Seroprevalence of Adeno-Associated Virus Neutralizing Antibodies in Males with Duchenne Muscular Dystrophy.

Adeno-associated virus (AAV)-based gene therapies are emerging strategies in Duchenne muscular dystrophy (DMD) treatment. Exposure to wild-type AAV can lead to development of neutralizing antibodies (NAbs) and blocking of AAV transduction, thereby limiting the delivery of AAV vector-based gene therapy. Therefore, it is imperative to check for the presence of AAV NAbs in a patient who is a candidate for gene therapy. We prospectively enrolled 101 genetically confirmed males with DMD (median age 11 years, 48% ambulatory and 59% on steroids) and performed AAV neutralization assays against AAV2, AAV8, AAV9, and AAVrh74 serotypes. The serotype analysis showed that AAV9 (36%) and AAVrh74 (32%) seroprevalence was lower compared with AAV2 (56%) and AAV8 (47%). Interestingly, age was not correlated with NAb titer for any of the capsids. NAb responses were observed at a higher frequency in African American participants and at a lower frequency in Caucasian participants for all four serotypes. Further analysis showed no significant differences in NAb titers regardless of serotype and whether participants were taking steroids or not. Finally, we observed higher AAV8, AAV9, and AAVrh74 seroprevalence and significantly higher AAV2 and AAV8 NAb titers in participants who were ambulatory compared with nonambulatory participants. Overall, these data identify AAV9 and AAVrh74 as the two serotypes with lower pre-existing NAb titers in this study's cohort of 101 males with DMD, possibly showing their utility in future gene therapy applications in treatment of this cohort of patients with DMD.

CSF IL-8 Associated with Response to Gene Therapy in a Case Series of Spinal Muscular Atrophy.

Gene therapies have greatly changed the outlook in spinal muscular atrophy (SMA), and this disorder provides a rare opportunity to study longitudinal biomarker changes correlated with reduced disease burden and improved clinical outcomes. Recent work suggests clinical response to correlate with declining cerebrospinal fluid (CSF) levels of the neurodegenerative marker neurofilament light chain (NfL) in children receiving serial anti-sense oligonucleotide therapy. However, change in CSF NfL levels is no longer a practical biomarker as more children undergo single-dose gene replacement therapy. Here we leverage serial CSF samples (median of 4 per child) collected in 13 children with SMA undergoing anti-sense oligonucleotide therapy to characterize the longitudinal profiles of NfL as well as inflammatory and neuronal proteins. In contrast to neurodegeneration in adults, we found NfL levels to first decrease following initiation of treatment but then increase upon further treatment and improved motor functions. We then examined additional CSF inflammatory and neuronal markers for linear association with motor function during SMA treatment. We identified longitudinal IL-8 levels to inversely correlate with motor functions determined by clinical examination (F(1, 47) = 12.903, p = 0.001) or electromyography in the abductor pollicis brevis muscle (p = 0.064). In keeping with this, lower baseline IL-8 levels were associated with better longitudinal outcomes, even though this difference diminished over 2 years in the younger group. We thus propose CSF IL-8 as a biomarker for baseline function and short-term treatment response in SMA, and a candidate biomarker for future treatment trials in other neurodegenerative disorders.

Endothelial nitric oxide synthase is regulated by ERK phosphorylation at Ser602.

eNOS (endothelial nitric oxide synthase) contains a MAPK (mitogen-activated protein kinase)-binding site associated with a major eNOS control element. Purified ERK (extracellular-signal-regulated kinase) phosphorylates eNOS with a stoichiometry of 2-3 phosphates per eNOS monomer. Phosphorylation decreases NO synthesis and cytochrome c reductase activity. Three sites of phosphorylation were detected by MS. All sites matched the SP and TP MAPK (mitogen-activated protein kinase) phosphorylation motif. Ser602 lies at the N-terminal edge of the 42-residue eNOS AI (autoinhibitory) element. The pentabasic MAPK-binding site lies at the opposite end of the AI, and other critical regulatory features are between them. Thr46 and Ser58 are located in a flexible region associated with the N terminus of the oxygenase domain. In contrast with PKC (protein kinase C), phosphorylation by ERK did not significantly interfere with CaM (calmodulin) binding as analysed by optical biosensing. Instead, ERK phosphorylation favours a state in which FMN and FAD are in close association and prevents conformational changes that expose reduced FMN to acceptors. The close associations between control sites in a few regions of the molecule suggest that control of signal generation is modulated by multiple inputs interacting directly on the surface of eNOS via overlapping binding domains and tightly grouped targets.

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.

The CB2 cannabinoid receptor-selective agonist O-3223 reduces pain and inflammation without apparent cannabinoid behavioral effects.

Although Δ(9)-tetrahydrocannabinol (THC) and other mixed CB(1)/CB(2) receptor agonists are well established to elicit antinociceptive effects, their psychomimetic actions and potential for abuse have dampened enthusiasm for their therapeutic development. Conversely, CB(2) receptor-selective agonists have been shown to reduce pain and inflammation, without eliciting apparent cannabinoid behavioral effects. In the present study, we developed a novel ethyl sulfonamide THC analog, O-3223, and compared its pharmacological effects to those of the potent, mixed CB(1)/CB(2) receptor agonist, CP55,940, in a battery of preclinical pain models. Competitive cannabinoid receptor binding experiments revealed that O-3223 was approximately 80-fold more selective for CB(2) than CB(1) receptors. Additionally, O-3223 behaved as a full CB(2) receptor agonist in [(35)S]GTPγS binding. O-3223 reduced nociceptive behavior in both phases of the formalin test, reduced thermal hyperalgesia in the chronic constriction injury of the sciatic nerve (CCI) model, and reduced edema and thermal hyperalgesia elicited by intraplantar injection of LPS. These effects were blocked by pretreatment with the CB(2) receptor-selective antagonist SR144528, but not by the CB(1) receptor antagonist, rimonabant. Unlike CP55,940, O-3223 did not elicit acute antinociceptive effects in the hot-plate test, hypothermia, or motor disturbances, as assessed in the rotarod test. These data indicate that the CB(2) receptor-selective agonist, O-3223, reduces inflammatory and neuropathic nociception, without affecting basal nociception or eliciting overt behavioral effects. Moreover, this compound can serve as a template to develop new CB(2) receptor agonists with increased receptor selectivity and increased potency in treating inflammatory and neuropathic pain.

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.

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.

Interlaboratory comparison study of serum total testosterone [corrected] measurements performed by mass spectrometry methods.

BACKGROUND: Though mass spectrometry (MS) assays are increasingly used for routine clinical measurements of serum total testosterone (TT), information about the variability of results is limited. This study assessed the variability of TT measurement results from routine MS assays. METHODS: Twenty serum samples (12 females, 8 males) were analyzed on 2 days by seven high performance liquid chromatography (HPLC), and one gas chromatography (GC)-tandem mass spectrometry (HPLC-MS/MS, GC-MS/MS) assays. Two samples (male and female) were provided in five replicates to assess the within-run variability. Results were compared against those obtained at National Institute of Standards and Technology (NIST). The within- and between-laboratory variability was assessed for each sample. Comparisons to the NIST results were performed using bias plot and Deming regression analysis. RESULTS: The overall coefficient of variation of the results obtained with MS assays was <15%CV at >1.53 nmol/L and <34%CV at 0.3 nmol/L. The between-assay variability was the major contributor to the overall variability. The assay precision was the highest (<3%CV) with assays using liquid-liquid extraction for sample preparation or GC-MS/MS. The mean percent difference to the reference assay was 11%. The slopes of Deming regression analysis of the MS assays were between 0.903 and 1.138 (correlation coefficient: >0.996). TT concentrations for one assay were above the measurement range. CONCLUSIONS: The variability of TT measurement results among MS assays is substantially smaller than that reported for immunoassays. The type of sample preparation may affect assay precision. Standardizing assays can further reduce the variability of measurement results.

Synthesis and pharmacological activity of a potent inhibitor of the biosynthesis of the endocannabinoid 2-arachidonoylglycerol.

Biosynthesis Inhibition: O-5596, a new inhibitor of the biosynthesis of the endocannabinoid, 2-arachidonoylglycerol, was synthesized and found to be potent (IC(50)=100 nM) and selective versus other proteins and enzymes of the endocannabinoid system in vitro and active in vivo at reducing food intake in mice.

Modulation of cannabinoid receptor activation as a neuroprotective strategy for EAE and stroke.

Recognition of the importance of the endocannabinoid system in both homeostasis and pathologic responses raised interest recently in the development of therapeutic agents based on this system. The CB(2) receptor, a component of the endocannabinoid system, has significant influence on immune function and inflammatory responses. Inflammatory responses are major contributors to central nervous system (CNS) injury in a variety of diseases. In this report, we present evidence that activation of CB(2) receptors, by selective CB(2) agonists, reduces inflammatory responses that contribute to CNS injury. The studies demonstrate neuroprotective effects in experimental autoimmune encephalomyelitis, a model of multiple sclerosis, and in a murine model of cerebral ischemia/reperfusion injury. In both cases, CB(2) receptor activation results in reduced white cell rolling and adhesion to cerebral microvessels, a reduction in immune cell invasion, and improved neurologic function after insult. In addition, administration of the CB(1) antagonist SR141716A reduces infarct size following ischemia/reperfusion injury. Administration of both a selective CB(2) agonist and a CB(1) antagonist has the unique property of increasing blood flow to the brain during the occlusion period, suggesting an effect on collateral blood flow. In summary, selective CB(2) receptor agonists and CB(1) receptor antagonists have significant potential for neuroprotection in animal models of two devastating diseases that currently lack effective treatment options.

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 Ca(2+)-activated K(+) currents in human embryonic kidney cells stably transfected with the alpha-subunit of the human, large conductance Ca(+)-activated K(+) (BK(Ca)) 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 (pEC(50), 5.49 and 5.14, respectively), whereas it caused further contraction of vessels preconstricted with KCl (pEC(50), 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 alpha-subunit of the human BK(Ca) channel cells, ARA-S and its enantiomer, N-arachidonoyl-D-serine, enhanced the whole-cell outward K(+) current with similar potency (pEC(50), 5.63 and 5.32, respectively). The potentiation was not altered by the beta(1) subunit or mediated by ARA-S metabolites, stimulation of known cannabinoid receptors, G proteins, protein kinases, or Ca(2+)-dependent processes; it was lost after patch excision or after membrane cholesterol depletion but was restored after cholesterol reconstitution. BK(Ca) currents were also enhanced by N-arachidonoyl ethanolamide (pEC(50), 5.27) but inhibited by another endocannabinoid, O-arachidonoyl ethanolamine (pIC(50), 6.35), or by the synthetic cannabinoid O-1918 [(-)-1,3-dimethoxy-2-(3-3,4-trans-p-menthadien-(1,8)-yl)-orcinol] (pIC(50), 6.59), which blocks ARA-S-induced vasodilation. We conclude the following. 1) ARA-S directly activates BK(Ca) channels. 2) This interaction does not involve cannabinoid receptors or cytosolic factors but is dependent on the presence of membrane cholesterol. 3) Direct BK(Ca) channel activation probably contributes to the endothelium-independent component of ARA-S-induced mesenteric vasorelaxation. 4) O-1918 is a BK(Ca) channel inhibitor.

N-arachidonyl maleimide potentiates the pharmacological and biochemical effects of the endocannabinoid 2-arachidonylglycerol through inhibition of monoacylglycerol lipase.

Inhibition of the metabolism of the endocannabinoids, anandamide (AEA) and 2-arachidonyl glycerol (2-AG), by their primary metabolic enzymes, fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), respectively, has the potential to increase understanding of the physiological functions of the endocannabinoid system. To date, selective inhibitors of FAAH, but not MAGL, have been developed. The purpose of this study was to determine the selectivity and efficacy of N-arachidonyl maleimide (NAM), a putative MAGL inhibitor, for modulation of the effects of 2-AG. Our results showed that NAM unmasked 2-AG activity in a tetrad of in vivo tests sensitive to the effects of cannabinoids in mice. The efficacy of 2-AG (and AEA) to produce hypothermia was reduced compared with Delta(9)-tetrahydrocannabinol; however, 2-AG differed from AEA by its lower efficacy for catalepsy. All tetrad effects were partially CB(1) receptor-mediated because they were attenuated (but not eliminated) by SR141716A [N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-H-pyrazole-3-carboxamide HCl] and in CB(1)(-/-) mice. In vitro, NAM increased endogenous levels of 2-AG in the brain. Furthermore, NAM raised the potency of 2-AG, but not AEA, in agonist-stimulated guanosine 5'-O-(3-[(35)S]thio)triphosphate binding assay, a measure of G-protein activation. These results suggest that NAM is an MAGL inhibitor with in vivo and in vitro efficacy. NAM and other MAGL inhibitors are valuable tools to elucidate the biological functions of 2-AG and to examine the consequences of dysregulation of this endocannabinoid. In addition, NAM's unmasking of 2-AG effects that are only partially reversed by SR141716A offers support for the existence of non-CB(1), non-CB(2) cannabinoid receptors.

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.

Synthesis of 2-(5Z,8Z,11Z,14Z)-Icosa-5,8,11,14-tetraenamidoethyl-d(4) Dihydrogen Phosphate, Tetra-deuterated pAEA.

A labile intermediate phospho-anandamide (2-(5Z,8Z,11Z,14Z)-icosa-5,8,11,14-tetraenamidoethyl dihydrogen phosphate, pAEA) has been identified in mouse brain and macrophages, but its precise quantitation was difficult because of its low concentration and chemical instability. We report the synthesis of tetra-deuterated pAEA from 2-aminoethyl dihydrogen phosphate-1,1,2,2-d(4) and (5Z,8Z,11Z,14Z)-2,5-dioxopyrrolidin-1-yl icosa-5,8,11,14-tetraenoate. The compound will be used to quantitate the pAEA necessary for a novel biosynthetic pathway.

Multiple pathways involved in the biosynthesis of anandamide.

Endocannabinoids, including anandamide (arachidonoyl ethanolamide) have been implicated in the regulation of a growing number of physiological and pathological processes. Anandamide can be generated from its membrane phospholipid precursor N-arachidonoyl phosphatidylethanolamine (NAPE) through hydrolysis by a phospholipase D (NAPE-PLD). Recent evidence indicates, however, the existence of two additional, parallel pathways. One involves the sequential deacylation of NAPE by alpha,beta-hydrolase 4 (Abhd4) and the subsequent cleavage of glycerophosphate to yield anandamide, and the other one proceeds through phospholipase C-mediated hydrolysis of NAPE to yield phosphoanandamide, which is then dephosphorylated by phosphatases, including the tyrosine phosphatase PTPN22 and the inositol 5' phosphatase SHIP1. Conversion of synthetic NAPE to AEA by brain homogenates from wild-type and NAPE-PLD(-/-) mice can proceed through both the PLC/phosphatase and Abdh4 pathways, with the former being dominant at shorter (<10 min) and the latter at longer (60 min) incubations. In macrophages, the endotoxin-induced synthesis of anandamide proceeds uniquely through the phospholipase C/phosphatase pathway.

Novel, potent THC/anandamide (hybrid) analogs.

The structure-activity relationship (SAR) of the end pentyl chain in anandamide (AEA) has been established to be very similar to that of Delta(9)-tetrahydrocannabinol (Delta(9)-THC). In order to broaden our understanding of the structural similarities between AEA and THC, hybrid structures 1-3 were designed. In these hybrids the aromatic ring of THC-DMH was linked to the AEA moiety through an ether linkage with the oxygen of the phenol of THC. Hybrid 1 (O-2220) was found to have very high binding affinity to CB1 receptors (K(i)=8.5 nM), and it is interesting to note that the orientation of the side chain with respect to the oxygen in the phenol is the same as in THCs. To further explore the SAR in this series the terminal carbon of the side chain was modified by adding different substituents. Several such analogs were synthesized and tested for their CB1 and CB2 binding affinities and in vivo activity (tetrad tests). The details of the synthesis and the biological activity of these compounds are described.

Significance of cannabinoid CB1 receptors in improgan antinociception.

UNLABELLED: Improgan is a congener of the H(2) antagonist cimetidine, which produces potent antinociception. Because a) the mechanism of action of improgan remains unknown and b) this drug may indirectly activate cannabinoid CB(1) receptors, the effects of the CB(1) antagonist/inverse agonist rimonabant (SR141716A) and 3 congeners with varying CB(1) potencies were studied on improgan antinociception after intracerebroventricular (icv) dosing in rats. Consistent with blockade of brain CB(1) receptors, rimonabant (K(d) = 0.23 nM), and O-1691 (K(d) = 0.22 nM) inhibited improgan antinociception by 48% and 70% after icv doses of 43 nmol and 25 nmol, respectively. However, 2 other derivatives with much lower CB(1) affinity (O-1876, K(d) = 139 nM and O-848, K(d) = 352 nM) unexpectedly blocked improgan antinociception by 65% and 50% after icv doses of 300 nmol and 30 nmol, respectively. These derivatives have 600-fold to 1500-fold lower CB(1) potencies than that of rimonabant, yet they retained improgan antagonist activity in vivo. In vitro dose-response curves with (35)S-GTPgammaS on CB(1) receptor-containing membranes confirmed the approximate relative potency of the derivatives at the CB(1) receptor. Although antagonism of improgan antinociception by rimonabant has previously implicated a mechanistic role for the CB(1) receptor, current findings with rimonabant congeners suggest that receptors other than, or in addition to CB(1) may participate in the pain-relieving mechanisms activated by this drug. The use of congeners such as O-848, which lack relevant CB(1)-blocking properties, will help to identify these cannabinoid-like, non-CB(1) mechanisms. PERSPECTIVE: This article describes new pharmacological characteristics of improgan, a pain-relieving drug that acts by an unknown mechanism. Improgan may use a marijuana-like (cannabinoid) pain-relieving mechanism, but it is shown presently that the principal cannabinoid receptor in the brain (CB(1)) is not solely responsible for improgan analgesia.

The role of fluorine substitution in the structure-activity relationships (SAR) of classical cannabinoids.

A facile synthesis of 1-fluoro-1-deoxy-Delta(8)-THC analogs with side chains seven carbons in length, in the alkane/ene/yne- series (6, 5, and 4), was achieved from 1-fluoro-3,5-dimethoxybenzene (1). In vitro studies show that substitution by a fluorine has a significant detrimental effect on CB1 binding which is supported by in vivo testing. The implications of these results on the SAR of classical cannabinoids are discussed.

Cannabinoid CB(2) receptor activation decreases cerebral infarction in a mouse focal ischemia/reperfusion model.

Cannabinoid CB(2) Receptor (CB(2)) activation has been shown to have immunomodulatory properties without psychotropic effects. The hypothesis of this study is that selective CB(2) agonist treatment can attenuate cerebral ischemia/reperfusion injury. Selective CB(2) agonists (O-3853, O-1966) were administered intravenously 1 h before transient middle cerebral artery occlusion (MCAO) or 10 mins after reperfusion in male mice. Leukocyte/endothelial interactions were evaluated before MCAO, 1 h after MCAO, and 24 h after MCAO via a closed cranial window. Cerebral infarct volume and motor function were determined 24 h after MCAO. Administration of the selective CB(2) agonists significantly decreased cerebral infarction (30%) and improved motor function (P<0.05) after 1 h MCAO followed by 23 h reperfusion in mice. Transient ischemia in untreated animals was associated with a significant increase in leukocyte rolling and adhesion on both venules and arterioles (P<0.05), whereas the enhanced rolling and adhesion were attenuated by both selective CB(2) agonists administered either at 1 h before or after MCAO (P<0.05). CB(2) activation is associated with a reduction in white blood cell rolling and adhesion along cerebral vascular endothelial cells, a reduction in infarct size, and improved motor function after transient focal ischemia.