1. Abstract  Objectives: We wish to determine the efficacy and mechanism of action of Sophora flavesence alkaloids mediated inhibition of airway constriction.  Methods: We will use a murine model of asthma to evaluate effects of Sophora flavesence alkaloids on airway hyperreactivity using invasive spirometry of in vivo treated asthmatic mice and ex vivo experiments utilizing myography of murine tracheal rings. Ex vivo studies with human bronchial rings are also planned.  Results/Anticipated Results: In preliminary results we have found that Sophora flavesence alkaloids are potent suppressors of acetylcholine induced contraction of murine tracheal rings (P<O.001 vs. vehicle). We anticipate that Sophora flavesence alkaloid activity will be independent of beta-adrenergic agonist activity, sensitive to COX-inhibition and mediated by PGE2 as we have observed previously for the parent anti-asthma TCM formula ASHMI. We also expect that human bronchial tissue will be responsive to Sophora flavesence alkaloid activity.  Discussion/Significance of Impact: We believe that Sophora f1avesence alkaloids have potential for development as asthma drugs for human use providing a non-beta adrenergic pathway for relief from bronchoconstriction. Investigation of Anti-bronchoconstrictive properties of Sophora flavasence Alkaloids Experimental Design and Methods KD Srivastava, HA Sampson and X-M Li. Pediatric Allergy & Immunology, The Icahn School of Medicine at Mount Sinai, New York, NY  6 week old female BALB/C mice were systemically sensitized with and challenged with Ovalbumin (OVA) plus alum and intratracheally challenged with OVA to generate asthmatic mice  Trachea were harvested from mice 48 hours after final OVA challenge and 3-4 mm rings were obtained and myography was performed using Myodac20 apparatus (ADInstruments)  Tracheal ring contraction to 10 -4 M Acetylcholine (ACh) was measured in the presence and absence of major alkaloid compounds present in S. flavesence (SFA-I, SFA-II and SFA-III)  In some experiments COX inhibition was included by addition of the non-selective COX blocker Indomethacin (5uM) to organ bath prior to pretreatment with test compounds Conclusion and Future Studies Results Aim of The Current Study To investigate ability of Sophora flavesence alkaloid compounds- SF-I, SF-II and SF-III to inhibit airway constriction Funding: Research Supported by grant PO1 AT002647 to X-M Li and KL2 Scholar award (KL2TR000069 ) to KD Srivastava  We have previously shown that the anti-asthma TCM herbal formula ASHMI directly inhibits Acetylcholine (ACh) induced constriction in murine tracheal rings. Zhang et al Phytother Res 2010  Anti-constrictive effects of ASHMI are independent of  - adrenergic agonist activity but dependent on COX- pathway mediated PGE2 activity on EP2/EP4 receptors Srivastava et al, Am J Physiol (manuscript in review)  Bioassay guided studies revealed that anti-constrictive properties of ASHMI were limited to the component herb Sophora flavesence (SF)  Alkaloid-rich fractions of the parent herbal formula ASHMI and SF were found to have robust anti- constrictive properties  Three Sophora flavesence alkaloid (SFA) compounds designated SF-I, SF-II and SF-III have been chosen for the current study Introduction We found that Sophora flavesence alkaloids significantly inhibited murine tracheal ring contraction to acetylcholine in the following order- SFA-I>SFA-III>SFA-II. We believe these compounds have potential for development as asthma drugs for humans. Further pre-clinical studies planned include:  Selection of lead compound and pre-clinical in vivo treatment experiments in murine models of asthma  Optimization of dose and route of administration  Safety studies  Pre-clinical ex vivo testing in human airway segments SFA-I SFA-III SFA-II *** ### *** ### Figure -3. Effect of COX inhibition on SFA-I, SFA-II and SFA-II activity. Tracheal rings were pretreated for 30 minutes with non- selective COX blocker Indomethacin (5uM) prior to addition of SFA compounds. Contraction to 10 -4 M Ach was normalized to percent of contraction to 60mM KCL. Pilot data are shown. Figure-2. Effect of SFA-I, SFA-II and SFA-II on tracheal ring responses to 10 -4 M Ach. Tracheal rings were pretreated for 30 mins. Contraction was normalized to percent of contraction to 60mM KCL. Data are shown as Mean±SD. N=3 rings from separate mice. ***, P,0.001 vs None; ###, P<0.001 vs SFA-II. Figure -1. Representative real time tracing of murine tracheal ring contractions. Rings were first stimulated with 60mM KCL to assess contractile potential. Compounds or PSS (vehicle) were added 30 minutes prior to stimulation with 10 -4 M Ach. Ring viability was tested 90 minutes after Ach washout by KCL stimulation