jhw-015 and Disease-Models--Animal

When Zika virus emerged as a public health emergency there were no drugs or vaccines approved for its prevention or treatment. We used a high-throughput screen for Zika virus protease inhibitors to identify several inhibitors of Zika virus infection. We expressed the NS2B-NS3 Zika virus protease and conducted a biochemical screen for small-molecule inhibitors. A quantitative structure-activity relationship model was employed to virtually screen ∼138,000 compounds, which increased the identification of active compounds, while decreasing screening time and resources. Candidate inhibitors were validated in several viral infection assays. Small molecules with favorable clinical profiles, especially the five-lipoxygenase-activating protein inhibitor, MK-591, inhibited the Zika virus protease and infection in neural stem cells. Members of the tetracycline family of antibiotics were more potent inhibitors of Zika virus infection than the protease, suggesting they may have multiple mechanisms of action. The most potent tetracycline, methacycline, reduced the amount of Zika virus present in the brain and the severity of Zika virus-induced motor deficits in an immunocompetent mouse model. As Food and Drug Administration-approved drugs, the tetracyclines could be quickly translated to the clinic. The compounds identified through our screening paradigm have the potential to be used as prophylactics for patients traveling to endemic regions or for the treatment of the neurological complications of Zika virus infection.

Topics: Animals; Antiviral Agents; Artificial Intelligence; Chlorocebus aethiops; Disease Models, Animal; Drug Evaluation, Preclinical; High-Throughput Screening Assays; Immunocompetence; Inhibitory Concentration 50; Methacycline; Mice, Inbred C57BL; Protease Inhibitors; Quantitative Structure-Activity Relationship; Small Molecule Libraries; Vero Cells; Zika Virus; Zika Virus Infection

Bone cancer pain (BCP) is a severe complication of advanced bone cancer. Although cannabinoid receptor 2 (CB2) agonists may have an analgesic effect, the underlying mechanism remains unclear. CB2 serves a protective role in various pathological states through the activation of autophagy. Therefore, the present study aimed to determine whether the analgesic effects of the selective CB2 agonist JWH015 was mediated by the activation of autophagy in BCP. BCP was induced by the intra‑femur implantation of NCTC2472 fibrosarcoma cells in C3H/HeN mice. The pain behaviors were assessed on the following postoperative days. The selective CB2 agonist JWH015 (1 and 2 µg) was intrathecally administered on day 14 following implantation. AM630 (1 µg), a CB2 antagonist, was injected 30 min before JWH015 administration. Lipopolysaccharide (LPS; 100 nM)‑stimulated primary neurons were treated with JWH015 (1 µM) and AM630 (1 µM) to further verify the mechanism by which CB2 affects autophagy. The results demonstrated that autophagy flux was impaired in spinal neurons during BCP, as indicated by the increased ratio of microtubule‑associated protein 1 light chain 3β (LC3B)‑II/LC3B‑I and increased expression of p62. Intrathecal administration of JWH015 attenuated BCP, which was accompanied by the amelioration of impaired autophagy flux (decreased LC3B‑II/LC3B‑I ratio and decreased p62expression). In addition, the activation of glia cells and upregulation of the glia‑derived inflammatory mediators, interleukin (IL)‑1β and IL‑6 were suppressed by JWH015. In LPS‑stimulated primary neurons, IL‑1β and IL‑6 were increased, and autophagy flux was impaired; whereas treatment with JWH015 decreased the expression of IL‑1β and IL‑6, LC3B‑II/LC3B‑I ratio and expression of p62. These effects were by pretreatment with the CB2‑selective antagonist AM630. The results of the present study suggested that the impairment of autophagy flux was induced by glia‑derived inflammatory mediators in spinal neurons. Intrathecal administration of the selective CB2 agonist JWH015 ameliorated autophagy flux through the downregulation of IL‑1β and IL‑6 and attenuated BCP.

Topics: Animals; Autophagy; Bone Neoplasms; Cancer Pain; Cannabinoid Receptor Agonists; Cytokines; Disease Models, Animal; Dose-Response Relationship, Drug; Indoles; Inflammation Mediators; Injections, Spinal; Male; Mice; Neuroglia; Neurons; Pain Management; Rats; Receptor, Cannabinoid, CB2; Spinal Cord

CB2 cannabinoid receptor (CB2R) gene is associated with depression. We investigated the gene-environment interaction between CB2R function and diverse stressors. First, anxiety-like behavior during chronic-mild-stress (CMS) was evaluated in C57BL/6JJmsSlc mice following treatment with CB2R agonist JWH015 or inverse-agonist AM630. Second, locomotor activity and anxiety-like behavior were measured following exposure to an immune poly I:C stressor. Gene expressions of HPA axis related molecules,

Topics: Animals; Anxiety; Brain-Derived Neurotrophic Factor; Cannabinoid Receptor Agonists; Corticotropin-Releasing Hormone; Depression; Disease Models, Animal; Gene Expression Regulation; Gene-Environment Interaction; Hippocampus; Hypothalamo-Hypophyseal System; Immunologic Factors; Indoles; Interleukin-1beta; Locomotion; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Pituitary-Adrenal System; Poly I-C; Receptor, Cannabinoid, CB2; Receptors, Glucocorticoid; Signal Transduction; Tacrolimus Binding Proteins

Breast cancer is the second leading cause of cancer deaths among women. Cannabinoid receptor 2 (CNR2 or CB2) is an integral part of the endocannabinoid system. Although CNR2 is highly expressed in the breast cancer tissues as well as breast cancer cell lines, its functional role in breast tumorigenesis is not well understood. We observed that estrogen receptor-α negative (ERα-) breast cancer cells highly express epidermal growth factor receptor (EGFR) as well as insulin-like growth factor-I receptor (IGF-IR). We also observed IGF-IR upregulation in ERα+ breast cancer cells. In addition, we found that higher CNR2 expression correlates with better recurrence free survival in ERα- and ERα+ breast cancer patients. Therefore, we analyzed the role of CNR2 specific agonist (JWH-015) on EGF and/or IGF-I-induced tumorigenic events in ERα- and ERα+ breast cancers. Our studies showed that CNR2 activation inhibited EGF and IGF-I-induced migration and invasion of ERα+ and ERα- breast cancer cells. At the molecular level, JWH-015 inhibited EGFR and IGF-IR activation and their downstream targets STAT3, AKT, ERK, NF-kB and matrix metalloproteinases (MMPs). In vivo studies showed that JWH-015 significantly reduced breast cancer growth in ERα+ and ERα- breast cancer mouse models. Furthermore, we found that the tumors derived from JWH-015-treated mice showed reduced activation of EGFR and IGF-IR and their downstream targets. In conclusion, we show that CNR2 activation suppresses breast cancer through novel mechanisms by inhibiting EGF/EGFR and IGF-I/IGF-IR signaling axes.

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Cell Transformation, Neoplastic; Disease Models, Animal; Epidermal Growth Factor; ErbB Receptors; Estrogen Receptor alpha; Female; Gene Expression; Heterografts; Humans; Indoles; Insulin-Like Growth Factor I; Mice; Prognosis; Receptor, Cannabinoid, CB2; Receptor, IGF Type 1; Signal Transduction

The misuse of prescription opiates is on the rise with combination therapies (e.g. acetaminophen or NSAIDs) resulting in severe liver and kidney damage. In recent years, cannabinoid receptors have been identified as potential modulators of pain and rewarding behaviors associated with cocaine, nicotine and ethanol in preclinical models. Yet, few studies have identified whether mu opioid agonists and CB2 agonists act synergistically to inhibit chronic pain while reducing unwanted side effects including reward liability. We determined if analgesic synergy exists between the mu-opioid agonist morphine and the selective CB2 agonist, JWH015, in rodent models of acute and chronic inflammatory, post-operative, and neuropathic pain using isobolographic analysis. We also investigated if the MOR-CB2 agonist combination decreased morphine-induced conditioned place preference (CPP) and slowing of gastrointestinal transit. Co-administration of morphine with JWH015 synergistically inhibited preclinical inflammatory, post-operative and neuropathic-pain in a dose- and time-dependent manner; no synergy was observed for nociceptive pain. Opioid-induced side effects of impaired gastrointestinal transit and CPP were significantly reduced in the presence of JWH015. Here we show that MOR + CB2 agonism results in a significant synergistic inhibition of preclinical pain while significantly reducing opioid-induced unwanted side effects. The opioid sparing effect of CB2 receptor agonism strongly supports the advancement of a MOR-CB2 agonist combinatorial pain therapy for clinical trials.

Topics: Analgesics, Non-Narcotic; Analgesics, Opioid; Animals; Cannabinoid Receptor Agonists; Chronic Pain; Constipation; Corpus Striatum; Disease Models, Animal; Dopamine; Dose-Response Relationship, Drug; Drug Synergism; Indoles; Male; Mice, Inbred ICR; Morphine; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB2; Receptors, Opioid, mu; Reward

Previous studies have shown that modulation of the receptor-mediated endocannabinoid system during ischemia injury can induce potent neuroprotective effects. However, little is known about whether cannabinoid-2 (CB2) receptor agonist would produce a protective effect on blood-spinal cord barrier (BSCB) during ischemia. Using an in vivo transient spinal cord ischemia model in rats, JWH-015 (1mg/kg, i.p.), a CB2 receptor selective agonist, or vehicles were injected 20 min before ischemia. The effects of JWH-015 on BSCB permeability, the major structural protein for the formation of caveolae, caveolin-1 (cav-1), tight junction (TJ) protein Occludin and zona occludens protein-1 (ZO-1) were examined at day 1, day 3 and day 7 of reperfusion after transient spinal cord ischemia in rats. Here we demonstrated that JWH-015 significantly down-regulated the expression of cav-1, up-regulated the expression of TJ proteins, and then decreased the permeability of BSCB compared with control group. In addition, using an in vitro BBB model, oxygen glucose deprivation (OGD) was applied to simulate spinal cord ischemia in vitro in Human brain microvascular endothelial cells (HBMECs). JWH-015 greatly increased the transepithelial electrical resistance (TEER) and changed the distribution of ZO-1 and Occludin. Moreover, JWH-015 induced the expression of p-PKB and p-FoxO1 protein and decreased the expression of cav-1, which were greatly reversed by ROS inhibitor or PI3K inhibitor. Taken together, all of these results suggested that JWH-015 might regulate the BSCB permeability and this effect could be related to paracellular and transcellular pathway. And pharmacological CB2R ligands offer a new strategy for BSCB protection during ischemic injury.

Topics: Animals; Blood-Brain Barrier; Capillary Permeability; Disease Models, Animal; Electric Impedance; Evans Blue; Glucose; Horseradish Peroxidase; Hypoxia; In Vitro Techniques; Indoles; Male; Occludin; Proto-Oncogene Proteins c-akt; Rats; Rats, Wistar; Receptor, Cannabinoid, CB2; Reperfusion Injury; Spinal Cord; Tight Junctions; Zonula Occludens-1 Protein

Interstitial cystitis is a debilitating bladder inflammation disorder. To date, the understanding of the causes of interstitial cystitis remains largely fragmentary and there is no effective treatment available. Recent experimental results have shown a functional role of the endocannabinoid system in urinary bladder. In this study, we evaluated the anti-inflammatory effect of selective cannabinoid CB1 and CB2 receptor agonists in a mouse model of interstitial cystitis. Bladder inflammation was induced in mice by lipopolysaccharide (LPS) and whole bladders were removed 24h later. LPS induced a significant increase of the contractile amplitude in spontaneous activity and a hypersensitivity to exogenous acetylcholine-induced contraction of whole-isolated bladder. Next, we evaluated the anti-inflammatory activity of cannabinoidergic compounds by pretreating mice with CB1 or CB2 selective agonist compounds, respectively ACEA and JWH015. Interestingly, JWH015, but not ACEA, antagonized LPS-induced bladder inflammation. Additionally, anti-inflammatory activity was studied by evaluation, leukocytes mucosa infiltration, myeloperoxidase activity, and mRNA expression of pro-inflammatory interleukin (IL-1α and IL-1β), tumor necrosis factor-alpha (TNF-α) and cannabinoid CB1 and CB2 receptors. JWH015 significantly decreased leukocytes infiltration in both submucosa and mucosa, as well as the myeloperoxydase activity, in LPS treated mice. JWH015 reduced mRNA expression of IL-1α, IL-1β, and TNF-α. LPS treatment increased expression of bladder CB2 but not CB1 mRNA. Taken together, these findings strongly suggest that modulation of the cannabinoid CB2 receptors might be a promising therapeutic strategy for the treatment of bladder diseases and conditions characterized by inflammation, such as interstitial cystitis.

Topics: Animals; Arachidonic Acids; Cannabinoids; Cystitis, Interstitial; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Indoles; Lipopolysaccharides; Male; Mice; Organ Culture Techniques; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2

Tolerance to the local antiallodynic effects of morphine, DPDPE ([D-Pen(2),D-Pen(5)]-Enkephalin) or JWH-015 ((2-methyl-1-propyl-1H-indol-3-yl)-1-naphthalenylmethanone) after their repeated administration during neuropathic pain was evaluated. The role of the nitric oxide-cGMP-protein kinase G (PKG)-c-Jun N-terminal kinase (JNK) signaling pathway on the peripheral morphine-induced tolerance after the chronic constriction of sciatic nerve in mice was also assessed. The mechanical and thermal antiallodynic effects produced by a high dose of morphine, DPDPE or JWH-015 subplantarly administered daily from days 10 to 20 after nerve injury were estimated with the von Frey filaments and cold plate tests. The antiallodynic effects of the repeated administration of morphine combined with a sub-analgesic dose of a selective inducible nitric oxide synthase (NOS2) (L-N(6)-(1-iminoethyl)-lysine; L-NIL), L-guanylate cyclase (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one; ODQ), PKG ((Rp)-8-(para-chlorophenylthio)guanosine-3',5'-cyclic monophosphorothioate; Rp-8-pCPT-cGMPs) or JNK (anthra[1,9-cd]pyrazol-6(2H)-one; SP600125) inhibitor from days 10 to 20 after injury were also evaluated. The repeated administration of morphine, but not DPDPE or JWH-015, produced a rapid development of tolerance to its mechanical and thermal antiallodynic effects in sciatic nerve-injured mice. The co-administration of morphine with L-NIL, ODQ, Rp-8-pCPT-cGMPs or SP600125 avoided the development of morphine antiallodynic tolerance after nerve injury. These findings reveal that the repeated local administration of DPDPE or JWH-015 did not induce antinociceptive tolerance after sciatic nerve injury-induced neuropathic pain. Our data also indicate that the peripheral nitric oxide-cGMP-PKG-JNK signaling pathway participates in the development of morphine tolerance after nerve injury and propose the inactivation of this pathway as a promising strategy to avoid morphine tolerance during neuropathic pain.

Topics: Analgesics, Opioid; Animals; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Tolerance; Enkephalin, D-Penicillamine (2,5)-; Hot Temperature; Hyperalgesia; Indoles; Male; MAP Kinase Signaling System; Mice; Mice, Inbred C57BL; Morphine; Nitric Oxide; Sciatic Neuropathy

Peripheral nerve injury generally results in spinal neuronal and glial plastic changes associated with chronic behavioral hypersensitivity. Spinal mitogen-activated protein kinases (MAPKs), eg, p38 or extracellular signal-regulated kinases (ERKs), are instrumental in the development of chronic allodynia in rodents, and new p38 inhibitors have shown potential in acute and neuropathic pain patients. We have previously shown that the cannabinoid type 2 receptor agonist JWH015 inhibits ERK activity by inducing MAPK phosphatase (MKP)-1 and MKP-3 (the major regulators of MAPKs) in vitro in microglial cells. Therefore, we decided to investigate the role of these phosphatases in the mechanisms of action of JWH015 in vivo using the rat L5 nerve transection model of neuropathic pain. We observed that peripheral nerve injury reduced spinal MKP-1/3 expression and activity and that intrathecal JWH015 reduced established L5 nerve injury-induced allodynia, enhanced spinal MKP-1/3 expression and activity, and reduced the phosphorylated form of p38 and ERK-1/2. Triptolide, a pharmacological blocker of MKP-1 and MKP-3 expression, inhibited JWH015's effects, suggesting that JWH015 exerts its antinociceptive effects by modulating MKP-1 and MKP-3. JWH015-induced antinociception and MKP-1 and MKP-3 expression were inhibited by the cannabinoid type 2 receptor antagonist AM630. Our data suggest that MKP-1 and MKP-3 are potential targets for novel analgesic drugs.. MAPKs are pivotal in the development of chronic allodynia in rodent models of neuropathic pain. A cannabinoid type 2 receptor agonist, JWH015, reduced neuropathic allodynia in rats by reducing MAPK phosphorylation and inducing spinal MAPK phosphatases 1 and 3, the major regulators of MAPKs.

Topics: 4-Nitrophenylphosphatase; Animals; Disease Models, Animal; Diterpenes; Dual Specificity Phosphatase 1; Dual Specificity Phosphatase 6; Epoxy Compounds; Gene Expression Regulation; Hyperalgesia; Immunosuppressive Agents; Indoles; Male; Mitogen-Activated Protein Kinase Phosphatases; Nerve Tissue Proteins; Neuralgia; Phenanthrenes; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB2; Signal Transduction; Spinal Cord; Time Factors

A series of potent indol-3-yl-tetramethylcyclopropyl ketones have been prepared as CB 2 cannabinoid receptor ligands. Two unsubstituted indoles ( 5, 32) were the starting points for an investigation of the effect of indole ring substitutions on CB 2 and CB 1 binding affinities and activity in a CB 2 in vitro functional assay. Indole ring substitutions had varying effects on CB 2 and CB 1 binding, but were generally detrimental to agonist activity. Substitution on the indole ring did lead to improved CB 2/CB 1 binding selectivity in some cases (i.e., 7- 9, 15- 20). All indoles with the morpholino-ethyl side chain ( 32- 43) exhibited weaker binding affinity and less agonist activity relative to that of their tetrahydropyranyl-methyl analogs ( 5- 31). Several agonists were active in the complete Freund's adjuvant model of chronic inflammatory thermal hyperalgesia ( 32, 15).

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Binding, Competitive; Cell Line; Disease Models, Animal; Drug Evaluation, Preclinical; Humans; Hyperalgesia; Indoles; Ketones; Ligands; Molecular Conformation; Rats; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Stereoisomerism; Structure-Activity Relationship