Nitric oxide (NO) is a potent and multifaceted bioregulatory agent. This project is aimed at a) finding ways to target NO to specific sites in the body for important research and/or therapeutic applications and b) characterizing the possible role of NO as a determinant of cancer risk. Our focus during the past year has been on elucidating the fundamental chemistry of the NO-releasing diazeniumdiolates (compounds containing the [N(O)NO] functional group) with an eye toward using our accumulating knowledge in this area to solve important research and clinical problems. Specifically, we are using the knowledge gained in our basic research investigations into the synthesis, structure, spectra, dissociation to NO, alkylation, arylation, and photodegradation of the diazeniumdiolate functional group to design prodrugs that are stable at physiological pH but that can be activated to generate NO by hydrolysis or enzymatic action. As one example of this approach to drug discovery, we have been working to design agents that can be activated for NO release by enzymes of the glutathione S-transferase (GST) family. The pi isoform of GST is overexpressed in some tumor cells, rendering them resistant to several lines of anticancer therapy, while the alpha isoform is vital to proper cell function. By exploiting the growing knowledge about the structures of the GSTs' active sites, we have gone from an initial drug candidate whose alpha:pi rate ratio was 100 to a third generation analogue with a ratio of 0.3. If enough additional selectivity can be designed into this series, a drug capable of irreversibly inhibiting the pi enzyme while sparing the alpha may be forthcoming. We have also discovered that this same class of agent rather potently induces apoptosis in NO-sensitive leukemia cells in vitro. We are currently synthesizing a growing library of such arylated diazeniumdiolates for screening against these cells. There has been considerable spinoff from our basic research effort into potential non-cancer clinical applications as well, because many life-threatening disorders arise from localized deficiencies of bioregulatory NO. (1) Two of our diazeniumdiolates have been demonstrated by others to cure cerebral vasospasm in monkeys and dogs; if one or both of these drugs can be shown to have similar effectiveness in humans, new therapies may become available for the several thousand patients per year in the United States who are stricken with this condition, one for which no reliable therapy currently exists. (2) A diazeniumdiolated protein we have prepared was shown to promote healing of coronary arteries in pigs when injected into the pericardium (whose contents bathe the surface of the heart) immediately before balloon angioplasty; if balloon-deployed stainless steel stents whose surfaces we have succeeded in diazeniumdiolating can be demonstrated to have a similar antiproliferative effect, a substantial reduction of restenosis risk in angioplasty patients may be forthcoming. (3) Insoluble polymers containing NO-generating diazeniumdiolate groups resist adhesion of blood platelets, making such polymers excellent candidates for use as thromboresistant coatings for a variety of implants, shunts, blood conduits, biosensors, catheters, vascular grafts, and other medical devices that come into contact with blood during use. Diazeniumdiolates we have designed have also been shown to: (4) induce erections in male cats, suggesting possible utility in treating impotence; (5) relieve pulmonary hypertension in rats receiving once-a-day doses in nebulized form, raising the possibility of ambulatory therapy for respiratory distress patients who are currently confined to critical care units so that they can be continuously treated with NO gas via their respirator air; and (6) protect the livers of rats from tumor necrosis factor-a-induced cell death, suggesting use in treating fulminant liver failure.
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