaluation of loxoprofen and its alcohol metabolites for potency and selectivity of inhibition of cyclooxygenase-2

Loxoprofen, its trans-alcohol and cis-alcohol metabolites were evaluated for selectivity of inhibition of COX-2 over COX-1. The (2S,1'R,2'S)-trans-alcohol derivative was found to be the most active metabolite and to be a potent and nonselective inhibitor of COX-2 and COX-1 in both enzyme and human whole blood assays.     

Synthesis, characterization, and activity of metabolites derived from the cyclooxygenase-2 inhibitor rofecoxib (MK-0966, Vioxx)

Metabolites of the COX-2 inhibitor rofecoxib (MK-0966, Vioxx) were prepared by synthetic or biosynthetic methods. Metabolites include products of oxidation, glucuronidation, reduction and hydrolytic ring opening. Based on an in vitro whole blood assay, none of the known human metabolites of rofecoxib inhibits COX-1 nor contributes significantly to the inhibition of COX-2.     

Cyclooxygenase and lipoxygenase metabolite synthesis by polymorphonuclear neutrophils: in vitro effect of dipyrone

Functional activity of polymorphonuclear neutrophils (PMN) is associated with the metabolism of Arachidonic Acid (AA) released from membrane phospholipids. In this study the in vitro effect of dipyrone, a non steroidal anti-inflammatory drug, on the production of AA metabolites through cyclooxygenase (CO) and lipoxygenase (LO) pathways by stimulated PMN has been investigated. PMN isolated by counterflow centrifuge elutriator were greater than 98% pure and viable. Metabolite production was evaluated by RIA of Thromboxane A2 (TxA2), Prostaglandin E2 (PGE2), Leukotriene B2 (LTB4) and Leukotriene C4 (LTC4) after PMN stimulation with calcium ionophore A 23187 (20 microM). The levels of beta-thromboglobulin (RIA) lower than 5 ng/ml allowed us to rule out activation of residual contaminant platelets. In these experimental conditions, in the absence of dipyrone the products (ng/10(6) cells) of AA metabolism were LTB4 (3.51 +/- 0.22), LTC4 (0.81 +/- 0.08), TxB2 (0.144 +/- 0.025) and PGE2 (0.150 +/- 0.017). Incubation with dipyrone induced changes of PGE2 and TXB2 production in a dose dependent fashion (r = 0.83 and r = 0.87, p less than 0.001), obtaining already at the lowest drug concentration (5 micrograms/ml) a significant inhibition (33 and 40% for TxB2 and PGE2 p less than 0.005). No significant changes of LTB4 and LTC4 production have been observed. The results of this study indicate that dipyrone relevantly affects CO metabolite synthesis by stimulated PMN at concentrations comparable to those reached in therapeutic use. The inhibition of PGE2 synthesis which is present in inflamed tissues and actively participates in inflammatory reactions, could contribute to the therapeutic anti-inflammatory action of dipyrone.     

Indolyl esters and amides related to indomethacin are selective COX-2 inhibitors

Previous studies from our laboratory have revealed that esterification/amidation of the carboxylic acid moiety in the nonsteroidal anti-inflammatory drug, indomethacin, generates potent and selective COX-2 inhibitors. In the present study, a series of reverse ester/amide derivatives were synthesized and evaluated as selective COX-2 inhibitors. Most of the reverse esters/amides displayed time-dependent COX-2 inhibition with IC₅₀ values in the low nanomolar range. Replacement of the 4-chlorobenzoyl group on the indole nitrogen with a 4-bromobenzyl moiety resulted in compounds that retained selective COX-2 inhibitory potency. In addition to inhibiting COX-2 activity in vitro, the reverse esters/amides also inhibited COX-2 activity in the mouse macrophage-like cell line, RAW264.7. Overall, this strategy broadens the scope of our previous methodology of neutralizing the carboxylic acid group in NSAIDs as a means of generating COX-2-selective inhibitors and is potentially applicable to other NSAIDs.     

COX-2 and prostanoid expression in micturition pathways after cyclophosphamide-induced cystitis in the rat

The purpose of this study was to determine the role of cyclooxygenase-2 (COX-2) and its metabolites in lower urinary tract function after induction of acute (4 h), intermediate (48 h), or chronic (10 day) cyclophosphamide (CYP)-induced cystitis. Bladders were harvested from euthanized female rats for analyses. Conscious cystometry was used to assess the effects of a COX-2-specific inhibitor, 5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulfonyl)phenyl2(5H)-furanone (DFU, 5 mg/kg sc), a disubstituted furanone, in CYP-induced cystitis. COX-2 mRNA was increased in inflamed bladders after acute (12-fold) and chronic (9-fold) treatment. COX-2 protein expression in inflamed bladders paralleled COX-2 mRNA expression. Prostaglandin D2-methoxime expression in the bladder was significantly (P < or = 0.01) increased in acute (3-fold) and chronic (5.5-fold) cystitis. Prostaglandin E2 was significantly (P < or = 0.01) increased (2-fold) in the bladder with intermediate (1.7-fold) and chronic (2.6-fold) cystitis. COX-2-immunoreactive cell profiles were distributed throughout the inflamed bladder and coexpressed histamine immunoreactivity. Conscious cystometry in rats treated with CYP + DFU showed increased micturition intervals 4 and 48 h after CYP treatment and decreased intravesical pressures during filling and micturition compared with rats treated with CYP + vehicle. These studies suggest an involvement of urinary bladder COX-2 and its metabolites in altered micturition reflexes with CYP-induced cystitis.     

Endocannabinoid 2-arachidonoylglycerol protects neurons by limiting COX-2 elevation

Endocannabinoids are involved in synaptic signaling and neuronal protection; however, our understanding of the mechanisms by which endocannabinoids protect neurons from harmful insults remains elusive. 2-Arachidonoylglycerol (2-AG), the most abundant endogenous cannabinoid and a full agonist for cannabinoid receptors (CB1 and CB2), is a substrate for cyclooxygenase-2 (COX-2) and can be metabolized by COX-2. Here we show, however, that 2-AG is also capable of suppressing elevation of hippocampal COX-2 expression in response to proinflammatory and excitotoxic stimuli. 2-AG prevents neurodegeneration from toxic assaults that elevate COX-2 expression and inhibits the COX-2 elevation-enhanced excitatory glutamatergic synaptic transmission. The action of 2-AG on suppression of COX-2 appeared to be mediated via the pertussis toxin-sensitive G protein-coupled CB1 receptor and MAPK/NF-kappaB signaling pathways. Our results reveal that 2-AG functions as an endogenous COX-2 inhibitor protecting neurons from harmful insults by preventing excessive expression of COX-2, which provides a mechanistic basis for opening up new therapeutic approaches for protecting neurons from inflammation- and excitotoxicity-induced neurodegeneration.     

Molecular biology of cannabinoid receptors

During the last decade, research on the molecular biology and genetics of cannabinoid receptors has led to a remarkable progress in understanding of the endogenous cannabinoid system, which functions in a plethora of physiological processes in the animal. At present, two types of cannabinoid receptors have been cloned from many vertebrates, and three endogenous ligands (the endocannabinoids arachidonoyl ethanolamide, 2-arachidonoyl glycerol and 2-arachidonoyl-glycerol ether) have been characterized. Cannabinoid receptor type 1 (CB(1)) is expressed predominantly in the central and peripheral nervous system, while cannabinoid receptor type 2 (CB(2)) is present almost exclusively in immune cells. Cannabinoid receptors have not yet been cloned from invertebrates, but binding proteins for endocannabinoids, endocannabinoids and metabolic enzyme activity have been described in a variety of invertebrates except for molting invertebrates such as Caenorhabditis elegans and Drosophila. In the central nervous system of mammals, there is strong evidence emerging that the CB(1) and its ligands comprise a neuromodulatory system functionally interacting with other neurotransmitter systems. Furthermore, the presynaptic localization of CB(1) together with the results obtained from electrophysiological experiments strengthen the notion that in cerebellum and hippocampus and possibly in other regions of the central nervous system, endocannabinoids may act as retrograde messengers to suppress neurotransmitter release at the presynaptic site. Many recent studies using genetically modified mouse lines which lack CB(1) and/or CB(2) finally could show the importance of cannabinoid receptors in animal physiology and will contribute to unravel the full complexity of the cannabinoid system.     

Tetranor PGDM, an abundant urinary metabolite reflects biosynthesis of prostaglandin D2 in mice and humans

Prostaglandin D(2) (PGD(2)) is a cyclooxygenase (COX) product of arachidonic acid that activates D prostanoid receptors to modulate vascular, platelet, and leukocyte function in vitro. However, little is known about its enzymatic origin or its formation in vivo in cardiovascular or inflammatory disease. 11,15-dioxo-9alpha-hydroxy-2,3,4,5-tetranorprostan-1,20-dioic acid (tetranor PGDM) was identified by mass spectrometry as a metabolite of infused PGD(2) that is detectable in mouse and human urine. Using liquid chromatography-tandem mass spectrometry, tetranor PGDM was much more abundant than the PGD(2) metabolites, 11beta-PGF(2alpha) and 2,3-dinor-11beta-PGF(2alpha), in human urine and was the only endogenous metabolite detectable in mouse urine. Infusion of PGD(2) dose dependently increased urinary tetranor PGDM > 2,3-dinor-11beta-PGF(2alpha) > 11beta-PGF(2alpha) in mice. Deletion of either lipocalin-type or hemopoietic PGD synthase enzymes decreased urinary tetranor PGDM. Deletion or knockdown of COX-1, but not deletion of COX-2, decreased urinary tetranor PGDM in mice. Correspondingly, both PGDM and 2,3-dinor-11beta-PGF(2alpha) were suppressed by inhibition of COX-1 and COX-2, but not by selective inhibition of COX-2 in humans. PGD(2) has been implicated in both the development and resolution of inflammation. Administration of bacterial lipopolysaccharide coordinately elevated tetranor PGDM and 2,3-dinor-11beta-PGF(2alpha) in volunteers, coincident with a pyrexial and systemic inflammatory response, but both metabolites fell during the resolution phase. Niacin increased tetranor PGDM and 2,3-dinor-11beta-PGF(2alpha) in humans coincident with facial flushing. Tetranor PGDM is an abundant metabolite in urine that reflects modulated biosynthesis of PGD(2) in humans and mice.     

The endocannabinoid arachidonyl ethanolamide (anandamide) increases pulmonary arterial pressure via cyclooxygenase-2 products in isolated rabbit lungs

Several cannabinoids elicit systemic vasodilation, mainly via CB1 cannabinoid and vanilloid receptors. However, effects in the pulmonary circulation are unknown. Using the isolated, ventilated, buffer-perfused rabbit lung, we have shown that the endocannabinoids arachidonyl ethanolamide (anandamide) and 2-arachidonyl glycerol (2-AG) dose-dependently increase pulmonary arterial pressure (+19.9 +/- 3.4 mmHg, 5 microM, and +39.5 +/- 10.8 mmHg, 0.4 microM, respectively). 2-AG induced lung edema. The CB1 receptor antagonist AM-251 (0.1 and 5 microM) and the VR1 vanilloid receptor antagonist capsazepine (10 microM) failed to reduce anandamide's effects. The metabolically stable anandamide and 2-AG analogs R-methanandamide and noladin ether, Delta9-tetrahydrocannabinol, and the synthetic cannabinoid HU-210, which is no arachidonic acid product, were without effect. The unspecific cyclooxygenase (COX) inhibitor aspirin (100 microM, P < 0.001) and the specific COX-2 inhibitor nimesulide (10 microM, P < 0.01) completely prevented pulmonary hypertension after 5 microM anandamide. COX-2 RNA was detected in rabbit lungs. The synthetic thromboxane receptor antagonist SQ 29,548 was without effect, but the specific EP1 prostanoid receptor antagonist SC-19220 (100 microM) inhibited the pressure increase after anandamide (P < 0.05). PCR analysis detected fatty acid amidohydrolase (FAAH), an enzyme that degrades endocannabinoids, in rabbit lung tissue. Furthermore, the specific FAAH inhibitor methyl arachidonyl fluorophosphonate (0.1 microM) blocked pressure effects of anandamide (P < 0.01). Finally, anandamide (99 +/- 55 pmol/g) and 2-AG (19.6 +/- 8.4 nmol/g) were found in native lungs. We conclude that anandamide increases pulmonary arterial pressure via COX-2 metabolites following enzymatic degradation by FAAH into arachidonic acid products.     

Diglyceride/monoglyceride lipases pathway is not essential for arachidonate release in thrombin-activated human platelets

Human platelets prelabeled with arachidonate exhibited a rapid and transient rise in arachidonoyl monoglyceride in addition to arachidonoyl diglyceride following thrombin stimulation. Substantial release of arachidonate and its metabolites also occurred at the early phase. Preincubation of labeled platelets with RHC 80267, a potent inhibitor of diglyceride lipase, prior to thrombin stimulation abolished the transient rise in monoglyceride but not the increase in diglyceride and the release of arachidonate and its metabolites. These results suggest that diglyceride does metabolize to monoglyceride and release arachidonate in intact platelets. However, the diglyceride/monoglyceride lipases pathway does not appear to be essential in releasing arachidonate during thrombin stimulation.     

Effects of shear stress on eicosanoid gene expression and metabolite production in vascular endothelium as studied in a novel biomechanical perfusion model

This study investigated the effects of shear stress on gene expression of prostacyclin synthesis-related enzymes cyclooxygenases (COX-1 and COX-2), prostacyclin synthase (PGS), and thromboxane synthase (TXS) and their metabolites prostaglandin (PGI(2)) and thromboxane A(2) (TXA(2)) in endothelium of intact conduit vessels. Paired human umbilical veins were perfused at high/low shear stress (25/<4 dyn/cm(2)) at identical intraluminal pressure (20 mmHg) for 1.5, 3, or 6 hours in a computerized vascular model. High shear perfusion induced a significant, monophasic upregulation of PGS and TXS gene expressions after 6 hours. COX-1 and COX-2 mRNA showed a biphasic response with peaks at 1.5 and 6 hours, with a nadir level at 3 hours. Shear-induced gene expression was associated with a significantly greater accumulation of 6-keto prostaglandin F(1alpha) and TXA(2) in the perfusion medium. Thus, shear stress independently of perfusion pressure alters the expression of prostacyclin synthesis-related enzymes and the biosynthesis of their vasoactive metabolites.     

Total biodegradation of 4-chlorobiphenyl (4 CB) by a two-membered bacterial culture

Summary Several bacterial strains that can oxidize mono- and dichlorinated biphenyls with one unsubstituted ring have already been described. The major route for this biodegradation leads ultimately to the corresponding chlorobenzoic acid, but several other minor chlorinated metabolites that might possibly be of concern for the environment have also been described previously. Since none of the bacterial strains that are able to oxidize these chlorinated biphenyls in pure culture are known to degrade chlorobenzoic acid, the oxidation of these substrates by axenic cultures always generates chlorobenzoates plus several other metabolites. In the present study, we have estimated the biodegradation of 4-chlorobiphenyl (4CB) by a two-membered bacterial culture containing one strain able to grow on 4CB and to transform it into 4-chlorobenzoate (4CBA) and one strain able to degrade 4CBA. The results were encouraging, since it was shown that the degradation of 4CB was more rapid and complete with the double bacterial culture.     

The pore-lining regions in cytochrome c oxidases: A computational analysis of caveolin,cholesterol and transmembrane helix contributions to proton movement

Cytochrome c oxidase (CcO) is the terminal enzyme in the electron transfer chain. CcO catalyzes a four electron reduction of O₂ to water at a catalytic site formed by high-spin heme (a₃) and copper atoms (Cu_B). While it is recognized that proton movement is coupled to oxygen reduction, the proton channel(s) have not been well defined. Using computational methods developed to study protein topology, membrane channels and 3D packing arrangements within transmembrane (TM) helix arrays, we find that subunit-1 (COX-1), subunit-2 (COX-2) and subunit-3 (COX-3) contribute 139, 46 and 25 residues, respectively, to channel formation between the mitochondrial matrix and intermembrane space. Nine of 12 TM helices in COX-1, both helices in COX-2 and 5 of the 6 TM helices in COX-3 are pore-lining regions (possible channel formers). Heme a₃ and the Cu_B sites (as well as the Cu_A center of COX-2) are located within the channel that includes TM-6, TM-7, TM-10 and TM-11 of COX-1 and are associated with multiple cholesterol and caveolin-binding (CB) motifs. Sequence analysis identifies five CB motifs within COX-1, two within COX-2 and four within COX-3; each caveolin containing a pore-lining helix C-terminal to a TM helix–turn–helix. Channel formation involves interaction between multiple pore-lining regions within protein subunits and/or dimers. PoreWalker analysis lends support to the D-channel model of proton translocation. Under physiological conditions, caveolins may introduce channel formers juxtaposed to those in COX-1, COX-2 and COX-3, which together with cholesterol may form channel(s) essential for proton translocation through the inner mitochondrial membrane.