Differential Effects of Endocannabinoids on Amyloid-Beta Aggregation and Toxicity.

The regulation and metabolism of the endocannabinoid system has received extensive attention for their potential neuroprotective effect in neurodegenerative diseases such as Alzheimer's disease (AD), which is characterized by amyloid ß (Aß) -induced cell toxicity, inflammation, and oxidative stress. Using in vitro techniques and two cell lines, the mouse hippocampus-derived HT22 cells and Chinese hamster ovary (CHO) cells expressing human cannabinoid receptor type 1 (CB1), we investigated the ability of endocannabinoids to inhibit Aß aggregation and protect cells against Aß toxicity. The present study provides evidence that endocannabinoids N-arachidonoyl ethanol amide (AEA), noladin and O-arachidonoyl ethanolamine (OAE) inhibit Aß42 aggregation. They were able to provide protection against Aß42 induced cytotoxicity via receptor-mediated and non-receptor-mediated mechanisms in CB1-CHO and HT22 cells, respectively. The aggregation kinetic experiments demonstrate the anti-Aß aggregation activity of some endocannabinoids (AEA, noladin). These data demonstrate the potential role and application of endocannabinoids in AD pathology and treatment.

Assessing the impact of a cannabis course on pharmacy students' understanding, beliefs and preparedness regarding medical and recreational cannabis.

BACKGROUND: With the legalization of cannabis in Canada in 2018, pharmacists are increasingly likely to encounter patients using this substance. The primary objective of this pre-post questionnaire study was to evaluate the impact of an accredited cannabis course on the understanding, beliefs, perceptions and knowledge of undergraduate PharmD students. METHODS: A 38-question, web-based survey generated in REDCap was administered to third-year PharmD students at the University of Waterloo, prior to and right after taking an accredited cannabis course. The pre- and postsurvey data were analyzed using SPSS version 25. Pearson chi-square tests were performed on questions in which answers consisted of qualitative categorical data. Two-sided t tests were performed to test the significance of mean differences of questions measuring continuous variables. RESULTS: In a class of 120 students, 110 completed the presurvey and 79 students completed the postsurvey. After the course, students were more likely to report being knowledgeable and prepared for patient encounters dealing with medical and recreational cannabis, understanding that medical cannabis should be prescribed for select (vs all) medical conditions, rating the quality of evidence as poor to moderate for medical use of cannabis, understanding that medical documents should be more prescriptive and understanding that cannabis should not be sold in pharmacies (p < 0.05). INTERPRETATION: With cannabis education a part of their curriculum, pharmacy students felt more prepared to engage patients using cannabis both medically and recreationally. Furthermore, students were more cautious regarding the potential use of cannabis therapeutically and indicated that more oversight should be in place. Can Pharm J (Ott) 2021;154:xx-xx.

The effects of heat and freeze-thaw cycling on naloxone stability.

PURPOSE: The availability of take home naloxone (THN) was increased for Canadians in 2016, including access to kits via pharmacies. Unlike typical over-the-counter (OTC) and prescription drugs, THN kits may be stored in non-standard conditions, including in vehicles, backpacks, and out of doors. To evaluate whether these non-standard storage conditions affect stability, we investigated the impact of heat and freeze-thaw cycling on naloxone hydrochloride stability. METHODS: To assess the effect of heat, naloxone hydrochloride ampoules were exposed to 80 °C in a temperature-controlled oven for 8 h followed by 16 h at room temperature. To assess the effect of freeze-thaw cycles, naloxone hydrochloride ampoules were exposed to - 20 °C for 16 h followed by 8 h at 4 °C. The impact of these conditions on naloxone hydrochloride stability was evaluated each day for 1 week and after 2 and 4 weeks. The concentration of remaining naloxone hydrochloride was quantified using high-performance liquid chromatography (HPLC). Naloxone hydrochloride ampoules stored at room temperature served as the experimental control. RESULTS: Naloxone hydrochloride ampoules exhibit no changes in drug concentration following exposure to heat or freeze-thaw cycles for up to 28 days compared to ampoules maintained at room temperature (as indicated in the product monograph). CONCLUSIONS: Naloxone hydrochloride remains chemically stable following exposure to heat or freeze-thaw cycles after 28 days. If THN kits are stored in non-standard conditions (for up to 28 days) the active naloxone is likely to remain stable. Despite this, pharmacists should continue to emphasize the importance of appropriate storage of THN kits to ensure optimal efficacy should naloxone administration be required in an emergency situation.

Fluoxetine-induced transactivation of the platelet-derived growth factor type ß receptor reveals a novel heterologous desensitization process.

Many G protein-coupled receptors (GPCRs), including serotonin (5-HT) receptors promote the activity of receptor tyrosine kinases (RTKs) via intracellular signaling pathways in a process termed transactivation. Although transactivation pathways are commonly initiated by a GPCR, a recent report demonstrated that serotonin-selective reuptake inhibitors (SSRIs) were able to block 5-HT-induced transactivation of the platelet-derived growth factor (PDGF) type ß receptor. We show that a 45 min pretreatment of SH-SY5Y cells with the SSRI fluoxetine indeed blocked 5-HT-induced transactivation of the PDGFß receptor. However, upon further examination, we discovered that during the pretreatment period, fluoxetine itself was transiently transactivating the PDGFß receptor via 5-HT2 receptor activation. After 45min, the increase in PDGFß receptor phosphorylation induced by fluoxetine had returned to baseline, but a subsequent transactivating stimulus (5-HT) failed to "re-transactivate" the PDGFß receptor. We further demonstrate that 45min, but not 3h, 5-HT pretreatment blocks dopamine-induced PDGFß receptor transactivation. This did not involve changes in PDGF receptor function, since ligand (PDGF)-induced PDGFß receptor activation was not inhibited by 5-HT pretreatment. To our knowledge this is the first demonstration of the heterologous desensitization of an RTK transactivation pathway and reveals a previously unknown short-term "blackout" period where no additional transactivation signaling is possible.

Preclinical development and ocular biodistribution of gemini-DNA nanoparticles after intravitreal and topical administration: towards non-invasive glaucoma gene therapy.

Gene therapy could offer improvement in the treatment of glaucoma compared to the current standard of lowering intraocular pressure. We have developed and characterized non-viral gemini surfactant-phospholipid nanoparticles (GL-NPs) for intravitreal and topical administration. Optimized GL-NPs (size range 150-180 nm) were biocompatible with rat retinal ganglion (RGC-5) cells with >95% viability by PrestoBlue™ assay. GL-NPs carrying Cy5-labeled plasmid DNA demonstrated distinct trafficking behavior and biodisposition within the eye in vivo after intravitreal or topical application with respect to pathways of movement and physicochemical stability. After intravitreal injection in mice, GL-NPs localized within the nerve fiber layer of the retina, whereas after topical application, GL-NPs were located in several anterior chamber tissues, including the limbus, iris and conjunctiva. GL-NPs were thermodynamically stable in the vitreous and tear fluid and were trafficked as single, non-aggregated particles after both types of administration. FROM THE CLINICAL EDITOR: In this paper, the development and characterization of non-viral gemini surfactant-phospholipid nanoparticles is reported with the goal of establishing a gene delivery system that addresses glaucoma in a non-invasive fashion. The authors found that after topical application, the concentration of these nanoparticles was higher in anterior chamber-related components of the eye, whereas intra-vitreal administration resulted in accumulation in the retinal nerve fibre layer.

Association Between Opioid-Related Mortality and History of Surgical Procedure: A Population-Based Case-Control Study.

Objective: This study examined whether there is an association between opioid-related mortality and surgical procedures. Methods: A case-control study design using deceased controls compared individuals with and without opioid death and their exposure to common surgeries in the preceding 4 years. This population-based study used linked death and hospitalization databases in Canada (excluding Quebec) from January 01, 2008 to December 31, 2017. Cases of opioid death were identified and matched to 5 controls who died of other causes by age (±4 years), sex, province of death, and date of death (±1 year). Patients with HIV infection and alcohol-related deaths were excluded from the control group. Logistic regression was used to determine if there was an association between having surgery and death from an opioid-related cause by estimating the crude and adjusted odds ratios (ORs) with the corresponding 95% confidence interval (CI). Covariates included sociodemographic characteristics, comorbidities, and the number of days of hospitalization in the previous 4 years. Results: We identified 11,865 cases and matched them with 59,345 controls. About 11.2% of cases and 12.5% of controls had surgery in the 4 years before their death, corresponding to a crude OR of 0.89 (95% CI: 0.83-0.94). After adjustment, opioid mortality was associated with surgical procedure with OR of 1.26 (95% CI: 1.17-1.36). Conclusions: After adjusting for comorbidities, patients with opioid mortality were more likely to undergo surgical intervention within 4 years before their death. Clinicians should enhance screening for opioid use and risk factors when considering postoperative opioid prescribing.

5-Hydroxytryptamine type 7 receptor neuroprotection against NMDA-induced excitotoxicity is PDGFß receptor dependent.

The serotonin (5-HT) type 7 receptor is expressed throughout the CNS including the hippocampus. Long-term (2-24 h) activation of 5-HT7 receptors regulates growth factor receptor expression, including the expression of platelet-derived growth factor (PDGF) ß receptors. Direct activation of PDGFß receptors in primary hippocampal and cortical neurons inhibits NMDA receptor activity and attenuates NMDA receptor-induced neurotoxicity. Our objective was to investigate whether the 5-HT7 receptor-induced increase in PDGFß receptor expression would be similarly neuroprotective. We demonstrate that 5-HT7 receptor agonist treatment in primary hippocampal neurons also increases the expression of phospholipase C (PLC) γ, a downstream effector of PDGFß receptors associated with the inhibition of NMDA receptor activity. To determine if the up-regulation of PDGFß receptors is neuroprotective, primary hippocampal neurons were incubated with the 5-HT7 receptor agonist, LP 12, for 24 h. Indeed, LP 12 treatment prevented NMDA-induced neurotoxicity and this effect was dependent on PDGFß receptor kinase activity. Treatment of primary neurons with LP 12 also differentially altered NMDA receptor subunit expression, reducing the expression of NR1 and NR2B, but not NR2A. These findings demonstrate the potential for providing growth factor receptor-dependent neuroprotective effects using small-molecule ligands of G protein-coupled receptors.

Search for lithium isotope effects in neuronal HT22 cells.

Lithium has been used as a treatment for bipolar disorder for over half a century, but there has thus far been no clinical differentiation made between the two naturally occurring stable isotopes (6Li and 7Li). While the natural lithium salts commonly used in treatments are composed of a mixture of these two stable isotopes (approximately 7.59% 6Li and 92.41% 7Li), some preliminary research indicates the above two stable isotopes of lithium may have differential effects on rat behaviour and neurophysiology. Here, we evaluate whether lithium isotopes may have distinct effects on HT22 neuronal cell viability, GSK-3-ß phosphorylation in HT22 cells, and GSK-3-ß kinase activity. We report no significant difference in lithium isotope toxicity on HT22 cells, nor in GSK-3-ß phosphorylation, nor in GSK-3-ß kinase activity between the two isotopes of lithium.

The Interplay Between Cholesterol and Amyloid-ß on HT22 Cell Viability, Morphology, and Receptor Tyrosine Kinase Signaling.

BACKGROUND: There is a lack of understanding in the molecular and cellular mechanisms of Alzheimer's disease that has hindered progress on therapeutic development. The focus has been on targeting toxic amyloid-ß (Aß) pathology, but these therapeutics have generally failed in clinical trials. Aß is an aggregation-prone protein that has been shown to disrupt cell membrane structure in molecular biophysics studies and interfere with membrane receptor signaling in cell and animal studies. Whether the lipid membrane or specific receptors are the primary target of attack has not been determined. OBJECTIVE: This work elucidates some of the interplay between membrane cholesterol and Aß42 on HT22 neuronal cell viability, morphology, and platelet-derived growth factor (PDGF) signaling pathways. METHODS: The effects of cholesterol depletion by methyl-ß-cyclodextrin followed by treatment with Aß and/or PDGF-AA were assessed by MTT cell viability assays, western blot, optical and AFM microscopy. RESULTS: Cell viability studies show that cholesterol depletion was mildly protective against Aß toxicity. Together cholesterol reduction and Aß42 treatment compounded the disruption of the PDGFα receptor activation. Phase contrast optical microscopy and live cell atomic force microscopy imaging revealed that cytotoxic levels of Aß42 caused morphological changes including cell membrane damage, cytoskeletal disruption, and impaired cell adhesion; cell damage was ameliorated by cellular cholesterol depletion. CONCLUSIONS: Cholesterol depletion impacted the effects of Aß42 on HT22 cell viability, morphology, and receptor tyrosine kinase signaling.

Development and evaluation of multifunctional agents for potential treatment of Alzheimer's disease: application to a pyrimidine-2,4-diamine template.

We investigated a group of 2-benzylpiperidin-N-benzylpyrimidin-4-amines with various electron-withdrawing or electron-donating groups (EWGs or EDGs, respectively) as multi-targeted Alzheimer's disease (AD) therapeutics. The synthesized derivatives were screened for anti-cholinesterase (AChE and BuChE), anti-Aß-aggregation (AChE- and self-induced) and anti-ß-secretase (BACE-1) activities in an effort to identify lead, multifunctional candidates as part of our multi-targeted approach to treat AD. Biological assessment revealed that the nature of the substituent on the C-4 benzylamine group (e.g., halogen vs methoxy-based) greatly affected the biological profile. In vitro screening identified N(2)-(1-benzylpiperidin-4-yl)-N(4)-(3,4-dimethoxybenzyl)pyrimidine-2,4-diamine (7h) as the lead candidate with a dual ChE (AChE IC(50)=9.9 µM; BuChE IC(50)=11.4 µM), Aß-aggregation (AChE-induced=59.3%; self-induced=17.4% at 100 µM) and BACE-1 (34% inhibition at 10 µM) inhibitory profile along with good cell viability (% neuroblastoma cell viability at 40 µM=81.0%). Molecular modeling studies indicate that a central pyrimidine-2,4-diamine ring serves as a suitable template to develop novel small molecule candidates to target multiple pathological routes in AD.

Drug-Drug Interaction of the Sodium Glucose Co-Transporter 2 Inhibitors with Statins and Myopathy: A Disproportionality Analysis Using Adverse Events Reporting Data.

INTRODUCTION: An increased risk of myopathy due to a potential interaction between sodium glucose co-transporter-2 inhibitors (SGLT-2i) and HMG-CoA reductase inhibitors (statins) has been suggested by case reports. OBJECTIVE: We aimed to assess if the reporting of myopathy is disproportionally higher among people using both SGLT-2i and statins compared to using either SGLT-2i or statins alone. METHODS: We conducted a disproportionality analysis using data from the US Food and Drug Administration Adverse Event Reporting System (FAERS). We included reports with at least one antihyperglycemic agent. We compared the proportion of myopathy cases to non-cases between those not using SGLT-2i or statins, using SGLT-2i only, statins only, or both. We calculated the reporting odds ratio and 95% confidence interval. We further stratified by individual SGLT-2i and selected statins (rosuvastatin or atorvastatin). RESULTS: We included 688,388 reports with at least one antihyperglycemic agent recorded, of which 9.80% had at least one SGLT-2i agent. Among all included reports, there were a total of 2202 myopathy cases with the majority, 61.26%, occurring among those using statins alone and only 2.72% of myopathy cases were among those using both SGLT-2i and statins together. Reporting of myopathy was not disproportionally higher among those reporting the use of SGLT-2i with statins (reporting odds ratio 2.95, 95% confidence interval 2.27-3.85) compared to statins alone (reporting odds ratio 6.41, 95% confidence interval 5.86-7.02). CONCLUSIONS: Reports of myopathy were not disproportionally higher among those using SGLT-2i with statins compared to SGLT-2i or statins alone at the class level. Further observational studies may be needed to better assess this interaction at the agent level.

The role of pharmacists in opioid stewardship: A scoping review.

BACKGROUND: The opioid epidemic is an international public health concern. Pharmacists are in a strategic position to promote and implement effective opioid stewardship due to both their central role on health care teams and frequent interaction with patients. Despite this integral role, pharmacists do not have harmonized scopes of practice in opioid stewardship. OBJECTIVES: This scoping review was conducted to identify and critically review the role of pharmacists in opioid stewardship and identify future areas of study. METHODS: The scoping review was conducted according to the methodological framework proposed by Arksey and O'Malley, which was further modified by the Joanna Briggs Institute. Six databases were searched for original, peer-reviewed research; PubMed (MEDLINE), Ovid Embase, Ovid International Pharmaceutical Abstracts, Scopus, Cochrane Library, and APA PsycInfo. RESULTS: In 92% of the included studies (n = 77), opioid stewardship interventions led by either a pharmacist or in an interdisciplinary team resulted in improvements in at least one outcome measure, with education and medication therapy adjustments being the most predominant activities. Other areas supported by evidence include community stakeholder education, policy and guideline setting, and risk assessment. CONCLUSION: This scoping review provides valuable insight into the various roles pharmacists can have in opioid stewardship. The findings from this review identified opioid stewardship activities that can make significant contributions towards reducing the impact of the opioid crisis. This review informs future research and has the potential to influence pharmacy practice on a national and international scale.

Postsynaptic clustering and activation of Pyk2 by PSD-95.

The tyrosine kinase Pyk2 plays a unique role in intracellular signal transduction by linking Ca(2+) influx to tyrosine phosphorylation, but the molecular mechanism of Pyk2 activation is unknown. We report that Pyk2 oligomerization by antibodies in vitro or overexpression of PSD-95 in PC6-3 cells induces trans-autophosphorylation of Tyr402, the first step in Pyk2 activation. In neurons, Ca(2+) influx through NMDA-type glutamate receptors causes postsynaptic clustering and autophosphorylation of endogenous Pyk2 via Ca(2+)- and calmodulin-stimulated binding to PSD-95. Accordingly, Ca(2+) influx promotes oligomerization and thereby autoactivation of Pyk2 by stimulating its interaction with PSD-95. We show that this mechanism of Pyk2 activation is critical for long-term potentiation in the hippocampus CA1 region, which is thought to underlie learning and memory.

What Is Known about Community Pharmacy-Based Take-Home Naloxone Programs and Program Interventions? A Scoping Review.

A variety of new sources describing community pharmacy-based take-home naloxone (THN) programs have emerged recently in the literature. There is a need to define the types of take-home naloxone programs being offered to support future research designs in implementing and evaluating standardized programs that fill pharmacist and patient knowledge gaps and lift current barriers for optimal community pharmacy naloxone provision. The objective of this paper is to summarize the literature on community pharmacy-based THN programs, including specific program interventions used to increase naloxone dispensing, naloxone availability and dispensing patterns, facilitators and barriers for the THN programs, and knowledge gaps. Online databases such as PubMed, EMBASE, Scopus, and International Pharmaceutical Abstracts (IPA) and a search of the grey literature were used to identify eligible sources. Sources were screened by two reviewers for eligibility in COVIDENCE software. Both reviewers compared screening results and resolved conflicts through discussion. A data extraction form for all identified full texts was completed by both reviewers and results were compiled through reviewer discussion. Fifty-two sources met the eligibility criteria. The top three barriers identified were: cost/coverage of naloxone, stigma, and education/training for pharmacists. THN program interventions included screening tools, checklists, pocket cards, patient brochures, and utilizing the pharmacy management system to flag eligible patients. Patient knowledge gaps included naloxone misinformation and lack of awareness, while pharmacists demonstrated administrative, clinical, and counselling knowledge gaps. Naloxone availability was found to be highly variable, where independent and rural pharmacies were less likely to stock or dispense naloxone. Further, pharmacies located in districts with higher rates of opioid overdose deaths and lower household income were also less likely to have naloxone available. This review identified multiple new programs, showcasing that the implementation and evaluation of THN programs are an expanding area of research. Future research should focus on implementing and evaluating a THN program through a randomized controlled trial design that incorporates solutions for the barriers and knowledge gaps identified in this study.