Clinical studies have demonstrated that inhaled nitric oxide (NO) gas is a selective pulmonary vasodilator and is useful in the treatment of diseases associated with pulmonary vascular hypertension. However, the delivery of NO gas requires complex delivery equipment and because of its short half-life, must be delivered continuously for abrupt withdrawal of therapy is often associated with rebound hypoxemia. The use of diazeniumdiolates, NO prodrugs, formed by reacting NO with specific amines, has been studied with increasing interest as an alternative treatment. These compounds spontaneously release NO at predictable rates when introduced into aqueous conditions. The primary investigator hypothesizes that a diazeniumdiolate could be designed to be retained in the lungs throughout the duration of NO release. Aerosol deliver of such a compound using a convenient intermittent dosing regimen would insure a sustained NO release directly to the pulmonary vasculature bed. Our studies have shown that the diazeniumdiolate, N,N-dimethylaminoethyl putreaninate diazeniumdiolate, DMAEP/NO selectively reduced pulmonary hypertension in both oleic acid and thromboxane agonist induced hypertension in pigs. It is believed that the selectivity of DMAEP/NO for the lung is due to strong interactions between choline or putrescence transporters on the apical side of the lung and the dimethylamino or aminobutyl functionality on DMAEP/NO. These intermolecular interactions slow the flux of the compound into the systemic circulation long enough for the released NO to be contained within the lungs. The ultimate goal of this study is to optimize the structural and NO releasing characteristics of a class of DMAEP/NO analogs that function as substrates/inhibitors for the choline/polyamine transporters found in the lungs.
In Specific Aim 1, the investigators synthesize a series of polyamine/choline based diazeniumdiolates which have 4-12 hour half-life NO release profiles. This procedure will enable them to increase selectively by taking advantage of the pulmonary transporters while at the same time provide the patient with the convenience of intermittent dosing. The major objective of Specific Aim 2 is to investigate the transport of these compounds through monolayers of primary lung epithelial and A549 cell cultures. Competitive assays will be used to determine if this transport involves the choline of polyamine transporters. Structures which provide substantial interaction with these transporters will be identified. Understanding more about the transport mechanism will allow for the design of a molecule with the characteristics necessary to limit its flux across the monolayer and isolate its activity to the lungs.
In Specific Aim 3, compounds that have been found to associate with the pulmonary transporters will be radio-labeled in order to thoroughly examine transport properties through cellular monolayers. Clearance of the NO carrier molecule is essential for the long term use of these compounds in a clinical setting.
