Larry K. Keefer

“NONOates” (1-substituted diazen-1-ium-1,2-diolates) as nitric oxide donors: Convenient nitric oxide dosage forms

Abstract 1-Substituted diazen-1-ium-1,2-diolates have proved useful as tools in enzymology and other pharmacological research applications in which spontaneous generation of nitric oxide according to a reasonably well-defined time course is required. This chapter summarizes relevant physicochemical data, including the NO release rates and product profiles, for a selection of these compounds. Guidelines for quality control and a systematic nomenclature scheme are also presented. It is hoped that, in summarizing this information here, our chapter will help those contemplating new applications of diazeniumdiolate technology as they seek to capitalize on what we believe are the inherent advantages of this compound type in research on the pharmacological properties of nitric oxide.

Nitric oxide (NO) donor molecules : effect of NO release rate on vascular smooth muscle cell proliferation in vitro

Nitric oxide (NO) inhibits vascular smooth muscle cell (SMC) growth in vitro. To determine the effects of release rate and exposure time on SMC growth inhibition by NO, we compared the activities of five NO donors that generate NO with half-lives of 2 min (DEA/NO, Et2N[N2O2]Na), 15 min (PAPA/NO, CH3(CH2)2N[N2O2]-(CH2)3NH3+), 39 min, (SPER/NO, H2N(CH2)3NH2+(CH2)4N[N2O2]-(CH2)3NH2), 3 h (DPTA/NO, H2N(CH2)3N[N2O2]-(CH2)3NH3+), and 20 h (DETA/NO, H2N(CH2)2N[N2O2]-(CH2)2NH3+). After 22-h treatment, rat aorta SMC (RA-SMC) DNA synthesis was inhibited with IC50 values of 180, 60, and 40 microM for SPER/NO, DPTA/NO, and DETA/NO, respectively. DEA/NO and PAPA/NO did not inhibit DNA synthesis significantly at any concentration tested (20-500 microM). The inhibitory effect of NO on RA-SMC DNA synthesis was thus greatest when a given molar dose of NO was delivered slowly throughout the 22-h period. The antiproliferative effect of DETA/NO was confirmed by measurement of cell numbers for 7 days. When RA-SMC were treated with 500 microM DETA/NO on days 1, 3, and 5, growth was completely suppressed. Cell viability was > 95%, confirming that DETA/NO was not cytotoxic. The results suggest that NO donors may be useful inhibitors of intimal hyperplasia and restenosis after vascular injury such as balloon angioplasty.

Preparation and characterization of hydrophobic polymeric films that are thromboresistant via nitric oxide release

The preparation of hydrophobic polymer films (polyurethane and poly(vinyl chloride)) containing nitric oxide (NO)-releasing diazeniumdiolate functions is reported as a basis for improving the thromboresistivity of such polymeric materials for biomedical applications. Several different approaches for preparing NO-releasing polymer films are presented, including: (1) dispersion of diazeniumdiolate molecules within the polymer matrix; (2) covalent attachment of the diazeniumdiolate to the polymer backbone; and (3) ion-pairing of a diazeniumdiolated heparin species to form an organic soluble complex that can be blended into the polymer. Each approach is characterized in terms of NO release rates and in vitro biocompatibility. Results presented indicate that the polymer films prepared by each approach release NO for variable periods of time (10-72 h), although they differ in the mechanism, location and amount of NO released. In vitro platelet adhesion studies demonstrate that the localized NO release may prove to be an effective strategy for improving blood compatibility of polymer materials for a wide range of medical devices.

Nitric oxide and nanotechnology: A novel approach to inhibit neointimal hyperplasia

OBJECTIVE Nitric oxide (NO) has been shown to inhibit neointimal hyperplasia after arterial interventions in several animal models. To date, however, NO-based therapies have not been used in the clinical arena. Our objective was to combine nanofiber delivery vehicles with NO chemistry to create a novel, more potent NO-releasing therapy that can be used clinically. Thus, the aim of this study was to evaluate the perivascular application of spontaneously self-assembling NO-releasing nanofiber gels. Our hypothesis was that this application would prevent neointimal hyperplasia. METHODS Gels consisted of a peptide amphiphile, heparin, and a diazeniumdiolate NO donor (1-[N-(3-Aminopropyl)-N-(3-ammoniopropyl)]diazen-1-ium-1,2-diolate [DPTA/NO] or disodium 1-[(2-Carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate [PROLI/NO]). Nitric oxide release from the gels was evaluated by the Griess reaction, and scanning electron microscopy confirmed nanofiber formation. Vascular smooth muscle cell (VSMC) proliferation and cell death were assessed in vitro by (3)H-thymidine incorporation and Personal Cell Analysis (PCA) system (Guava Technologies, Hayward, Calif). For the in vivo work, gels were modified by reducing the free-water content. Neointimal hyperplasia after periadventitial gel application was evaluated using the rat carotid artery injury model at 14 days (n = 6 per group). Inflammation and proliferation were examined in vivo with immunofluorescent staining against CD45, ED1, and Ki67 at 3 days (n = 2 per group), and graded by blinded observers. Endothelialization was assessed by Evans blue injection at 7 days (n = 3 per group). RESULTS Both DPTA/NO and PROLI/NO, combined with the peptide amphiphile and heparin, formed nanofiber gels and released NO for 4 days. In vitro, DPTA/NO inhibited VSMC proliferation and induced cell death to a greater extent than PROLI/NO. However, the DPTA/NO nanofiber gel only reduced neointimal hyperplasia by 45% (intima/media [I/M] area ratio, 0.45 +/- 0.07), whereas the PROLI/NO nanofiber gel reduced neointimal hyperplasia by 77% (I/M area ratio, 0.19 +/- 0.03, P < .05) vs control (injury alone I/M area ratio, 0.83 +/- 0.07; P < .05). Both DPTA/NO and PROLI/NO nanofiber gels significantly inhibited proliferation in vivo (1.06 +/- 0.30 and 0.19 +/- 0.11 vs injury alone, 2.02 +/- 0.20, P < .05), yet had minimal effect on apoptosis. Only the PROLI/NO nanofiber gel inhibited inflammation (monocytes and leukocytes). Both NO-releasing nanofiber gels stimulated re-endothelialization. CONCLUSIONS Perivascular application of NO-releasing self-assembling nanofiber gels is an effective and simple therapy to prevent neointimal hyperplasia after arterial injury. Our study demonstrates that the PROLI/NO nanofiber gel most effectively prevented neointimal hyperplasia and resulted in less inflammation than the DPTA/NO nanofiber gel. This therapy has great clinical potential to prevent neointimal hyperplasia after open vascular interventions in patients.

Estrogen Metabolism and Risk of Breast Cancer in Postmenopausal Women

BACKGROUND Estrogens are recognized causal factors in breast cancer. Interindividual variation in estrogen metabolism may also influence the risk of breast cancer and could provide clues to mechanisms of breast carcinogenesis. Long-standing hypotheses about how estrogen metabolism might influence breast cancer have not been adequately evaluated in epidemiological studies because of the lack of accurate, reproducible, and high-throughput assays for estrogen metabolites. METHODS We conducted a prospective case-control study nested within the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial (PLCO). Participants included 277 women who developed invasive breast cancer (case subjects) and 423 matched control subjects; at PLCO baseline, all subjects were aged 55-74 years, postmenopausal and not using hormone therapy, and provided a blood sample. Liquid chromatography-tandem mass spectrometry was used to measure serum concentrations of 15 estrogens and estrogen metabolites, in unconjugated and conjugated forms, including the parent estrogens, estrone and estradiol, and estrogen metabolites in pathways defined by irreversible hydroxylation at the C-2, C-4, or C-16 positions of the steroid ring. We calculated hazard ratios (HRs) approximating risk in highest vs lowest deciles of individual estrogens and estrogen metabolites, estrogens and estrogen metabolites grouped by metabolic pathways, and metabolic pathway ratios using multivariable Cox proportional hazards models. All statistical tests were two-sided. RESULTS Nearly all estrogens, estrogen metabolites, and metabolic pathway groups were associated with an increased risk of breast cancer; the serum concentration of unconjugated estradiol was strongly associated with the risk of breast cancer (HR = 2.07, 95% confidence interval [CI] = 1.19 to 3.62). No estrogen, estrogen metabolite, or metabolic pathway group remained statistically significantly associated with the risk of breast cancer after adjusting for unconjugated estradiol. The ratio of the 2-hydroxylation pathway to parent estrogens (HR = 0.66, 95% CI = 0.51 to 0.87) and the ratio of 4-hydroxylation pathway catechols to 4-hydroxylation pathway methylated catechols (HR = 1.34, 95% CI = 1.04 to 1.72) were statistically significantly associated with the risk of breast cancer and remained so after adjustment for unconjugated estradiol. CONCLUSIONS More extensive 2-hydroxylation of parent estrogens is associated with lower risk, and less extensive methylation of potentially genotoxic 4-hydroxylation pathway catechols is associated with higher risk of postmenopausal breast cancer.

Diazeniumdiolates:: Pro- and antioxidant applications of the “NONOates”

Diazeniumdiolates are compounds containing the X-[N(O)NO](-) structural unit that as a class offer many advantages as tools for probing the roles of nitric oxide (NO) in biological redox processes. Available examples in which X is a secondary amine group spontaneously generate up to two molecules of NO per [N(O)NO](-) unit when dissolved in aqueous media; their half-lives range from 2 s (for X = L-prolyl) to 20 h [for X = (H(2)NCH(2)CH(2))(2)N] at pH 7. 4 and 37 degrees C, and are in general relatively little influenced by medium effects or metabolism. When X = O(-) (Angelis salt), first-order dissociation produces NO(-) rather than NO, but the ion becomes an NO source on 1-electron oxidation; diazeniumdiolate-derived NO can also be used to generate reactive nitrogen/oxygen species with higher nitrogen oxidation states (+3 and +4) in the presence of selected oxidizing agents. The advantages of diazeniumdiolates in biomedical research are briefly illustrated with examples from the recent literature probing NOs role in inhibiting oxidative drug metabolism, radical-induced lipid oxidation, the cytotoxicity of reactive oxygen species, and ischemia-induced vascular reoxygenation injury. Future work with this compound class should provide further insight into the mechanisms of NOs involvement in pro- and antioxidant processes, and may well lead to important medicinal advances, including reversal of cerebral vasospasm and radiosensitization of hypoxic tumors.

Mechanism of vascular relaxation induced by the nitric oxide (NO)/nucleophile complexes, a new class of NO-based vasodilators.

Compounds formed by reacting nitric oxide (NO) with various nucleophiles have been shown to dilate aortic segments with a potency that correlates strongly with the amount of NO they release spontaneously in aqueous buffers. We performed experiments aimed at confirming their mechanism of action and using the data to design improvements in their pharmacologic properties. That the vasorelaxant action these agents induce is endothelium-independent was demonstrated by exposure of denuded versus intact aortic segments to the diethylamine/NO complex (DEA/NO); denudation had no significant effect on potency. Similarly, NG-monomethyl-L-arginine, an NO synthase inhibitor, did not affect the action of DEA/NO. However, both the vasorelaxant potency of DEA/NO and the amount of cyclic guanosine monophosphate it induced were significantly diminished by the guanylate cyclase inhibitor, methylene blue. The results support the view that the NO/nucleophile adducts induce vasodilation by spontaneously releasing NO, which then activates guanylate cyclase. This mechanistic conclusion suggests that not only potency but also duration of action, a clinically relevant parameter not studied in the previous investigation, might also be controllable by structural modification. We tested this hypothesis by comparing DEA/NO and the spermine/NO adduct (SPER/NO), whose half-lives (t1/2) are 2.1 and 39 min, respectively, for persistence of their dilatory effects. The response to DEA/NO rapidly peaked (maximum at 5 min) and receded during the 60-min observation period; SPER/NO required 15 min to reach peak relaxation but maintained this level throughout the experiment.(ABSTRACT TRUNCATED AT 250 WORDS)

A liquid chromatography-mass spectrometry method for the quantitative analysis of urinary endogenous estrogen metabolites

The ability to measure estrogen metabolites (EMs) quantitatively is important for investigating their individual roles in cancer screening, treatment and prevention, as well as in a host of other hormone-related disorders. In this protocol we describe a method that is capable of quantitating 15 distinct EMs in urine. Endogenous EMs are quantitatively measured using a liquid chromatography–tandem mass spectrometry method in which the spectrometer operates in a selected reaction monitoring mode. This method is capable of quantifying estrone and its 2-, and 4- and 16α-hydroxy and its 2-, 4-methoxy derivatives, and 2-hydroxyestrone-3-methyl ether; 17β-estradiol and its 2-hydroxy, and 2- and 4-methoxy derivates, and estriol, 16-epiestriol, 17-epiestriol, and 16-ketoestradiol. The method requires only 0.5 ml of urine and approximately 60 urine samples can be quantitatively analyzed per week.

Structure Mechanism Insights and the Role of Nitric Oxide Donation Guide the Development of Oxadiazole-2-Oxides as Therapeutic Agents against Schistosomiasis

Schistosomiasis is a chronic parasitic disease affecting hundreds of millions of individuals worldwide. Current treatment depends on a single agent, praziquantel, raising concerns of emergence of resistant parasites. Here, we continue our explorations of an oxadiazole-2-oxide class of compounds we recently identified as inhibitors of thioredoxin glutathione reductase (TGR), a selenocysteine-containing flavoenzyme required by the parasite to maintain proper cellular redox balance. Through systematic evaluation of the core molecular structure of this chemotype, we define the essential pharmacophore, establish a link between the nitric oxide donation and TGR inhibition, determine the selectivity for this chemotype versus related reductase enzymes, and present evidence that these agents can be modified to possess appropriate drug metabolism and pharmacokinetic properties. The mechanistic link between exogenous NO donation and parasite injury is expanded and better defined. The results of these studies verify the utility of oxadiazole-2-oxides as novel inhibitors of TGR and as efficacious antischistosomal agents.

Augmentation of Intrapericardial Nitric Oxide Level by a Prolonged-Release Nitric Oxide Donor Reduces Luminal Narrowing After Porcine Coronary Angioplasty

Background—Nitric oxide (NO) is a potent vasodilator and antiplatelet agent that suppresses vascular smooth muscle cell proliferation. Hypothesizing that generating NO in the pericardial space would reduce luminal narrowing after coronary angioplasty without affecting systemic hemodynamics, we have determined the effect of a novel NO donor on vascular healing after balloon overstretch. Methods and Results—Diazeniumdiolated bovine serum albumin (D-BSA; molecular weight 74 kDa, half-life for NO release 20 days) was radioiodinated and found by intravital &ggr;-imaging to have a longer residence time in pig pericardium than a low-molecular-weight (0.5 kDa) analogue (22 versus 4.6 hours, respectively). Intrapericardial injection of D-BSA immediately before 30% overstretch of normal left anterior descending and left circumflex coronary arteries dose dependently reduced the intimal/medial area ratio by up to 50% relative to controls treated with underivatized albumin when measured 2 weeks after intervention. Positive remodeling was also noted, which increased luminal area relative to control. Conclusions—Perivascular exposure of coronary arteries to NO via intrapericardial D-BSA administration reduced flow-restricting lesion development after angioplasty in pigs without causing significant systemic effects. The data suggest that intrapericardial delivery of NO donors for which NO release rates and pericardial residence times are matched and optimized might be a beneficial adjunct to coronary angioplasty.