Topical nitrite reverses nitroglycerin tolerance.

PROJECT SUMMARY Nitroglycerin (NTG) has been the first-line medication for treating angina for the past 150 years. However, the clinical utility of NTG is greatly limited by its loss of efficacy upon repeated use, i.e. `NTG tolerance'. Recent advances in understanding the mechanism underlying NTG-mediated vasodilation have led to the discovery that the tolerance phenomenon can be eliminated by concomitant administration of nitrite together with NTG. Leveraging this discovery, NANOS, Inc. has patented the method of co- administration of NTG and nitrite and will use it to improve and expand the therapeutic use of NTG. Our experiments in rats demonstrate that intra-peritoneal injection of nitrite eliminates NTG tolerance induced by repeated application of NTG transdermal patches. Nitrite, an endogenous molecule which is also FDA-approved for human use, has high safety margins and numerous cardiovascular benefits that are attributed to its ability to increase nitric oxide bioavailability. We hypothesize that a topical nitrite ointment will avert nitroglycerin tolerance in rodents and swine. Our long-term goal is to develop an easy-to-use transdermal patch product for the combined application of nitrite and NTG that averts the problem of NTG tolerance in patients. The objective of this proposal is to establish the feasibility and efficacy of topical nitrite application for this purpose in rats and pigs. First, we will formulate an ointment for topical application of nitrite. Topically applied nitrite dissolved in aqueous buffer readily crosses the skin into the bloodstream, where it decays with a half-life of 30 min. We will develop a water-in-oil (W/O) ointment to enable slow uptake of topical nitrite into the circulation to prolong its therapeutic effects, and determine its physicochemical and biopharmaceutical parameters. Second, we will induce NTG tolerance in rats by repeated application of NTG patches and subsequently measure the dilatory response of coronary arteries to acute doses of NTG, both in vivo via ultrasound measurements and ex vivo via Langendorff perfusion of isolated hearts. We will determine the time- and dose-dependent effects of nitrite on eliminating NTG tolerance and correlate it with pharmacokinetic (PK) parameters of nitrite measured in plasma. Third, we will measure coronary artery dilatory responses to NTG infusion in pigs via surgical instrumentation. We will confirm the efficacy of topical nitrite ointment at reversing nitroglycerin tolerance in pigs and determine the toxicity and PK of the nitrite ointment. Completion of this project will generate requisite proof-of-concept data for clinical development of a combined NTG/nitrite formulation that will resolve the century-old problem of nitroglycerin tolerance.