BackgroundK2 products are synthetic cannabinoid-laced, marijuana-like drugs of abuse, use of which is often associated with clinical symptoms atypical of marijuana use, including hypertension, agitation, hallucinations, psychosis, seizures and panic attacks. JWH-018, a prevalent K2 synthetic cannabinoid, is structurally distinct from Δ9-THC, the main psychoactive ingredient in marijuana. Since even subtle structural differences can lead to differential metabolism, formation of novel, biologically active metabolites may be responsible for the distinct effects associated with K2 use. The present study proposes that K2's high adverse effect occurrence is due, at least in part, to distinct JWH-018 metabolite activity at the cannabinoid 1 receptor (CB1R).Methods/Principal FindingsJWH-018, five potential monohydroxylated metabolites (M1–M5), and one carboxy metabolite (M6) were examined in mouse brain homogenates containing CB1Rs, first for CB1R affinity using a competition binding assay employing the cannabinoid receptor radioligand [3H]CP-55,940, and then for CB1R intrinsic efficacy using an [35S]GTPγS binding assay. JWH-018 and M1–M5 bound CB1Rs with high affinity, exhibiting Ki values that were lower than or equivalent to Δ9-THC. These molecules also stimulated G-proteins with equal or greater efficacy relative to Δ9-THC, a CB1R partial agonist. Most importantly, JWH-018, M2, M3, and M5 produced full CB1R agonist levels of activation. CB1R-mediated activation was demonstrated by blockade with O-2050, a CB1R-selective neutral antagonist. Similar to Δ9-THC, JWH-018 and M1 produced a marked depression of locomotor activity and core body temperature in mice that were both blocked by the CB1R-preferring antagonist/inverse agonist AM251.Conclusions/SignificanceUnlike metabolites of most drugs, the studied JWH-018 monohydroxylated compounds, but not the carboxy metabolite, retain in vitro and in vivo activity at CB1Rs. These observations, combined with higher CB1R affinity and activity relative to Δ9-THC, may contribute to the greater prevalence of adverse effects observed with JWH-018-containing products relative to cannabis.

In recent years, synthetic analogues of naturally occurring cathinone have emerged as psychostimulant-like drugs of abuse in commercial 'bath salt' preparations. 3,4-Methylenedioxypyrovalerone (MDPV) is a common constituent of these illicit products, and its structural similarities to the more well-known drugs of abuse 3,4-methylenedioxymethamphetamine (MDMA), and methamphetamine (METH) suggest that it may have similar in vivo effects to these substances. In these studies, adult male NIH Swiss mice were trained to discriminate 0.3 mg/kg MDPV from saline, and the interoceptive effects of a range of substitution doses of MDPV, MDMA, and METH were then assessed. In separate groups of mice, surgically implanted radiotelemetry probes simultaneously monitored thermoregulatory and locomotor responses to various doses of MDPV and MDMA, as a function of ambient temperature. We found that mice reliably discriminated the MDPV training dose from saline and that cumulative doses of MDPV, MDMA, and METH fully substituted for the MDPV training stimulus. All three drugs had similar ED 50 values in this procedure. Stimulation of motor activity was observed following administration of a wide range of MDPV doses (1-30 mg/kg), and the warm ambient temperature potentiated motor activity and elicited profound stereotypy and self-injurious behavior at 30 mg/kg. In contrast, MDPV-induced hyperthermic effects were observed in only the warm ambient environment. This pattern of effects is in sharp contrast to MDMA, where ambient temperature interacts with thermoregulation, but not locomotor activity. These studies suggest that although the interoceptive effects of MDPV are similar to those of MDMA and METH, direct effects on thermoregulatory processes and locomotor activity are likely mediated by different mechanisms than those of MDMA.

Drug-elicited head-twitch behavior is a useful model for studying hallucinogen activity at 5-HT 2A receptors in the mouse. Chemically diverse compounds active in this assay yield biphasic dose-effect curves, but there is no compelling explanation for the "descending" portion of these functions. A set of experiments was designed to test the hypothesis that the induction of head-twitch behavior is mediated by agonist actions at 5-HT 2A receptors, whereas the inhibition of head-twitch behavior observed at higher doses results from competing agonist activity at 5-HT 2C receptors. The effects of the phenethylamine hallucinogen R (

Mu-opioid and CB1-cannabinoid agonists produce analgesia; however, adverse effects limit use of drugs in both classes. Additive or synergistic effects resulting from concurrent administration of low doses of mu- and CB1-agonists may produce analgesia with fewer side effects. Synergism potentially results from interaction between mu-opioid receptors (MORs) and CB1 receptors (CB1Rs). AM-251 and rimonabant are CB1R antagonist/inverse agonists employed to validate opioid-cannabinoid interactions, presumed to act selectively at CB1Rs. Therefore, the potential for direct action of these antagonists at MORs is rarely considered. This study determined if AM-251 and/or rimonabant directly bind and modulate the function of MORs. Surprisingly, AM-251 and rimonabant, but not a third CB1R inverse agonist AM-281, bind with mid-nanomolar affinity to human MORs with a rank order of affinity (Ki) of AM-251 (251 nM) > rimonabant (652 nM) > AM281 (2135 nM). AM-251 and rimonabant, but not AM-281, also competitively antagonize morphine induced G-protein activation in CHO-hMOR cell homogenates (Kb = 719 or 1310 nM, respectively). AM-251 and rimonabant block morphine inhibition of cAMP production, while only AM-251 elicits cAMP rebound in CHO-hMOR cells chronically exposed to morphine. AM-251 and rimonabant (10 mg/kg) attenuate morphine analgesia, whereas the same dose of AM-281 produces little effect. Therefore, in addition to high CB1R affinity, AM-251 and rimonabant bind to MORs with mid-nanomolar affinity and at higher doses may affect morphine analgesia via direct antagonism at MORs. Such CB1-independent actions of these antagonists may contribute to reported inconsistencies when CB1/MOR interactions are examined via pharmacological methods in CB1-knockout versus wild-type mice.

Collagen is a major component of the extracellular matrix and its integrity is essential for connective tissue and organ function. The importance of proteins involved in intracellular collagen post-translational modification, folding and transport was recently highlighted from studies on recessive forms of osteogenesis imperfecta (OI). Here we describe the critical role of SC65 (Synaptonemal Complex 65, P3H4), a leprecan-family member, as part of an endoplasmic reticulum (ER) complex with prolyl 3-hydroxylase 3. This complex affects the activity of lysyl-hydroxylase 1 potentially through interactions with the enzyme and/or cyclophilin B. Loss of Sc65 in the mouse results in instability of this complex, altered collagen lysine hydroxylation and cross-linking leading to connective tissue defects that include low bone mass and skin fragility. This is the first indication of a prolyl-hydroxylase complex in the ER controlling lysyl-hydroxylase activity during collagen synthesis.

Marijuana substitutes often contain blends of multiple psychoactive synthetic cannabinoids (SCBs), including the prevalent SCBs (1-pentyl-1H-indole-3-yl)-1-naphthalenyl-methanone (JWH-018) and (1-butyl-1H-indole-3-yl)-1-naphthalenyl-methanone (JWH-073). Because SCBs are frequently used in combinations, we hypothesized that coadministering multiple SCBs induces synergistic drug-drug interactions. Drug-drug interactions between JWH-018 and JWH-073 were investigated in vivo for D 9 -tetrahydrocannabinol (D 9 -THC)-like discriminative stimulus effects, analgesia, task disruption, and hypothermia. Combinations (JWH-018:JWH-073) of these drugs were administered to mice in assays of D 9 -THC discrimination, tail-immersion, and food-maintained responding, and rectal temperatures were measured. Synergism occurred in the D 9 -THC discrimination assay for two constant dose ratio combinations (1:3 and 1:1). A 1:1 and 2:3 dose ratio induced additivity and synergy, respectively, in the tail-immersion assay. Both 1:1 and 2:3 dose ratios were additive for hypothermia, whereas a 1:3 dose ratio induced subadditive suppression of food-maintained responding. In vitro drug-drug interactions were assessed using competition receptor-binding assays employing mouse brain homogenates and cannabinoid 1 receptor (CB1R)-mediated inhibition of adenylyl cyclase activity in Neuro2A wild-type cells. Interestingly, synergy occurred in the competition receptorbinding assay for two dose ratios (1:5 and 1:10), but not in the adenylyl cyclase activity assay (1:5). Altogether, these data indicate that drug-drug interactions between JWH-018 and JWH-073 are effect-and ratio-dependent and may increase the relative potency of marijuana substitutes for subjective D 9 -THC-like effects. Combinations may improve the therapeutic profile of cannabinoids, considering that analgesia but not hypothermia or task disruption was potentiated. Importantly, synergy in the competition receptor-binding assay suggests multiple CB1R-SCB binding sites.

Osteocytes are long‐lived, highly interconnected, terminally differentiated osteoblasts that reside within mineralized bone matrix. They constitute about 95% of adult bone cells and play important functions including in the regulation of bone remodeling, phosphate homeostasis, and mechanical stimuli sensing and response. However, the role of osteocytes in the pathogenesis of congenital diseases of abnormal bone matrix is poorly understood. This study characterized in vivo transcriptional changes in osteocytes from Crtap KO and oim/oim mouse models of osteogenesis imperfecta (OI) compared with wild‐type (WT) control mice. To do this, RNA was extracted from osteocyte‐enriched cortical femurs and tibias, sequenced and subsequently analyzed to identify differentially expressed transcripts. These models were chosen because they mimic two types of OI with different genetic mutations that result in distinct type I collagen defects. A large number of transcripts were dysregulated in either model of OI, but 281 of them were similarly up‐ or downregulated in both compared with WT controls. Conversely, very few transcripts were differentially expressed between the Crtap KO and oim/oim mice, indicating that distinct alterations in type I collagen can lead to shared pathogenic processes and similar phenotypic outcomes. Bioinformatics analyses identified several critical hubs of dysregulation that were enriched in annotation terms such as development and differentiation, ECM and collagen fibril organization, cell adhesion, signaling, regulatory processes, pattern binding, chemotaxis, and cell projections. The data further indicated alterations in important signaling pathways such as WNT and TGF‐β but also highlighted new candidate genes to pursue in future studies. Overall, our study suggested that the osteocyte transcriptome is broadly dysregulated in OI with potential long‐term consequences at the cellular level, which deserve further investigations. © 2019 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

Osteogenesis imperfecta (OI) is characterized by osteopenia and bone fragility, and OI patients during growth often exhibit high bone turnover with the net result of low bone mass. Recent evidence shows that osteocytes significantly affect bone remodeling under physiological and pathological conditions through production of osteoclastogenic cytokines. The receptor activator of nuclear factor kappa-B ligand (RANKL) produced by osteocytes for example, is a critical mediator of bone loss caused by ovariectomy, low-calcium diet, unloading and glucocorticoid treatment. Because OI bone has increased density of osteocytes and these cells are embedded in matrix with abnormal type I collagen, we hypothesized that osteocyte-derived RANKL contributes to the OI bone phenotype. In this study, the conditional loss of RANKL in osteocytes in oim/oim mice (oim-RANKL-cKO) resulted in dramatically increased cancellous bone mass in both the femur and lumbar spine compared to oim/oim mice. Bone cortical thickness increased significantly only in spine but ultimate bone strength in the long bone and spine was minimally improved in oim-RANKL-cKO mice compared to oim/oim mice. Furthermore, unlike previous findings, we report that oim/oim mice do not exhibit high bone turnover suggesting that their low bone mass is likely due to defective bone formation and not increased bone resorption. The loss of osteocyte-derived RANKL further diminished parameters of formation in oim-RANKL-cKO. Our results indicate that osteocytes contribute significantly to the low bone mass observed in OI and the effect of loss of RANKL from these cells is similar to its systemic inhibition.