Ions of structure X[N(O)NO]- display broad-spectrum pharmacological activity that correlates with the rate and extent of their spontaneous, first-order decomposition to nitric oxide when dissolved. We report incorporation of this functional group into polymeric matrices that can be used for altering the time course of nitric oxide release and/or targeting it to tissues with which the polymers are in physical contact. Structural types prepared include those in which the [N(O)NO]- group is attached to heteroatoms in low molecular weight species that are noncovalently distributed throughout the polymeric matrix, in groupings pendant to the polymer backbone, and in the polymer backbone itself. They range in physical form from films that can be coated onto other surfaces to microspheres, gels, powders, and moldable resins. Chemiluminescence measurements confirm that polymers to which the [N(O)NO]- group is attached can serve as localized sources of nitric oxide, with one prototype providing sustained NO release for 5 weeks in pH 7.4 buffer at 37 degrees C. The latter composition, a cross-linked poly-(ethylenimine) that had been exposed to NO, inhibited the in vitro proliferation of rat aorta smooth muscle cells when added as a powder to the culture medium and showed potent antiplatelet activity when coated on a normally thrombogenic vascular graft situated in an arteriovenous shunt in a baboon's circulatory system. The results suggest that polymers containing the [N(O)NO]- functional group may hold considerable promise for a variety of biomedical applications in which local delivery of NO is desired.

We have used structure-based design techniques to introduce the drug O 2 -[2,4-dinitro-5-(N-methyl-N-4-carboxyphenylamino) phenyl] 1-N,N-dimethylamino)diazen-1-ium-1,2-diolate (PABA/NO), which is efficiently metabolized to potentially cytolytic nitric oxide by the isoform of glutathione S-transferase, an enzyme expressed at high levels in many tumors. We have used mouse embryo fibroblasts (MEFs) null for GST (GST Ϫ/Ϫ ) to show that the absence of GST results in a decreased sensitivity to PABA/NO. Cytotoxicity of PABA/NO was also examined in a mouse skin fibroblast (NIH3T3) cell line that was stably transfected with GST and/or various combinations of ␥-glutamyl cysteine synthetase and the ATP-binding cassette transporter MRP1. Overexpression of MRP1 conferred the most significant degree of resistance, and in vitro transport studies confirmed that a GST-activated metabolite of PABA/NO was effluxed by MRP1 in a GSHdependent manner. Additional studies showed that in the absence of MRP1, PABA/NO activated the extracellular-regulated and stress-activated protein kinases ERK, c-Jun NH 2 -terminal kinase (JNK), and p38. Selective inhibition studies showed that the activation of JNK and p38 were critical to the cytotoxic effects of PABA/NO. Finally, PABA/NO produced antitumor effects in a human ovarian cancer model grown in SCID mice.

Two novel strategies for synthesizing stable polyurethanes (PUs) capable of generating bioactive nitric oxide (NO) are described. The methods rely on covalently attaching diazeniumdiolate (N(2)O(2)(-)) groups onto secondary amine nitrogens at various positions within the polymer chain such that, when in contact with water or physiological fluids, only the two molecules of NO available from each diazeniumdiolate moiety are released into the surrounding medium, with potential byproducts remaining covalently bound to the matrix. Extensive analysis of the NO(x)() products released from the polymers was employed to develop appropriate strategies to better stabilize the diazeniumdiolate-based polymer structures. In one approach, diazeniumdiolate groups are attached to secondary amino nitrogens of alkane diamines inserted within the diol chain extender of a PU material. Oxidative loss of NO was minimized by blending the polymer with a biocompatible, relatively nonnucleophilic salt before exposing solutions of the polymer to NO during the diazeniumdiolation step. Fluxes of molecular NO from such materials during immersion in physiological buffer reached levels as high as 19 pmol x cm(-2) x s(-1) with a total recovery of 21 nmol of NO/mg of PU. A second general synthetic strategy involved omega-haloalkylating the urethane nitrogens and then displacing the halide from the resulting polymer with a nucleophilic polyamine to form a PU with pendent amino groups suitable for diazeniumdiolation. Commercially available Pellethane 2363-80AE that was bromobutylated and then reacted with diethylenetriamine and further exposed to gaseous NO proved stable in solid form for several months, but released NO with a total recovery of 17 nmol/mg upon immersion in physiological buffer. This material showed an initial NO flux of 14 pmol x cm(-2) x s(-1) when immersed in pH 7.4 buffer at 37 degrees C, with gradually decreasing but still observable fluxes for up to 6 days.