Raj K. Razdan

Levels, metabolism, and pharmacological activity of anandamide in CB1 cannabinoid receptor knockout mice: Evidence for non-CB1, non-CB2 receptor-mediated actions of anandamide in mouse brain

Abstract: Anandamide [arachidonylethanolamide (AEA)] appears to be an endogenous agonist of brain cannabinoid receptors (CB1), yet some of the neurobehavioral effects of this compound in mice are unaffected by a selective CB1 antagonist. We studied the levels, pharmacological actions, and degradation of AEA in transgenic mice lacking the CB1 gene. We quantified AEA and the other endocannabinoid, 2‐arachidonoyl glycerol, in six brain regions and the spinal cord by isotope‐dilution liquid chromatography‐mass spectrometry. The distribution of endocannabinoids and their inactivating enzyme, fatty acid amide hydrolase, were found to overlap with CB1 distribution only in part. In CB1 knockout homozygotes (CB1‐/‐), the hippocampus and, to a lesser extent, the striatum exhibited lower AEA levels as compared with wild‐type (CB1+/+) controls. These data suggest a ligand/receptor relationship between AEA and CB1 in these two brain regions, where tonic activation of the receptor may tightly regulate the biosynthesis of its endogenous ligand. 2‐Arachidonoyl glycerol levels and fatty acid amide hydrolase activity were unchanged in CB1‐/‐ with respect to CB1+/+ mice in all regions. AEA and Δ9‐tetrahydrocannabinol (THC) were tested in CB1‐/‐ mice for their capability of inducing analgesia and catalepsy and decreasing spontaneous activity. The effects of AEA, unlike THC, were not decreased in CB1‐/‐ mice. AEA, but not THC, stimulated GTPγS binding in brain membranes from CB1‐/‐ mice, and this stimulation was insensitive to CB1 and CB2 antagonists. We suggest that non‐CB1, non‐CB2 G protein‐coupled receptors might mediate in mice some of the neuro‐behavioral actions of AEA.

Inhibition of monoacylglycerol lipase and fatty acid amide hydrolase by analogues of 2-arachidonoylglycerol.

The pharmacology of monoacylglycerol lipase (MAGL) is not well understood. In consequence, the abilities of a series of analogues of 2‐arachidonoylglycerol (2‐AG) to inhibit cytosolic 2‐oleoylglycerol and membrane‐bound anandamide hydolysis by MAGL and fatty acid amide hydrolase (FAAH), respectively, have been investigated. 2‐AG and its 1‐regioisomer (1‐AG) interacted with MAGL with similar affinities (IC50 values 13 and 17 μM, respectively). Shorter homologues of 2‐AG (2‐linoleoylglycerol and 2‐oleoylglycerol) had affinities for MAGL similar to 2‐AG. This pattern was also seen when the arachidonoyl side chain of arachidonoyl trifluoromethylketone was replaced by an oleoyl side chain. Arachidonoyl serinol (IC50 value 73 μM) was a weaker inhibitor of MAGL than 2‐AG. The IC50 values of noladin ether towards MAGL and FAAH were 36 and 3 μM, respectively. Arachidonoyl glycine interacted with FAAH (IC50 value 4.9 μM) but only weakly interacted with MAGL (IC50 value >100 μM). α‐Methyl‐1‐AG had similar potencies towards MAGL and FAAH (IC50 values of 11 and 33 μM, respectively). O‐2203 (1‐(20‐cyano‐16,16‐dimethyl‐eicosa‐5,8,11,14‐tetraenoyl) glycerol) and O‐2204 (2‐(20‐hydroxy‐16,16‐dimethyl‐eicosa‐5,8,11,14‐tetraenoyl) glycerol) were slightly less potent, but again affected both enzymes equally. α‐Methyl‐1‐AG, O‐2203 and O‐2204 interacted only weakly with cannabinoid CB1 receptors expressed in CHO cells (Ki values 1.8, 3.7 and 3.2 μM, respectively, compared with 0.24 μM for 1‐AG) and showed no evidence of central cannabinoid receptor activation in vivo at doses up to 30 mg kg−1 i.v. It is concluded that compounds like α‐Methyl‐1‐AG, O‐2203 and O‐2204 may be useful as leads for the discovery of selective MAGL inhibitors that lack direct effects upon cannabinoid receptors.

New potent and selective inhibitors of anandamide reuptake with antispastic activity in a mouse model of multiple sclerosis

We previously reported that the compound O‐2093 is a selective inhibitor of the reuptake of the endocannabinoid anandamide (AEA). We have now re‐examined the activity of O‐2093 in vivo and synthesized four structural analogs (O‐2247, O‐2248, O‐3246, and O‐3262), whose activity was assessed in: (a) binding assays carried out with membranes from cells overexpressing the human CB1 and CB2 receptors; (b) assays of transient receptor potential of the vanilloid type‐1 (TRPV1) channel functional activity (measurement of [Ca2+]i); (c) [14C]AEA cellular uptake and hydrolysis assays in rat basophilic leukaemia (RBL‐2H3) cells; (d) the mouse ‘tetrad’ tests (analgesia on a hot plate, immobility on a ‘ring’, rectal hypothermia and hypolocomotion in an open field); and (e) the limb spasticity test in chronic relapsing experimental allergic encephalomyelitis (CREAE) mice, a model of multiple sclerosis (MS). O‐2093, either synthesized by us or commercially available, was inactive in the ‘tetrad’ up to a 20u2003mgu2003kg−1 dose (i.v.). Like O‐2093, the other four compounds exhibited low affinity in CB1 (Ki from 1.3 to >10u2003μM) and CB2 binding assays (1.310u2003μM), very low potency as fatty acid amide hydrolase (FAAH) inhibitors (IC50>25u2003μM) and were inactive in the ‘tetrad’ up to a 30u2003mgu2003kg−1 dose (i.v.). While O‐2247 and O‐2248 were poor inhibitors of [14C]AEA cellular uptake (IC50>40u2003μM), O‐3246 and O‐3262 were quite potent in this assay. O‐3246, which exhibits only a very subtle structural difference with O‐2093, is the most potent inhibitor of AEA uptake reported in vitro under our experimental conditions (IC50=1.4u2003μM) and is 12‐fold more potent than O‐2093. When injected intravenously O‐3246 and O‐3262, again like O‐2093 and unlike O‐2247 and O‐2248, significantly inhibited limb spasticity in mice with CREAE. These data confirm the potential utility of selective AEA uptake inhibitors as anti‐spasticity drugs in MS and, given the very subtle chemical differences between potent and weak inhibitors of uptake, support further the existence of a specific mechanism for this process.

SR-141716A-induced stimulation of locomotor activity: A structure–activity relationship study

The central cannabinoid receptor (CB(1)) antagonist, SR-141716A, has been used extensively to ascertain that cannabinoids interact with the CB(1) receptor. SR-141716A has been shown to produce effects opposite of cannabinoids when administered alone. It has been theorized that SR-141716A may act as an inverse agonist at the CB(1) receptor or by disinhibiting an endogenous cannabinoid tone. In an effort to ascertain the exact interaction between SR-141716A and the CB(1) receptor, we have conducted a structure-activity relationship study to compare CB(1) receptor affinity of SR-141716A analogs with their ability to produce an increase in locomotor activity. SR-141716A produced a significant increase in locomotor activity in mice within the first hour of administration. Twenty SR-141716A analogs from five different chemical series were also tested. Our data implicate particular regions of the SR-141716A molecule that may be involved in stimulation and depression of locomotor activity. When the K(I) of the analogs was plotted against the percent stimulation that each analog produced, it is evident that there is no correlation between the ability of the analogs to stimulate locomotor activity and their affinity for the CB(1) receptor. [35S]GTPgammaS binding data indicate that SR-141716A and five of the analogs are inverse agonists. However, none of the analogs demonstrating inverse agonism produce stimulation of locomotor activity. It is therefore concluded that the SR-141716A-induced stimulation in locomotor activity is not the result of inverse agonist activity at the CB(1) receptor or by disinhibition of an endogenous tone.

Pharmacological evaluation of aerosolized cannabinoids in mice

The reemergence on the debate of the use of marijuana for medicinal purposes has been the impetus for developing an acceptable delivery form of aerosolized cannabinoids. The goals of the present study were to: (1) develop and characterize the physical properties of an aerosolized form of Delta(9)-tetrahydrocannabinol (Delta(9)-THC), the major psychoactive constituent present in marijuana; and (2) assess the pharmacological effects of cannabinoid inhalation in mice. A Small Particle Aerosol Generator (SPAG) nebulizer, used to generate the aerosol, had an output of approximately 0.154 mg/l of aerosolized Delta(9)-THC with a 2.0 microm mass median aerodynamic diameter and a 2.2 geometric standard deviation (GSD). Virtually all the particles were less than 5.0 microm in diameter suggesting that they were sufficiently small to penetrate deeply into the lungs. Inhalation exposure to aerosolized Delta(9)-THC in mice elicited antinociceptive effects that were dependent on concentration and exposure time with an estimated Delta(9)-THC dose of 1.8 mg/kg. On the other hand, inhalation exposure to Delta(9)-THC failed to produce two other indices indicative of cannabinoid activity, hypothermia and decreases in spontaneous locomotor activity. The antinociceptive effects occurred within 5 min of exposure and lasted approximately 40 min in duration. The cannabinoid receptor antagonist N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2, 4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide HCl (SR 141716A), but not naloxone, blocked these antinociceptive effects (AD(50)=0.09 mg/kg) indicating a cannabinoid receptor mechanism of action. Similarly, inhalation exposure to a water soluble cannabinoid analog, 3-(5-cyano-1, 1dimethylheptyl)-1-(4-N-morpholinobutyrloxy)-Delta(8)-te trahydrocann abinol (O-1057), produced antinociception that was blocked by SR 141716A. These results demonstrate that the development of an aerosolized form of cannabinoids for human medicinal use is feasible.

Behavioral comparisons of the stereoisomers of tetrahydrocannabinols

Abstract The potencies of (−)- trans -Δ9-THC, (+)- trans -Δ9-THC, (+)- cis -Δ9-THC, (−)- trans -Δ8-THC and (+)- trans -Δ8-THC were compared in several different species. (−)- trans -Δ9-THC was 100 times more potent than (+)- trans -Δ9-THC in depressing schedule-controlled responding in monkeys. The (+)- trans isomers were less effective than their corresponding (−)- trans isomers in the dog static-ataxia test, but potency ratios could not be determined due to a lack of dose-responsiveness of the (+)- trans isomers. However, it appeared that their potency differed by at least ten fold. The potency of (+)- cis -Δ9-THC in the dog static-ataxia test was comparable to that of (+)- trans -Δ9-THC. The hypothermia in mice produced by the (−) isomers of trans -Δ9-THC and trans -Δ8-THC were 9.1 and 30.4 times greater than that produced by their respective (+)-isomers. Also, the potency ratio of the (+)- and (−)- trans -Δ9-THC was 5.6 as measured by depression of spontaneous activity in mice. The magnitude of the potency ratios of the THC stereo-isomers is dependent upon the species and the pharmacological test used.

A cannabinoid with cardiovascular activity but no overt behavioral effects

Abnormal-Δ8-tetrahydrocannabidiol (ABN-Δ8-THC) failed to elicit central nervous system and cardiovascular effects in laboratory animals. Abnormal-cannabidiol (ABN-CBD) was also devoid of overt behavioral effects but produced marked hypotension with only slight bradycardia in anesthesized dogs.

The Endogenous Brain Constituent N-Arachidonoyl l-Serine Is an Activator of Large Conductance Ca2+-Activated K+ Channels

The novel endocannabinoid-like lipid N-arachidonoyl l-serine (ARA-S) causes vasodilation through both endothelium-dependent and -independent mechanisms. We have analyzed the vasorelaxant effect of ARA-S in isolated vascular preparations and its effects on Ca2+-activated K+ currents in human embryonic kidney cells stably transfected with the α-subunit of the human, large conductance Ca+-activated K+ (BKCa) channel [human embryonic kidney (HEK) 293hSlo cells]. ARA-S caused relaxation of rat isolated, intact and denuded, small mesenteric arteries preconstricted with (R)-(-)-1-(3-hydroxyphenyl)-2-methylaminoethanol hydrochloride (pEC50, 5.49 and 5.14, respectively), whereas it caused further contraction of vessels preconstricted with KCl (pEC50, 5.48 and 4.82, respectively). Vasorelaxation by ARA-S was inhibited by 100 nM iberiotoxin. In human embryonic kidney cells stably transfected with the α-subunit of the human BKCa channel cells, ARA-S and its enantiomer, N-arachidonoyl-d-serine, enhanced the whole-cell outward K+ current with similar potency (pEC50, 5.63 and 5.32, respectively). The potentiation was not altered by the β1 subunit or mediated by ARA-S metabolites, stimulation of known cannabinoid receptors, G proteins, protein kinases, or Ca2+-dependent processes; it was lost after patch excision or after membrane cholesterol depletion but was restored after cholesterol reconstitution. BKCa currents were also enhanced by N-arachidonoyl ethanolamide (pEC50, 5.27) but inhibited by another endocannabinoid, O-arachidonoyl ethanolamine (pIC50, 6.35), or by the synthetic cannabinoid O-1918 [(-)-1,3-dimethoxy-2-(3-3,4-trans-p-menthadien-(1,8)-yl)-orcinol] (pIC50, 6.59), which blocks ARA-S-induced vasodilation. We conclude the following. 1) ARA-S directly activates BKCa channels. 2) This interaction does not involve cannabinoid receptors or cytosolic factors but is dependent on the presence of membrane cholesterol. 3) Direct BKCa channel activation probably contributes to the endothelium-independent component of ARA-S-induced mesenteric vasorelaxation. 4) O-1918 is a BKCa channel inhibitor.

Water-Soluble Derivatives of Δ1 Tetrahydrocannabinol

Δ1-Tetrahydrocannabinol, which is resinous and insoluble in water and therefore difficult to study pharmacologically, can be converted to a watersoluble derivative without loss of its biological activity. This has been achieved by preparing esters bearing a nitrogen moiety with the use of carbodiimide as the condensing agent. The availability of such water-soluble derivatives will allow the evaluation of Δ1-tetrahydrocannabinol in self-administration studies in monkeys for its addiction liability potential in man. This technique of water solubilization is also applicable to other compounds of chemical and biological significance.

Evaluation of Prevalent Phytocannabinoids in the Acetic Acid Model of Visceral Nociception

Considerable preclinical research has demonstrated the efficacy of Delta(9)-tetrahydrocannabinol (Delta(9)-THC), the primary psychoactive constituent of Cannabis sativa, in a wide variety of animal models of pain, but few studies have examined other phytocannabinoids. Indeed, other plant-derived cannabinoids, including cannabidiol (CBD), cannabinol (CBN), and cannabichromene (CBC) elicit antinociceptive effects in some assays. In contrast, tetrahydrocannabivarin (THCV), another component of cannabis, antagonizes the pharmacological effects of Delta(9)-THC. These results suggest that various constituents of this plant may interact in a complex manner to modulate pain. The primary purpose of the present study was to assess the antinociceptive effects of these other prevalent phytocannabinoids in the acetic acid stretching test, a rodent visceral pain model. Of the cannabinoid compounds tested, Delta(9)-THC and CBN bound to the CB(1) receptor and produced antinociceptive effects. The CB(1) receptor antagonist, rimonabant, but not the CB(2) receptor antagonist, SR144528, blocked the antinociceptive effects of both compounds. Although THCV bound to the CB(1) receptor with similar affinity as Delta(9)-THC, it had no effects when administered alone, but antagonized the antinociceptive effects of Delta(9)-THC when both drugs were given in combination. Importantly, the antinociceptive effects of Delta(9)-THC and CBN occurred at lower doses than those necessary to produce locomotor suppression, suggesting motor dysfunction did not account for the decreases in acetic acid-induced abdominal stretching. These data raise the intriguing possibility that other constituents of cannabis can be used to modify the pharmacological effects of Delta(9)-THC by either eliciting antinociceptive effects (i.e., CBN) or antagonizing (i.e., THCV) the actions of Delta(9)-THC.