Conference Program November 8th-10th, 2019 Richmond, VA Hosted by:

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Introduction Conference Locations:

• Reception (Fri) & Dinner (Sat): Hampton Inn and Suites Downtown Richmond 700 E. Main St., Richmond, VA 23219 2nd Floor Meeting Room

• Welcome, Food, Breaks, Posters Hunton Student Center 1110 E. Broad St, Richmond, VA 23219 (Entrance through garden off N. 12th St.)

• Lecture and Keynote Talks: Sanger Hall, Room 1-044 1101 E. Marshall St., Richmond, VA 23219 (Entrance via garden off N. 12th St)

CCC website: https://rampages.us/ccc2019/ Host: Joel Schlosburg (Joel.Schlosburg@vcuhealth.org) Note: The Saturday evening banquet (6:30 – 8:30 pm) will have food and beverage (sorry, no alcohol on Friday). For those looking for some additional “merriment” and socialization, below are a handful of suggested local gathering spots:

1. Kabana Rooftop: outdoor rooftop lounge with cocktails, bites, and local DJ sets on weekends. May be a bit nippy 20 floors up on the roof of the hotel, so dress accordingly.

2. Belle: also located within the lobby area of the hotel, this small upscale cocktail and southern cuisine spot is somewhat small but very convenient.

3. Capital Ale House: boasting hundreds of beers on tap and draught, wine, and local-sourced bar bites, and right across the street from the hotel on Main St. Occasional local music, large gathering space, Richmond staple for almost 20 years.

4. Penny Lane Pub: for those looking for cozy British pub, this place has been serving pub food and drinks since about 1980, a favorite with many VCU labs for happy hours. Two blocks down to 5th St, and one block uphill to Franklin on the corner.

5. The Tobacco Company: recently rebuilt and reopened, this fine dining bar/club/restaurant has anchored downtown since the 1970s. Main floor features a large bar area, basement a club (covers and bottle service, if that’s your style). Bit of a walk to 12th St. and Cary.

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Overhead of the walk from the hotel to the conference site:

Sponsors: We would also like to thank the contributions and sponsorship of our partner institutions whose stewardship of previous CCC meetings has allowed continued success:

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Carolina Cannabinoid Collaborative 2019 Schedule Food and Posters: Hunton Student Center, North 12th St and Broad St. Lectures: Sanger Hall Room 1-044, entry via Patterson Memorial Garden Friday, November 8th, 2019

6:30 pm to 8:30 pm

Registration and Reception (platters, hors d'oeuvres, flatbreads) Capital Ballroom, 2nd floor, Hampton Inn and Suites Downtown 700 E. Main St., Richmond, VA 23219

Saturday, November 9th, 2019 6:30 am to 9:00 am Hot Breakfast (for hotel guests), Hampton Inn Lobby

9:00 am to 9:30 am Welcome and Registration, Hunton Student Center Lobby 1110 E. Broad St (entrance via rear garden area)

9:30 am to 9:40 am Welcoming remarks and info, Joel Schlosburg, VCU Sanger Hall 1-044

9:40 am to 10:00 am

Somnath Mukhopadhyay, NC Central University “CB1R allosteric modulator PSNCBAM1 blocks adolescent ethanol and cannabinoid co-exposure-induced sustained impairment of neurogenesis and hippocampus-dependent functions during adulthood”

10:00 am to 10:20 am Martin Kaczocha, Stony Brook University “FABP5 coordinates lipid signaling that promotes prostate cancer metastasis”

10:20 am to 10:40 am

Lois Akinola, VCU “Inhibition of monoacylglycerol lipase reduces nicotine reward in the conditioned place preference test in mice”

10:40 am to 11:20 am Break, Hunton Student Center

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11:20 am to 11:40 am

Heather Bradshaw, Indiana University “GPR55 activity regulates CNS prostaglandins: mounting evidence for the interplay between cannabinoids and prostaglandins”

11:40 am to 12:00 pm

Rukiuyah Van Dross, East Carolina University “15-deoxy-prostamide J2, a metabolite of AEA, elicits cytotoxic and immune cell-mediated antitumor activity via the endoplasmic reticulum (ER) stress pathway”

12:00 pm to 12:20 pm

Tory Spindle, Johns Hopkins University “Pharmacodynamic and pharmacokinetic effects of vaporized and oral cannabidiol (CBD) and vaporized CBD-dominant cannabis in male and female infrequent cannabis users”

12:20 pm to 12:40 pm

Justin Poklis, VCU “Unregulated and adulterated cannabidiol (CBD) products and their unexpected effects on an unsuspecting public”

12:40 pm to 1:40 pm Lunch, Hunton Student Center Lobby

1:40 pm to 2:00 pm Dai Lu, Texas A&M University “CB1 positive allosteric ligands: new development and clinical relevance for developing novel pain relievers”

2:00 pm to 2:20 pm

Julien Dodu, VCU “The CB1 positive allosteric modulator, ZCZ011, attenuates naloxone-precipitated withdrawal signs in oxycodone-dependent mice”

2:20 pm to 3:00 pm Keynote Speaker: Ruth Ross, University of Toronto “CB1 NAMs: Paradox and Promise”

3:00 pm to 5:00 pm Break & Poster Session, Hunton Student Center 2nd floor lounge

6:00 pm to 8:00 pm

Buffet Dinner (with hosted beer/wine bar) Capital Ballroom, 2nd floor, Hampton Inn and Suites Downtown 700 E. Main St., Richmond, VA 23219

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Sunday, November 10th, 2019 6:30 am to 9:00 am Hot Breakfast (for hotel guests), Hampton Inn Lobby

9:20 am to 9:40 am Joel Schlosburg, VCU “Linking the resistance to opioid addiction by FAAH inactivation to stress mechanisms”

9:40 am to 10:20 am Keynote Speaker: Bradley Alger, Univ. of Maryland (Emeritus) “Thinking About Cannabinoids”

10:20 am to 10:40 am

Denise Valenti, Immad LLC “Marijuana and driving: your retina and brain”

10:40 am to 11:00 am

Aron Lichtman, VCU “A mouse in vivo screen of CB1 receptor allosteric modulators”

11:00 am to 11:20 am Concluding remarks, business meeting, and coffee break

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Poster Presentations – Saturday 3-5PM 1. Abby Pondelick, VCU “ZCZ011 analog does not act as an allosteric modulator of the CB1 receptor in vivo” 2. Mohammed Mustafa, VCU “Investigation of the novel anandamide analog (13S,1′R)-dimethylanandamide (AMG315) in the mouse drug discrimination paradigm” 3. Ian Jacobs, UNC - Chapel Hill “Attenuation of HIV-1 tat induced increases in sEPSC frequency by FAAH inhibition and upregulation of CB1R associated with inhibitory control deficits” 4. Kennedy Goldsborough, VCU “In vitro evaluation of diacylglycerol lipase inhibition: effects on the tumor suppressive capacity of paclitaxeI” 5. Ariel Myers, East Carolina University “Defining an orthotopic, syngeneic animal model for discovering the effect of endocannabinoids and their metabolites on cancer metastasis” 6. Clare Diester, VCU “Effects of Δ9-THC and monoacylglycerol lipase inhibitors on pain-stimulated and pain-depressed acute pain behaviors in mice” 7. Thomas Gamage, RTI “Promoting PAM versus agonist effects through structural changes in CB1 allosteric modulators” 8. Rosamond Goodson, VCU “Cannabidiol does not affect oxycodone self-administration or morphine somatic withdrawal in male Wistar rats” 9. Megan Key, UNC - Chapel Hill “Tat transgenic mice show altered synaptic integrity with a potential compensatory mechanism via cannabinoid type 1 receptors” 10. Daniel Barrus, RTI “Evaluation of CB1 positive allosteric modulator (PAM)-antagonists for probe dependence” 11. Ahmed Elhassanny, East Carolina University “The endocannabinoid metabolite, 15-deoxy,Δ12,14-prostamide J2, induces endoplasmic reticulum stress-dependent damage-associated molecular pattern (DAMP) expression in melanoma”

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12. Aly Rudy, VCU “Latent class analysis of young adult current cannabis users” 13. Alexis League, UNC - Chapel Hill “Monoacylglycerol lipase inhibition alters inhibitory control in tat transgenic mice” 14. Logan Neel, Auburn University “Computational design and synthesis of novel cannabinoid receptor 2 selective agonists” 15. Shanequa Taylor, RTI “Route of administration effects on Δ9-tetrahydrocannabinol discrimination in rats” 16. Barkha Yadav, UNC - Chapel Hill “CNS region-dependent increases in neuroprotective eCB-like lipids accompany microgliosis in morphine tolerant HIV-1 tat transgenic mice” 17. Erin Martin, Medical University of South Carolina “Assessment of impairment following oral and vaporized cannabis administration in infrequent users” 18. Changqing Xu, UNC - Chapel Hill “Effects of endocannabinoid enzyme inhibitors on neurotransmission in the prefrontal cortex of tat transgenic mice” 19. Alisha Eversole, Johns Hopkins University “Brain imaging of cannabinoid type 1 receptors in male and female non-users and female chronic users”

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CB1R ALLOSTERIC MODULATOR PSNCBAM1 BLOCKS ADOLESCENT ETHANOL AND CANNABINOID CO-EXPOSURE-INDUCED SUSTAINED IMPAIRMENT OF NEUROGENESIS AND HIPPOCAMPUS-

DEPENDENT FUNCTIONS DURING ADULTHOOD

Dal Khatri1, Bhupendra Nath1, Andrew Olivares 1,and Somnath Mukhopadhyay1, 2, 3,4

1 Neuroscience Research Program, Biomedical Biotechnology Research Institute, 2Department of Chemistry and Biochemistry, North Carolina Central University, Durham, NC; 3Bowles Alcohol

Research Center, 4Department of Pharmacology, UNC-Chapel Hill School of Medicine, Chapel Hill NC

Background: A large body of research has indicated the deleterious effects of both ethanol and cannabinoids on adolescent brain development and behavior. However, in spite of the increasing trend of coexposure to ethanol and cannabinoid during adolescence, very little is known about the effects of this combined exposure during adolescence in adult brain neurogenesis and hippocampal related learning and memory tasks in adulthood. The purpose of the current study is to determine a) if adolescence coexposure to ethanol and cannabinoid cause a significant change in neurogenesis and hippocampus related functions that persist during adulthood and b) if CB1R allosteric regulator can modify these effects in vivo. Method: Adolescent male Wistar rats at post-natal day (PND) 28 (body wt. 200-300 gm) were treated with vehicle or ethanol (3 mg/kg/day, ig) or CB1R agonist ACEA (0.3 mg/kg/day, ip,) or JZL195 (0.3 mg/kg/day, ip; dual inhibitor of endocannabinoid degrading enzymes FAAH& MAGL), on a 2 day on/2 day off paradigm alone (AIE: adolescent Intermittent ethanol(AIE) and AIC : adolescent Intermittent cannabinoid) or in combination (AIE+AIC) till PND 48 (11 treatments) in the presence and absence of CB1R allosteric regulator PSNCBAM1 (PSNC;0.3 mg/kg/day, i.p). For combination treatment ethanol and cannabinoid drug doses were reduced to half and PSNC was administered 30 min prior to ethanol, ACEA or JZL195 treatment. Body weight was taken every alternate day and 24 hr. following the last treatment, animals were subjected to Y-maze or NOR (Novel Object Recognition) tasks respectively using standard protocols. The animals were then allowed to grow to adulthood (PND 90) without any drug treatment except food and water ad libitum. At PND 91, the animals were again subjected to Y-maze or NOR tasks. After 24 hours of the last trial for both the tests, animals were sacrificed and brains were collected. Immunohistochemical analysis was carried out to assess for changes in neurogenesis, (DCX+IR), cell proliferation (Ki67) & apoptosis (cleaved caspase-3). Result: We found that adolescent exposure to ethanol, cannabinoids or their combination significantly impaired both spatial memory and novel object recognition during adolescence and these effects remain persistent when the same tests were performed during adulthood following a 6 weeks of abstinence. We also found that ethanol or CB1R agonist ACEA or endocannabinoid inactivation enzyme inhibitor alone or in combination significantly reduced doublecortin positive neurons (DCX+IR) with concomitant increase in cleaved caspase-3 positive cell in dentate gyrus of hippocampus. Pretreatment with PSNC significantly blocked the ethanol/cannabinoid-induced impairment of spatial memory function and novel object recognition ability and reversed ethanol-induced inhibition of adult neurogenesis and cell death. Interestingly PSNC did not produced any effect when administered alone. Conclusion: Together, the results from these studies revealed the following important phenomenon a) combined exposure to ethanol and cannabinoid during adolescence significantly blocked hippocampal-dependent spatial memory and object recognition function during adolescence, b) these effects persist even in the adulthood as evident from the reduced spatial memory and novel object recognition score measured during adulthood ; c) the impairment of the behavioral functions may be correlated with the reduced hippocampal neurogenesis and increased cell death; d) combined exposure produced a significantly greater damage than the drugs alone as the reduced doses used during combined treatment produced almost the same deleterious effects in memory functions and neurogenesis and e) CB1R allosteric modulator PSNC can attenuate the ethanol/ cannabinoid-mediated effects highlighting the possible therapeutic potential of the compound and/or its analogs.

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FABP5 COORDINATES LIPID SIGNALING THAT PROMOTES PROSTATE CANCER METASTASIS

Gregory Carbonetti1,2, Tessa Wilpshaar1,2, Jessie Kroonen1,2, Cynthia Converso2, Keith Studholme1, Simon D’Oelsnitz2 and Martin Kaczocha1,2

Departments of 1Anesthesiology, and 2Biochemistry and Cell Biology,

Stony Brook University, Stony Brook, NY, 11794, USA Prostate cancer (PCa) remains the second leading cause of cancer-related death in men in the United States. PCa is characterized by dysregulated lipid metabolism and signaling, which fuels the growth and metastasis of prostate tumors. Previous work has demonstrated that overexpression of monoacylglycerol lipase (MAGL) and fatty acid synthase (FASN), enzymes that generate free fatty acid pools, increases the metastatic potential of PCa. Fatty acid binding proteins (FABPs) are a family of intracellular proteins that bind to cellular lipids and facilitate their transport to diverse cellular compartments, including the nucleus. The healthy prostate does not express any FABPs. In contract, aggressive metastatic prostate tumors display robust upregulation of FABP5, which promotes tumor growth and metastasis. Here, we test the hypothesis that the pro-metastatic effects of FABP5 are dependent upon lipid pools generated by MAGL or FASN, and conversely that the products of these enzymes rely upon FABP5 for transport to nuclear receptors to enhance PCa metastasis. Overexpression of MAGL or FASN failed to increase migration or invasion in the poorly metastatic LNCaP cells (which do not natively express FABP5) in vitro. In contrast, co-expression of MAGL or FASN with FABP5 significantly increased cellular metastatic potential. Overexpression of MAGL or FASN in the aggressive PC3 cell-line (which natively expresses FABP5) enhanced migration and invasion while FABP5 knockdown suppressed this metastatic phenotype. Conversely, the ability of FABP5 to enhance migration and invasion was attenuated upon FASN or MAGL inhibition, suggesting a mutual dependency between FABP5 and MAGL/FASN to promote PCa metastasis. Mechanistically, the pro-metastatic effects elicited by MAGL/FASN and FABP5 overexpression were mediated by the nuclear peroxisome proliferator-activated receptor gamma (PPARγ) as evidenced by increased nuclear accumulation of FABP5, enhanced interactions between FABP5 and PPARγ, potentiation of PPARγ transcriptional activity, and attenuation of metastasis upon PPARγ inhibition. Implantation of PC3 cells into the ventral lobe of the prostate induced tumor formation that metastasized to femurs and lungs, which was suppressed upon FABP5 knockdown. Overexpression of MAGL or FASN enhanced primary tumor growth and metastasis, which was attenuated upon concomitant knockdown of FABP5. These results indicate that the pro-metastatic effects elicited by MAGL and FASN in vivo are critically dependent upon FABP5 expression. Collectively, our data position FABP5 as an integral component of a lipid signaling network that drives PCa metastasis and suggest that FABP5 may represent a promising target for the development of novel therapeutics to treat aggressive metastatic PCa. Acknowledgements: This work was funded by the Stony Brook University TRO Grant and the Department of Anesthesiology

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INHIBITION OF MONOACYLGLYCEROL LIPASE REDUCES NICOTINE REWARD IN THE CONDITIONED PLACE PREFERENCE TEST IN MICE

Lois S. Akinola1, Pretal P. Muldoon1, Joel Schlosburg1, Laura J. Sim-Selley1, Anu Mahadevan2,

Benjamin F. Cravatt3, Aron H. Lichtman1, and M. Imad Damaj1

1Department of Pharmacology and Toxicology, Medical College of Virginia Campus, Virginia

Commonwealth University, 1217 E Marshall St, Richmond, VA 23298, USA; 2Organix Inc., 240 Salem Street, Woburn, MA 01801, USA; 3Department of Chemistry, The Skaggs Institute for

Chemical Biology, The Scripps Research Institute, La Jolla , CA 92037, USA.

Nicotine, the main psychoactive component in tobacco, plays a major role in the initiation and maintenance of tobacco dependence and addiction, a leading cause of preventable death worldwide. Although a variety of nicotine cessation therapies has been developed, the efficacy of these treatments remains quite modest. Consequently, an essential need exists for more effective pharmacotherapies than current existing treatments. We have previously reported that pharmacological inhibition of monoacylglycerol lipase (MAGL), the primary catabolic enzyme of the endocannabinoid, 2-arachindonoylglycerol (2-AG), attenuates nicotine withdrawal. However, the consequences of MAGL inhibition on nicotine reward remain unknown. Therefore, we used the selective MAGL enzyme inhibitor, JZL184, to elevate endogenous 2-AG levels and found that it reduced nicotine conditioned place preference (CPP) model in mice. We also assessed if any observed effects were mediated via a CB1 receptor-dependent mechanism. Our data indicate that MAGL inhibition reduces nicotine CPP through a non-CB1 receptor mediated mechanism of action. Overall, the results of the present study suggest that MAGL inhibition represents a promising target for the development of nicotine therapies for the treatment of nicotine dependence.

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GPR55 ACTIVITY REGULATES CNS PROSTAGLANDINS: MOUNTING EVIDENCE FOR THE INTERPLAY BETWEEN CANNABINOIDS AND PROSTAGLANDINS

Heather B Bradshaw, Indiana University

Converging data supports a strong relationship between cannabinoid (CB) signaling and prostaglandin (PG) signaling in the CNS. Through lipidomics analysis of CNS tissue in a wide-range of CB-related proteins, our group has demonstrated that PGs are significantly increased in CB1, CB2, NAPE-PLD, and ABHD12 KOs, decreased in MAGL KOs, and had no change in FAAH KO CNS tissue. In addition, we recently showed that treatment with THC drives changes in PG levels in most brain areas, wherein PGs were predominately increased in adolescent mice and predominately decreased in the adult with no effect in some brain areas. By contrast, cannabidiol (CBD) caused significant decreases in PGs in all brain areas. We followed up these whole tissue analyses with treatment of THC, CBD, or CBD:THC in CNS-derived cell lines for microglia (BV2), astrocytes (C6 glioma), and neurons (N18). THC alone caused significant increases in PGs in BV2s and N18; whereas CBD caused significant decreases in PGs in BV2 and N18. THC:CBD treatment caused a mosaic response in PGs (combination of increases and decreases) in BV2s and N18. PGs were undetected in all treatment groups in C6 glicoma cells suggesting that baseline production of PGs are well below our detection level in these cells. (Unpublished data from our lab did measure PGs in C6 Gliomas after LPS stimulation illustrating that these CB-responsive cells also make PGs.) Here, we performed lipidomics analysis on GPR55 KO mice and found significant decreases in PGs in 6 of 8 brain areas. We then stimulated BV2 cells for two hours with the GPR55 agonist, O-1602 and caused an increase of PGs. 2-hour stimulation with the GPR55 antagonist ML-193 had no effect on PGs; however, 24-hour treatment with ML-193 did cause a decrease in PGs. Recent data demonstrates that CBD acts as a potent GPR55 antagonist, so future work in our lab is investigating if the reduction in PGs measured after CBD treatment is GPR55-dependent. Each of these lines of evidence illustrate the relationship with CB signaling and PG signaling, which is an understudied though important relationship that likely plays a role in both the medicinal and psychoactive activity of Cannabis.

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15-DEOXY-PROSTAMIDE J2, A METABOLITE OF AEA, ELICITS CYTOTOXIC AND IMMUNE CELL-MEDIATED ANTITUMOR ACTIVITY VIA THE ENDOPLASMIC RETICULUM (ER) STRESS PATHWAY

1,2Rukiyah Van Dross, 1Hussam Albassam, 1Ahmed Elhassanny, 1Ariel Myers, 1Kathleen Thayne,

3Daniel Ladin

1Department of Pharmacology and Toxicology, 2Center for Health Disparities, 3Department of Medicine, Brody School of Medicine, East Carolina University, Greenville, NC, USA

Cancer is the second leading cause of death in the United States. If diagnosed at the early stage, cancer is effectively managed with surgical approaches. However advanced stage disease, which has spread to other parts of the body, is more difficult to treat and requires intervention with cytotoxic and/or immunostimulatory antineoplastic agents. Immunotherapeutic agents have greatly improved clinical outcomes for different types of cancer. Unfortunately, these agents and the cytotoxic therapeutics cause significant, often life-threatening adverse effects. These toxicities are typically due to the damaging effect of the drug on non-tumor cells. Therefore, agents that harm cancer cells and have minimal impact on non-cancer cells are needed to reduce the side effects that are associated with antitumor drugs. Previous data from our laboratory showed that arachidonoyl ethanolamide (AEA) was cytotoxic towards different cancer cell lines. We determined that the antineoplastic activity of AEA was actually mediated by 15dPMJ2, a unique metabolic product of AEA that is produced in tumor cells that overexpress cyclooxygenase-2 (COX-2). According to our data, 15dPMJ2 exhibited greater toxicity towards non-melanoma skin cancer (NMSC), melanoma, and colon cancer cells compared to their non-tumor, cell type matched counterparts. This tumor selectivity was reliant upon the induction of the endoplasmic reticulum (ER) stress pathway. Interestingly, the ER stress pathway is also essential for activation of damage associated molecular patterns (DAMPs). After exposure to select cytotoxic agents, DAMPs are released from or found on the surface of tumor cells. DAMP signaling then elicits an antitumor immune response that leads to the development of T cell memory against the tumor cell type. Our data show that 15dPMJ2 increased DAMP expression selectively in tumor cells. Furthermore, in our in vivo studies, 15dPMJ2 inhibited melanoma tumor growth and it increased the quantity of tumor infiltrating immune cells. Taken together, the data demonstrate that 15dPMJ2 is a small molecule DAMP inducer that not only has highly selective cytotoxicity but also functions as an immune modulator. Therefore, 15dPMJ2 may provide long-lasting antitumor activity against advanced stage disease while affording considerable safety.

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PHARMACODYNAMIC AND PHARMACOKINETIC EFFECTS OF VAPORIZED AND ORAL CANNABIDIOL (CBD) AND VAPORIZED CBD-DOMINANT CANNABIS IN MALE AND FEMALE

INFREQUENT CANNABIS USERS

Spindle T.1, Goffi E.1, Cone, E.J. 1, Mitchell, J.2, Bigelow, G.E.1, Flegel, R.3, & Vandrey R.1

1Behavioral Pharmacology Research Unit, Johns Hopkins University School of Medicine

2RTI International, 3Substance Abuse and Mental Health Services Administration (SAMHSA)

The use and availability of oral and inhalable cannabidiol (CBD) products has increased drastically in recent years. Commercial CBD products often contain low levels of Δ-9-tetrahydrocannabinol (THC). Controlled clinical research on CBD products, with and without low levels of THC, is extremely scare. This study evaluated and compared the acute pharmacokinetics and pharmacodynamics of oral CBD, vaporized CBD, and vaporized cannabis containing high CBD and low THC. Eighteen healthy adults (9 men; 9 women) completed four, double-blind, double dummy, acute drug administration sessions on separate days (separated by 1 week). The four dosing conditions were: 100mg oral CBD, 100mg vaporized pure CBD, CBD-dominant cannabis (100mg CBD; 3.7mg THC), and placebo. Subjective drug effects, vital signs, and cognitive/psychomotor performance were evaluated at baseline and for 8 hours post-dosing. Whole blood, oral fluid, and urine samples were collected repeatedly over 3 days after each drug administration; urine results are currently only available for the first 6 completers. Vaporized CBD and CBD-dominant cannabis increased subjective ratings of “Drug Effect”, “Pleasant Drug Effect”, and “Like Drug Effect” relative to placebo. Women reported higher ratings than men for “Pleasant Drug Effect” and “Like Drug Effect” in the Vaporized CBD and CBD-dominant cannabis conditions. Subjective drug effects did not differ between oral CBD and placebo. CBD did not increase subjective ratings for items typically associated with acute cannabis exposure (e.g., “Paranoid”; “Heart Pounding”; “Anxious”). Impairment of cognitive/psychomotor performance was not observed in any dosing condition. THC and its principal metabolites (11-OH-THC, THCCOOH) were not detected in blood following pure CBD administration (both oral and vaporized). Whole blood CBD concentrations were higher following inhalation of CBD-dominant cannabis compared to pure CBD in crystalline powder form. Pure CBD (oral or vaporized) did not increase urinary concentrations of THCCOOH (target analyte in urine drug tests for cannabis), but 2/6 participants tested positive for cannabis using federal workplace urine drug testing guidelines. Overall, these results demonstrate that vaporization of pure CBD produces discriminable subjective drug effects, but does not produce “THC-like” subjective effects or cognitive/psychomotor impairment. In addition, CBD products that are contaminated with low levels of THC may produce positive urine results for cannabis. Additional research is needed to characterize the pharmacodynamic and pharmacokinetic effects of oral and vaporized CBD at higher doses, alone and in combination with varying THC doses, and also to explore the effects of chronic CBD use on drug testing outcomes. Acknowledgements: Funded by the Substance Abuse and Mental Health Services Administration

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UNREGULATED AND ADULTERATED CANNABIDIOL (CBD) PRODUCTS AND THEIR UNEXPECTED EFFECTS ON AN UNSUSPECTING PUBLIC

Justin L. Poklis1, Alaina K. Friedrich2, Kimberly Karin1, Haley A. Mulder2, Michelle R. Peace2

Departments of 1Pharmacology & Toxicology and 2Forensic Science, Virginia Commonwealth

University, Richmond, VA, USA Purpose: Cannabidiol (CBD) is a significant active ingredient of C. sativa and C. indica and has been purported to have anti-convulsant, anti-nociceptive, and anti-psychotic properties. A single CBD formulation has been approved by the United States Food and Drug Administration. The Farm Bill recently legalized hemp production in the United States, from which CBD can be extracted. A federally unregulated consumer-driven market of CBD products has created public health and safety challenges. The purpose of this research was to identify unlabeled psychoactive compounds in eight CBD products received from individuals who had purchased CBD products for therapeutic uses and reported untoward effects. Methods: Components of the CBD products were identified using a Shimadzu QP2020 Gas Chromatography Mass Spectrometry and a Direct Analysis in Real Time AccuTOFTM mass spectrometry. Results: Seven of eight products contained CBD. The eighth sample contained MMB-FUBICA. The other 7 contained 5F-ADB, dextromethorphan, AMB-FUBINACA, or other cannabimimetics. Other hemp related compounds were identified. Discussion: Reported use of these products led to statements such as “I have not been able to leave my apartment for four days”, “What happened next I can only describe as the situation rapidly devolving into the scariest night of my life – I felt like I was dissociating from reality” and “a 79 year old grandmother just wanted to be pain-free, but she had severe hallucinations”. All persons were surprised by the effects but were unsure of what action could be taken to report companies or distributors. Several were concerned that any attention would have adverse recourse, personally and/or professionally. Conclusions: Analysis of the eight samples revealed a number of unexpected novel psychoactive compounds that accounted for the untoward effects the individuals described. The federally unregulated CBD market has created a significant public health and safety problem in the United States. Acknowledgements: National Institutes of Health [P30DA033934] and National Institute of Justice, Office of Justice Programs [2016-DN-BX-0150]. The opinions, findings, and conclusions or recommendations expressed in this abstract are those of the authors and do not necessarily reflect those of the Department of Justice.

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CB1 POSITIVE ALLOSTERIC LIGANDS: NEW DEVELOPMENT AND CLINICAL RELEVANCE FOR DEVELOPING NOVEL PAIN RELIEVERS

Dai Lu,1* Debra Kendall,2 Aron Lichtman3

1Department of Pharmaceutical Sciences, Rangel College of Pharmacy, Texas A&M University;

2 Department of Pharmaceutical Science, University of Connecticut; 3Department of Medicinal Chemistry, Virginia Common Wealth University

ZCZ011 and GAT211 are the two leading CB1 positive allosteric ligands derived from 2-phenylindole scaffold. These compounds exhibited promising analgesic effects in preclinical models of neuropathic and inflammatory pain. To translate these early findings into novel pain relievers, optimization of these lead compounds are essential for providing experimental drugs to explore the complex pharmacology of CB1 allostery and conduct proof-of concept investigations. The recent development of Gi-protein biased allosteric agonists of μ-opioid receptor suggested that the analgesic effect and untoward side effects may be separated through the functional selectivity of allosteric ligands. However, ZCZ011 and GAT211 are racemates with one chiral center and an aliphatic nitro group, both of which are significant hurdles for structural optimization of the scaffold 2-phenylindole. We carried a SAR study to optimize the substituents on the indole ring, and developed a new scaffold by introducing an amino group at the C-3 position of the indole ring to create opportunities for removing the chiral center and conducting further structural optimization. In this study, several Gi-biased CB1 allosteric modulators were identified. Preliminary study showed that the compounds showed no significant cannabimimetic effects, and exhibited allosteric modulation properties. Characterization of their analgesic properties is on-going. The functional selectivity of these newly developed ligands and the new scaffold developed from this study are valuable for discovery of biased CB1 PAMs as novel pain relievers.

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THE CB1 POSITIVE ALLOSTERIC MODULATOR, ZCZ011, ATTENUATES NALOXONE-PRECIPITATED WITHDRAWAL SIGNS IN OXYCODONE-DEPENDENT MICE

Julien C. Dodu1, Joel E. Schlosburg1, Dai Lu2 and Aron H. Lichtman1

1Dept. of Pharmacology & Toxicology, Virginia Commonwealth University, Richmond, VA

2College of Pharmacy, Texas A&M, College Station, TX, USA In 2018, over 1.5 million American individuals were diagnosed with opioid use disorder (OUD). Although methadone and buprenorphine effectively ameliorate opioid withdrawal, a prominent issue of OUD, these drugs carry limitations including abuse liability and low to moderate compliance rates. Therefore, a great need exists to develop alternative and adjunct treatments for OUD. Stimulation of the cannabinoid 1 (CB1) receptor attenuates opioid withdrawal signs in pre-clinical models of opioid dependence; however, CB1 receptor agonists also elicit side effects, including cannabimimetic effects as well as tolerance and dependence following repeated administration. In contrast, CB1 allosteric modulators (AMs), specifically positive AMs (PAMs), target a topographically distinct site from the CB1 orthosteric site that induces a conformational change that enhances orthosteric ligand binding. CB1 PAMs produce antinociceptive effects in mouse models of neuropathic and inflammatory pain, without the development of tolerance or dependence. CB1 PAMs also attenuate cannabinoid withdrawal signs in mice. Therefore, we hypothesize that CB1 PAMs will dose-dependently attenuate naloxone-precipitated somatic withdrawal signs in oxycodone dependence in mice. In the present study, male and female CD-1 mice were administered daily subcutaneous (s.c.) injections of oxycodone (dissolved in 0.9% saline) for eight days with increasing doses of 9, 17.8, 23.7, and 33 mg/kg oxycodone b.i.d. (~7 h separating injections) on days 1-2, 3-4, 5-6, 7-8, respectively. On the morning of day 9, mice were administered 33 mg/kg oxycodone, 2 h later administered s.c. naloxone (1 mg/kg), and immediately recorded for 30 min. An observer blinded with respect to condition scored somatic withdrawal signs including jumping, paw flutters, head shakes, digging, backing, diarrhea, and decrease of body weight. We next evaluated whether an intraperitoneal injection of the CB1 PAM, ZCZ011 (0, 5, 10, 20, or 40 mg/kg), administered 75 min prior to naloxone injection would attenuate somatic withdrawal signs. ZCZ011 (20 and 40 mg/kg) significantly reduced paw flutters, head shakes, and body weight loss, and 40 mg/kg reduced diarrhea. Ongoing studies are testing whether the anti-withdrawal effects of ZCZ011 require CB1 receptors. These findings support the hypothesis that the CB1 PAM, ZCZ011, dose-dependently attenuated naloxone-precipitated somatic withdrawal signs in oxycodone-dependent mice.

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LINKING THE RESISTANCE TO OPIOID ADDICTION BY FAAH INACTIVATION TO STRESS MECHANISMS

Joel E. Schlosburg, Erica N. Golden, Rosamond M. Goodson

Department of Pharmacology & Toxicology, Virginia Commonwealth University, Richmond, VA Over the past several years, our lab has presented research on the reduced escalation of opiate intake due to FAAH inactivation, as well as reduced motivation to self-administer opiates under extended-access conditions. After extensive testing of differential sensitivity to acute heroin, and non-drug rewards, as well as rewarding properties in the context of escalating use, it appears the reductions in opioids are not primarily due to alterations in reward. We are instead examining these findings primarily in the theoretical context of stress regulation, and aiming to test the hypothesis that the reduced vulnerability in transitioning to a compulsive, dependent state are due to a reduction of stress activation under drug-taking conditions, and reduced stress activity and aversiveness in withdrawal states. Using a combination of PF3845 (2mg/kg iv or 5mg/kg sc/ip) or FAAH knockout rats, we examined serum corticosterone (CORT) levels in extended-access heroin self-administering subjects to determine the timeline of stress activation and negative feedback, as well as during withdrawal. We also tested the sensitivity of naloxone to suppress operant responding for sucrose pellets following the induction of opioid dependence via morphine pellet implantation (2x75mg). FAAH inactivation reversed the cycle of heroin intake elevating CORT levels with escalating use, followed by a crash in CORT response likely due to negative feedback inhibition. Importantly, while both showed some increase in stress activation during modest withdrawal, it was greatly reduced in the FAAH knockout subjects. We also found that FAAH knockout rats were far less sensitive to the naloxone withdrawal-induced suppression of responding for sucrose pellets. This suggests that FAAH inhibition may reduce opioid addiction risk through mechanisms related to the stressfulness and aversiveness of repeated opioid withdrawal. Acknowledgments: Funding provided by the VCU School of Medicine and NIH grant K99/R00DA037344.

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MARIJUANA AND DRIVING: YOUR RETINA AND BRAIN

Dr. Denise A. Valenti, OD, FAAO

Optometrist, IMMAD, Quincy, MA

Background Dysfunction of the retina with marijuana use can result in an impairment to drive. Cannabinoid receptors are found throughout the human retina. Marijuana use causes dysfunction in retinal ganglion cells with both acute and chronic use. The variety of retinal receptors indicate the potential for different actions of cannabinoids; TCH, CBD, CBG and CBN. Users consuming marijuana acutely, describe a tunnel vision effect. However, the only report of peripheral dysfunction in literature, describe reductions along the only meridian measured, 180 degrees (Moskowitz). An animal model shows that cannabis can impair the lateral geniculate nucleus-LGN, and functional brain imaging in humans demonstrate similar deficits. The LGN and occipital lobe changes indicate potential dysfunction elsewhere in addition to the retina. Processing upstream also has the potential of lateralization of functional impairment. Method The participants presented in this study were part of a National Institutes of Health and National Institutes on Drug Abuse, Small Business Innovation Research (SBIR) Phase I contract. The intent of the Phase I protocol was to develop an objective technology in a goggle/smartphone system with a Bluetooth response button. The system is a visual field test using square striped temporal targets of variable contrast. The prototype goggle system is shown below. This IRB approved project uses opportunistically dosed participants. Each volunteer is seen for an initial un-dosed visit. The participant is administered an informed consent concerning their participation in the study, and fills out a Daily Use Of Marijuana Questionnaire to gather demographic information. They then advise the research team of a date and time when they plan to consume marijuana by vaping or inhalation of their own legal marijuana. They are not required to use any specific product or provide labels. All participants have a best corrected visual acuity of at least 20/40 in each eye. We utilized a Zeiss 710 Frequency Doubling Technology (FDT) visual field test during the un-dosed and dosed visits as well. FDT uses a variable contrast low spatial frequency grating of 10° presented at 1 of 16 test locations, and a 5° central target, to test the central-midperipheral forty degrees of vision. The grating alternates at 25 Hz. FDT has twelve reliability measures; six fixation tests, three false positives and three false negatives. An image of an FDT is below right. Discussion Using opportunistically dosed participants for marijuana studies is problematic, but does allow for an understanding of how marijuana impacts the visual system. The individual case using the FDT data shown on the far right, is a 24 year old male. This case is demonstrating the more common response we have found in a number of participants, with a trend of impairment in the superior left quadrant. While we hypothesize that the neuroprocessing dysfunction is at the retinal level and this is supported by electrodiagnostic studies in acute and chronic users, the lateralization points to changes further upstream. We hypothesize the LGN. The significant points identified in this work did not support lateralization. Cannabinoids are diverse and retinal functioning related to cannabis is complex. Studies with greater numbers of participants, control over the products used and the amount consumed will allow us to better characterize the dysfunction in the retina and further upstream. However, even in this small study, the implications for impairment to drive are evident. More research is certainly warranted. Acknowledgements: IMMAD - Impairment Measurement Marijuana and Driving and is funded through an NIH NIDA SBIR contract.

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A MOUSE IN VIVO SCREEN OF CB1 RECEPTOR ALLOSTERIC MODULATORS

Aron H. Lichtman1, Mohammed Mustafa1, Guilia Donvito1, Amelia Swafford1, Julien Dodu1, Lauren Moncayo1, Rebecca Moncayo1, Dai Lu2, and Debra A. Kendall3

1Dept. of Pharmacology & Toxicology, Virginia Commonwealth University, Richmond, VA 2Rangel College of Pharmacy Health Science Center, Texas A&M University, Kingsville, TX

3Department of Pharmaceutical Sciences, University of Connecticut Storrs, CT The endogenous cannabinoid system possesses multiple potential therapeutic targets, including two cannabinoid receptors (CB1 and CB2) and enzymes regulating synthesis and degradation of endogenous cannabinoids. Ligands that bind CB1 show promise in a variety of preclinical models of pain, anxiety, nausea and emesis, and obesity; however, their distinct array of side effects dampen enthusiasm for wide spread therapeutic development. Alternatively, ligands binding putative CB1 allosteric sites modify receptor conformation, which leads to changes in binding and/or functional properties/efficacies of orthosteric ligands. Accordingly, CB1 allosteric modulators are hypothesized to alter CB1 binding/efficacy of the endogenous cannabinoids to elicit potential therapeutic effects (e.g., antinociception), but with a reduced side effect profile. Although a growing number of CB1 allosteric modulators have been identified through functional reporter-based in vitro assays, relatively few of these ligands have been adequately tested for in vivo activity. During this presentation, we will describe the use of a battery of tests in behaving mice to identify in vivo activity of CB1 allosteric modulators. Whereas most of ligands failed to elicit in vivo effects consistent with CB1 allosteric activity, three structurally related CB1 positive allosteric modulators (ZCZ011, GAT211, ABD1236) found to be positive in screening assay also produced antinociceptive effects in neuropathic pain models. These antinociceptive effects occurred in the absence of cannabimimetic side effects and did not undergo tolerance following repeated administration. Taken together, these findings provide proof of principle that CB1 allosteric modulators offer promise as a potential therapeutic strategy to treat neuropathic pain. This research was supported in part by NIH grant DA039942.

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IN VIVO EFFECTS OF THE CB1 RECEPTOR ALLOSTERIC MODULATOR LDK1730 IN MICE 1Abby Pondelick, 1Julien Dodu, 1Mohammed Mustafa, 2Debra Kendall, 3Dai Lu, and 1Aron

Lichtman

1Dept. of Pharmacology & Toxicology, Virginia Commonwealth University, Richmond, VA 2Department of Pharmaceutical Sciences, University of Connecticut Storrs, CT

3Rangel College of Pharmacy Health Science Center, Texas A&M University, Kingsville, TX

The CB1 receptor has become a popular focus of study since activation of the receptor results in an array of pharmacological and behavioral effects, including but not limited to antinociceptive and anti-stress responses, changes in feeding behavior and energy storage, and alterations of motor performance. However, the therapeutic potential of the CB1 receptor is limited by the adverse side effects, such as psychoactive intoxication, dependence, and hinderance to short-term memory, associated with receptor activation by agonists (e.g. delta-9-tetrahydrocannabinol (THC)). Allosteric modulators present a promising therapeutic approach, since they bind to a secondary (allosteric) site that is distinct from the primary (orthosteric) binding site used by agonists such as THC; therefore, inducing conformational changes of the receptor to increase (positively allosteric modulator: PAM) or decrease (negative allosteric modulator: NAM) the potency and efficacy of orthosteric ligands without producing unwanted side effects. The CB1 allosteric modulator, ZCZ011, has previously been shown in vivo to produce no common cannabimimetic effects while augmenting the actions of CB1 orthosteric ligands and producing antinociception in models of inflammatory and neuropathic pain. However, little is known about the relationship between the structure of CB1 allosteric modulators and the resulting effects in vivo, underscoring the importance of studying structural analogs to better understand how differing functional groups act upon the receptor. The present study explored whether the CB1 receptor allosteric modulator LDK1730, an analog of ZCZ011, elicits common cannabimimetic side effects or alters responses to an orthosteric ligand. To first test whether LDK1730 exhibited any pharmacological effects when given alone, mice were injected with LDK1730 (40 mg/kg) or vehicle and assessed on the tetrad assay which measures catalepsy, antinociception, hypothermia, and locomotor activity. All measurements occurred 30, 60, and 120-min post-injection, except locomotor activity which was only tested at 30-min post injection. Similar to ZCZ011, LDK1730 resulted in no significant changes across all tetrad measures. We next examined if this analog altered the potency or efficacy of an orthosteric agonist, by testing LDK1730 (40 mg/kg) or vehicle in combination with the potent synthetic cannabinoid, CP55,940 (0.1, 0.3 1.0, 3.0 mg/kg) on measurements of catalepsy, antinociception, and hypothermia. LDK1730 failed to produce any shift in the CP55,940 dose-response curve on all measures. The present results suggest LDK1730 does not produce any common cannabimimetic effects, but the analog is also not acting as a PAM or NAM of the CB1 receptor in vivo. These findings and future studies of different ZCZ011 analogs aid the understanding and exploration of how allosteric modulators act at the CB1 receptor and how the structure effects behavioral responses. Acknowledgements: This work is supported by NIH grant #’s R01DA039942 and P30DA033934

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INVESTIGATION OF THE NOVEL ANANDAMIDE ANALOG (13 S,1′R)-DIMETHYLANANDAMIDE (AMG315) IN THE MOUSE DRUG DISCRIMINATION PARADIGM

Mohammed Mustafa1, Aron Lichtman1, Lipin Ji2, Yingpend Liu2, Alexandros Makriyannis2,

Spyridon Nikas2

1 Dept. of Pharmacology & Toxicology, Virginia Commonwealth University, Richmond, VA. 2 Center for Drug Discovery and Department of Pharmaceutical Sciences, Northeastern

University, Boston, MA. The endogenous cannabinoid anandamide modulates a wide variety of physiological processes through its interaction with CB1 and CB2 receptors, in addition to other classes of receptors such as the transient receptor potential cation channel subfamily V member 1. Anandamide is a low-efficacy agonist at the CB1 receptor and is rapidly degraded by fatty acid amide hydrolase (FAAH). This rapid deactivation of anandamide limits its utility as a therapeutic agent or as a probe for investigating the endocannabinoid system in whole organisms. Although FAAH inhibitors are available for research purposes, have been tested in clinical trials, and elevate anandamide levels in brain, these inhibitors also increase brain levels of other fatty acid amide signaling molecules. Thus, the pharmacological effects of FAAH inhibitors may be mediated by anandamide, other fatty acid amides, or an interaction of these myriad lipid signaling molecules. In contrast, metabolically stable analogs of anandamide with increased affinity and efficacy at the CB1 receptor represent useful tools for structure activity relationship studies. A newly developed chiral anandamide analog, (13 S,1′R)-dimethylanandamide (AMG315) is resistant to hydrolysis by FAAH as well as the oxidative enzyme COX-2. In competition binding experiments using [3H]CP55,940 as the radioligand AMG315 possesses high affinity for the CB1 receptor (Ki ± SEM = 7.8 ± 1.4 nM) and is a potent agonist of CB1 in vitro (EC50 ± SEM = 0.6 ± 0.2 nM). In addition, AMG315 produces an antinociceptive effect in the complete Freund’s adjuvant model of inflammatory pain (Liu et al. 2018). In the present study, we report that AMG315 dose-dependently substitutes for CB1 agonists and produces response rate suppression in the mouse drug discrimination paradigm. Male C57BL/6J mice were trained to discriminate the high efficacy CB1 receptor agonist CP55,940 (0.1 mg/kg) from vehicle, and male FAAH (-/-) mice were trained to discriminate anandamide (6 mg/kg) from vehicle. AMG315 (0.3 – 6 mg/kg) dose-dependently substituted for drug-like responding and dose-dependently reduced operant response rates in both groups of mice. This study demonstrates that AMG315, a novel chiral anandamide analog, produces in vivo subjective effects similar to CB1 receptor agonists in the mouse drug discrimination paradigm. Acknowledgements: This work supported by National Institutes of Health (Grant numbers R01DA039942 and P30DA033934)

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ATTENUATION OF HIV-1 TAT INDUCED INCREASES IN sEPSC FREQUENCY BY FAAH INHIBITION AND UPREGULATION OF CB1R ASSOCIATED WITH INHIBITORY CONTROL DEFICITS

Ian R. Jacobsa, Changqing Xua, Douglas J. Hermesa, Callie Xua, Micah J. Niphakisb, Benjamin F.

Cravattb, Ken Mackiec, Aron H. Lichtmand, Bogna M. Ignatowska-Jankowskae and Sylvia Fittinga

aDepartment of Psychology & Neuroscience, University of North Carolina at Chapel Hill,

Chapel Hill, NC 27599, USA; bDepartment of Chemical Physiology, Scripps Research Institute, La Jolla, CA 92037, USA; cDepartment of Psychological & Brain Science, Indiana University,

Bloomington, IN 47405, USA; dDepartment of Pharmacology & Toxicology, Virginia Commonwealth University, Richmond, VA 23298, USA; eOkinawa Institute of Science and

Technology, Neuronal Rhythms in Movement Unit, Okinawa 904-0495, Japan As individuals with HIV live longer lives, owing to cART therapy, a new mild form of cognitive impairment known as HIV-1 Associated Neurocognitive Disorder (HAND) has emerged. The transactivator of transcription (Tat) acts directly on neurons but also indirectly via its actions on glia to mediate the neurotoxic effects in HAND. The Go/No-Go (GNG) task was used to examine HAND in the Tat transgenic mouse model. The GNG task involves subjects discriminating between two stimuli sets in order to know whether to inhibit a previously trained response or not. Results reveal inhibitory control deficits in female Tat(+) mice (p = .048) and an upregulation of cannabinoid type 1 receptors (CB1R) in the infralimbic (IL) cortex in the same female Tat(+) group (p = .023). A significant negative correlation was noted between inhibitory control and IL CB1R expression (r = -.543, p = .045), with CB1R expression predicting 30% of the variance of inhibitory control (R2 = .295, p = .045). Furthermore, there was a significant increase in spontaneous excitatory postsynaptic current (sEPSC) frequencies in Tat(+) compared to Tat(-) mice (p = .008, combined sex). The increase in sEPSC frequency was significantly attenuated by bath application of PF3845, a fatty acid amide hydrolase (FAAH) enzyme inhibitor (p < .001). Overall, the GNG task is a viable measure to assess inhibitory control deficits in the Tat transgenic mouse model and results suggest a potential therapeutic treatment for the observed deficits using drugs that modulate endocannabinoid enzyme activity. Acknowledgements: We gratefully acknowledge the support from the National Institute on Drug Abuse (NIDA R21 DA041903, R01 DA045596, T32 DA007244, R01 DA039942, P30 DA033934, and R21 AG042745). Bogna M. Ignatowska-Jankowska was supported by the fellowship from the Japan Society for Promotion of Science (JSPS) and Grant-in-Aid for JSPS fellows 17F17388.

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IN VITRO EVALUATION OF DIACYLGLYCEROL LIPASE INHIBITION: EFFECTS ON THE TUMOR SUPPRESSIVE CAPACITY OF PACLITAXEL

Kennedy Goldsborough, Lauren Kyte, Valerie Carpenter, Nipa Patel, Giulia Donvito, David

Gewirtz, and Aron Lichtman

Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond, VA, USA

Paclitaxel is an effective chemotherapeutic agent for lung, breast, and ovarian cancers, which also produces significant debilitating side effects, particularly chemotherapy-induced peripheral neuropathy (CIPN). CIPN features include numbness, tingling, loss of proprioception, hyperalgesia, and allodynia, and often persists long after the end of treatment. As there are no treatments to prevent this condition, patients are often forced to alter their cancer treatment because CIPN can substantially reduce quality of live. Several components within the endocannabinoid system represent propitious targets for the prevention or treatment of CIPN, because of their antinociceptive and anti-inflammatory properties in rodent models. Our ongoing work revealed that inhibition of the endocannabinoid synthetic enzymes diacylglycerol (DAGL)-α and-β ameliorate nociceptive behavior in an established paclitaxel mouse model of CIPN. As DAGL is a viable target to treat neuropathic pain associated with chemotherapy, we sought to examine whether the DAGL inhibitors DO34 and KT109 would alter paclitaxel-induced cytotoxicity or alter tumor growth on their own in two non-small cell lung cancer (NSCLC) lines, H460 and A549, by quantifying cell viability and apoptosis. To assess cell viability, H460 and A549 cells were treated with vehicle, DO34 (500 nM), or KT109 (500 nM) in combination with vehicle or paclitaxel (100 nM) for 24 hours. Cell cultures were then washed to remove the drug, and counted via trypan blue exclusion on days 1, 3, 5, and 7 post-treatment. In order to assess apoptosis, H460 and A549 cells were exposed to the aforementioned drug conditions for 48 hours. Cells were then stained with Annexin V / Propidium Iodide, incubated, and evaluated via flow cytometry. Neither KT109 nor DO34 altered NSCLC cell proliferation and apoptosis alone or interfered with paclitaxel-induced growth inhibition of NSCLC cells. While additional studies are needed to examine KT109 and DO34 in mouse xenograft mouse models of cancer, the present findings suggest that DAGL inhibitors lack intrinsic effects on tumor growth alone as well as do not interfere with the anti-proliferative effects of paclitaxel. Accordingly, these data support the concept that targeting DAGL to prevent or treat CIPN may not affect cancer growth or paclitaxel treatment of NSCLCs.

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DEFINING AN ORTHOTOPIC, SYNGENEIC ANIMAL MODEL FOR DISCOVERING THE EFFECT OF ENDOCANNABINOIDS AND THEIR METABOLITES ON CANCER METASTASIS

1Ariel Myers, 1Kathleen Thayne, 2Gina Murray and 1,3Rukiyah Van Dross

1Department of Pharmacology and Toxicology; 2Department of Pathology, 3Center for Health Disparities, Brody School of Medicine at East Carolina University; Center for Health Disparities

Greenville, NC, USA

Metastatic melanoma (MM) is the deadliest form of skin cancer in the United States with one person dying from the disease every hour. Metastasis occurs when cells from the primary tumor enter the systemic circulation and grow at local and distant organ sites. The most common sites of melanoma metastasis are the lymph nodes, bone, brain, and lungs. Melanoma that has metastasized is not effectively treated with cytotoxic therapeutics that are currently available, although immunotherapeutic and targeted agents produce more favorable outcomes. Despite the availability of these effective agents, MM is still associated with high mortality rates. In order to develop new and highly effective agents that robustly eliminate MM, current models of metastatic disease in immunocompetent animals must be improved. Several in vivo models, including those that look at endocannabinoids and their metabolites, focus on injecting tumor cells directly into the circulation via the tail vein to allow lesions to form within the lungs. While these models permit rapid metastasis at a relevant sites, they lack primary tumor formation and therefore, do not mimic the clinical disease. Other MM models employ subcutaneous tumor inoculation to permit primary tumors to spontaneously metastasize. However, the subcutaneous space is not an orthotopic site for melanoma. Therefore, well-established models of spontaneous metastasis from orthotopic sites must be improved to more accurately, recapitulate MM and study the effect of endocannabinoids and their metabolites in this disease. The goal of the current study is to modify an existing syngeneic, orthotopic model of spontaneous MM to more precisely reproduce clinical features of metastasis. In this model, B16F10 melanoma cells that expressed firefly luciferase were inoculated on the dorsal ear of C57BL/6 mice. Spontaneous metastasis was then facilitated by removing the primary lesion to prolong the duration before humane endpoints were reached. Metastatic tumor formation in the draining lymph node and at distant sites were observed by using the IVIS Lumina imaging system and by gross examination of target organs after euthanasia. According to our preliminary data, primary tumors developed after an average of 2-3 weeks and metastatic lesions formed in the draining lymph node approximately 8 weeks after inoculation. Phenotypic characteristics that served as markers of metastasis and determinants of primary tumor removal were also identified. Histopathologic analysis of the primary and metastatic tumors revealed that the morphological appearance of the lesions was consistent with patient melanoma. Further optimization of this model will allow us to mimic MM more precisely. Animal tumor models with greater translational value will provide a more comprehensive understanding of the metastatic process and ultimately, the effects endocannabinoids and their metabolites in MM.

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A STRATEGY TO TEST CANDIDATE ANALGESIC DRUG EFFECTS ON ACUTE PAIN-STIMULATED AND PAIN-DEPRESSED PAIN BEHAVIORS

IN MICE

Clare Diester, Matthew Banks, S. Stevens Negus

Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond, VA, USA

Introduction. Preclinical assays of pain and analgesia often yield false-positive effects with candidate analgesics. This study evaluated a strategy to improve predictive validity by comparing drug effects on complementary endpoints of pain-stimulated and pain-depressed behaviors. Methods: Intraperitoneal administration of dilute lactic acid (IP acid) served as an acute noxious stimulus in male and female ICR mice to produce four pain-related behaviors: stimulation of (1) stretching and (2) facial grimace, and depression of (3) rearing and (4) nesting. Studies compared effects of two clinically-effective positive controls (ketoprofen and oxycodone), an active negative control (diazepam), and two cannabinoid drugs under consideration as candidate analgesics [the cannabinoid receptor agonist ∆9-tetrahydrocannabinol (THC) and the monoacylglycerol lipase inhibitor MJN110]. Drug effects on control nesting in the absence of IP acid were also determined to assess drug potency and effectiveness to produce general behavioral disruption. Results. IP acid stimulated stretching and facial grimace and depressed rearing and nesting. Drug potencies and efficacies to block IP acid effects and to produce general behavioral disruption of control nesting were used to generate antinociception profiles and rank drugs for apparent analgesic effectiveness and safety: ketoprofen > oxycodone > MJN110 > THC > diazepam. At the extremes, ketoprofen blocked all IP acid effects at doses that had no effect on control nesting, whereas diazepam reduced only IP acid-stimulated stretching and grimace and only at a dose that also disrupted control nesting. Discussion. This approach provides a strategy to evaluate drug effects on a panel of complementary behavioral endpoints and prioritize drugs for promotion to clinical testing. Acknowledgements: NIH grants T32DA007027 and R01DA030404

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PROMOTING PAM VERSUS AGONIST EFFECTS THROUGH STRUCTURAL CHANGES IN CB1 ALLOSTERIC MODULATORS

Thomas F. Gamage, Charlotte E. Farquhar, Daniel Barrus, Tony Landavazo, Joseph Wilson,

Brian F. Thomas, Bruce E. Blough, and Jenny L. Wiley

RTI International, Research Triangle Park, North Carolina, USA Positive allosteric modulators (PAMs) of the cannabinoid type-1 (CB1) receptor have shown promise in preclinical models of pain (e.g. ZCZ011, GAT211)1,2 while producing minimal undesirable cannabinoid effects which typically occur with orthosteric ligands. Mechanisms through which CB1 PAMs may be producing these effects through the CB1 receptor include 1) enhancement of endogenous cannabinoid signaling via positive binding cooperativity (α), a bidirectional vector through which both ligands increase the affinity of the other for the receptor; 2) promotion of receptor conformations through which endogenous cannabinoids can exert enhanced efficacy (β); and/or 3) allosteric agonism in which the modulator activates the receptor in the absence of orthosteric ligand (τB). These can be observed when an allosteric modulator is co-applied in an agonist concentration response curve as 1) leftward shifts in the agonist pEC50; 2) increases in Emax; and 3) increases in the bottom of the concentration-response curve3. In order to examine how structure may impact these three allosteric properties, SAR studies were conducted using the 3-(2-nitro-1-phenylethyl)-2-phenylindole scaffold with a focus on substitutions of the nitro and phenyl groups on the 3-ethyl moiety. Effects of 10 µM of modulator on CP55,940s’ pharmacology in [35S]GTPγS binding were conducted. Many of the compounds tested retained allosteric activity as determined by significant differences in CP55,940’s pEC50 [F(49,111)=39.3 p< 0.0001, Emax [F(49,106)=9.24 p< 0.0001] and curve bottom [F(49,110)=46.60 p<0.0001]. The percent increase in [3H]CP55,940 binding by the select compounds predicted shifts in the bottom, [p<0.001; r=0.90 (0.81 – 0.95)], pEC50 [p<0.001; r= 0.91 (0.83 – 0.95)] and Emax [p<0.001; r= 0.59 (0.32 – 0.78)] parameters of CP55,940’s concentration-response curve. An interesting pattern emerged from substitutions on the 1-phenylethyl group where ortho- substitutions enhanced alpha, meta- substitutions were mostly tolerated, but para- substitutions reduced alpha, suggesting that the conformation of the phenyl ring plays a significant role in how a ligand induces allostery and/or potentially interacts with the orthosteric site. Notably, halogen substitutions at the ortho position exhibited enhanced allosteric agonism whereas oxygen containing groups promoted PAM effects with less agonism. Carbamate substitutions for the 2-nitro group promoted NAM-like activity as CP55,940 pEC50 values were mostly right-shifted by these compounds, particularly with inclusion of para-substitutions on the 1-phenylethyl group. These data provide insight into structural elements of the 2-phenylindole scaffold that could enhance allosteric parameters and/or affinity at the CB1 receptor. Acknowledgements: R01 DA-03672, R01 DA-40460, K01 DA-045752

1. Ignatowska et al. (2015). Neuropsychopharm, 40, 2948-2959. 2. Slivicki et al. (2018). Biol Psychiatry, 84(10):722-733 3. Kenakin, TP & Miller, LJ (2010). Pharmacol Rev, 62(2), 265-304.

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CANNABIDIOL (CBD) DOES NOT AFFECT OXYCODONE SELF-ADMINISTRATION OR MORPHINE SOMATIC WITHDRAWAL IN MALE WISTAR RATS

Rosamond M. Goodson, Erica N. Golden, Joel E. Schlosburg

Department of Pharmacology & Toxicology, Virginia Commonwealth University, Richmond,

VA, USA

Cannabidiol (CBD), a non-psychoactive phytocannabinoid present in Cannabis sativa, has been widely promoted as a potential treatment for epilepsy (recently receiving FDA approval for that purpose), chronic pain, cancer, and other health problems, including drug use disorders. It’s pervasiveness and over-the-counter expansion necessitates validation of potential benefits. Several preclinical studies on cocaine, methamphetamine, ethanol, and opioids suggest that CBD may attenuate drug self-administration, reinstatement, and/or withdrawal. Recent studies from the group of Yasmin Hurd in rodents and humans show that acute CBD can modestly reduce cue-induced reinstatement in rodents, and cue-induced craving and anxiety for heroin in patients with opioid use disorder. Those same studies also showed no changes in baseline intake in rodents, or overall craving and relapse ratings in humans. To date, there is little in the literature on the effects of CBD administration on opioid self-administration in dependent subjects. The purpose of the present study was to examine CBD’s effects on extended-access oxycodone self-administration and somatic withdrawal in male Wistar rats. Using doses like those previously published (30 mg/kg), we examined the intake of heroin in separate groups of 2hr-access and 12-hr access oxycodone self-administering rats. Whether stable non-dependent users or dependent escalated users, CDB pretreatment did not alter self-administration, especially when compared to our positive control buprenorphine. Similarly, in a classical naloxone-precipitated (1 mg/kg) withdrawal model in rats dependent on morphine implanted pellets (2x75 mg), CBD failed to reduce weight loss and general somatic signs compared to a FAAH inhibitor (PF3845) and Δ9-THC. This suggests that CBD, at active doses known to reduce cue-induced craving, is unlikely to significantly reduce relapse or excessive intake risks associated with opioid dependence. Acknowledgments: Funding provided by the VCU School of Medicine and NIH grant K99/R00DA037344.

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TAT TRANSGENIC MICE SHOW ALTERED SYNAPTIC INTEGRITY WITH A POTENTIAL COMPENSATORY MECHANISM VIA CANNABINOID TYPE 1 RECEPTORS

Megan C. Key1, Hong Yuan2, Zibo Li3, Douglas J. Hermes4, Ian R. Jacobs4, Ken Mackie5, Aron H.

Lichtman6, Bogna M. Ignatowska-Jankowska7, and Sylvia Fitting4

1Curriculum in Neuroscience, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA; 2Biomedical Research Imaging Center, Department of Radiology, University of North Carolina

Chapel Hill, Chapel Hill, NC, USA; 3Biomedical Research Imaging Center, Department of Radiography, University of North Carolina Chapel Hill, Chapel Hill, NC, USA; 4Department of Psychology & Neuroscience, University of North Carolina Chapel Hill, Chapel Hill, NC, USA; 5Department of Psychological & Brain Sciences, Indiana University, Bloomington, IN, USA;

6Department of Pharmacology & Toxicology, Virginia Commonwealth University, Richmond, VA, USA; 7Neuronal Rhythms in Movement Unit, Okinawa Institute of Science and Technology,

Okinawa, Japan HIV-1 associated neurocognitive disorders (HAND) affect 30-50% of HIV+ patients. Although combined anti-retroviral therapies (cART) have worked to decrease the progression of HIV-1 towards AIDS and death, its limited penetration into the CNS is inefficient to treat HAND. Therefore, it is necessary to research and develop neuroprotective therapies that target the CNS. HAND is characterized by severe and minor cognitive decline linked to synaptodendritic neuronal injury. Using a Tat transgenic mouse model, previous work in our lab has demonstrated that CNS expression of HIV-1 Tat protein alone is able to recapitulate the neurodegenerative effects of HAND. In the present study we analyzed ubiquitous synaptic densities in the Tat transgenic mouse model via positron-emission tomography (PET) imaging using the synaptic vesicle glycoprotein 2A (SV2A) selective radiotracer, 11C-UCB-J. Experiments were conducted in a longitudinal study, assessing SV2A at baseline, 2 weeks, and 3 months of Tat induction. Our data indicate a significant downregulation of SV2A in the cerebrum of Tat(+) transgenic mice over time compared to Tat(-) controls (p = 0.047). Interestingly, overexpression of Tat also results in the altered expression of synaptotagmin II, a key calcium sensing synaptic vesicle protein known to cotraffick with SV2A at the presynapse. Lastly, our immunohistochemistry data demonstrate a significant upregulation of CB1R expression in female Tat(+) mice compared to same sex Tat(-) controls (p = 0.023). These findings suggest there is a decrease in synaptic integrity due to Tat, which the endocannabinoid system can potentially be compensating for with the upregulation of CB1R expression. Further manipulation of the endocannabinoid system may be critical to understanding its therapeutic potential for HAND.

Acknowledgements: Funded by IMSD R25 GM055336; NCI P30 CA016086-40; and NIDA R21 DA041903, R01 DA045596, T32 DA007244, UNC CFAR P30 AI50410, R01 DA032933, and P30 DA033934.

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EVALUATION OF CB1 POSITIVE ALLOSTERIC MODULATOR (PAM)-ANTAGONISTS FOR PROBE DEPENDENCE

Daniel Barrus, Thuy Nguyen, Brian Thomas, Yanan Zhang, Jenny Wiley and Thomas Gamage

Discovery Sciences, RTI International, Research Triangle Park, NC, USA

Inhibition of the cannabinoid type-1 receptor (CB1) has been identified as a potential pharmacotherapeutic route for the treatment of drug addiction and obesity. However, the use of CB1 inverse agonists/antagonists like SR141716 is associated with significant adverse effects due to their suppression of CB1’s considerable constitutive activity. Targeting CB1 via allosteric modulation represents a promising approach for producing the therapeutic outcomes of CB1 inhibition while circumventing these adverse effects. Allosteric modulators bind to receptor sites distinct from the orthosteric site and stabilize the receptor in a conformation in which an orthosteric ligand has altered affinity and/or function. One of the earliest identified CB1 allosteric modulators, PSNCBAM-1, is classified as a positive allosteric modulator (PAM)-antagonist because it increases agonist affinity but decreases agonist efficacy. Structure-activity relationship studies of PSNCBAM-1 have identified several promising CB1 PAM-antagonists, but little research has evaluated the effects of these compounds on endogenous ligands. Such research is necessary because the relationship between allosteric and orthosteric ligands can exhibit probe dependence; i.e. an allosteric modulator’s effects vary based on the orthosteric ligand being used to probe receptor function. We tested PSNCBAM-based PAM-antagonists for probe-dependent differences in their potency to modulate the functional effects of CP55,940 and anandamide, as measured in the [35S]GTPγS binding assay. Additionally, we evaluated the affinity and binding cooperativity of these compounds using [3H]SR141716. Determining the probe dependence of allosteric modulators, especially regarding endogenous ligands, is critical in predicting the effects these compounds might produce in vivo. Acknowledgements: R01DA040693, R01DA003672, R01DA040460, K01DA045752

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THE ENDOCANNABINOID METABOLITE, 15-DEOXY, Δ12,14-PROSTAMIDE J2, INDUCES ENDOPLASMIC RETICULUM STRESS-DEPENDENT DAMAGE-ASSOCIATED MOLECULAR PATTERN (DAMP)

EXPRESSION IN MELANOMA

1Ahmed Elhassanny, 1Rene Escobedo, 2Daniel Ladin and 1,3Rukiyah Van Dross

1Department of Pharmacology and Toxicology, 2Department of Medicine, 3Center for Health Disparities, Brody School of Medicine, East Carolina University, Greenville, NC, USA Melanoma is the most lethal form of skin cancer in the United States. Current trends show

that melanoma incidence has increased over the last 30 years. Melanoma treatment depends on the stage of the cancer. Early stages of melanoma are often managed by surgical excision; however, advanced stages are treated with chemotherapy. Cytotoxic chemotherapeutics for advanced stage metastatic melanoma have limited efficacy. The use of immune-modulating drugs such as checkpoint inhibitors has significantly improved therapeutic outcomes, however these agents can cause life-threatening autoimmune side effects. Therefore, immunotherapeutic agents that can preferentially eliminate melanoma cells are needed. Agents that induce the release of damage-associated molecular patterns (DAMPs) can stimulate antitumor immunity. DAMPs are produced by damaged or dying cancer cells and include signals such as expression of cell surface calreticulin (CRT) and the secretion of ATP. DAMPs can recruit and activate immune cells such as dendritic cells which in turn stimulate tumor-specific cytotoxic T cells to kill cancer cells in a process referred to as immunogenic cell death. DAMP expression depends on endoplasmic reticulum stress (ER) and oxidative stress. We recently found that the arachidonoyl ethanolamide (AEA) metabolite, 15-deoxy, Δ12,14-prostamide J2 (15dPMJ2), induces DAMPs in melanoma. In addition, we determined that other cannabinoids induce DAMP expression in tumorigenic melanocytes. The purpose of this study was to investigate: 1) the selectivity of DAMP induction in melanoma by 15dPMJ2, 2) the effect of 15dPMJ2-treated melanoma cells on the activation of bone marrow-derived dendritic cells (BMDCs), and 3) the role of ER stress and oxidative stress in this process.

To determine if 15dPMJ2 causes tumor-selective activation of BMDCs, we examined the

expression of cell surface calreticulin and the release of ATP in B16F10 melanoma cells and Melan-A non-tumorigenic melanocytes. Our results showed that 15dPMJ2 significantly increased DAMP expression in tumorigenic but not in non-tumorigenic melanocytes. In addition, 15dPMJ2-treated B16F10 cells induced maturation of BMDCs as manifested by an increase in the expression of MHCII, CD80 and CD86. However, 15dPMJ2-treated Melan-A cells did not activate BMDCs. In this study, we also tested the role of ER stress and oxidative stress in 15dPMJ2-induced DAMP expression. Our results showed that the ER stress inhibitor GSK2606414 inhibited DAMP induction and BMDC activation by 15dPMJ2-treated B16F10 cells. On the other hand, the antioxidant Trolox slightly decreased 15dPMJ2 induced calreticulin and ATP release in B16F10 cells. However, Trolox did not inhibit the activation of BMDCs by 15dPMJ2-treated B16F10 cells. These results suggest that ER stress, but not oxidative stress is required for DAMP induction and the activation of BMDCs by 15dPMJ2. In conclusion, 15dPMJ2 can selectively induce DAMPs in melanoma which results in activation of BMDCs by an ER stress dependent mechanism. Therefore, 15dPMJ2 is a potential immunotherapeutic agent for melanoma.

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LATENT CLASS ANALYSIS OF YOUNG ADULT CURRENT CANNABIS USERS

Alyssa Rudy1, Andrew Barnes2, & Caroline O. Cobb1

1 Department of Psychology, Virginia Commonwealth University, Richmond, VA. 2 Department of Health and Policy, School of Medicine, Virginia Commonwealth University, Richmond, VA Despite the variety of cannabis administration methods (i.e., vaporizer, bong, edibles), cannabis users are often considered a homogenous group. The current study aimed to identify subgroups of cannabis users based on use frequency of cannabis administration methods using a latent class analysis (LCA). Past 30-day cannabis users were selected from an online survey of undergraduates collected in 2017 (n=208/446). LCA indicators were frequency of cannabis administration methods in the past 30 days with joint, blunt, pipe/bowl, and bong using a 3-point scale (0, 1-10, 10+ times) and edible and rig/vaporizer using a binary scale (yes, no). Bivariate analyses were conducted among sub-groups with the following variables: gender, race/ethnicity, school year, past 30-day tobacco use, cannabis dependence, cannabis harm perceptions, and average days of cannabis use per month. Latent Gold and IBM SPSS were used for statistical analyses. LCA identified four classes: Low-Blunt, Low-Bong, Mod-Poly, and High-Poly. The Low-Blunt class was the largest (60%) and was characterized by blunt use at low frequencies (1-10 times). The Low-Bong class (24%) primarily involved bong use at low frequencies (1-10 times). The Mod-Poly class (13%) was characterized by use of all cannabis methods at various frequencies. The High-Poly class was the smallest class (4%) and included participants who used all cannabis methods at high frequencies. Among the three largest classes, there were significant demographic differences. In the Low-Blunt class, 73.8% were female, whereas females comprised 58.3% in the Low-Bong class and 53.8% in the Mod-Poly class (p<0.05). Race/ethnicity significantly differed among the three largest classes (p<0.05), such that the Mod-Poly and Low-Bong classes were mostly White (61.5% and 56.3%, respectively), whereas the Low-Blunt class was more diverse with 36.9% White and 25.4% Black. The Mod-Poly class had significantly higher past 30 day tobacco use compared to the Low-Blunt class (73.1% vs. 45.9%, respectively; p<0.05). The Mod-Poly class had significantly higher cannabis dependence compared to the Low-Blunt class (M=7.4, SD=2.7 vs. M=5.8, SD=1.7, respectively; p<0.01). Average days of cannabis use per month significantly differed across all three of the largest classes with the Mod-Poly class having the highest average days (M=25.2, SD=7.7), compared to Low-Bong (M=12.2, SD=11.3) and Low-Blunt classes (M=6.4, SD=7.7; p<0.001). Cannabis users differed in their administration method use and these use patterns were associated with differential demographic and cannabis dependence characteristics. These results can inform interventions for different types of cannabis users.

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MONOACYLGLYCEROL LIPASE INHIBITION ALTERS INHIBITORY CONTROL IN TAT TRANSGENIC MICE

Alexis F. League*1, Ian R. Jacobs1, Benjamin L. Gorman1, Justin L. Poklis2, Micah J. Niphakis3,

Benjamin F. Cravatt3, Aron H. Lichtman2, Bogna M. Ignatowska-Jankowska4, and Sylvia Fitting1

1Department of Psychology & Neuroscience, University of North Carolina Chapel Hill,

Chapel Hill, NC, USA; 2Department of Pharmacology & Toxicology, Virginia Commonwealth University, Richmond, VA, USA; 3Department of Chemistry, Scripps

Research Institute, La Jolla, CA, USA; 4Okinawa Institute of Science and Technology, Neuronal Rhythms in Movement Unit, Okinawa, Japan

HIV-associated neurocognitive disorders are characterized by an array of behavioral phenotypes, several of which involve inhibitory control deficits and motivation dysregulation. While combination antiretroviral therapy (cART) has increased the expected lifespan of individuals living with HIV-1, residual proteins such as transactivator of transcription (Tat) are poorly suppressed and drive neurocognitive deficits which persist beyond currently available treatments. Given previous work demonstrating the neuroprotective role of targeted cannabinoid modulation of 2-arachidonoylglycerol (2-AG) in vitro, the present study aimed to investigate the therapeutic utility of blocking 2-AG hydrolysis in vivo using monoacylglycerol lipase inhibitor MJN110 in a Tat transgenic mouse model. A novel behavioral assay was used to examine olfactory discriminative stimulus learning and cognitive flexibility, wherein animals were trained to approach a particular odor which predicted a salient reinforcer. Ultrahigh performance liquid chromatography tandem mass spectrometry (UPLC-MS) was employed to elucidate brain region-specific protein expression differences by genotype and treatment. While Tat(+) subjects acquired the discrimination task significantly faster than Tat(-) controls, daily administration of MJN110 (1 mg/kg) abolished this effect in reversal training. UPLC-MS revealed significantly increased prefrontal and striatal 2-AG levels in both genotypes treated with MJN110. Further experiments will clarify the striatum’s role in the MJN110-driven effects observed, and ultimately seek to identify whether the obtained results implicate endocannabinoid activity in regulation of reward-related motivated behavior.

Acknowledgements: Funded by NIH T32 DA007244, R21 DA041903, R01 DA045596, R01 DA039942, and P30 DA033934; and the Japan Society for Promotion of Science (JSPS)

17F17388.

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COMPUTATIONAL DESIGN AND SYNTHESIS OF NOVEL CANNABINOID RECEPTOR 2 SELECTIVE AGONISTS

Logan T. Neel and Forrest T. Smith

Department of Drug Discovery and Development, Auburn University Harrison School of

Pharmacy, Auburn, AL, USA

The cannabinoid receptor 2 (CB2) is a promising therapeutic target for neuropathic and inflammatory pain, in disease states such as rheumatoid arthritis, asthma and tuberculosis. Pain is a disease that affects more people than cancer, heart disease and diabetes combined, indicating there is a need for the treatment of pain that will not lead to the drug dependence that is commonly seen with opioids. The majority of the known agonists for the CB2 receptor are not selective and will activate both the CB1 and CB2, simultaneously. While activation of CB2 by an agonist is desirable, activation of CB1 receptors is associated with psychotropic effects. The studies of CB2 are limited due to the unknown crystal structure for this G-protein coupled receptor. However, there is a crystal structure for the CB1 receptor, which was utilized as a template for the development of a homology model of CB2. In this project, a CB2 homology model was built and then validated to ensure the accuracy of the G Protein Coupled Receptor. The model was validated by using SP and XP glide ligand docking, molecular dynamic simulations, field based QSAR models, enrichment studies of decoys and pharmacophore studies of CB2 agonists with known binding affinities. The binding pocket of CB2 and CB1 will be analyzed and compared in order to develop CB2 selective agonists. The synthetic cannabinoid, Win-55,212-2, has high affinity for the cannabinoid receptors which has led to the studies of amino alkyl indole derivatives being potent cannabinoid agonists. In this project, in silico methods will be utilized to optimize the indole scaffold to increase selectivity at CB2. The indole scaffold will not be the only option considered when trying to design CB2 selective agonists. The database, Enamine (https://enamine.net/library-synthesis/real-compounds/real-database), will be evaluated as well, which contains over 7 million synthesizable compounds, all of which follow the Lipinski’s Rule of Five and Verber criteria. This database will then be subjected to a tool known as Shape Screen, which is associated within Schrodinger modeling software. This tool is known for analyzing the shape query of an active compound that binds to the desired receptor and then compares other molecules based on the shape similarity to the active compound. The top scoring molecules will then be synthesized within the laboratory and pharmacological evaluations will be done thereafter. Acknowledgements: Funded by the American Foundation for Pharmaceutical Education (AFPE) Fellowship and Center for Neuroscience Initiative (CNSi)

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ROUTE OF ADMINISTRATION EFFECTS ON Δ9-TETRAHYDROCANNABINOL DISCRIMINATION IN RATS

Shanequa I. Taylor, Timothy W. Lefever, and Jenny L. Wiley

RTI International, Research Triangle Park, NC 27709 Although smoking and vaping remain the most common methods of cannabis use, a substantial proportion of users report consumption of cannabis in the form of edibles. To date, however, most preclinical research has employed the use of injected Δ9-tetrahydrocannabinol (THC), the primary psychoactive ingredient of cannabis, as a proxy for cannabis use. This practice fails to consider route of administration as an important variable that may alter the behavioral effects observed after THC administration and/or may contribute to their magnitude or duration. The goal of the present study was to examine the effects of route of administration on THC-like psychoactivity in drug discrimination, an animal model of the subjective effects of THC intoxication. To this end, female and male Long Evans rats were trained to discriminate between 3 mg/kg intraperitoneal (i.p.) THC and vehicle in a standard two-lever drug discrimination procedure under a FR10 for food reinforcement. Following acquisition, dose-effect curves were determined with THC after i.p. or oral (p.o.) administration. Subsequently, a time course was examined using a dose that produced > 80% substitution with each route of administration to determine how long THC-like discriminative stimulus effects lasted for each route. Results showed that, over a dose range of 0.1 to 10 mg/kg, i.p. THC produced dose-dependent increases in responding on the THC-associated lever, with maximal substitution greater than 80% at doses of 1 mg/kg and higher in females and at doses of 3 and 10 mg/kg in males. Response rates were decreased in males at higher doses (as compared to vehicle), but were stable across doses for females; however, females also exhibited lower responding at vehicle baseline compared to males. In females, maximal THC-like responding with the 3 mg/kg THC training dose occurred at 30-120 min whereas in males maximal THC-like effects of the same dose were observed at 15-60 min. Response rates in males showed a corresponding decrease during this period, with rebound increase as THC’s discriminative stimulus effects wore off. Response rates in females were lower but were also more consistent across time. THC also produced dose-dependent increases in THC-lever responding following oral administration. Maximal substitution was greater than 80% at doses of 1-10 mg/kg in females and at doses of 3 and 10 mg/kg in males. Orally administered THC did not affect response rates up to a dose of 10 mg/kg in either sex, although responding was significantly lower overall in females (vs. males). In both sexes, the onset of maximal THC-like effects occurred over the course of 30 min after p.o. administration of 3 mg/kg THC and THC-like responding began decreasing after 120 min, with parallel decreases for both sexes. Overall response rates were lower in females (vs males). These results suggest that route of administration alters the time course of THC-like discriminative stimulus effects in rats and that this alteration is sex-dependent, with females showing quicker onset and offset after i.p. administration (vs p.o.). Future research will examine concentration-effect and time course of other routes of administration (e.g., vaping). Acknowledgements: This study was funded by NIH/NIDA grant DA-045003.

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CNS REGION-DEPENDENT INCREASES IN NEUROPROTECTIVE eCB-LIKE LIPIDS ACCOMPANY MICROGLIOSIS IN MORPHINE TOLERANT HIV-1 TAT TRANSGENIC MICE

Douglas J. Hermes1, Ian R. Jacobs1, Megan C. Key1, Alexis F. League1, Changqing Xu1, Barkha J. Yadav-Samudrala1, Rick Meeker2, Bogna Ignatowska-Jankowska3, Aron H. Lichtman4, Zibo

Li5, Zhanhong Wu5, Hong Yuan5, Pamela E. Knapp4,6, Kurt F. Hauser4,6, and Sylvia Fitting1*

1Department of Psychology & Neuroscience, University of North Carolina, Chapel Hill, NC, USA; 2 Department of Neurology, University of North Carolina, Chapel Hill, NC, USA; 3Okinawa

Institute of Science and Technology, Neuronal Rhythms in Movement Unit, Okinawa 904-0495, Japan; 4Department of Pharmacology & Toxicology, Virginia Commonwealth University, Richmond, VA, USA; 5Department of Radiology, School of Medicine, University of North

Carolina, Chapel Hill, NC, USA; 6Department of Anatomy & Neurobiology, Virginia Commonwealth University, Richmond, VA, USA

Inflammation in the central nervous system plays an important role in opiate and human immunodeficiency virus type-1 (HIV-1) comorbidity as it has been recognized to worsen disease progression and HIV-1-related neuropathology. This is an important issue as opioids are commonly prescribed for treatment of HIV-1-related pain. In the present study, we assessed morphine-induced antinociceptive tolerance at both spinal and supraspinal sites using the HIV-1 Tat transgenic mouse model that expresses the HIV-1 Tat protein in a GFAP-regulated, doxycycline (DOX)-inducible manner. Neuroinflammation was assessed by positron emission tomography (PET) imaging using the [18F]-PBR111 ligand and by immunohistochemistry, using the well-established microglia marker, Iba-1. Significant increases in antinociceptive tolerance were noted in the tail-flick and partially in the hot-plate assays in Tat positive transgenic mice [Tat(+) mice] compared to Tat negative controls [Tat(−) mice]. Increased tolerance was accompanied by microgliosis assessed by Iba-1+ cell counts in the spinal cord and striatum, but not by increases in neuroinflammation as assessed by PET imaging of [18F]-PBR111 binding. Data analysis for Iba-1 and 3-NT double staining to observe activated microglia is currently ongoing. Chronic morphine and Tat exposure had differential effects on endocannabinoid (eCB) levels and eCB-like neuroprotective lipids in a central nervous system (CNS) region-dependent manner. In the striatum, eCB-like lipids were significantly increased by chronic morphine exposure, including peroxisome proliferator activator receptor alpha (PPAR-α) ligands N-oleoyl ethanolamide (OEA) and N-palmitoyl ethanolamide (PEA). No effects were seen in the spinal cord for eCBs or eCB- like lipids. Acknowledgements: This work was supported by the National Institute on Drug Abuse (NIDA R01 DA045596 (SF), R21 DA041903 (SF), T32 DA007244 (IRJ, AFL), R01 DA039942 (AHL), K02 DA027374 (KFH), and R01 DA045588 (KFH)).

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ASSESSMENT OF IMPAIRMENT FOLLOWING ORAL AND VAPORIZED CANNABIS ADMINISTRATION IN INFREQUENT USERS

Erin Martin1, Tory Spindle2, Megan Grabenauer3, Michael Milburn4 & Ryan Vandrey2

1Medical University of South Carolina, Charleston, SC; 2Johns Hopkins University School of Medicine, Baltimore, MD; 3RTI International, Research Triangle Park, NC; 4DRUIDApp Inc.,

Cambridge, MA

Background: Recent shifts in policy have led to an increase in cannabis availability in the absence of necessary public safety research. Alternative routes of administration, such as oral ingestion and vaporized inhalation, have also increased in prevalence, and their associated pharmacokinetics and pharmacodynamics have not yet thoroughly been explored. The aims of this study are to characterize oral and vaporized cannabis doses likely to cause impairment, and to identify markers that can reliably detect this impairment.

Methods: Twenty infrequent cannabis users (≥ 30 days since last use) completed six, double-blind outpatient sessions separated by at least 1 week, in which they consumed cannabis brownies (0, 10, or 25mg THC) or inhaled vaporized cannabis (0, 5, or 20mg THC). In each session, blood, urine, and oral fluid were collected and subjective, cognitive, and psychomotor effects were assessed prior to cannabis administration and for 8hrs thereafter. The Digit Symbol Substitution Task (DSST), Paced Serial Addition Task (PASAT), Divided Attention Task (DAT), field sobriety tests (results not yet available), and DRUID Application measured cognitive/psychomotor performance.

Results: For oral and vaporized cannabis, ratings of “Drug Effect” increased significantly in a dose-dependent manner. For oral cannabis, 10mg qualitatively impaired performance on the DAT and PASAT, while 25mg significantly impaired performance on the DAT, PASAT, and DRUID compared with placebo. For vaporized cannabis, 20mg significantly impaired performance on the DAT, PASAT, and DRUID compared with placebo, but 5mg did not. Mean peak DRUID scores for the 20mg vaporized dose and the 25mg oral dose were similar to alcohol exposure at a .08 BAC in a prior study. Drug effects/impairment on these tasks often persisted after blood and oral fluid THC concentrations returned to zero, and mean urinary THC-COOH concentrations remained elevated for the 10 and 25mg oral doses and 20mg vaporized dose when these effects had abated. Conclusions: 10 and 25mg oral and 20mg vaporized doses increased subjective drug effects and impaired cognitive/psychomotor performance on most tasks compared to placebo; 5mg vaporized cannabis produced discriminative drug effects without significant impairment. The DAT and DRUID appeared to be the most robust measures for identification of impairment. Reliable markers of cannabis intoxication/impairment are needed given the expanding legal cannabis market. Support: National Institute of Justice

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EFFECTS OF ENDOCANNABINOID ENZYME INHIBITORS ON NEUROTRANSMISSION IN THE PREFRONTAL CORTEX OF TAT TRANSGENIC MICE

Changqing Xu1, Douglas J. Hermes1, Ian R. Jacobs1, Megan C. Key2, Alexis F. League1, Callie Xu1, Micah J. Niphakis3, Benjamin F. Cravatt3, Ken Mackie4, Aron H. Lichtman5, Bogna M.

Ignatowska-Jankowska6 and Sylvia Fitting1

1Department of Psychology & Neuroscience, University of North Carolina Chapel Hill, Chapel Hill, NC, USA; 2Curriculum in Neuroscience, University of North Carolina Chapel Hill, Chapel Hill,

NC, USA; 3Department of Chemical Physiology, Scripps Research Institute, La Jolla, CA, USA; 4Department of Psychological & Brain Sciences, Indiana University, Bloomington, IN, USA;

5Department of Pharmacology & Toxicology, Virginia Commonwealth University, Richmond, VA, USA; 6Okinawa Institute of Science and Technology, Neuronal Rhythms in Movement Unit,

Okinawa, Japan

Human immunodeficiency virus type 1 (HIV-1) infects the brain and, despite combined antiretroviral therapy (cART), many infected individuals suffer from HIV-1-associated neurocognitive disorders (HAND). The HIV-1 transactivator of transcription (Tat) protein plays a paramount role in the development of chronic neuroinflammatory conditions and neuronal damage found in HIV-1 associated neurocognitive disorders (HAND). It has been shown that endocannabinoids (eCBs) have protective properties in several central nervous system (CNS) disease models, but their effects in HAND remain unknown. To address this issue, we investigated the actions of the eCBs N-arachidonoylethanolamine (anandamide/AEA) and 2-arachidonoylglycerol (2-AG) on glutamatergic and GABAergic neurotransmission in the medial prefrontal cortex (mPFC) using brain slices of HIV-1 Tat transgenic mice. We demonstrate that pretreatment with respective highly selective and potent inhibitors of their primary hydrolytic enzyme inhibitors (PF3845, MJN110) have neuroprotective effects against Tat-induced alteration in synaptic neurotransmission. Results indicate that PF3845 and MJN110 significantly decrease the mean frequency but not the mean amplitude of glutamatergic and GABAergic currents (EPSCs and IPSCS, respectively) for transgenic Tat(+) and Tat(-) brain slices for both sex (females and males). Interestingly, the mean frequency of spontaneous EPSCs (sEPSCs) was significantly increased in transgenic Tat(+) brain slices compared to Tat(-) control slices. Understanding the effect of the HIV-1 Tat protein on cognitive functioning, such as inhibitory control has the potential to identify novel therapeutic interventions to benefit individuals suffering from HAND and other cognitive impairments. Acknowledgements: Funded by NIDA R21 DA041903, R01 DA045596, UNC CFAR P30 AI50410, T32 DA007244, R01 DA039942, and K05 DA021696.

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BRAIN IMAGING OF CANNABINOID TYPE 1 RECEPTORS IN MALE AND FEMALE NON-USERS AND FEMALE CHRONIC USERS.

Eversole, A., Spindle, T., Vandrey, R., Kuwabara, H., Guarda, A., Wong, D.F., Weerts, E.

Department of Psychiatry and Behavioral Sciences, School of Medicine, Johns Hopkins

University

Most cannabis effects result from actions on CB1 receptors. Females have greater sensitivity to cannabis effects and show an accelerated trajectory from first use to onset of cannabis use disorder (CUD) compared to males. Additional research is needed to determine whether these observed sex differences are the result of differences in CB1 receptor availability. With positron emission tomography (PET) and the radiotracer 11C-OMAR, CB1 receptor distribution volume (VT) was characterized in female chronic cannabis users (N=10) after 3 days of cannabis abstinence and age-similar male (N=6) and female (N=8) non-cannabis users. CB1 receptors were characterized in brain regions associated with reward, mood, motor function, and learning. Cannabis users also completed two controlled cannabis smoking sessions. In the first session, participants smoked placebo cannabis and in the second session, they smoked active cannabis (25 mg THC). Pharmacodynamic effects (subjective drug effects; cognitive performance) were measured during both sessions. Following the second session, participants completed a 4 day/3 night inpatient stay, during which cannabis withdrawal symptoms were measured; thus time since last cannabis use was held constant for all participants. Participants completed the PET scan on the third day of cannabis abstinence. Among non-cannabis users, females had higher CB1 VT in several brain regions (e.g. amygdala, frontal lobe, hippocampus) compared to males. For females, cannabis users tended to have lower CB1 VT than non-users in all brain regions that were assessed, likely reflecting CB1 downregulation due to repeated cannabis use. CB1 receptor availability tended to be positively correlated with the magnitude of subjective drug effects (e.g., “like drug”). Overall, this study highlights that there are inherent differences in CB1 receptor availability between men and women that may underlie sex differences in cannabis sensitivity and progression to CUD. Moreover, these data suggest chronic cannabis use can lead to downregulation of CB1 receptors. Future research should prospectively study CB1 receptor availability prior to long-term cannabis cessation in order to determine whether, and for how long, CB1 downregulation persists in cannabis users. Acknowledgements: Funded by the National Institutes of Health (Grant no. 5R21DA043963)

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Notes:

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Notes: