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Abstract A1Targeting αvβ6-integrins using micellar nanoparticles for delivery of PNKP inhibitors to non small cell lung cancer (NSCLC)?Igor M. Paiva1, Sams Sadat1, Mohammad R. Vakili1, Marco Paladino3, Dennis G. Hall3, Michael Weinfeld2,? Afsaneh Lavasanifar1.?1Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, AB, Canada T6G 2E1 2Department of Oncology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada T6G 1Z2 3Department of Chemistry, Faculty of Science, University of Alberta, Edmonton, AB, Canada T6G 2G2?Purpose: In the present study we developed NSCLC-targeted nanoparticles encapsulating a relatively?potent novel PNKP inhibitor, namely A83B4C63, and assess the anticancer activity of this formulation?alone or in combination with irinotecan. Methods: Nanoparticles of poly(ethylene oxide)-poly(α-benzyl? carboxylate-ε-caprolactone) (PEO-PBCL) with maleimide group on their PEO segment were developed? and used for conjugation of H2009.1 peptide with a terminal cysteine group. Cy5.5-labelled?nanoparticles were used to evaluate cell uptake using flow cytometry. The cytotoxicity of A83B4C63 in?NSCLC cell lines expressing different levels of PTEN expression with and without irinotecan treatment?was assessed by MTS and colony-forming assays. Results: H2009.1-modified nanoparticles showed?higher interaction with NSCLC cells overexpressing αvβ6-integrin in comparison with their unmodified?counterparts. The PTEN-positive NSCLC cells (i.e., H1975 cells) did not show loss of viability upon?treatment with A83B4C63 monotherapy. The PTEN-deficient H1299 cells, on the other hand, showed less growth and colony formation following treatment with A83B4C63 monotherapy. Treatment with?A83B4C63 made both cells, more sensitive to Irinotecan. The sensitization effect of A83B4C63 upon?combination therapy with irinotecan was significantly enhanced for H1299 over H1975 cells. Conclusion: The results imply a potential for polymeric micellar formulations of A83B4C63 as mono-therapeutics in?NSCLC cells with low PTEN expression and/or as targeted sensitizers to topoisomerase I inhibitors?against NSCLC tumors.Abstract A2Panitumumab modified polymer-based nano-delivery system for targeting of non-small cell lung cancer (NSCLC)Nasim Sarrami, Igor Paiva, Afsaneh LavasanifarFaculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, AB, CanadaPurpose: Lung cancer is the leading cause of cancer deaths. Among lung cancer cases, non-small cell lung cancer (NSCLC) is more common. It is reported that 65-90% of NSCLC express EGFR and for this phenotype, monoclonal antibodies (mAb)s such as Panitumumab that target EGFR are treatment options. The purpose of this study is to prepare PEO-PBCL micelles modified on their surface with panitumumab and assess this delivery system for targeted drug delivery to NSCLC. Methods: Maleimide-PEO-PBCL was prepared and mixed with methoxy PEO-PBCL at 1:1 ratio. Both block copolymers and their mixture were self-assembled to nanostructures by a co-solvent evaporation method. Micellar size and polydispersity index (PDI), and morphology were assessed. Panitumumab was thiolated though reaction with 2-iminothiolane. Thiolated panitumumab was then reacted with maleimide micelles. This was followed by reaction with 2-mecrcaptoethanol to neutralize remaining free thiol groups on the antibody.? The obtained micelles were purified through elusion from Sepharose? CL-6B column by PBS. The eluted fractions were characterized by dynamic light scattering and absorption spectroscopy at 280 nm. Developed micelles will be loaded with a fluorescent dye and used to assess interaction with H1975 and H1299 NSCLC cells expressing different levels of EGFR. Results: The size distribution results reported one peak at 40.1 nm diameter with 100% intensity and PDI of 0.181 for plain micelles. For panitumumab micelles, one peak at 59.8 nm with 98.5% intensity and PDI of 0.335 was seen. The results of transmission electron microscopy pictures were in line with the results of size measurements by dynamic size scattering. The conjugation efficiency of panitumumab was ? 80% corresponding to a molar ratio of 1:100 of PEO-PBCL. Conclusions: The results show successful development of panitumumab attached micelles to targeted EGFR positive NSCLC.Abstract A3 (oral presentation, poster not judged)Nano-Encapsulation of Novel Inhibitors of ERCC1-XPF for Targeted Sensitization of Colorectal Cancer to Platinum-Based Chemotherapeutics?Parnian Mehinrad1, Sams M. A. Sadat1, Ahmed H. Elmenoufy3,6, Michael Weinfeld2,4, Feridoun Karimi-Busheri2, Frederick G. West3, Afsaneh Lavasanifar1,51 Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, AB, Canada2 Department of Oncology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada3 Department of Chemistry, Faculty of Science, University of Alberta, Edmonton, AB, Canada4 Department of Experimental Oncology, Cross Cancer Institute, Edmonton, AB, Canada5 Department of Chemical and Material Engineering, University of Alberta, Edmonton, AB, Canada6 Department of Pharmaceutical Chemistry, College of Pharmacy, Misr University for Science and Technology, P.O. Box 77, 6th of October City 12568, EgyptPurpose: The ERCC1-XPF nuclease is a hetero-dimeric enzyme that plays a role in nucleotide excision repair, double strand break repair, and inter-strand crosslink repair. Cells with disabled ERCC1-XPF are particularly sensitive to DNA damage by platinum-based drugs. Our research team has developed a novel potent inhibitor of ERCC1-XPF heterodimerization, namely A4 (IC50 0.33 ± 0.12 nM; KD 100 ± 5 nM). Systemic inhibition of ERCC1-XPF, however, can lead to the sensitization of normal cells as well as cancer cells to DNA damaging chemotherapeutics leading to detrimental side effects. For this reason, we propose to develop a nano-delivery system for the encapsulation of A4 to provide tumor targeted delivery and investigate the effect of free and nano-encapsulated A4 in sensitization of colorectal cancer cells to carboplatin. Methods: Three different self associating block copolymers, i.e., poly (ethylene oxide)-poly (-benzyl carboxylate--caprolactone) (PEO-PBCL) PEO-poly(-caprolactone) (PEO-PCL) and PEO-poly (D, L-lactide) (PEO-PDLA) were used for the encapsulation of A4. Developed nano-delivery systems were characterized for their average diameter, A4 encapsulation, and in vitro release. The cytotoxicity of A4 as free and encapsulated in PEO-PBCL nanoparticles (NP)s was assessed against HCT116, SW620, and HT29 cells alone or upon co-treatment with carboplatin using the MTT assay. Results: Among NP formulations of A4, PEO-PBCL provided the best physiochemical characteristics (average diameter of 45.19 ± 0.32 nm, PDI: 0.120 ± 0.008, 83.06 ± 5.8% encapsulation efficiency; < 50% drug release in 24 h). The results of cytotoxicity study showed a significant reduction in the viability of the HCT116 and SW620 cell line following co-treatment of 0.5 ?M of A4 (as free and particularly NP form) with carboplatin (25-100?M). The SW620 cell line was more sensitive to the effect of A4. Conclusions: The results indicate a potential for PEO-PBCL NP formulations of A4 for chemosensitization of CRC to platinum chemotherapeutics. Support: Alberta Cancer foundation?Abstract A4Liposome Encapsulating Polymeric Micelle (Lipocell): A Novel Nano-material for Enhanced Drug Delivery and TargetingSirazum Munira, Afsaneh LavasanifarUniversity of Alberta, Edmonton, Alberta, Canada.Purpose: Liposome and polymeric micelle are used to deliver hydrophilic drugs and hydrophobic drugs, respectively, but not efficient in delivering drugs with opposite characteristics. This research aims to develop a novel drug delivery system combining both liposome and polymeric micelle for the co-delivery of a hydrophilic and a hydrophobic drug, and to study different factors on the properties of liposomes loaded with polymeric micelle, namely Lipocells. Method: A total of nine di-block copolymers of varying degrees of polymerization (DP) were synthesized based on poly(ethylene glycol) (PEG) (DP= 12, 17, 45) and poly(?-caprolactone) (PCL) (DP= 7, 15, 23). Near-infrared (NIFR) fluorophore Cy 5.5 was attached at the end of the copolymers to make the self-associated micelles formed from these block copolymers traceable. Liposomes with different molar ratios of DSPC, DPPC, DSPE-PEG and cholesterol are being prepared. Polymeric micelles are loaded in liposomes following either incubation above the transition temperature of the liposome, hydration of the dried phospholipid film with the micelle suspension, or the freeze-thaw method. Effects of the molecular weights of the copolymers, their hydrophilic-lipophilic balance (HLB), transition temperatures of the phospholipids, and loading conditions on the size and morphology of hybrid lipid/polymer nanoparticles as well as level of polymer encapsulation within the liposomes will be assessed. Result: 1H NMR results showed the successful synthesis of PEO-PCL block copolymers at the 12-7, 12-15, 12-23, 17-7, 17-15, 17-23, 45-7, 45-15 and 45-23 DPs for PEG-PCL blocks. NIFR Cy5.5 was attached to selected polymer structures within this library successfully. The sizes of micelles were around 31 nm. Liposomes were prepared with sizes ranging from 84 nm to 250 nm. Conclusion: The results so far showed successful labeling of PEO-PCL polymers with Cy5.5 dye at different DPs of PEG and PCL. Support: This research is funded by NSERC Discovery and NSERC CREATE PoND.Abstract A5 (oral presentation, poster not judged)BCS-based biowaiver using biphasic dissolution test?Daniela Amaral Silva1, Katherine Curo Melo1,2, Neal M. Davies1, Nadia Bou-Chacra2, Humberto G. Ferraz2, Raimar L?benberg1.?1?Faculty of Pharmacy & Pharmaceutical Sciences, University of Alberta, Edmonton, AB T6G 2E1, Canada;?2 Faculty of Pharmaceutical Sciences, University of Sao Paulo, Sao Paulo 05508-000, Brazil.Purpose: Biowaivers based on BCS class can be used to establish therapeutic equivalence based on dissolution tests. Such tests are used as a surrogate to determine if two pharmaceutical equivalent products are interchangeable/bioequivalent. The objective of the present study was to compare a biphasic dissolution system with compendial methods in examining the in vitro performance of widely used drug products. We hypothesized that where in vitro equivalence was not achieved in compendial methods, the partitioning profile to the organic phase in the biphasic system could signal bioequivalence among the drug products and comparator pharmaceutical product (CPP). Methods: Five commercial metronidazole tablets were tested in compendial Simulated Intestinal Fluid (SIF), as well as in physiological buffer capacity (5mM phosphate buffer at pH 6.8, 900 mL). The tablets were also tested in a biphasic dissolution system in which the aqueous layer was composed of 200 mL of 5 mM phosphate buffer (pH 6.8) with 100 mL of n-octanol on top. Results: None of the tested metronidazole products were in vitro equivalent to the CPP or to other manufacturers in compendial buffer. The tested metronidazole products followed a similar pattern than that obtained in the compendial buffer in the aqueous phase of the biphasic system. However, this was not the case for the organic phase partition profiles. All the tested products had a good correlation to the CPP, which could indicate in vitro equivalence between these products. This could potentially allow for a biowaiver application. Conclusion: The application of biphasic dissolution to highly soluble drugs might be beneficial to estimate the product’s in vivo behavior.Abstract A6 (oral presentation, poster not judged)In vitro and in vivo performance of enteric coated formulationsDaniela Amaral Silvaa, Neal M. Daviesa, Michael R. Doschaka, JozefAl-Gousousb, Nadia Bou-Chacrac, Raimar L?benberga.a Faculty of Pharmacy & Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta, Canadab Johannes Gutenberg Universit?t Mainz, Mainz, Germanyc Faculty of Pharmaceutical Sciences, University of Sao Paulo, Sao Paulo, SP, BrazilPurpose: Over the last 70 years several cases of in vivo failure of enteric coated (EC) formulations have been reported. The observed failures seem to be due to the slower than expected in vivo performance of EC products. Upon reaching the intestinal lumen, the dosage form is exposed to a bicarbonate buffered environment at much lower buffering capacity compared to those applied in compendial phosphate buffers. Hence, there is an urgent need to understand the behavior of EC products in bicarbonate buffer (BCB) and to revaluate the current dissolution methods used for such products. We hypothesized that the performance of EC products in BCB would be different compared with compendial phosphate buffer, giving more physiological insight with regards to the observed clinical failures. Methods: The current study mechanistically investigated the performance of five EC products available in the Canadian market. The evaluated parameters were the buffer system (bicarbonate buffer vs. phosphate buffer), buffer capacity, medium pH and formulations parameters (coat material and thickness). All dissolution tests were performed using an USP apparatus 2, 900 mL dissolution media, 75 rpm rotation speed and temperature set at 37.0°C. The coat thickness was assessed using Micro-CT scan. Results: All formulations displayed a fast release in phosphate buffer and complied with the compendial performance specifications. On the other hand, they all had a much slower drug release in bicarbonate buffer and failed the USP acceptance criteria. Also, the nature of the drug (acid vs base) impacted the dissolution behavior in BCB more than the coat thickness. Conclusion: This pilot study indicates that compendial dissolution test for enteric coated tablets lacks physiological relevance and it needs to be reconsidered. Bicarbonate buffer seems to be a clinically relevant dissolution medium for EC products. Thus, an in vivo relevant performance method for EC products is needed.?Reference:?D.A.?Silva,?N.M.?Davies,?M.R.?Doschak,?J.?Al-Gousous,?N.?Bou-Chacra,?R.L?benberg. Mechanistic understanding of underperforming enteric coated products: opportunities to add clinical relevance to the dissolution test. J. Contr. Release,?325?(2020), pp.?323-334Abstract B1 (oral presentation, poster not judged)An analytical method for quantification of cycloheximide in blood fluids of rats using liquid?chromatography with tandem mass spectrometry detection?Hamdah Al Nebaihi and Dion Brocks?Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, AB, Canada?Cycloheximide (CHX) is commonly used for in vitro studies for inhibition of protein synthesis. It has also?been intraperitoneally dosed to rats to block the in vivo formation of intestinal lymph in drug absorption?studies. However it can be toxic, and its pharmacokinetic behaviour is unknown. Purpose: To develop a?sensitive and specific method for CHX in rat blood fluids using liquid chromatography with tandem mass?spectrometry detection. Methods: CHX and colchicine (internal standard, IS) were extracted from 0.1 mL rat blood or plasma using 3 mL of n-hexane:dichloromethane:isopropanol (300:150:15). The mixture was?vortex-mixed then centrifuged for 10 min. The organic phase was transferred to new glass tubes and?evaporated in vacuo. The dried samples were reconstituted in methanol:water (10:90) and injected into the? instrument. The mobile phase was a combination of 10 mM ammonium acetate:methanol (15:85). The?detector was operated under positive multiple reaction monitoring mode for analyte. Compound-dependent?parameters and instrument conditions were optimized automatically using LabSolutions software and further optimized manually. To test applicability, blood was assayed from male Sprague-Dawley rats given?CHX 5 mg/kg orally. Results: The assay exhibited excellent linearity in peak area response over the?concentration ranges of 4-1000 ng CHX /mL blood fluid. The intra- and inter-run coefficients of variation?and percent error were ≤13% and the recovery was ≥97%. All samples tested for stability were ±15% of the nominal concentration. CHX concentration was quantifiable up to 24 h after oral doses of 5 mg/kg to?adult male Sprague-Dawley rats. Conclusion: The method displayed high calibers of sensitivity and?selectivity for detecting very low concentrations of CHX in rats.Abstract B2Determination of ropivacaine and its major metabolite pipecoloxylidide in rat plasma using high?performance liquid chromatography (HPLC)??Shamima Parvin, Dion R. Brocks??Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, AB, Canada??Purpose: To develop a selective and sensitive HPLC method for the determination of ropivacaine and its?major metabolite pipecoloxylidide (PPX) in rat plasma. Methods: The extraction of ropivacaine, PPX and? racemic bupivacaine (internal standard, IS) from rat plasma (0.1mL) was achieved using 3ml diethyl ether? under alkaline conditions using 0.08 ml NaOH. After liquid–liquid extraction, the separation of analytes?was accomplished using reverse phase chromatography. The mobile phase, a combination of acetonitrile and monobasic potassium phosphate solution (25 mM KH2PO4-3 mM sulfuric acid-3.6 mM triethylamine) in a ration of 20:80 (v/v) was pumped isocratically through a C18 analytical column. The UV wavelength?was at 237 nm for PPX, and subsequently changed to 210 nm for ropivacaine and the IS. Results: The assay?exhibited excellent linearity (r2 > 0.999) in peak response over the concentration ranges of 20–2500 ng/mL?ropivacaine and PPX in rat plasma. The intra- and inter-day coefficients of variation of ropivacaine in the?plasma were <7% at the lowest, and <15% at other concentrations. The percent error values were less than?20%. On the other hand, for the PPX, the intra-and-inter day coefficients of variance was < 3% for the lowest concentration and <9% for the other concentrations, and the mean errors were <20%. Conclusions:? In conclusion, the assay was sensitive and selective to use in measuring concentrations of ropivacaine and?PPX in rat plasma.?Abstract B3 (oral presentation, poster not judged)Population Pharmacokinetics of Mycophenolic Acid in Pediatric Patients: A Literature ReviewYan Rong, Heajin Jun, Tony KL KiangFaculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, AB, CanadaPurpose: Mycophenolic acid (MPA) is an immunosuppressant commonly prescribed in pediatric kidney transplant patients and sometimes used in other indications, e.g. glomerulonephritis, lupus nephritis, nephrotic syndrome, liver transplant, hematopoietic stem cell transplant, and autoimmune disorders. Population pharmaco-kinetic/-dynamic modeling has been frequently used to characterize the fixed, random, and covariate effects of MPA in adult patients. However, MPA population pharmacokinetic data in the pediatric population have not been systematically summarized. The objective of this review was to provide an up-to-date critique of currently available pediatric MPA population pharmacokinetic models, with emphases on modelling techniques, pharmacological findings, and clinical relevance. Methods: PubMed and EMBASE were searched from inception of database to May, 2020. The inclusion criteria were: 1) papers were peer-reviewed; 2) papers reported population pharmacokinetic model of MPA; and 3) papers recruited subjects with mean or median age <18-year old. Results: A total of 11 studies have been identified representing kidney transplant (n=4), liver transplant (n=1), hematopoietic stem cell transplant (n=1), idiopathic nephrotic syndrome (n=2), systemic lupus erythematosus (n=2), and a combined population consisted of kidney, liver, and haematopoietic stem cell transplant patients (n=1). Critical analyses were provided in the context of MPA absorption, distribution, metabolism, excretion, and bioavailability in this database. Comparisons to adult patients were also provided. With respect to clinical utility, Bayesian estimation models (n=6) with acceptable accuracy and precision for MPA exposure determination have also been identified and systematically evaluated. Overall, our analyses have identified unique features of MPA clinical pharmacology in the pediatric population, while recognizing several gaps that still warrant further investigations. Conclusions: This review can be used by pharmacologists and clinicians for improving MPA pharmacokinetic-pharmacodynamic modeling and patient care. (This work has been published: Rong Y, Jun H, Kiang TKL. Population pharmacokinetics of mycophenolic acid in paediatric patients. Br J Clin Pharmacol. 2021 Apr;87(4):1730-1757).Abstract C1A Comparison of Angiostatin Levels in Hospitalized Covid-19 Patients: Preliminary Results Aleksandra Franczak1, Paul Jurasz1,2?1Department of Pharmacology, Faculty of Medicine and Dentistry, University of?Alberta, Edmonton, AB, Canada?2Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta,??Edmonton, AB, Canada??Purpose: Infection with the SARS-CoV-2 virus and subsequent Covid-19 disease have overwhelmed health systems around the world. Severe Covid-19 is associated with an increased risk of thrombotic complications including venous thromboembolism, disseminated intravascular coagulation and stroke. A potential biomarker that may contribute to Covid-19 etiology, disease progression and outcome is the blood protein angiostatin. Angiostatin is a platelet-derived break-down product of plasmin(ogen) with pro-apoptotic and necrotic properties under conditions of hypoxia and acidosis, conditions often? associated with SARS-CoV-2 infections. Moreover, unbiased weighted gene correlation network?analysis of transcriptional responses in mice following SARS-CoV infection demonstrated that an?enhanced urokinase-plasminogen pathway regulates lethal vs. sublethal infection. Therefore, the aim?of the study was to determine if there is a difference in plasma angiostatin levels between hospitalized?Covid-19 survivors vs. non-survivors, as its elevation can indicate fibrinolysis shut down, thrombosis,?and potential contribution to organ failure. Methods: The study was approved by UAlberta Health?Research Ethics Board and the Northern Alberta Clinical Trials and Research Centre. Archived frozen age- and sex-matched plasma samples (nsurvivors = 65, nnon-survivors = 64) were obtained from Alberta?Precision Laboratories. Angiostatin and plasminogen were quantified by immunoblot and statistical?analysis was carried out using the Mann-Whitney U test. Results: 64.3% of samples were from males.? The mean age was 75 ± 13 yrs. No significant difference was observed in angiostatin concentration?between survivors and non-survivors (135.9 ± 5.2 and 141.6 ± 6.0 μg/ml, respectively; p = 0.659).? Compared to survivors, non-survivors had a significantly higher angiostatin-to-plasminogen ratio?(0.067 ± 0.003 vs 0.088 ± 0.006; p = 0.016). There were no associations between?angiostatin/angiostatin-to-plasminogen and age. Conclusions: The results suggest that an elevated?angiostatin-to-plasminogen ratio is associated with fatal outcome in Covid-19. The potential negative consequences of angiostatin action may be aggravated by low fibrinolytic activity.Abstract C2Dichloroacetate Increases Myocardial Pyruvate Dehydrogenase ActivityWithout Improving Cardiac Abnormalities in a Mouse Model of Human Barth Syndrome?Amanda A. Greenwell1,2,3, Christina T. Saed1,2,3, Keshav Gopal1,2,3,Seyed Amirhossein Tabatabaei Dakhili1,2,3, Jennifer Kruger4, Farah Eaton1,2,3, John R. Ussher1,2,31Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, AB, Canada2Cardiovascular Research Centre, University of Alberta, Edmonton, AB, Canada3Women and Children’s Health Research Institute, University of Alberta, Edmonton, AB, Canada4Health Sciences Laboratory Animal Services, University of Alberta, Edmonton, AB, Canada?Purpose: Heart failure presents as the leading cause of infant mortality in individuals with Barth syndrome (BTHS), a rare genetic disorder first described by Peter Barth and colleagues in 1983. The causative mutation underlying the BTHS phenotype has been mapped to the tafazzin (TAZ) gene, which encodes for a phospholipid transacylase critical for the remodelling of the mitochondrial phospholipid, cardiolipin. Perturbations in myocardial energy metabolism may contribute to the development and progression of cardiomyopathy in BTHS. Given that cardiac glucose oxidation is impaired in a mouse model of human BTHS, we aimed to assess whether pharmacological enhancement of glucose oxidation via dichloroacetate (DCA) treatment could mitigate BTHS-related cardiac dysfunction. Methods: DCA was added to the drinking water (84 mM) of 6-week-old mice with experimental BTHS (doxycycline inducible Taz knockdown (TazKD) mice) for 6 weeks. In vivo cardiac function was assessed through ultrasound echocardiography prior to and following the treatment period. Hearts were extracted from TazKD mice and their wild-type (WT) littermates for protein expression profiling at the end of the treatment protocol.? Results: Prior to treatment at 4-5 weeks of age, TazKD mice exhibited hypertrophic cardiomyopathy as demonstrated by an increase in the ratio between left ventricular (LV) anterior and posterior wall thickness during diastole and lean body mass. TazKD mice also exhibited impaired LV systolic and diastolic volumes. Treatment with DCA significantly decreased inhibitory phosphorylation of pyruvate dehydrogenase, the rate-limiting enzyme of glucose oxidation, in ventricular extracts from TazKD mice compared to WT littermates, thus indicative of increased myocardial glucose oxidation. However, despite improvement of cardiac energy metabolism, DCA-treated TazKD mice exhibited similar cardiac structural and functional abnormalities as control-treated TazKD mice.? Conclusions: Our findings suggest that optimization of myocardial glucose oxidation may not be a feasible strategy in BTHS for improving its associated cardiomyopathy.?Abstract C3Platelets Stimulate Programmed Death-Ligand 1 Expression by Cancer Cells:? Inhibition by Anti-Platelet Drugs?A. Asgari, G. Lesyk, E. Poitras, N. Govindasamy, K. Terry, Rachel To, V. Back, J.K. Rudzinski, J.D. Lewis, and P.Jurasz?Introduction: Platelets help facilitate hematogenous metastasis in part by promoting cancer cell immunoevasion, although our understanding of platelet function in modulating the adaptive immune system in cancer is limited. A major negative regulator of the adaptive response is the?immune checkpoint protein Programmed Death Ligand 1 (PD-L1).??Aim: As platelets secrete factors that may increase PD-L1 expression, we investigated whether?they up-regulate cancer cell PD-L1, thus promoting immunoevasion, and whether common anti platelet drugs inhibit this process.?Methods: Platelets were isolated from human volunteers. A549 lung, PD-L1 null A549, and 786- O renal cancer cells were incubated with and without platelets, and cancer cell PD-L1 expression?was measured by qPCR and flow cytometry. Additionally, platelet-cancer cell incubations were?performed in the presence of common anti-platelet drugs, and with growth factor neutralizing?antibodies. Following incubation with platelets, A549 cells were co-cultured with Jurkat cells and?interleukin-2 (IL-2) levels were measured by flow cytometry as a marker of T-cell activation. Results: Platelets increased PD-L1 mRNA and surface protein expression by A549 and 786-0?cells. Combined neutralization of VEGF and PDGF prevented the platelet-induced up-regulation?of PD-L1 by A549 cells, as did the anti-platelet drug eptifibatide. A549 incubated with platelets?demonstrated a reduced ability to activate Jurkat cells and this effect was reversed by eptifibatide. Conclusion: As platelets promote immunoevasion of the adaptive immune response by?increasing cancer cell PD-L1 expression and as anti-platelet drugs prevent this immunoevasive?response, the investigation of anti-platelet drugs as adjuvant therapy to immune checkpoint inhibitors may be warranted in the treatment of cancer.?Funding Source: This work was funded by grants from the Canadian Institutes of Health?Research MOP-130289 and MOP-162128 to P.J.?Conflict of Interest: The authors have no conflicts of interest to declare.?- Approval for the study was obtained from the University of Alberta Human Research Ethics?Board.Abstract C4Assessing the chemo-sensitization of colorectal cancer cells by inhibitors of ERCC1-XPF or PNKPBahareh Laribi1, Sams Sadat1, Marco Paladino2, Dennis Hall2, Fredrick West2, Michael Weinfeld3, Afsaneh Lavasanifar11Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, AB, Canada2 Department of Chemistry, Faculty of Science, University of Alberta, Edmonton, AB, Canada3Department of Oncology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, CanadaPurpose: Chemotherapeutics causing DNA damage, e.g., oxaliplatin (OXP) and irinotecan (IRI), are important treatment options in colorectal cancer (CRC). However, the high capacity of the cells in DNA repair may make cancer cells resistant to these anti-cancer agents. Our research team has identified inhibitors of two DNA repair enzymes, namely PNKP and ERCC1-XPF, that are expected to sensitize cancer cells to the topoisomerase I inhibitors and platinum-based chemotherapeutics, respectively. In this study we investigated whether A83B4C63 (an inhibitor of PNKP) and Q4 (an inhibitor of ERCC1-XPF) can sensitize CT26 murine CRC cells to IRI and OXP or Doxorubicin (DOX), respectively. Methods: In vitro cytotoxicity of DOX, OXP, IRI, A83B4C63 and Q4 alone or DOX/Q4, OXP/Q4, and A83B4C63/IRI combination was assessed in CT26 cells using MTT assay. Dose-response curve and IC50 values were calculated by Graphpad prism software. The synergism between combination treatments were determined by calculation of combination index (CI) using CompuSyn or Combenefit software. Results: Treatment of CT26 cells with Q4 (15 μM)/OXP (0.08 μM) combination for 48 h resulted in a significant reduction in cell viability compared to treatment with each compound alone. The CI for combinational therapy was 0.44 which indicates synergistic effect. The CI value for Q4 (15 μM)/ DOX (0.008 μM) combination was 0.35 indicating synergistic effects. The CI for A83B4C63 (2.5 μM)/IRI (30 μM) combination was 0.87 in this cell line, again pointing to a synergistic effect. The highest synergy was observed at molar ratios of 2:3 and 1:5 for combinations of OXP/Q4 and A83B4C63/IRI, respectively, based on the Bliss model in Combenefit software. Conclusions: Our preliminary findings indicate that A83B4C63 and Q4 can act as chemosensitizer for IRI and OXP in CRC, respectively, and show synergistic effect at certain molar ratios of chemosensitizer to chemotherapeutic agent.Abstract C5Investigating whether the anti-anginal drug ranolazine mitigates non-alcoholic fatty liver disease associated with type 2 diabetes?Christina T. Saed1,2,3, Amanda A. Greenwell1,2,3, Seyed Amirhossein Tabatabaei Dakhili1,2,3, Keshav? Gopal1,2,3, Farah Eaton 1,2,3, John R. Ussher1,2,3?1Faculty of Pharmacy and Pharmaceutical Sciences.?2Alberta Diabetes Institute.?3Cardiovascular Research Centre.?Purpose: Non-alcoholic fatty liver disease (NAFLD) is defined as the presence of excess fat in the liver of individuals that are not drinking alcohol. NAFLD is a devastating disease that greatly increases a person’s?risk for both diabetes and cardiovascular disease. Unfortunately, there is no treatment for this devastating?disease. Nearly 7 million people in Canada suffer from NALFD, and it is therefore of great importance that?we better understand its pathology so that we can develop potential pharmacotherapies. Of interest, we have?shown that ranolazine, a second-line agent for angina, can also mitigate NAFLD in obesity. Our aim was to determine whether ranolazine has these same actions in NAFLD associated with type 2 diabetes (T2D).? Methods: T2D was induced in 8-week-old male C57BL/6J mice (Jackson Laboratories) by 12-weeks of?high-fat diet supplementation with a single low-dose injection of streptozotocin [75 mg/kg] at 4-weeks.? Lean control mice were fed a low-fat diet for 12 weeks. At 8-weeks, lean and T2D mice treated with either?vehicle or ranolazine (50 mg/kg) once daily via oral gavage for 30-days. Following which we assessed?hepatic steatosis and glucose homeostasis. Results: Contrasting our previous findings in obesity-related?NAFLD, ranolazine treatment did not improve glycemia or hepatic steatosis in T2D-related NAFLD, as indicated by similar glucose tolerance and hepatic triacylglycerol content as their vehicle treated T2D counterparts. Conclusion: Our results suggest that ranolazine’s potential utility as a therapy for NAFLD?may be limited to obese individuals but not those with T2D, and it will be important for future research to?identify the reasons explaining these discrepancies.Abstract C6 (oral presentation, poster not judged)Genetic deletion of soluble epoxide hydrolase preserves cardiac function and limits inflammation in?acute lipopolysaccharide injury?Deanna K. Sosnowski1, Lockhart Jamieson1, Artiom Gruzdev2, Darryl C. Zeldin2, John M. Seubert1, 3 ?1Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta, Canada,? 2National Institute of Environmental Health Sciences, NIH, RTP, NC, USA, 3Department of? Pharmacology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada?Purpose: Acute inflammatory syndromes, such as endotoxemia, elicit detrimental multi-organ responses resulting in cardiac dysfunction often leading to death. Emerging evidence suggests epoxylipids can exert cardioprotective effects by modulating the NLRP3 inflammasome pathway. However, these beneficial?epoxylipids may be metabolized by soluble epoxide hydrolase (sEH). This study investigated whether cardiomyocyte-specific sEH-knockdown can attenuate inflammation and cardiac dysfunction in a model?of acute lipopolysaccharide (LPS) injury via modulation of the NLRP3 inflammasome pathway. Methods: Cardiomyocyte-targeted sEH-knockdown mice were produced by crossing Ephx2-floxed and Cre recombinase expressing mice. Male sEHFl/Fl (knockdown) and sEH+/+ (Cre control) mice were given tamoxifen (45 mg/kg, 6 i.p injections over 8 days) 5 weeks prior to LPS injection (10mg/kg, i.p.). Wild type (WT) and global sEH-null mice were subjected to LPS treatment as comparators. Echocardiography?was conducted pre-injection and 6 or 24hr post-LPS. Plasma cytokine levels were determined with multi plex assays. Neonatal rat cardiomyocytes were treated with LPS (1?g/mL), 19, 20- epoxydocosapentaenoic acid (EDP, 1?M) or sEH inhibitor, trans-4-[4-(3-adamantan-1-yl-ureido)- cyclohexyloxy]-benzoic acid (tAUCB, 10?M), for 6hr. NLRP3 and pro-IL-1β expression was assessed? using Western immunoblotting. Results: sEH deletion preserved cardiac function 24hr post-LPS exposure indicating an early decline at 6hr which was recovered in sEHFl/Fl and sEH-null but not WT mice. Plasma levels of pro-inflammatory cytokines post-LPS exposure were attenuated in mice lacking sEH. Cardiomyocytes treated with LPS had increased NLRP3 inflammasome and pro-IL-1β expression, which was not attenuated by co-treatment with EDP or tAUCB. However, plasma levels of IL-1β were reduced?in both sEHFl/Fl and global sEH-null mice following LPS exposure. Conclusion: Deletion of sEH protects?cardiac function and limits pro-inflammatory responses post-LPS exposure. Preliminary data suggest?epoxylipids do not prevent the formation of the inflammasome complex in cardiomyocytes but may impact downstream effectors. Thus, limiting the NLRP3 inflammasome cascade to reduce LPS-induced?cardiac and inflammatory injury. Support: NSERCAbstract C7Studying the Aryl Hydrocarbon Receptor (AhR) ligand-binding domain in a computational?approach??Farag Mosa1, Ayman El-Kadi1 and Khaled Barakat1?1Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, AB, Canada?Purpose: The aryl hydrocarbon receptor (AhR) is a ligand-activated transcriptional factor.? It regulates various genes' expression and plays a pathophysiological function in numerous?diseases, including cancer progression and cardiovascular diseases. The full-length AhR encompasses various domains, including bHLH, PAS A, PAS B & transactivation domains. The PAS B domain plays a crucial role in regulating the activity of AhR b?interacting with small molecules through its ligand-binding domain (LBD). Here we used computational modelling to study LBD structure and understand how AhR ligands interact?with residues in LBD. Methods: The crystal structure of the PAS B domain from mouse?and human is not available; hence, we used different homology modelling algorithms to?build their 3-dimensional (3-D) structures. The models were then refined using molecular dynamics (MD) simulations, followed by data mining and clustering analysis to extract their most dominant conformations. Compounds were then tested for their binding to the LBD using molecular docking simulations. Results: Structures for human hypoxia inducible factors (HIF-2α) and (HIF-1α) were used as templates to build different models?for the PAS B domain. HIF-1/2α showed ~31% sequence similarities to PAS B. 17? different PAS B models were built using different homology modelling algorithms and? were evaluated using the Ramachandran plot. The most dominant conformations from human and mouse models represent 83 % and 65%, respectively. Two models showed?acceptable scores and were used to test Several AhR modulators' binding containing Dioxin?and CH-223191 in the AhR- LBD. Conclusions: Molecular docking predicts how AhR LBD interacts with AhR modulators in their binding pocket and identifies the important residues that interact with the different AhR ligands. Our study can advance the?development of new AhR antagonists for the treatment of cancer and cardiovascular diseases.Abstract C8Identifying Novel Allosteric Sites and Analyzing the Hydrogen Bond Networks and Correlated Dynamics in the SARS-CoV-2 RNA Polymerase?Khaled Barakat, Marawan Ahmed, Yasser Tabana, Minwoo Ha?Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, AB, Canada?Purpose: SARS-CoV-2 is the causative agent of COVID-19. Inhibition of the SARS-CoV-2 replicase machinery has been proven recently as a promising approach in combating the virus. Despite this, there are still several aspects related to the structure, function, and dynamics of the CoV-2 polymerase that need to be addressed. This study aims to further characterize the polymerase complex using in-silico analysis. Methods: Recently resolved SARS-CoV-2 polymerase cryo-EM structures were used as a starting point to construct the RdRp assembly. Active site-bound systems of the polymerase complex were constructed representing the incorporation of RNA nucleotides in the nascent chain. After adjusting for physiological conditions, classical MD simulations (~100 ns) and accelerated MD simulations were conducted. Statistical analysis of the collected coordinates was done to characterize the conformational dynamics, hydrogen bonding, water hydration shell, free energy, and druggability of the systems. Results: This is the first study to build the SARS-CoV-2 replicase complex, providing deep insight into its dynamicity. Our findings provide a detailed analysis of the hydrogen bond networks at various parts of the polymerase structure and suggest possible nucleotide substitutions that can be tolerated by the polymerase complex. Three druggable allosteric sites within the nsp12 RdRp that can be targeted by small molecule inhibitors are reported. Our correlated motion analysis shows that the dynamics within one of the newly identified sites are linked to the active site, indicating that targeting this site can significantly impact the catalytic activity of the SARS-CoV-2 polymerase.Conclusions: MD simulations and subsequent state-of-the-art data analysis were used to study the properties of the various polymerase subdomains under physiological conditions. This study provides a rational basis for the discovery of new drug interventions against SARS-CoV-2. Support: We acknowledge the NSERC Discovery grant, funding from the Alberta Cancer Foundation, and computing facilities provided by Compute Canada.Abstract C916-(R/S)-Hydroxyeicosatetraenoic acids (HETEs) induce human CYP1B1 through transcriptional?and allosteric mechanisms?Rahmat Hidayat, Sherif M Shoieb, Mahmoud A. El-Ghiaty, Mohammed A. Alqahtani, Ayman O.S. El Kadi?Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, AB, Canada?Purpose: Several reports from our laboratory and others demonstrated the direct contribution of cytochrome P450 1B1 (CYP1B1) enzyme and its associated cardiotoxic mid-chain, hydroxyeicosatetraenoic acid (HETEs) metabolites in the development of cardiac hypertrophy and heart failure. Besides, we have?previously reported that the subterminal HETE, 16-HETE, confer cardioprotection against cardiovascular?diseases. Therefore, we investigated the effect of 16-(R/S)-HETE on CYP1B1 at mRNA, protein, and catalytic activity levels. Methods: We incubated human fetal ventricular cardiomyocytes (RL-14) cell lines,? recombinant human CYP1B1, and human liver microsomes in the presence and the absence of 16-(R/S)-?HETE. Thereafter, real-time PCR, Western blot analysis, and CYP1B1-dependant EROD Assay were?performed to determine the level of CYP1B1 mRNA, protein, and catalytic activity levels, respectively. To?examine whether a similar effect will happen with other CYP that is regulated by the same transcription?factors, aryl hydrocarbon receptor, we tested the effect of 16-(R/S)-HETE on CYP1A2 catalytic activity in?human recombinant CYP1A2 using MROD assay. Results: Our results showed that 16-HETE significantly upregulated CYP1B1 at mRNA and protein levels in RL-14 cell lines. Surprisingly, 16-(R/S)-HETE significantly increased CYP1B1 activity in RL-14 cells, recombinant human CYP1B1, and human liver?microsomes. On the contrary, 16-(R/S)-HETE significantly inhibited CYP1A2 catalytic activity mediated?by the recombinant human CYP1A2 and human liver microsomes. Conclusions: Our study provides the first evidence that 16-(R/S)-HETE increases CYP1B1 gene expression, protein at least in part, through a?transcriptional mechanism, and CYP1B1 catalytic activity in recombinant and liver microsome possibly through an allosteric mechanism. Supports: This work was supported by a grant from the Canadian? Institutes of Health Research [CIHR PS 168846] to A.O.S.E. R.H is the recipient of the Indonesia? Endowment Fund for Education Scholarship.?Abstract C10 (oral presentation, poster not judged)Fishing the target of a potent small molecule immunomodulator for cancer?immunotherapy?Yasser Tabana1, Dinesh Babu1, Marawan Ahmed1, Garett Dunsmore3, Shima Shahbaz2, R? Piragasam4, Richard Fahlman4, Tae Chul moon1, Shokrollah Elahi2,3, Arno Siraki1, Khaled? Barakat1,*?1Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, AB,?Canada?2Department of Dentistry, 3Department of Medical Microbiology and Immunology, University of?Alberta, Edmonton, AB, Canada?4Department of Biochemistry, Faculty of Medicine & Dentistry, University of Alberta,?Edmonton, AB T6G 2H7, Canada.?*Corresponding author: Dr. Khaled Barakat, kbarakat@ualberta.ca?Background: Cancer immunotherapy has emerged as the fourth pillar of cancer treatment along?with surgery, radiation, and chemotherapy. Immunotherapeutic approaches utilize components of?a patient’s own immune system to selectively target cancer cells. Our lab has identified a small molecule (Compound-A) that boost T-cell proliferation and cytokine production. A comprehensive investigation is ongoing to identify and validate the target(s) and pathway(s) of?this compound that contribute to its activity on T-cell proliferation and cytokine production. ?Methods: Effect of compound-A on its ability to produce cytokines (IFNγ and IL-2) and increase the T-cell proliferation in peripheral blood mononuclear cell (PBMCs) were measured by ELISA and CFSE staining, respectively. The genomic and proteomic changes were analyzed using RNA sequencing and label-free quantitative proteomics. The microsomal stability, and toxicity of?Compound-A against PBMCs were determined. Identification of the possible target(s) using pull?down was also conducted. Results: Compound-A increased T-cell proliferation and IL-2 secretion.? Compound-A showed that 59.6% was remaining after 60 min of incubation with human hepatic microsomes. The CC50 observed against PBMCs was 20.6 ?M. After treating PBMCs with Compound-A for 12 h, a total of 792 differentially expressed genes (DEGs) were identified?including 377 upregulated and 415 downregulated genes. Also, a total of 863 DEGs were identified?after 24 h treatment, including 444 upregulated and 419 downregulated genes. GO and genome pathway analysis showed that these DEGs were enriched in signaling pathways associated with?response to IFNγ. Plausible targets were obtained by pull-down assay, although they need further confirmation. Conclusion: Our study showed the immunostimulatory activities of Compound-A?with possible immunological targets. A future direction will be to validate the molecular targets?responsible for its immunological activities.Abstract C11Cardiac cytochrome P450-derived oxylipins and the NLRP3 inflammasome in macrophage-like cell?models??Robert Valencia1, John M. Seubert1,2?1Department of Pharmacology, Faculty of Medicine and Dentistry, 2Faculty of Pharmacy and?Pharmaceutical Sciences, University of Alberta, Edmonton, AB, Canada?Purpose: Macrophages are major propagators of inflammation during cardiac homeostasis and disease.? The NLRP3 inflammasome mediates inflammatory cytokine secretion and lytic cell death in response to?mitochondrial damage, oxidative stress, and other stimuli associated with cardiac insults. N-3 and -6?polyunsaturated fatty acids (PUFAs) are metabolized by cytochrome-P450 (CYP) epoxygenases to numerous lipid mediators which are readily converted to vicinal diols by soluble epoxide hydrolase (sEH). The docosahexaenoic acid (DHA) epoxide, 19,20-epoxydocosapentaenoic acid (19,20-EDP) and? linoleic acid (LA) vicinal diol, 12,13-dihydroxyoctadecanoic acid (12,13-DiHOME) exhibit protective? and deleterious effects on mitochondria, respectively. We hypothesize that these metabolites differentially?modulate macrophage mitochondria impacting pro-inflammatory signaling. The aim of this study is to investigate mechanistic insights into how CYP-derived PUFA metabolites regulate cardiac homeostasis and disease through effects in non-parenchymal cardiac cells such as macrophages. Methods: We utilized? macrophage-like cell models (RAW 264.7, THP-1) to investigate the roles of 19,20-EDP and 12,13- DiHOME in NLRP3 inflammasome priming and activation and prototypical “M1'' macrophage? polarization. Neonatal rat cardiomyocytes (NRCMs) were used in a macrophage-conditioned media?(MCM) model. We assessed GPR120, a fatty acid receptor with anti-inflammatory and NLRP3-inhibiting?activity. Results: Preliminary data indicate 12,13-DiHOME exacerbates pro-IL-1beta upregulation in? lipopolysaccharide (LPS)-induced RAW 264.7 or M1-polarized THP-1 macrophages. NLRP3?upregulation in MCM-treated NCRMs was mildly but significantly reduced by pre-treating macrophages?with 19,20-EDP. LPS modulated GPR120 levels/localization in macrophages. Conclusions: Early data?suggests these oxylipins differentially affect NLRP3 inflammasome priming and activation and GPR120?is dynamically regulated following LPS treatment in macrophages.?Abstract C12 (oral presentation, poster not judged)Repurposing diphenylbutylpiperidines for the potential treatment of type 2 diabetesSeyed Amirhossein Tabatabaei Dakhili1,2,3, Amanda A. Greenwell1,2,3, Christina T. Saed1,2,3,Rami Al Batran1,2,3, Jadin J. Chahade1,2,3, Keshav Gopal1,2,3, Farah Eaton1,2,3, John R. Ussher1,2,31Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, AB, Canada2Alberta Diabetes Institute, University of Alberta, Edmonton, AB, Canada3Cardiovascular Research Centre, University of Alberta, Edmonton, AB, CanadaPurpose: T2D is a tremendous global health problem that nearly 400 million people worldwide are living with, including >2.5 million people specifically within Canada, mostly attributed to underlying obesity. Despite the significant progress in the understanding of T2D pathophysiology and the introduction of new treatment options, the disease is still considered a challenge to effectively treat. Hence, more effective and safer treatments are critical for improving the hyperglycemic profile and quality of life in patients suffering from T2D. We previously demonstrated that pimozide, a diphenylbutylpiperidine (DPB) based antipsychotic agent, can protect against obesity-induced hyperglycemia by inhibiting a key enzyme in ketone body oxidation known as succinyl CoA:3-ketoacid CoA transferase (SCOT). Follow-up in silico molecular modeling determined that a wide range of DPBs can theoretically inhibit SCOT activity. Hence, our goal was to determine whether DPBs, in general, can be repurposed for the treatment of T2D. Methods:?8-week-old male C57BL/6J mice (Jackson Laboratories) was subjected to experimental obesity via consuming a high-fat/high-sucrose diet for 12-weeks, whereas the lean mice received supplementation with a low-fat/low-sucrose diet for 12-weeks. At 10-weeks into the protocol, lean and obese mice were treated with DPBs (penfluridol, fluspirilene, pimozide (10 mg/kg)) or vehicle control (corn oil) once every 2 days via oral gavage for 14-days following which we assessed circulating ketone body levels and glucose homeostasis. Results:?Consistent with our previous observations with pimozide, treatment with either fluspirilene or penfluridol improved glucose tolerance but not insulin sensitivity, which was associated with a marked increase in circulating ketone body levels.?This improvement was independent of changes in food intake, body weight, and overall adiposity. Conclusions:?Our findings suggest all DPBs may have glucose-lowering actions via SCOT inhibition, indicating that they may have potential utility in being repurposed for the treatment of T2D.Abstract C13An HPLC method to determine the stability of Lidocaine and Ketoprofen compounded individually, and in combination, with topical gel formulations.Shadab Alam1, 2, Miguel Herrera Rueda1, Leanne Hahn2, Michael Doschak1, Rakesh Bhat1, 21 University of Alberta, Faculty of Pharmacy and Pharmaceutical Sciences, Edmonton, AB, Canada2 Applied Pharmaceutical Innovation, Edmonton, AB, CanadaPurpose: Pluronic lecithin organogel (or PLO gel) cream base is used in compounding pharmacies to formulate various drug combinations, to meet patient-specific needs. Regulatory requirements for compounding pharmacies now include documented stability?of any compounded preparations under specified mixing conditions. As such, the purpose of this project is to establish extraction procedures and develop high-performance liquid chromatography (HPLC) assay protocols for a variety of small molecule analgesic drugs, in order to confidently assign the shelf-life to respective preparations of compounded of active pharmaceutical ingredients under usual storage conditions. Here we report a common extraction and analytical HPLC method to determine the stability of Lidocaine and Ketoprofen in PLO gel compounded separately and in combination at different time period. Method: To extract the drug molecules 1:1 mixture of acetonitrile and water showed the best recovery and no solvent peak during the HPLC run. Gemini? 5?, C18 column were used to resolve the drug and quantify the recovery. Result: Extraction of active drug from all the three combinations (lidocaine + PLO, ketoprofen + PLO and lidocaine + ketoprofen + PLO) showed 100 % recovery with less than 5% RSD during the course of study. Conclusion: Consistent absolute recovery of these drugs revealed their ongoing activity within the formulation for the study duration of 12 months. The developed method is suitable to assay Lidocaine and Ketoprofen with PLO gel and may prove useful for other dosage forms, such as transdermal patches, ointments, etc.Abstract C14 (oral presentation, poster not judged)Fluconazole Protects against Abdominal Aortic Constriction-Induced Cardiac Hypertrophy in Rats Sherif M. Shoieb1, Jody Levasseur2, Heidi Silver2, Jason R. B. Dyck2, Ayman O.S. El-Kadi1 1 Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta, Canada?2 Cardiovascular Research Centre, Department of Pediatrics, Faculty of Medicine and Dentistry, University of? Alberta, Edmonton, Alberta, Canada?Purpose: Cytochrome P450 1B1 (CYP1B1) is known to be involved in the pathogenesis of several cardiovascular? diseases such as cardiac hypertrophy, through the formation of cardiotoxic metabolites named as midchain? hydroxyeicosatetraenoic acids (HETEs). Recently, we have demonstrated that fluconazole, an antifungal agent,?decreases thelevel of mid-chain HETEs in human liver microsomes, inhibits human recombinant CYP1B1 activity?and protects against angiotensin II-induced cellular hypertrophy in H9c2 cells. Therefore, the overall objectives of?the present study were to elucidate the potential cardioprotective effect of fluconazole against cardiac hypertrophy induced by abdominal aortic constriction (AAC). Methods: Male Sprague-Dawley rats were randomly assigned into four groups; sham control rats, fluconazole-treated (20 mg/kg daily for 4 weeks, intraperitoneal) sham rats, AAC rats and fluconazole-treated (20 mg/kg) AAC rats. Baseline and 5 weeks post-AAC echocardiography were performed. Gene and protein expression were measured using real-time PCR and WB analysis, respectively. The level of mid-chain HETEs was determined using liquid chromatography–mass spectrometry (LC/MS). Results: Echocardiography results showed that fluconazole significantly reversed the AAC-induced left ventricular?hypertrophy, as it ameliorated the AAC-mediated increase in left ventricular mass and several wall measurements.? Also, fluconazole significantly prevented the AAC-mediated increase of hypertrophic markers. The anti-hypertrophic effect of fluconazole was associated with a significant inhibition of CYP1B1 at the gene and protein?levels and a reduction in the formation rate of midchain HETEs. Conclusions: The current study demonstrates that?fluconazole protects against left ventricular hypertrophy. The findings of the present work highlight the potential repurposing of fluconazole as a CYP1B1 inhibitor for the protection against cardiac hypertrophy and a possible treatment for heart failure. Support: This work was supported by a grant from the Canadian Institutes of Health?Research [PS168846] to A.O.S.E. S.M.S. is the recipient of Alberta Innovates Graduate Student Scholarship and Alberta Graduate Excellence Scholarship.Abstract C15Staurosporine-induced cleavage of apoptosis-inducing factor in human fibrosarcoma cells is?independent of matrix metalloproteinase-2 or calpain?Wesam Bassiouni1, John M. Seubert1,2, Richard Schulz1,3?1 Department of Pharmacology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB,?Canada?2Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, AB, Canada 3Department of Pediatrics, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB,? Canada?Purpose: Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein which mediates staurosporine?(STS)-induced cell death. To mediate apoptosis, AIF must first be cleaved and translocated to the cytosol and then to the nucleus. AIF cleavage is thought to be calpain-1-dependent as calpain inhibitors reduced AIF proteolysis. However, many calpain inhibitors also inhibit matrix metalloproteinase-2 (MMP-2)?activity, an important intracellular and extracellular protease which is also implicated in apoptosis. Whether MMP-2 contributes to AIF cleavage is unknown. Here we investigated whether MMP-2 activity is affected?in response to STS and how it affects AIF cleavage. Methods: Human fibrosarcoma HT1080 cells were? treated with STS (0.1 ?M, 0.25-24 hr) followed by LDH cytotoxicity assay, gelatin zymography for MMP 2 activity and western blotting for measuring AIF level in mitochondrial-enriched and cytosolic cellular? fractions. The MMP-2 preferring inhibitors (ARP-100 or ONO-4817), calpain inhibitor (ALLM) or nonselective MMP-2/calpain inhibitor (PD150606) were then given 2 hr prior to STS treatment followed by western blotting. Results: A significant increase in cellular MMP-2 activity was seen by gelatin?zymography after 6 hr STS treatment, a time point prior to induction of cell necrosis. Western blot showed the time-dependent appearance of two forms of AIF (~60 and 45 kDa) in the cytosol which were?significantly increased at 6 hr compared to vehicle. No significant change in mitochondrial AIF level was?observed. Surprisingly, when treated with MMP or calpain inhibitors, none of the inhibitors prevented the?increase in the levels of cytosolic AIF fragments caused by STS. Conclusions: These results show that?MMP-2 activity is rapidly increased in response to STS. However, the cytosolic release of AIF may be independent of the proteolytic activities of MMP-2 or calpain. Support: Grants# 143299 and 156393, the?Canadian Institutes of Health Research, Alberta Innovates Graduate Student Scholarship, AB, Canada.Abstract C16Pharmacological Characterization of eNOS-based Megakaryocyte Subpopulations and?Its Implications for their Platelet ProgenyAmir Asgari and Paul Jurasz?Introduction: Platelets play a key role in atherothrombosis and its associated inflammation. Recently, we identified platelet and megakaryocyte/blast subpopulations based on endothelial nitric oxide synthase (eNOS) and nitric oxide (NO) production. We showed that eNOS-negative (eNOSneg) platelets initiate aggregate formation, while eNOS?positive (eNOSpos) platelets limit thrombus growth. However, it’s unknown how these?platelet and megakaryocyte subpopulations arise and the impact of anti-atherothrombotic drugs on their formation. Therefore, we aimed to (1) investigate whether pro-inflammatory cytokines known to down-regulate eNOS expression decrease the ratio of eNOSpos to?eNOSneg megakaryocytes/blasts and resulting platelets; and (2) whether anti?atherothrombotic drugs, with known anti-inflammatory properties would counteract the? effects of pro-inflammatory cytokines on eNOS expression and eNOS-based megakaryocyte? and platelet subpopulation formation.?Methods: Meg-01 were cultured in the presence of thrombopoietin and pro-inflammatory cytokines (IL-1β, IL-6,TNF-α, IFN-γ) and anti-atherothrombotic drugs acetylsalicylic acid?(ASA 10 – 30 mM) and atorvastatin (0.03 – 1 mM). The levels of eNOSneg and eNOSpos Meg 01 and platelet-like particles was measured using flow cytometry.?Results: Compared to control the cytokine cocktail significantly increased the percentage of?eNOSneg Meg-01 and this effect was inhibited by 30 mM ASA and 0.1 mM atorvastatin (Control 7.4 ± 0.9% vs cytokines 11.6 ± 1.2% vs cytokines & ASA and Control 9.4 ± 1.1% vs?cytokines 8.0 ±0.6 % vs cytokines & atorvastatin). Similarly, although fewer in their relative number compared to their parent Meg-01, platelet-like particles released from?eNOSpos Meg-01 cells decreased in response to inflammatory cytokines and this effect was?reversed by ASA and atorvastatin.?Conclusions: The generation of eNOSneg and eNOSpos megakaryocytes and platelets may?be counter-regulated by inflammatory status. Conversely, anti-atherothrombotic drugs ASA?and atorvastatin may promote an anti-thrombotic phenotype, in part, by increasing the?formation of eNOSpos megakaryocytes and platelets.?Abstract C17Discovery of Neutral Allosteric SHP2 Inhibitors through Ensemble-Based Consensus Molecular Docking, Endpoint and Absolute Binding Free Energy CalculationsAnna Jutla1 , Marawan Ahmed1 , Jitendra Kumar2 , Yasser Tabana2 , Kareen Hassan2 , Shokrollah Elahi2 , Michael Overduin2 and Khaled H. Barakat1,4* 1Faculty of Pharmacy and Pharmaceutical Sciences, University Of Alberta, Canada. 2Faculty of Medicine and Dentistry, University Of Alberta, Canada. 3Li Ka Shing Institute of Virology, University Of Alberta, Canada.^Equal First Authors*Corresponding Author Email: kbarakat@ualberta.ca; Ph: 1-780-492-5783.The abnormal activation of SHP2 (Src homology-2 domain-containing protein tyrosine phosphatase-2) is associated with multiple developmental pathologies and the progression of several malignancies. Consequently, SHP2 has attracted extensive interest as a potential therapeutic target, with identification of SHP2-selective inhibitors enabling the development of drug leads for treatment of these diseases. However, current lead candidates tend to be charged and bind off-targets. Here, we applied various computational tools to screen over 6 million compounds against the allosteric site within SHP2. Hits identified from this screening approach were validated for their binding to SHP2 using a protein thermal shift and a surface plasmon resonance assays. Our search identified three active molecules; Molport-005-593-606 (#1), Molport-005-718-898 (#2) and Molport-003-051-027 (#3), and binding kinetic analysis demonstrated that these molecules have nanomolar affinity towards SHP2. Our findings provide insights into the development of new therapeutics that bind to alternative sites of SHP2. The compounds and computational workflow developed here provide tools for designing novel allosteric inhibitors against multiple malignancies, which currently do not have a cure. Abstract D1Evaluation of Canadian Pharmacists’ Knowledge and Comfort in the Management of Epilepsy and Antiepileptic DrugsAkshita Chandok, Sherif Hanafy MahmoudFaculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, AB, CanadaPurpose: As antiepileptic drugs (AEDs) remain the mainstay of epilepsy management, pharmacists have the potential to play an integral role in the management. The goal of our study was to characterize Canadian pharmacists’ knowledge and comfort in managing epilepsy and AEDs and identify areas of need for the development of educational support tools.?Methods: An electronic survey with multiple-choice questions was designed and distributed to Canadian pharmacists through professional organizations and social media outlets. The survey consisted of four sections including demographics, knowledge, comfort and needs assessment around epilepsy management and AEDs.?Results: Of a total of 903 responses that were collected, only complete responses (n=605) were included. Nearly two-thirds of the participants were females (61.6%) and most reported more than 10 years of practice experience (61.6%). Majority of participants listed BSc Pharmacy (71.5%) as their highest level of pharmacy-related academic achievement and obtained their degree from Canada (72.8%). For comfort, majority of the participants responded agree or strongly agree to statement inquiring about the comfort in checking prescriptions, answering questions about drug interactions and counselling on AEDs. Conversely, over 50% of the participants selected disagree or strongly disagree when asked about their comfort regarding interpreting therapeutic drug monitoring and assisting patients withdraw from AEDs. For the knowledge section, the overall average score was 57.4±19.7% (n=477). Our analysis shows hospital practice, recent graduation and neurology experience as independent predictors of higher scores. Many participants indicated a need for tools addressing newer AEDs and monitoring of therapy with significant interest in support tools such as pocket summaries.?Conclusions: While Canadian pharmacists displayed knowledge and comfort in certain aspects of epilepsy management, some clear knowledge and comfort gaps are prevalent. These findings indicate a need for the development of epilepsy educational support tools.Abstract D2Development of Professional Identity in Pharmacy Students: A National SurveyAyush Chadha, Theresa Charrois, Jill HallFaculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, AB, CanadaPurpose: Professional identity is who we are in the context of our chosen profession. Professional identity formation is a complex and dynamic process and has not been well studied in pharmacy students. The purpose of this study was to describe which elements of curricular and non-curricular activities in pharmacy student life may contribute to professional identity formation.?Methods: This anonymous cross-sectional survey was administered to pharmacy students in years 1-5 at all 10 Canadian pharmacy schools with the help of local Canadian Association of Pharmacy Students and Interns (CAPSI) representatives. Students were asked to define professionalism and professional identity and what experiences were meaningful in the development of professional identity. Both an inductive and deductive approach were used for thematic analysis of written responses.?Results: A total of 172 students responded to at least 1 question in the survey. Most respondents were from the University of Alberta (45%) and were evenly distributed through years 1-4 of pharmacy. Key themes emerged: I) for the responses related to the definition of professionalism, a traditional view including the six tenets defined by the American College of Clinical Pharmacy and an expanded view that included responsibility and accountability; II) many students equated being nice with being professional; III) some students believe professionalism is acting professional where some believe it is being professional; and IV) most pharmacy students are unable to define professional identity but acknowledge teaching related opportunities, professional development and role-modelling by both faculty and student associations play a key role in developing professional identities.?Conclusions: Most pharmacy students struggle to define professional identity and distinguish it from professionalism. In order to nurture its development, both pharmacy faculty and student associations must intentionally design opportunities for student growth.Abstract D3Inclusion of Geriatric and Pediatric Learning Activities in Canadian Pharmacy ResidencyProgramsCody Thompson, Morgan Bharadia, Cheryl A. SadowskiFaculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, AB, CanadaPurpose: The purpose of this study was to describe the geriatric and pediatric training experiences acrossPGY1 and PGY2 Canadian Pharmacy Residency Programs. The objectives of the study were to: determine the proportion of PGY1 and PGY2 residency programs in Canada that include geriatric and pediatric rotations, describe the way geriatrics and pediatrics are included in residency programs, identify barriers for residency coordinators (RCs) to include geriatrics and pediatrics in residency programs, and identify gaps and opportunities in residency training related to geriatrics and pediatrics.?Methods: RCs from programs across Canada were surveyed through a semi-structured interview. The questions included requirements for rotations, preceptors, resources, and future planning for geriatrics and pediatrics. The study was approved by the University of Alberta Research Ethics Board.?Results: A total of 42 RC’s were invited to participate, representing 44 programs. 36 out of 44 programs participated. Of the 36 programs, there were 29 PGY1 and 7 PGY2. Out of the 29 PGY1 programs, 19 offered a geriatrics rotation (17 elective, 2 mandatory). 23 PGY1 programs offered a pediatrics rotation (6 mandatory). Of the 7 PGY2 programs, 2 mandated geriatrics. 6 out of the 7 PGY2 programs offered a pediatrics rotation (1 mandatory). Of the 10 PGY1 not offering geriatrics, 7 did not integrate learning objectives involving geriatrics into other rotations and 9 had no plans for expansion regarding geriatrics. Of the 6 PGY1 programs not offering a pediatrics rotation, 5 did not have specific learning objectives involving pediatrics and 3 had no plans for expansion regarding pediatrics.?Conclusion: Geriatric and pediatric rotations are commonly offered as elective learning opportunities for pharmacy residents. The incorporation of geriatrics and pediatrics was passive during exposure in other rotations. There is a lack of urgency to incorporate learning objectives involving geriatric and pediatric populations.Abstract D4Providing medication reviews for patients through community pharmacists in Canada: Findingsfrom a scoping reviewDamilola Olufemi-Yusuf1, Janice Kung2, Lisa Guirguis11 Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, AB, Canada2 Public Services Librarian, John W. Scott Health Sciences Library, University of Alberta, Edmonton,AB, CanadaPurpose: Within Canada and around the world, medication review services have been introduced as an important component of pharmacists’ patient care services to address medication-related problems and potentially improve patient health outcomes. The aim of this scoping review is to systematically map the existing Canadian literature on medication reviews provided by community pharmacists according to research designs, themes, and key findings. A secondary objective was to identify gaps that could inform future research directions.?Methods: We conducted the study following a scoping review framework and the PRISMA guidelines for Scoping Reviews. Three electronic databases (Medline, Embase, and CINAHL) were searched for peer-reviewed literature published between January 2000 until August 2020.?Results: Of the included 41 articles, 29 were quantitative, 10 were qualitative and two were mixed-method studies. The majority of studies were conducted in Ontario (n=21). Five major themes identified were program uptake, patient health outcomes, stakeholder beliefs and attitudes, processes and collaboration, and pharmacy workplace culture, which varied considerably. Factors influencing the uptake and implementation were interrelated (e.g. use of technology to identify eligible patients facilitated uptake while insufficient collaboration contributed to negative attitudes regarding medication reviews). Variable patient experiences and health outcomes may relate to different types of medication review and pharmacist practice across Canada. Few researchers evaluated eligibility criteria, strategies to engage patients and healthcare professionals, or compared practice models of medication reviews. About twelve percent of the research applied a theoretical framework.?Conclusion: Publicly funded medication reviews in Canadian community pharmacies reduce medication-related problems and potentially improve patient health outcomes. Changes in policy and practice could consider approaches to address barriers to the uptake, process, and outcomes of care. Future research could consider exploring models for sustainable delivery of these programs internationally and other identified gaps.Abstract D5 (oral presentation, poster not judged)The Impact of the COVID-19 Pandemic on Pharmacy Students’ Personal and Professional LearningDanielle K Nagy, Jill J Hall, Theresa L CharroisFaculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, AB, CanadaPurpose: The coronavirus disease of 2019 (COVID-19) pandemic impacted both healthcare delivery and the education of healthcare students, with a shift to remote delivery of coursework and assessment alongside the expansion of the scope of practice of Alberta pharmacists. The objective of this research was to understand how the learning of pharmacy students at the University of Alberta was impacted by the COVID-19 pandemic.?Methods: A cross-sectional survey was distributed to 397 pharmacy students in years 1-3. Students responded to three short-answer reflection questions: 1) How has the COVID-19 pandemic situation affected your learning? 2) From a pharmacy and pharmacy school perspective, what have you learned since the COVID-19 pandemic began? 3) From a personal perspective, what have you learned about yourself since the COVID-19 pandemic began? A thematic analysis was undertaken of students’ responses to these reflection questions.?Results: A total of 53 students responded to the survey(response rate of 13%). Two major themes were identified across all three reflection questions, with several subthemes: remote learning (learning environment, knowledge transfer, knowledge retention, assessment) and mental health (appreciation, stress, extroversion, motivation). Adaptability, routine, professional identity, and the role of the pharmacist were also identified as less prevalent themes.Conclusions: Pharmacy students’ responses led to the identification of several themes related to their learning given the changes brought about by the COVID-19 pandemic. This increased understanding of student perceptions has the potential to improve the remote delivery of education, support increased university-wide mental health resourcing, and shape pharmacy curriculum development.Abstract D6Long-Acting Reversible Contraception (LARC) Services by Pharmacists and Other HealthcareProfessionals: A Scoping ReviewEmma Bedard1, Nicole Kremer1, Janice Kung2, Nese Yuksel11Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, AB, Canada2John W. Scott Health Sciences Library, University of Alberta, Edmonton, AB, CanadaPurpose: Long-acting reversible contraception (LARC), including intrauterine methods, implants, and injections, is the most effective form of reversible contraception. Increasing LARC use is one method for preventing unintended pregnancies. Several studies have looked at ways to increase LARC use, but a review of the existing literature has not been published. Our objective is to describe LARC services that have been provided by healthcare professionals including pharmacists, physicians, nurses, and other providers.?Methods: A comprehensive search was run by a medical librarian on the MEDLINE, Embase,CINAHL, Cochrane Library, and Google Scholar databases (from inception to January 6, 2021). Title, abstract, full text screening, and data extraction was completed by two reviewers using the online software Covidence, and discrepancies were resolved by consensus. Studies were included if they described healthcare professional-led LARC services for contraception and included an evaluation of the service.?Results: After removal of duplicates, a total of 1551 articles were identified by the search. After full text screening, 27 studies were eligible for inclusion. Screening and data extraction is ongoing.Preliminary data suggests that studies addressed a wide variety of healthcare professionals (e.g., physicians, pharmacists, nurses, community health workers), interventions (e.g., training, education, counselling, provision of contraception), settings (e.g., health centers, hospitals, community settings), and countries (e.g., United States, India, Rwanda). Patient populations varied and often focused on adolescents, non-pregnant women, or women delivering at a hospital. Several studies involve multiple professional types and multi-pronged interventions. LARC types offered included intrauterine methods, injections, or implants alone or a combination of these options.?Conclusions: Knowing what strategies have been implemented and their effects on LARC uptake will assist in creating future pharmacist-led services aimed at reducing unintended pregnancies.?Support: Graduate research assistant fellowship, Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta.Abstract D7 (oral presentation, poster not judged)Prevalence and Risk Factors of Augmented Renal Clearance: A Systematic Review and Meta- analysisFatma Hefny1, Anna Stuart1, Janice Y Kung2, Sherif Hanafy Mahmoud11Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta, Canada2Public Services Librarian, John W. Scott Health Sciences Library, University of Alberta, Edmonton,Alberta, CanadaPurpose: Critical illness is uniquely complex and requires a multidisciplinary approach to provide comprehensive care. Assessment of kidney function in the critically ill conventionally overlooks the possibility for hyperfunctioning kidneys now known as Augmented Renal Clearance (ARC) which could explain a range of therapeutic failures in the ICU. The aim of this research is to conduct a systematic review and meta-analysis of the available literature on ARC and attempt to provide a step forward towards the early identification of those at risk for ARC allowing timely medication optimization by providing pooled estimates of ARC prevalence and contributing risk factors in various critically ill populations.?Methods: We developed comprehensive database searches related to ARC in critical care retrieving a total of 3,455 results; 1,761 were screened via Covidence yielding 107 studies included in the meta-analysis of prevalence and risk factors. We included human studies conducted in critically ill adult populations that reported the prevalence of or risk factors with estimates of effect. We excluded studies that focused on pediatrics or patients with altered baseline renal elimination. The statistical analysis was performed using R Statistical Software. We generated a random-effects meta- analytic model using the Inverse variance method and visualized the pooled estimates using forest plots.Results: 80 studies reported prevalence, the meta-analysis pooled prevalence was 38.9%. Pooled prevalence from Mixed, Neuro, Sepsis, Trauma ICUs, and Others were 35.9%, 74.1%, 27.9%, 57.5%, and 30.1% respectively. Twenty-seven studies were included in the meta-analysis of risk factors ofARC, namely age, male gender, trauma, SOFA, APACHEII, weight and DM. Pooled ORs were 0.95,2.37, 2.6, 0.86, 1, 1.6, 1.2.?Conclusions: ARC is a detectable commonly occurring phenomenon in critically ill patients that needs to receive more attention. More research is needed to establish a better understanding of ARC and its appropriate management.Abstract D8 (oral presentation, poster not judged)Cannabis Use in Menopause: A Survey of Usage and Perceptions Among Women in AlbertaKatherine Babyn1, Sue Ross2, Nese Yuksel11Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, AB, Canada2Department of Obstetrics and Gynecology, Faculty of Medicine and Dentistry, University of Alberta,Edmonton, AB, CanadaPurpose: Use of cannabis has increased in Canada since legalization, with growing interest to manage health issues. Midlife women may be using cannabis to help with symptoms overlapping with menopause. However, it is unclear how many women in Alberta are currently using cannabis, specifically for medical purposes related to menopause. As part of a mixed methods study, the purpose of the survey was to characterize cannabis use patterns and perceptions in a population of midlife women in Alberta.?Methods: This was a cross-sectional, web-based survey designed by the research team. Survey questions included self-reported menopause status, symptoms, and questions on cannabis use, reasons for use, information resources and overall perceptions. Women (ages 35 and over, residing in Alberta) were recruited through social media (Facebook, Instagram, Twitter). Descriptive statistics was used to describe the data.?Results: A total of 1,761 responses were collected between October and December 2020, of which 1,495 were included for analysis. Of the survey respondents, 486 (33%) women were in perimenopause, while 522 (35%) in post-menopause. Mean age was 49.0 years (SD=8.0). Of the 499 current cannabis users, 75% reported use for medical purposes and 213 (43%) used at least once daily. Reasons for current use included sleep issues (65%), anxiety (45%) and muscle/joint achiness (33%). Edibles (52%) and oils (47%) were the most common currently used formulations. More than 60% of women surveyed indicated ever using cannabis. Common sources of cannabis information for medical purposes were internet searches (46%) or family/friends (34%).?Conclusions: Midlife women are using cannabis. Most are using it for medical purposes on symptoms that often overlap with menopause, yet healthcare providers are not a common source of information for this. Current qualitative work in capturing the experiences of women using cannabis for medical purposes will provide further insight to these results.Abstract D9 (oral presentation, poster not judged)Identifying Evidence-Based Pharmacy Practices for the Implementation of PharmacogenomicsThrough A Scoping ReviewMeagan Hayashi1, Dalia A. Hamdy1,2, Sherif Hanafy Mahmoud11 Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta, Canada2 AbEx Health Services LTD, Fort Saskatchewan, Alberta, CanadaPurpose: Pharmacogenomics (PGx) can provide valuable pharmacokinetic and pharmacodynamics information for the pharmacist’s assessment of drug therapy, especially within medication therapy management (MTM) services. However, no review has comprehensively mapped pharmacists’ use of PGx in practice-based research. Doing so would allow future researchers, practitioners, and policy-makers to identify the ideal populations and settings for PGx implementation within the pharmacy. The purpose of this review is to identify the evidence to date of PGx use in pharmacy practice.?Methods: A scoping review was conducted to find all studied non-oncologic pharmacy practices incorporating PGx testing. Search terms were applied to 5 databases and relevant journals. Characteristics of patients, pharmacy settings, genetic tests, and outcomes were summarized to determine models most likely to benefit patients.?Results: The search identified 43 studies on the use of PGx by pharmacists published between 2007 and 2020. CYP2C19 testing with antiplatelets was the most studied model, found in both community and institutional settings. It also was the most actionable test: approximately 30% of patients have polymorphisms indicating a need for alternative antiplatelets, and identifying these patients can reduce morbidity and mortality by more than 50%. As technology shifts, broader studies using multi-gene panel tests within MTM demonstrate an approximate 50% decrease in emergency visits and hospitalizations in elderly polypharmacy patients. Clinical benefit or drug-gene interactions are also found in other cardiovascular, psychiatric, analgesic, and gastrointestinal indications. No evaluations of actual costs or of pharmacist prescribing were found in included literature. Facilitators towards successful PGx implementation included pharmacist education, collaboration with other healthcare providers, and the use of clinical decision software.?Conclusions: PGx has demonstrated feasibility and improved medication outcomes in many indications within pharmacy practice. Further PGx research should be directed towards pharmacist prescribing, education, and pharmacoeconomics.?Support: Pharmacy Practice Innovation Grant, Alberta Pharmacists’ Association, Edmonton, Canada.Abstract D10 (oral presentation, poster not judged)Re-examining the Role of Sulfonylureas in the Management of Type 2 DiabetesDanielle K Nagy, Scot H SimpsonFaculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, AB, CanadaPurpose: The interest in using sulfonylureas for treatment of type 2 diabetes has been reignited since publication of the CAROLINA trial because it showed the risk of cardiovascular events was no worse for glimepiride compared to linagliptin. However, application of the CAROLINA trial results to Canadian clinical practice is questionable because these agents are not commonly used. In fact, it is unclear how often sulfonylureas are used in clinical practice today. Therefore, the objective of this study is to illustrate trends in antihyperglycemic drug choice to intensify metformin monotherapy with a focus on sulfonylurea use.?Methods: This retrospective, cohort study was conducted using pharmacy dispensation records provided by Alberta Health between April 2009 and March 2018. New metformin users were followed until a second antihyperglycemic drug was started. This treatment intensification instance was categorized as either sulfonylurea-based or non-sulfonylurea-based and logistic regression will be used to determine predictors of drug therapy choice. The duration of sulfonylurea use will be examined over the next year.Results: Of 165,056 adult new metformin users, 55,909 (34%) had a second antihyperglycemic drug started an average of 1.3 years later (mean age was 55 years, 62% male). A total of 26,429 (47%) people started sulfonylurea-based therapy; 24,793 as a single agent and 1,636 in combination with otherantihyperglycemic drugs. The next most common drug classes at treatment intensification wereDPP4-inhibitors (n=11,698), followed by insulin (n=5,261), SGLT2-inhibitors (n=3,316), and GLP-1 receptor agonists (n=2,101). Analysis is ongoing.?Conclusion: Preliminary results show that sulfonylureas are commonly added to metformin therapy. These observations confirm that sulfonylureas remain an important treatment option in type 2 diabetes and that further research into the cardiovascular safety of these drugs is warranted.Abstract E1Modulation of aryl hydrocarbon receptor (AhR)-regulated genes expression by monomethylmonothioarsonic acid (MMMTAV) in Hepa1c1c7 cellsMahmoud A. El-Ghiaty, Mohammed A. Alqahtani, and Ayman O.S. El-KadiFaculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton,Alberta, CanadaBackground: Arsenic is a ubiquitous occupational and environmental contaminant that imposes threat to humans through its toxicity and carcinogenicity. Thiolated arsenic metabolites, such as monomethylmonothioarsonic acid (MMMTAV), have been detected in the urine of arsenic-exposed humans and animals suggesting their involvement in arsenic toxicity. Both inorganic arsenic and its methylated metaboliteshave been shown to modulate aryl hydrocarbon receptor (AhR)-regulated phase I and phase II xenobiotic metabolizing enzymes which are involved in the carcinogenic andcytoprotective pathways, respectively. However, little is known about thioarsenicals.Purpose: examining the impact of MMMTAV on AhR phase I and phase II enzymes typified by cytochrome P450 1a1 (Cyp1a1) and NADPH:quinone oxidoreductase(Nqo1), respectively.Methods: Murine hepatoma (Hepa1c1c7) cells were treated with MMMTAV (1, 5 and10 μM) in the absence or presence of 1 nM of the archetypal AhR ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Cyp1a1 and Nqo1 expression was determined at both mRNA and protein levels using qPCR and Western blot analysis, respectively.MTT Assay was used to assess cell viability.Results: Cell viability was not affected by all concentrations of MMMTAV used.MMMTAV decreased the constitutive Cyp1a1 mRNA, while, only at 10 μM, it decreased TCDD-mediated induction of Cyp1a1 mRNA. MMMTAV didn’t confer changes in constitutive or inducible levels of Cyp1a1 protein. On the other hand, 10 μMMMMTAV significantly increased Nqo1 TCDD-mediated induction at mRNA and protein levels.Conclusions: our study demonstrated that MMMTAV differentially modulated theAhR-regulated Cyp1a1 and Nqo1 enzymes with subsequent implications on cellular response to xenobiotics co-exposure. However, further studies are required to explain the mechanism of this behavior.Support: This work was supported by Natural Sciences and Engineering ResearchCouncil of Canada (NSERC) Discovery Grant RGPIN 250139 to A.O.S.E. M.A.E. is the recipient of Pharmacy PhD Alumni Graduate Student Scholarship.Abstract E2 (oral presentation, poster not judged)The activation of drug detoxifying enzyme NQO1 in the presence of clozapine-induced toxicity and oxidative stressMd Harunur Rashid1,2, Mahmoud A. El-Ghiaty1, Ayman El-Kadi1, Arno G. Siraki11Faculty of Pharmacy & Pharmaceutical Sciences, University of Alberta, Edmonton, Canada2Institute of Food and Radiation Biology, Bangladesh Atomic Energy Commission, BangladeshPurpose: Clozapine is an important atypical antipsychotic drug and is the only medication for treatment-resistant schizophrenia. However, it is thought to be metabolized by neutrophils to electrophilic reactive metabolite clozapine nitrenium ion (CNI) that may irreversibly damage critical neutrophil proteins, which may responsible for serious blood disorders such as agranulocytosis (potentially lethal neutropenia). Currently, the mechanism(s) for this toxicity is unknown but may involve the modulation of phase II detoxifying enzymes. Rationale: Patients treated with clozapine that experienced agranulocytosis have a lower frequency of the phase II drug detoxifying enzyme dihydronicotinamide riboside (NRH) quinone oxidoreductase 2 (NQO2, wild type) compared to patients that did not exhibit agranulocytosis but polymorphic NQO2 (NQO2 1541 AA, mutant) higher frequency. Moreover, NAD(P)H: quinone oxidoreductase-1 (NQO1) and NQO2 are homologs. Furthermore, certain electrophilic reactive metabolites are known to induce the expression of NQO1, through the antioxidant response element (ARE). Hypothesis: Since clozapine supposedly forms CNI, we hypothesize that clozapine/CNI induces NQO1 expression and activity.?Methods: 1. Clozapine cytotoxicity test for selecting concentrations (IC50); 2. Enzymatic activity assay to detect NQO1 activity; 3. Western blotting for the detection of NQO1 protein expression; 4. Reverse transcription-polymerase chain reaction (RT-PCR) assay for the detection of NQO1 gene expression.Results: A concentration-dependent cytotoxicity (IC50≈48 μM) was observed after clozapine treatment in HL-60 cells. Also, Clozapine treatment (10–50 μM) induced NQO1 activity, protein, and mRNA expression.?Conclusions: The induction of NQO1 enzymatic activity, protein, and gene expression indicate clozapine is responsible for the activation of NQO1 via a stress response. In the future, studies will be performed for determining if NQO1 activation is a protective mechanism against clozapine-induced cellular toxicity.?Abstract E3Methylmercury modulates the expression of AhR-regulated genes in Hepa1c1c7 cellsMohammed A. Alqahtani and Ayman O.S. El-KadiFaculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, AB, CanadaPurpose: Heavy metals and polyaromatic hydrocarbons (PAHs) are persistent environmental pollutants involved in the modulation of aryl hydrocarbon (AhR) gene battery, including cytochrome P450 (CYP) genes. Such modulation is linked to inducing carcinogenesis and cellular injury. The growing risk of exposure to these environmental toxicants, including methylmercury and PAHs typified by TCDD, increased the demand for further research to elucidate their individual and combined effects. We recently investigated the co-exposure effect to methylmercury in the presence of TCDD in vivo. Thus, the current study aimed to examine the co-exposure effect to MeHg and TCDD on AhR-regulated genes (Cyp1a1 and Ho-1) in vitro.Methods: Therefore, murine hepatoma cells (Hepa-1c1c7) were used to investigate the concentration-dependant effect of MeHg in the presence and absence of TCDD at multiple transcriptional levels of Cyp1a1 and Ho-1 genes. Real-time PCR, Western blot and 7- ethoxyresorufin O-deethylation (EROD) assays were used to determine mRNA, protein and enzymatic activity levels, respectively.Results: The increasing concentrations of MeHg in the presence of TCDD significantly inhibited the CYP1A1 catalytic activity. Accordingly, MeHg significantly induced Ho-1 at mRNA and protein levels, which was coincided with a decreased Cyp1a1 activity by 1-fold implying the Ho-1 potential inhibitory role.Conclusion: These findings together showed that the organic form of mercury, MeHg, can modulate the expression of TCDD-mediated Cyp1a1 induction in Hepa1c1c7 cells and its inhibitory role on Cyp1a1 activity is possibly through Ho-1 expression.?Support: This work was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant RGPIN 250139–107 to A.O.S.E. MA is the recipient of Saudi Government Scholarship.?Abstract E4 (oral presentation, poster not judged)Characterization of human sulfotransferases catalyzing the formation of p-cresol sulfate and identification of mefenamic acid as a potent metabolism inhibitor and potential therapeutic agent for detoxificationYan Rong, Tony KL KiangFaculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, AB, CanadaPurpose: p-Cresol sulfate (pCS) is the primary metabolite of p-cresol. Extensive in vitro and clinical studies provided evidences demonstrating pCS-associated toxicities. The objectives of this study were toi) characterize the contributions of human liver/kidney sulfotransferases (SULT) catalyzing pCS formation; and ii) determine the potencies and mechanisms of therapeutic inhibitors capable of attenuating the production of pCS.?Methods: The enzyme kinetics of pCS formation were determined using human recombinant (hr) enzymes (hrSULT1A1, 1A3, 1B1, 1E1, 2A1, and 1A1*2), pooled human liver cytosols, and pooled human kidney cytosols. The inhibitory effects of 14 chemical inhibitors were characterized in hrSULT1A1; the potencies and mechanisms of inhibition were determined for the identified most potent inhibitors using human liver and kidney cytosols.?Results: hrSULT1A1 was the primary enzyme responsible for the formation of pCS (maximum reaction rate [V max ]=789.5±101.7nmol/mg/min, substrate concentration at half of V max [K m ]=0.19±0.02?M, dissociation constant [K si ]=2458.0±332.8?M, mean±SD, n=3), while SULT1A3, SULT1B1, SULT1E1, and SULT2A1 contributed negligible or minor roles at toxic p-cresol concentrations. Moreover, hrSULT1A1*2 exhibited reduced enzyme activities (V max =230.6±17.7nmol/mg/min, K m =81.5±31.4?M, K si =986.0±434.4?M) compared to the wild type. The sulfonation of p-cresol was characterized by Michaelis-Menten kinetics in liver cytosols (V max =1.5±0.2nmol/mg/min, K m =14.8±3.4?M) and substrate inhibition in kidney cytosols (V max =0.19±0.05nmol/mg/min, K m =0.29±0.02?M, K si =911.7±278.4?M). Of the 14 investigated therapeutic inhibitors, mefenamic acid (inhibition constant [K i ]=2.4±0.1nM [liver], K i =1.2±0.3nM [kidney]) was the most potent in reducing the formation of pCS, exhibiting noncompetitive inhibition in human liver cytosols and hrSULT1A1, and mixed inhibition in human kidney cytosols.?Conclusions: Our novel findings indicated that SULT1A1 contributed an important role in p-cresol sulfonation in liver and kidneys, and mefenamic acid may be utilized as a potential therapeutic agent to attenuate the generation of pCS as an approach for detoxification.?Support: This work was supported by a start-up grant and an NSERC discovery grant awarded to Dr. Tony Kiang.Abstract E5Development of a High-Throughput and Sensitive Ultra-High Performance Liquid Chromatography with Florescence Detection Assay for the Quantification of p-Cresol and Its MetabolitesYashita Singh, Yan Rong, Tony KL KiangFaculty of Pharmaceutical Sciences, University of Alberta, Edmonton, AB, CanadaPurpose: p-Cresol is a uremic toxin that is accumulated to high concentrations in patients with renal dysfunction, which is primarily metabolized to p-cresol sulfate (pCS) and p-cresol glucuronide (pCG). Both p-cresol and its metabolites are associated with multi-organ toxicities. The objective of this project is to develop high-throughput and sensitive methods for quantifying p-cresol, pCS, and pCG within a single assay.?Methods: The assay was developed on a Shimadzu? ultra-high performance liquid chromatography with ultraviolet detector (UPLC, LC-2040C Plus), coupled with a fluorescence detector (RF-20Axs). Our approach was to optimize the instrument configuration (e.g., the detection methods and detector parameters) initially; followed by optimizations of chromatographic conditions including the percentage of organic solvent in the mobile phase, additive concentration, flow rate, and injection volume. The parameters generating the highest peak area counts were defined as the optimized conditions. 2’,6’ Dimethyl phenol was utilized as the internal standard.?Results: In terms of system configuration, fluorescence detector was ultimately selected due to the 5-25 folds higher sensitivity for all analytes as compared to the ultraviolet detector (under optimized absorbance settings identified using Shimadzu? UV-2600i spectrophotometer). The optimized excitation/emission wavelengths were 220/300 nm. With respect to chromatographic conditions, the best separation conditions (i.e., symmetrical, separated peaks / baselines with optimal intensity) were achieved using an isocratic flow of 73:27 methanol:water supplemented with 0.4 mM ammonium acetate and 0.02% formic acid at 0.5 mL/min flow rate. The injection volume was 10 μL, with a run time per injection of 12 minutes. Using the optimized conditions, the calibration ranges were determined to be 0.08-4.34μg/mL (p-cresol), 1.93-49.38μg/mL (pCS), and 0.11-4.34μg/mL (pCG), respectively. Conclusions: We have successfully developed a high throughput and sensitive UPLC-fluorescence detection assay for quantifying p-cresol, pCS and pCG. We are currently in the process of validating this assay in human blood and dried- blood-spot samples.Abstract E6 (poster not judged)Studying the effects of selective calcium channel blockers on ions’ permeation through the Cav1.2 ion channelFarag E.S. Mosa, SuryanarayananC1, Tianhua Feng, Khaled BarakatFaculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, AB, CanadaPurpose: Selective calcium channel antagonists are commonly used in the treatment of cardiovascular disorders. They are mostly classified into 1,4-dihydropyridine (1,4-DHPs) and non-DHPs. The non-DHPs class is further classified into phenylalkylamines (PAAs) and benzothiazepines (BZTs) derivatives. These blockers are used for the treatment of hypertension, angina pectoris, and cardiac arrhythmias. Despite their well-established efficacy, the structural basis behind their activity is not very clear.?Methods: In this study, we used a near-open confirmation (NOC) model of the Cav1.2 cardiac ion channel to examine the mode of binding of these antagonists within the pore domain and the fenestration of the pore-forming domains. Effects of calcium ion permeation in the presence of drug molecules were assessed using steered molecular dynamics (SMD) simulations.?Results: These studies reveal that nicardipine, a DHP derivative, shows a strong Cav1.2 blocking activity, requiring more than 2500 piko Newton (pN) force to pull calcium ions toward the channel’s pore in the presence of the compound. Similar blocking activity was observed for verapamil, a PAA derivative, requiring almost 2300 pN of force. The least blocking activity was observed for Diltiazem, a BZT derivative.?Conclusions: Our results explain the structural basis and the binding details of 1,4-DHPs, PAAs, and BZTs at their distinct Cav1.2 sites and offer detailed insights into their mechanism of action in modulating the Cav1.2 channel.Abstract F1Saliva based detection and quantification of SARS-CoV-2 by direct RT-qPCR that circumventsDNA/RNA ExtractionArchana Koul 1, Siddhartha Biswas 1, Rakesh Bhat 1,21 Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, AB, Canada2 Applied Pharmaceutical Innovation, Edmonton, AB, CanadaPurpose: SARS-CoV-2 detection and quantification using simple and robust method is the key to stop viral transmission. Currently RNA isolation from patient sample is the rate limiting step and purpose of my research is to develop a robust and stable buffer system that can be used to directly perform qPCR on patient samples without nucleic acid extraction step. In order to standardize buffer system for the collection and processing of SARS-CoV2 in the absence of BSL-3 facility, we simulated the entire procedure using Hepatitis B virus (HBV).?Methods: Saliva samples were spiked with HBV followed by addition of different buffers containing varied additives. Virus was heat inactivated at 65 ?C for 30 minutes in the collection tube, followed by direct qRT-PCR of two important genes of HBV i.e. X andHBeAg. A total of 39 combinations of buffers and additives were evaluated to improve viral RNA stability for direct qRT-PCR during the present study.?Results: Comparable Ct values were detected between TBE and PBS buffer, however, TBE buffer depicted the best Ct value and resulted in remarkably clustered data. Contrastingly, saliva with two commercially available buffers i.e. DNA/RNA Shield andRNA Later led to the abrogated detection of HBV. Additives like Tween 20, Triton X-100 and NP-40 and other RNA stabilizing agents like glycogen, proteinase K, BSA, DTT, also provided stability and virus detection without interfering with one step RT-qPCR. Addition of Tween-20 showed best results in combination with different buffer systems.?Conclusions: Heating of saliva samples beforehand represents a very simple method to inactivate the virus without opening the collection tubes which also confers added biosafety and thus reduces biohazard risks. Also, direct qRT-PCR resulted in time-efficient and cost-effective detection of the HBV. We are in a process to test our developed protocol to SARS-CoV-2 and other viruses.Abstract F2 (oral presentation, poster not judged)Proteasome-assisted degradation of the oncogenic FOXM1 transcription factorAntonio Vega-Medina, Carlos Velázquez-Martínez, Khaled BarakatFaculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, AB, CanadaPurpose: Despite the relatively good in vitro inhibitory profile that several experimental FOXM1 inhibitors exert on triple negative-breast cancer cells, their potency (in the 10-40 ?M range) is still a significant limitation. Therefore, through docking and molecular dynamics (MD) simulations, we studied the feasibility of using the protein cereblon (CRBN) to induce targeted protein degradation of FOXM1 via the ubiquitin system as a novel approach to target this oncoprotein.?Methods: We carried out a blind ligand-mediated protein-protein docking between (a) FOXM1, containing either FDI-6 or TFI-2 drug molecules, and (b) CRBN, containing lenalidomide. Then, we included a heptyl linker to bridge lenalidomide with FDI-6 to form a degrader; we followed the stability of the ternary complex (FOXM1- degrader-CRBN) via MD simulations. Finally, we adapted the methods reported by Castillo et al. and Huet al. to synthesize two molecules, namely F7L and T7L.?Results: After a 250 ns MD simulation, the root mean square deviation (RMSD) values showed good stability for both proteins when we modelled either drug, F7L (about 0.6 nm after 150 ns) or T7L (about 0.5 nm after 10 ns). Similarly, F7L and T7L showed good stability at about 0.15 nm throughout the trajectory and 0.35 nm after 140 ns, respectively, suggesting a better profile for F7L. The calculated root mean square deviation (RMSF) values also suggest a better binding mode for the drug T7L. We noticed that both proteins interacted through a network of salt bridges and hydrogen bonds across the protein-protein interface. Finally, to validate this theoretical approach we recently synthesized both molecules with good to moderate yield.?Conclusions: These results suggest that both ligands form stable ternary complexes and both molecules (F7L and T7L) may potentially induce ubiquitin assisted FOXM1 degradation; we are currently screening both drugs using SPR and WB assays.Abstract F3?Generation of replicon system for evaluation and antiviral screening of drugsSiddhartha Biswas1, Neal Davies1 and Rakesh Bhat1,21Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, AB, Canada2Applied Pharmaceutical Innovation, Edmonton, AB, CanadaThe SARS-CoV-2 outbreak has so far not been confined due to the unpreparedness and unsuccessful development of antiviral drugs against SARS-CoV-2. The development of a SARS- CoV-2 replicon will enable us to work with this pathogen in low-level of biocontainment laboratory (level-II) and importantly will allow us to introduce targeted mutations in the viral genome, that will lay foundation to the investigation into its biology and underlying disease etiology. Furthermore, the replicon system can serve as a platform to develop reporter viruses essential for rapid screening of antivirals. Truncated versions of the genomes, where key structural proteins are substituted with various reporter genes and/or antibiotic resistance gene(s) will further be developed to preclude the necessity of BSL3 facility and facilitate research projects in the BSL2 laboratories. The coronaviruses encode the largest genomes among RNA viruses and had raised some unique challenges in developing a molecular clone in the past and present. In current study, we are trying to overcome these problems by adopting two distinct approaches i) use of bacterial artificial chromosomes (BACs) and ii) in vitro ligation of cDNA fragments followed by generation of full-length RNA by in vitro transcription. ................
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