ItemStudy on the Antidepressant-like Effect of an Estrogenic Compound(2019-03-05) Nguyen, Vien; de la Cruz, Daniel; Prokai, Laszlo; Prokai-Tatrai, Katalin; Edwards, SarahPurpose: Estrogens have numerous beneficial effects on brain health; they impact learning, memory, and mood. We utilized the latter effect of estrogens to study an estrogenic compound’s antidepressant-like effect in experimental animals upon systemic (subcutaneous) drug administration. Method: The Porsolt Swim Test (PST) is a well-known model to survey the antidepressant-like effect of potential CNS agents. In this model, a mouse is placed in a water-filled container for a set period of time. When the animal ceases swimming and only makes enough movement to keep its head above the water, also known as immobility, this corresponds to a “depressive mood.” We used 30 ovariectomized CD-1 mice lacking endogenous estrogens to avoid inference with the exogenously added estrogenic compound. The mice were divided into 5 groups of 6, with one group being designated as the control. Test agent was administered at various doses to the other 4 groups in corn oil vehicle for 5 days daily, and experiment started 30 minutes after the last injection for each mouse. At the highest dose, ICI 182,780 (an estrogen antagonist) was also co-administered. The movement of the animals were taped for 6 minutes. Two blinded observers independently determined the immobility times for the last 4 minutes of the videotaped experiment. At the end of the experiment, the mice were sacrificed and tissues were harvested for future drug quantification. Uterus wet weights were also measured. Mice were only exposed to these experimental conditions on the day of the experiment. Results: Compared to the corn oil vehicle control, a dose-dependent and statistically significant reductions in immobility time were observed in animals treated with the estrogenic compounds. ICI 182,780 completely reversed the antidepressant-like effect of the test agent. Wet uterus weights were also statistically significantly different from those measured for the control group, indicating the uterotropic effect of the estrogenic test compound. Conclusion: We have showed that an estrogenic test compound produced a dose-dependent antidepressant-like effect in PST, and this effect was completely reversible by the co-administration of an estrogen antagonist, implicating that the genomic effect of the estrogenic compound played a pivotal role in the observed CNS effect. At the same time, we also showed that the uterus of animals receiving the estrogenic test compound became very large due to fluid imbibition, which is a typical detrimental peripheral side-effect of estrogens exogenously administered for the purpose of neurotherapy. In the future, drug content in the brain and blood of the experimental animals will be determined and correlated with the obtained neuropharmacological effect. Acknowledgement: Research reported in this publication was supported in part by the National Heart, Lung, and Blood Institute and the National Cancer Institute of the National Institutes of Health under Award Numbers R25HL125447 (to Dr. J.K. Vishwanatha) and R01CA215550 (to L.P.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. ItemMapping the binding site mediating carisoprodol’s direct activation of GABAA receptors(2019-03-05) Huang, Ren-Qi; Dillon, Glenn; Liu, Jin; Hayatshahi, Hamed; Emmitte, Kyle; Claudio, CarmenCarisoprodol (CSP) is a centrally-acting prescription muscle relaxant that can directly activate and allosterically modulate the GABAA receptor. GABAA receptors are the target of many different clinically prescribed compounds. Our previous studies have shown that CSP differentially potentiates GABAA receptor subtypes via allosteric modulation and direct activation. It has been reported that a single amino acid residue, L415, located at the top of the fourth transmembrane domain (TM4) in the a1-subunit of the GABAA receptor is critical to CSP’s direct gating effect. However, whether the residue is involved in CSP binding remains unsolved. The purpose of the present study is to explore the binding site mediating CSP’s direct action with in-silico docking, site-directed mutagenesis and whole-cell electrophysiology. Initial simulated docking of CSP at the GABAA receptor suggested that the CSP binding pocket may be formed by residues from the TM4, pre-TM1 and cys-loop regions of the a-subunit. In whole-cell electrophysiology studies, specific modifications of CSP’s molecular structure produced greater direct action on GABAA receptors. The role of the residues predicted as a CSP binding site in docking analysis are being verified with mutagenesis and patch clamp studies. It is expected that the results will not only enhance our understanding of CSP pharmacology but also the structure-function relationship at the GABAA receptor. ItemEvaluating Safety and Efficacy of Combination Therapy with Short-Contact Topical 5-Fluorouracil 5% and Calcipotriene for Actinic Keratoses(2019-03-05) Moore, Angela MD; Nguyen, Madalyn OMS-III; Hirapara, Bhavik OMS-IIBackground: Actinic Keratoses (AKs) are precancerous lesions to squamous cell carcinomas of the skin, affecting roughly 35% of adults over the age of 40. The lesions are directly linked to the cumulative exposure to UV radiation and arise from keratinocyte dysplasia. Standard treatment includes liquid nitrogen (LN2), topical 5-Fluorouracil creams (5-FU), or combination therapy. Vitamin D derivatives have demonstrated anti-proliferative properties in cancer treatment via stimulation of the vitamin D3 receptor and, therefore, may be efficacious in the treatment of AKs. However, these treatments are limited by side effects such as pruritus, erythema, dryness, and irritation. Objective: The purpose of this study is to evaluate the safety and efficacy of short-contact combination therapy of topical 5-FU 5% cream and vit D foam after LN2. Methods: This was a retrospective study on adults diagnosed with AKs in a private clinical dermatology office setting. Charts from 139 patients, 49% males and 51% females, with a mean age of 57.4, were examined. All patients were first treated with LN2 at baseline. Short-contact therapy of topical 5-FU and/or vit D foam consisted of a thin application nightly to the face for 5 nights and to other treatment areas for 7 nights, and then no application for 2 weeks. The cycles were repeated every 3 weeks. The patients were divided into six treatment groups: 1) 5-FU 5% after LN2, 2) vit D foam after LN2, 3) 5-FU 1% after LN2, 4) 5-FU 5% and vit D foam after LN2, 5) 5-FU 1% and vit D foam after LN2, and 6) LN2 alone. AKs of the of the face, scalp, chest, upper extremities, back, and lower extremities for each patient were documented at baseline and follow up visits at 20-50, and 51-100 days. An analysis of covariance (ANCOVA) model was used to compare post-treatment lesion counts between treatment groups at 95% confidence intervals. Results/Conclusions: Greater irritation was observed with the 5-FU 5% cream compared to other treatment groups. Short-contact combination therapy with 5-FU cream and Vit D foam after LN2 demonstrates increased efficacy over LN2 alone. Studies with an increased sample size for a longer duration should be performed to evaluate efficacy and safety.