TARGETING NITRIC-OXIDE (NO) DELIVERY IN-VIVO - DESIGN OF A LIVER-SELECTIVE NO DONOR PRODRUG THAT BLOCKS TUMOR NECROSIS FACTOR-ALPHA-INDUCEDAPOPTOSIS AND TOXICITY IN THE LIVER

We have designed a drug that protects the liver from apoptotic cell de ath by organ-selective pharmacological generation of the bioregulatory agent, nitric oxide (NO). The discovery strategy involved three steps : identifying a diazeniumdiolate ion (R2N[N(O)NO](-), where R2N = pyrr olidinyl) that spontaneously decomposes to NO with a very short half-l ife (3 s) at physiological pH; converting this ion to a series of pote ntial prodrug derivatives by covalent attachment of protecting groups that we postulated might be rapidly removed by enzymes prevalent in th e liver; and screening the prodrug candidates in vitro and in vivo to select a lead and to confirm the desired activity. Of five cell types examined, only cultured hepatocytes metabolized O-2-vinyl 1-(pyrrolidi n-1-yl)diazen-1-ium-1,2-diolate (V-PYRRO/NO) to NO, triggering cyclic guanosine 3',5'-monophosphate (cGMP) synthesis and protecting the hepa tocytes from apoptotic cell death induced by treatment with tumor necr osis factor-alpha (TNF alpha) plus actinomycin D. In vivo, V-PYRRO/NO increased Liver cGMP levels while minimally affecting systemic hemodyn amics, protecting rats dosed with TNF alpha plus galactosamine from ap optosis and hepatotoxicity. The results illustrate the potential utili ty of diazeniumdiolates for targeting NO delivery in vivo and suggest a possible therapeutic strategy for hepatic disorders such as fulminan t liver failure.