Receptor Pharmacology & Drug Delivery

Permanent URI for this collectionhttps://hdl.handle.net/20.500.12503/21770

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    DIETARY SUPPLEMENTS INFLUENCE ACTIVITY OF ACID-SENSING ION CHANNELS
    (2014-03) Agharkar, Amruta S.; Gonzales, Eric B.
    Dietary supplements or nutraceutical industry contributes billions of dollars to Unites States economy every year. The dietary supplement we intend to study is one of the most commonly used nutraceutical available in the market over the counter. It is used primarily by athletes and body builders to build lean body mass. Studies have shown that DS is effective in neuroprotection after stroke and also helps in improving muscle strength in patients suffering from muscle weakness or osteoarthritis. We are studying the effect of DS on acid-sensing ion channels (ASICs) which are the major contributors to neuronal damage after ischemia and pain. Determining the activity of DS on ASICs will give the new preventive measure for stroke and pain. Purpose (a): Dietary supplements and nutraceuticals have been the focus of research to identify novel therapeutics for a variety of pathologies, including the prevention of long-term consequences of stroke and reducing pain. Ion channels offer a growing group of molecular targets for treatment, which include the acid-sensing ion channels (ASICs). Acid-sensing ion channels (ASICs) are sodium channels that are sensitive to changes in extracellular pH, specifically those changes following injury and ischemia. These channels are expressed most prominently in peripheral and central nervous system. Their role in physiology is yet to be fully understood, but these channels have been implicated in pain sensation and centrally in the neurodegeneration following ischemic stroke. We identified an over-the-counter dietary supplement (DS) that shares similarity to guanidine compounds that selectively modulate acid-sensing ion channels. Thus, we hypothesize that this dietary supplement inhibits channel activity in acid-sensing ion channels. Methods (b): We will utilize whole cell patch-clamp electrophysiology technique to determine the intrinsic activity of DS on ASICs. The current elicited in absence and presence of DS at various pH will be normalized to maximum peak current obtained with control. Results (c): Our preliminary data show that DS decreased the ASIC1a pH sensitivity by shifting the observed proton activation profile to the right. Furthermore, we observed a change in the Hill coefficient of the DS influenced ASIC1a steady-state desensitization profile. Conclusions (d): Based on our preliminary data, we can conclude that DS influences ASIC current amplitude and steady state desensitization profile. Future experiments will focus on determining the influence of DS on other acid-sensing ion channel subtypes and identifying the DS binding site with the protein structure.
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    NOVEL GABAA-RHO1 INTERACTIONS WITH ACID SENSING ION CHANNEL LIGANDS
    (2014-03) Snell, Heather D.; Gonzales, Eric B.
    γ- amino butyric acid (GABA) is the major inhibitory neurotransmitter in the vertebrate brain, and targets the ionotropic GABAA receptors. GABAC, or GABAA-rho, is a subclass of GABAA receptors located in the retina. A group of ligands, which possess a guanidine group, have been shown to influence classical GABAA receptors. Many, however, have not yet been tried on the GABAA-rho receptor subclass. Our experiments show that these compounds have contrasting effects on the GABAA-rho1 receptor, which could lead to a novel binding site, and explain many side effects of certain drugs containing the guanidine group. Purpose (a): γ- amino butyric acid (GABA) is the major inhibitory neurotransmitter in the vertebrate brain, and targets the ionotropic GABAA receptors. GABAC, or GABAA-rho, is a subclass of GABAA receptors composed entirely of rho (ρ) subunits and are located on the axonal terminal of retinal bipolar cells, where it not only exhibits a tonic inhibitory current, but also regulates the GABA-A and other GABAA-rho synaptic currents. GABAA-rho exhibits unique properties, such as insensitivity to select antagonists of the heteromeric GABAA receptors. A group of ligands, which possess a guanidine group, have been shown to influence GABAA receptors. These compounds, such as (S)-2-Guanidinopropionic acid and guanidine acetic acid were competitive antagonists for the GABAA-rho1 receptor. Other guanidine compounds that are acid sensing ion channel (ASIC) ligands, might also exhibit unique effects on the GABAA-rho1 receptor.We hypothesize that these ASIC ligands will exhibit unique intrinsic activities on the GABAA-rho1 receptor, which is different from that of the heteromeric GABAA receptor. Methods (b): The human GABAA-rho1 receptors were expressed in HEK-293T cells, and activity was analyzed using whole cell patch-clamp electrophysiology. Results (c): When co-applied with GABA and compared to the GABA concentration profile, one ligand was found to decrease the maximal response, with no change in the GABA EC50,while a different ligand with the same guandine group, shifted the GABA EC50 to lower GABA concentrations. When applied alone, it failed to directly activate GABAA-rho1 receptors. Conclusions (d): These contrasting effects suggest that these ligands act at two binding sites within the GABAA-rho architecture. Future experiments will focus on additional characterization of these novel effects on GABAA-rho receptors and offer a novel chemical structure to design novel GABAA-rho therapeutics.
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    EVALUATION OF A "TROJAN HORSE" STRATEGY TO COMBAT NEUROBLASTOMA
    (2014-03) Vitug, Gaile; Sabnis, Nirupama; Shi, Yi; Di Pasqua, Anthony; Bowman, W. Paul; Lacko, Andras G.
    Neuroblastoma (NB) is the most common type of cancer in children less than a year old and stems from early neural cells that fail to differentiate into neurons or adrenal medulla cells. Upon initial diagnosis, 67% of cases show metastases to the lymph nodes or other organs; therefore, chemotherapy effectiveness is of particular concern. Currently, there are no FDA approved treatments or designs specifically available for NB patients and most are designed for different types of adult cancers with dose-limiting toxicities as a significant concern. Present study evaluates the therapeutic potential of a novel drug delivery system utilizing reconstituted high density lipoprotein (rHDL) containing hydrophobic analogue of Cisplatin (CisX) nanoparticles that act as a “Trojan horse” towards tumor cells. The characterization studies of CisX reveal sizes of the particles ranging from 14 nm to 164.9 nm with a mean diameter of 69.46 nm. Encapsulation efficiency was observed to be 26.34%. The cytotoxicity studies of free CisX vs rHDL-CisX towards NB cell line SJ-N-KP show 5.1 times lower IC50 value for the rHDL-CisX. This study reveals the potential of rHDL as a novel delivery method for chemotherapeutic drugs in the treatment of NB and warrants more investigation. Purpose (a): Neuroblastoma (NB) is the most common type of cancer in children less than a year old and stems from early neural cells that fail to differentiate into neurons or adrenal medulla cells. Upon initial diagnosis, 67% of cases show metastases to the lymph nodes or other organs; therefore, chemotherapy effectiveness is of particular concern. Currently, there are no FDA approved treatments or designs specifically available for NB patients and most are designed for different types of adult cancers with dose-limiting toxicities as a significant concern. Present study evaluates the therapeutic potential of a novel drug delivery system utilizing reconstituted high density lipoprotein (rHDL) containing hydrophobic analogue of Cisplatin (CisX) nanoparticles that act as a “Trojan horse” towards tumor cells. Methods (b): rHDL-CisX nanoparticles was prepared by lyophilization followed by cholate dialysis. Chemical composition of rHDL-CisX nanoparticles was determined using BCA protein assay kit and enzymatic reagent assay kits (phospolipid C and cholesterol E). Encapsulation efficiency of CisX was determined by Inductively Coupled Plasma Mass Spectrometry (ICPMS). Size estimations were determined using dynamic light scattering. The Neuroblastoma cell line, SJ-N-KP, was plated in 96 well plates and incubated at 5% CO2 at 37ºC for 72 hours. Comparative cytotoxicity was examined by CCK-8 assay (Dojindo) using Free CisX and rHDL-CisX nanoparticles at various concentrations. Results (c): The characterization studies of CisX reveal sizes of the particles ranging from 14 nm to 164.9 nm with a mean diameter of 69.46 nm. An encapsulation efficiency was observed to be 26.34%. The cytotoxicity studies of free CisX vs rHDL-CisX towards NB cell line SJ-N-KP show 5.1 times lower IC50 value for the rHDL-CisX. Conclusions (d): This study reveals the potential of rHDL as a novel delivery method for chemotherapeutic drugs in the treatment of NB and warrants more investigation. Additional studies using rHDL towards normal cells are also needed to further evaluate their therapeutic potential.