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Nitroxyl (HNO) donors have been shown to elicit a variety of pharmacological responses, ranging from tumoricidal effects to treatment of heart failure. Isopropylamine-based diazeniumdiolates have been shown to produce HNO on decomposition under physiological conditions. Herein, we report the synthesis and HNO release profiles of primary alicyclic amine-based diazeniumdiolates. These compounds extend the range of known diazeniumdiolate-based HNO donors. Acetoxymethyl ester-protected diazeniumdiolates were also synthesized to improve purification and cellular uptake. The acetoxymethyl derivative of cyclopentylamine diazeniumdiolate not only showed higher cytotoxicity toward cancer cells as compared to the parent anion but was also effective in combination with tamoxifen for targeting estrogen receptor α-negative breast cancer cells.

The nitric oxide (NO) prodrug JS-K, a promising anti-cancer agent, consists of a diazeniumdiolate group necessary for the release of NO as well as an arylating ring. In this study, we research the mechanism by which JS-K kills a murine erythroleukemia cell line and determine the roles of NO and arylation in the process. Our studies indicate that JS-K inhibits the PI 3-kinase/Akt and MAP kinase pathways. This correlates with the activation of the tumor suppressor FoxO3a and increased expression of various caspases, leading to apoptosis. The arylating capability of JS-K appears to be sufficient for inducing these biological effects. Overall, these data suggest that JS-K kills tumor cells by arylating and inactivating signaling molecules that block the activation of a tumor suppressor.

We introduce a strategy for generating mixtures of nitric oxide (NO) and nitroxyl (HNO) at tunable rates in physiological media. The approach involves converting a spontaneously HNO/NO-generating ion to a caged (prodrug) form that is essentially stable in neutral media, but that can be activated for HNO/NO release by adding an enzyme capable of efficiently opening the cage to regenerate the ion. By judiciously choosing the enzyme, substrate, and reaction conditions, unwanted scavenging of the HNO and NO by the protein can be minimised and the catalytic efficiency of the enzyme can be maintained. We illustrate this approach with a proof-of-concept study wherein the prodrug is Gal-IPA/NO, a diazeniumdiolate of structure iPrHN–N(O) NOR, with R = β- d -galactosyl. Escherichia coli-derived β- d -galactosidase at concentrations of 1.9–15 nM hydrolysed 56 μM substrate with half-lives of 140–19 min, respectively, producing the IPA/NO anion (iPrHN–N(O) NO−, half-life ∼3 min), which in turn spontaneously hydrolysed to mixtures of HNO with NO. Using saturating substrate concentrations furnished IPA/NO generation rates that were directly proportional to enzyme concentration. Consistent with these data, the enzyme/substrate combination applied to ventricular myocytes isolated from wild-type mouse hearts resulted not only in a significant positive inotropic effect, but also rescued the cells from the negative inotropy, hypercontractions, and occasional cell death seen with the enzyme alone. This mechanism represents an alternate approach for achieving controlled fluxes of NO/HNO to investigate their biological actions.

Amine-based diazeniumdiolates (NONOates) have garnered widespread use as nitric oxide (NO) donors, and their potential for nitroxyl (HNO) release has more recently been realized. While NO release rates can vary significantly with the type of amine, half-lives of seconds to days under physiological conditions, there is as yet no way to determine a priori the NO or HNO production rates of a given species, and no discernible trends have manifested other than that secondary amines produce only NO (i.e., no HNO). As a step to understanding these complex systems, here we describe a procedure for modeling amine-based NONOates in water solvent that provides an excellent correlation (R(2) = 0.94) between experimentally measured dissociation rates of seven secondary amine species and their computed NO release activation energies. The significant difference in behavior of NONOates in the gas and solvent phases is also rigorously demonstrated via explicit additions of quantum mechanical water molecules. The presented results suggest that the as-yet unsynthesized simplest amine-based NONOate, the diazeniumdiolated ammonia anion [H2N-N(O)═NO(-)], could serve as an unperturbed HNO donor. These results provide a step forward toward the accurate modeling of general NO and/or HNO donors as well as for the identification of tailored prodrug candidates.

Nitric oxide (NO) released from NO donors can be cytotoxic in tumor cells and can enhance the transport of drugs into brain tumors by altering blood-tumor barrier permeability. The NO donor JS-K [ O 2 -(2,4-dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate] releases NO upon enzymatic activation selectively in cells overexpressing glutathione-S-transferases (GSTs) such as gliomas. Thus, JS-K-dependent NO effects – especially on cell viability and vascular permeability – were investigated in U87 glioma cells in vitro and in an orthotopic U87 xenograft model in vivo by magnetic resonance imaging (MRI). In vitro experiments showed dose-dependent antiproliferative and cytotoxic effects in U87 cells. In addition, treatment of U87 cells with JS-K resulted in a dose-dependent activation of soluble guanylate cyclase and intracellular accumulation of cyclic guanosine monophosphate (cGMP) which was irreversibly inhibited by the selective inhibitor of soluble guanylate cyclase ODQ (1H-[1,2,4]oxadiazolo(4,3a)quinoxaline-1-one). Using dynamic contrast enhanced MRI (DCE-MRI) as a minimally invasive technique, we demonstrated for the first time a significant increase in the DCE-MRI read-out initial area under the concentration curve (iAUC 60 ) indicating an acute increase in blood-tumor barrier permeability after i.v. treatment with JS-K. Repeated MR imaging of animals with intracranial U87 gliomas under treatment with JS-K (3.5 μmol/kg JS-K 3×/week) and of untreated controls on day 12 and 19 after tumor inoculation revealed no significant changes in tumor growth, edema formation or tumor perfusion. Immunohistochemical workup of the brains showed a significant antiproliferative effect of JS-K in the gliomas. Taken together, in vitro and in vivo data suggest that JS-K has antiproliferative effects in U87 gliomas and opens the blood-tumor barrier by activation of the NO/cGMP signaling pathway. This might be a novel approach to facilitate entry of therapeutic drugs into brain tumors. DCE-MRI is a non-invasive, repeatable imaging modality to monitor biological effects of NO donors and other experimental therapeutics in intracranial tumor models.

JS-K is a nitric oxide (NO)-releasing prodrug of the O2-arylated diazeniumdiolate family that has demonstrated pronounced cytotoxicity and antitumor properties in a variety of cancer models both in vitro and in vivo. The current study of the metabolic actions of JS-K was undertaken to investigate mechanisms of its cytotoxicity. Consistent with model chemical reactions, the activating step in the metabolism of JS-K in the cell is the dearylation of the diazeniumdiolate by glutathione (GSH) via a nucleophilic aromatic substitution reaction. The resulting product (CEP/NO anion) spontaneously hydrolyzes, releasing two equivalents of NO. The GSH/GSSG redox couple is considered to be the major redox buffer of the cell, helping maintain a reducing environment under basal conditions. We have quantified the effects of JS-K on cellular GSH content, and show that JS-K markedly depletes GSH, due to JS-K's rapid uptake and cascading release of NO and reactive nitrogen species. The depletion of GSH results in alterations in the redox potential of the cellular environment, initiating MAPK stress signaling pathways, and inducing apoptosis. Microarray analysis confirmed signaling gene changes at the transcriptional level and revealed alteration in the expression of several genes crucial for maintenance of cellular redox homeostasis, as well as cell proliferation and survival, including MYC. Pre-treating cells with the known GSH precursor and nucleophilic reducing agent N-acetylcysteine prevented the signaling events that lead to apoptosis. These data indicate that multiplicative depletion of the reduced glutathione pool and deregulation of intracellular redox balance are important initial steps in the mechanism of JS-K's cytotoxic action., Graphical abstract Highlights ► JS-K is a NO-releasing prodrug, with anticancer activity in vitro and in vivo. ► JS-K metabolism results in the arylation of cellular glutathione (GSH). ► Micromolar levels of JS-K in media significantly deplete the cellular GSH reserve. ► Deregulation of cells' redox balance is a key contributor to JS-K toxicity.

Attachment of glutathione (GSH) to cysteine residues in proteins (S-glutathionylation) is a reversible post-translational modification that can profoundly alter protein structure and function. Often serving in a protective role, for example, by temporarily saving protein thiols from irreversible oxidation and inactivation, glutathionylation can be identified and semiquantitatively assessed using anti-GSH antibodies, thought to be specific for recognition of the S-glutathionylation modification. Here, we describe an alternate mechanism of protein glutathionylation in which the sulfur atoms of the GSH and the protein's thiol group are covalently bound via a cross-linking agent, rather than through a disulfide bond. This form of thiol cross-linking has been shown to occur and has been confirmed by mass spectrometry at the solution chemistry level, as well as in experiments documenting the potent antiproliferative activity of the bis-diazeniumdiolate Double JS-K in H1703 cells in vitro and in vivo. The modification is recognized by the anti-GSH antibody as if it were authentic S-glutathionylation, requiring mass spectrometry to distinguish between them.

We previously demonstrated that vascular smooth muscle cells (VSMC) proliferation and development of neointimal hyperplasia as well as the ability of nitric oxide (NO) to inhibit these processes is dependent on sex and hormone status. The aim of this study was to evaluate the role of estrogen receptor (ER) in mediating proliferation in male and female VSMC.Proliferation was assessed in primary rat aortic male and female VSMC using (3)H-thymidine incorporation in the presence or absence of ER alpha (α) inhibitor methyl-piperidino-pyrazole, the ER beta (β) inhibitor (R,R)-5,11-Diethyl-5,6,11,12-tetrahydro-2,8-chrysenediol, the combined ERαβ inhibitor ICI 182,780, and/or the NO donor DETA/NO. Proliferation was also assessed in primary aortic mouse VSMC harvested from wildtype (WT), ERα knockout (ERα KO), and ERβ knockout (ERβ KO) mice in the presence or absence of DETA/NO and the ERα, ERβ, and ERαβ inhibitors. Protein levels were assessed using Western blot analysis.Protein expression of ERα and ERβ was present and equal in male and female VSMC, and did not change after exposure to NO. Inhibition of either ERα or ERβ had no effect on VSMC proliferation in the presence or absence of NO in either sex. However, inhibition of ERαβ in rat VSMC mitigated NO-mediated inhibition in female but not male VSMC (P0.05). Evaluation of proliferation in the knockout mice revealed distinct patterns. Male ERαKO and ERβKO VSMC proliferated faster than male WT VSMC (P0.05). Female ERβKO proliferated faster than female WT VSMC (P0.05), but female ERαKO VSMC proliferated slower than female WT VSMC (P0.05). Last, we evaluated the effect of combined inhibition of ERα and ERβ in these knockout strains. Combined ERαβ inhibition abrogated NO-mediated inhibition of VSMC proliferation in female WT and knockout VSMC (P0.05), but not in male VSMC.These data clearly demonstrate a role for the ER in mediating VSMC proliferation in both sexes. However, these data suggest that the antiproliferative effects of NO may be regulated by the ER in females but not males.

Glutathione S-transferases (GSTs) control multidrug resistance and are upregulated in many cancers, including malignant gliomas. The diazeniumdiolate JS-K generates nitric oxide (NO) on enzymatic activation by glutathione and GST, showing promising NO-based anticancer efficacy.To evaluate the role of NO-based antitumor therapy with JS-K in U87 gliomas in vitro and in vivo.U87 glioma cells and primary glioblastoma cell lines were exposed to JS-K and a variety of inhibitors to study cell death by necrosis, apoptosis, and other mechanisms. GST expression was evaluated by immunocytochemistry, polymerase chain reaction, and Western blot, and NO release from JS-K was studied with a NO assay. The growth-inhibitory effect of JS-K was studied in a U87 xenograft model in vivo.Dose-dependent inhibition of cell proliferation was observed in human U87 glioma cells and primary glioblastoma cells in vitro. Cell death was partially induced by caspase-dependent apoptosis, which could be blocked by Z-VAD-FMK and Q-VD-OPH. Inhibition of GST by sulfasalazine, cGMP inhibition by ODQ, and MEK1/2 inhibition by UO126 attenuated the antiproliferative effect of JS-K, suggesting the involvement of various intracellular death signaling pathways. Response to JS-K correlated with mRNA and protein expression of GST and the amount of NO released by the glioma cells. Growth of U87 xenografts was reduced significantly, with immunohistochemical evidence for increased necrosis and apoptosis and reduced proliferation.Our data show for the first time the potent antiproliferative effect of JS-K in gliomas in vitro and in vivo. These findings warrant further investigation of this novel NO-releasing prodrug in gliomas.

Promising drug candidates of the diazeniumdiolate (NONOate) chemical family include several types of thiol modification among their mechanisms of action: 1) drugs designed to release nitric oxide (NO) on reaction with the thiol group of glutathione (GSH) arylate the GSH, a step that removes reducing equivalents from the cell; (2) a similar reaction of the drug with the thiol group of a protein changes its structure, leading to potentially impaired function and cell death; (3) the NO generated as a byproduct in the above reactions can undergo oxidation, leading to S-nitrosylation and S-glutathionylation; and (4) diazeniumdiolates can also generate nitroxyl, which reacts with thiol groups to form disulfides or sulfinamides.

Here I show that a "pure" research project, seemingly totally lacking in practical application when it was first published, can years later spark a whole new scientific field with the potential to revolutionize clinical practice. A 1961 publication describing adducts of nitric oxide (NO) with certain nucleophiles attracted little notice at the time, but later work showing that the adducts could be hydrolyzed to regenerate the NO in bioactive form has provided the foundation for a host of biomedical applications. Crucial to the discovery of widely used tools for studying NO's chemical biology as well as for the design of a variety of promising therapeutic advances has been the increasingly detailed understanding of the physicochemical properties of these "diazeniumdiolates" (also known as NONOates).

Arsenic is a cancer chemotherapeutic but hepatotoxicity can be a limiting side effect. O(2)-vinyl 1-[2-(carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate (V-PROLI/NO) is a nitric oxide (NO) donor prodrug and metabolized by liver cytochromes P450 (CYP450) to release NO. The effects of V-PROLI/NO pretreatment on the toxicity of arsenic (as NaAsO(2)) were studied in a rat liver cell line (TRL 1215). The cells acted upon the prodrug to release NO, as assessed by nitrite levels, in a time-dependent fashion to maximal levels of 8-fold above basal levels. Pretreatment with V-PROLI/NO markedly reduced arsenic cytolethality which was directly related to the level of NO produced by V-PROLI/NO treatment. Cyp1a1 expression was directly related to the level of NO production and to reduced arsenic cytotoxicity. V-PROLI/NO pretreatment markedly reduced arsenic-induced apoptosis and suppressed phosphorylation of JNK1/2. V-PROLI/NO pretreatment facilitated additional increases in arsenic-induced metallothionein, a metal-binding protein important in arsenic tolerance. Thus, V-PROLI/NO protects against arsenic toxicity in rat liver cells, reducing cytolethality, apoptosis and dysregulation of MAPKs, through generation of NO formed after metabolism by liver cell enzymes, possibly including Cyp1a1. CYP450 required for NO production from V-PROLI/NO treatment in the rat and human appears to differ as we have previously studied the ability of V-PROLI/NO to prevent arsenic toxicity in human liver cells where it reduced toxicity apparently through a CYP2E1-mediated metabolic mechanism. None-the-less, it appears that both rat and human liver cells act upon V-PROLI/NO via a CYP450-related mechanism to produce NO and subsequently reduce arsenic toxicity.

Periadventitial delivery of the nitric oxide (NO) donor PROLI/NO following arterial injury effectively inhibits neointimal hyperplasia. Given the short half-life of NO release from PROLI/NO, our goal was to determine if inhibition of neointimal hyperplasia by PROLI/NO was due to NO, or its metabolites nitrite and nitrate.In vitro, the NO donor DETA/NO inhibited proliferation of rat aortic vascular smooth muscle cells (RASMC), but neither nitrite nor nitrate did. In vivo, following rat carotid artery balloon injury or injury plus the molar equivalents of PROLI/NO, nitrite, or nitrate (n=8-11/group), PROLI/NO was found to provide superior inhibition of neointimal hyperplasia (82% inhibition of intimal area, and 44% inhibition of medial area, p0.001). Only modest inhibition was noted with nitrite or nitrate (45% and 41% inhibition of intimal area, and 31% and 29% inhibition of medial area, respectively, p0.001). No effects on blood pressure were noted with any treatment groups. In vivo, only PROLI/NO inhibited cellular proliferation and increased arterial lumen area compared to injury alone (p0.001). However, all three treatments inhibited inflammation (p0.001).PROLI/NO was more effective at inhibiting neointimal hyperplasia following arterial injury than nitrite or nitrate. However, modest inhibition of neointimal hyperplasia was observed with nitrite and nitrate, likely secondary to anti-inflammatory actions. In conclusion, we have demonstrated that the efficacy of NO donors is primarily due to NO production and not its metabolites, nitrite and nitrate.

Nitric oxide (NO)-based therapies decrease neointimal hyperplasia; however, studies have been performed only in male animal models. Thus, we sought to evaluate the effect of NO on vascular smooth muscle cells (VSMC) in vitro and neointimal hyperplasia in vivo based on sex and hormone status. In hormone-replete medium, male VSMC proliferated at greater rates than female VSMC. In hormone-depleted medium, female VSMC proliferated at greater rates than male VSMC. However, in both hormone environments, NO inhibited proliferation and migration to a greater extent in male compared to female VSMC. These findings correlated with greater G₀/G₁ cell cycle arrest and changes in cell cycle protein expression in male compared to female VSMC after exposure to NO. Next, the rat carotid artery injury model was used to assess the effect of NO on neointimal hyperplasia in vivo. Consistent with the in vitro data, NO was significantly more effective at inhibiting neointimal hyperplasia in hormonally intact males compared to females using weight-based dosing. An increased weight-based dose of NO in females was able to achieve efficacy equal to that in males. Surprisingly, NO was less effective at inhibiting neointimal hyperplasia in castrated animals of both sexes. In conclusion, these data suggest that NO inhibits neointimal hyperplasia more effectively in males compared to females and in hormonally intact compared to castrated rats, indicating that the effects of NO in the vasculature may be sex- and hormone-dependent.

The synthesis of poly(diol-co-citrate) elastomers that are biocompatible with vascular cells and can modulate the kinetics of the NO release based on the diol of selection is reported. NO-mediated cytostatic or cytotoxic effects can be controlled depending on the NO dose and the exposure time. When implanted in vivo in a rat carotid artery injury model, these materials demonstrate a significant reduction of neointimal hyperplasia. This is the first report of a NO-releasing polymer fabricated in the form of an elastomeric perivascular wrap for the treatment of neointimal hyperplasia. These elastomers also show promise for other cardiovascular pathologies where NO-based therapies could be beneficial.

Nitric oxide (NO) signaling in tumors and endothelial cells regulates vascular permeability and blood flow and therefore influences tumor uptake and response to therapeutic compounds. As delivery and efficacy of chemotherapy is impaired in CNS neoplasms due to a partially intact blood–brain barrier (BBB), we studied the effects of NO released by the short-acting NO donor disodium 1-[2-(carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate methanolate (PROLI/NO) on BBB integrity and blood flow in C6 gliomas using [14C]-aminoisobutyric acid (AIB) and [14C]-iodoantipyrine quantitative autoradiography. PROLI/NO selectively increased intratumoral uptake of [14C]AIB and [14C]sucrose when given as a 3-minute intracarotid infusion or a 15-minute i.v. infusion (AIB: tumor, K1 = 68.7 ± 3.2 vs 24.9 ± 0.9 µL g−1 min−1, P < .0001; sucrose, K1 = 16.9 ± 0.9 vs 11.5 ± 0.9 µL g−1 min−1, P = .0007). This effect was achieved without significant changes in cerebral and tumor blood flow or arterial blood pressure, which indicates that the effect on vascular permeability is independent of changes in vascular tone induced by NO. This effect was mediated by activation of the NO/3',5'-cyclic guanosine monophosphate (cGMP) pathway, as it was blocked by guanylate cyclase inhibition by LY83583 and reproduced by the delivery of 8-bromoguanosine 5'-monophosphate or inhibition of cGMP degradation by the phosphodiesterase inhibitor zaprinast. Inhibition of inducible NO synthase by aminoguanidine or cyclooxygenase inhibition by indometacin or dexamethasone did not reduce the blood–tumor barrier (BTB) response to PROLI/NO. PROLI/NO, and perhaps other NO-donating compounds, can be used to selectively increase BTB permeability in gliomas through the NO/cGMP pathway at doses that do not cause unwanted vasodilatory changes in blood flow and that do not affect the systemic circulation.

Here we describe a novel caged form of the highly reactive bioeffector molecule, nitroxyl (HNO). Reacting the labile nitric oxide (NO)- and HNO-generating salt of structure iPrHN-N(O)═NO(-)Na(+) (1, IPA/NO) with BrCH(2)OAc produced a stable derivative of structure iPrHN-N(O)═NO-CH(2)OAc (2, AcOM-IPA/NO), which hydrolyzed an order of magnitude more slowly than 1 at pH 7.4 and 37 °C. Hydrolysis of 2 to generate HNO proceeded by at least two mechanisms. In the presence of esterase, straightforward dissociation to acetate, formaldehyde, and 1 was the dominant path. In the absence of enzyme, free 1 was not observed as an intermediate and the ratio of NO to HNO among the products approached zero. To account for this surprising result, we propose a mechanism in which base-induced removal of the N-H proton of 2 leads to acetyl group migration from oxygen to the neighboring nitrogen, followed by cleavage of the resulting rearrangement product to isopropanediazoate ion and the known HNO precursor, CH(3)-C(O)-NO. The trappable yield of HNO from 2 was significantly enhanced over 1 at physiological pH, in part because the slower rate of hydrolysis for 2 generated a correspondingly lower steady-state concentration of HNO, thus, minimizing self-consumption and enhancing trapping by biological targets such as metmyoglobin and glutathione. Consistent with the chemical trapping efficiency data, micromolar concentrations of prodrug 2 displayed significantly more potent sarcomere shortening effects relative to 1 on ventricular myocytes isolated from wild-type mouse hearts, suggesting that 2 may be a promising lead compound for the development of heart failure therapies.

V-PYRRO/NO is a well studied nitric oxide (NO) prodrug which has been shown to protect human liver cells from arsenic, acetaminophen, and other toxic assaults in vivo. Its proline-based analogue, V-PROLI/NO, was designed to be a more biocompatible form that decomposes to the naturally occurring metabolites of proline, NO, and glycolaldehyde. Like V-PYRRO/NO, this cytochrome P450-activated prodrug was previously assumed to passively diffuse through the cellular membrane. Using (14)C-labeled proline in a competition assay, we show that V-PROLI/NO is transported through proline transporters into multiple cell lines. A fluorescent NO-sensitive dye (DAF-FM diacetate) and nitrite excretion indicated elevated intracellular NO release after metabolism over V-PYRRO/NO. These results also allowed us to predict and design a more permeable analogue, V-SARCO/NO. We report a proline transporter-based strategy for the selective transport of NO prodrugs that may have enhanced efficacy and aid in development of further NO prodrugs with increased permeability.

Objectives Glutathione S-transferases (GSTs) play an important role in multidrug resistance and are upregulated in multiple cancers. We have designed a prodrug class that releases nitric oxide on metabolism by GST. O2-(2,4-Dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate (JS-K, a member of this class) has potent antineoplastic activity. Methods We studied the effect of JS-K on angiogenesis in human umbilical vein endothelial cells (HUVECs), OPM1 multiple myeloma cells, chick aortic rings and in mice. Key findings JS-K inhibited the proliferation of HUVECs with a 50% inhibitory concentration (IC50) of 0.432, 0.466 and 0.505 μm at 24, 48 and 72 h, respectively. In the cord formation assay, JS-K led to a decrease in the number of cord junctions and cord length with an IC50 of 0.637 and 0.696 μm, respectively. JS-K inhibited cell migration at 5 h using VEGF as a chemoattractant. Migration inhibition occurred with an IC50 of 0.493 μm. In the chick aortic ring assay using VEGF or FGF-2 for vessel growth stimulation, 0.5 μm JS-K completely inhibited vessel growth. JS-K inhibited tumour angiogenesis in vivo in NIH III mice implanted subcutaneously with OPM1 multiple myeloma cells. Conclusions JS-K is a potent inhibitor of angiogenesis in vitro and tumour vessel growth in vivo. As such, it establishes a new class of antineoplastic agent that targets the malignant cells directly as well as their microenvironment.

Immunotherapy with IL-2 and anti-CD40 induces the expression of NOS2 in tumor-associated macrophages, and its expression is required for the inhibition of tumor metastasis., Using an orthotopic model of renal cell carcinoma, we showed previously that IL-2/anti-CD40 immunotherapy resulted in synergistic anti-tumor responses, whereas IL-2 or α-CD40 alone mediated partial transient anti-tumor effects. We now show that treatment of tumor-bearing mice with IL-2/α-CD40, but not IL-2 or α-CD40, induced significant nitric oxide synthase (NOS) 2 expression in tumor-associated macrophages. In control-treated mice (low NO), NOS2 inhibition reduced tumor burden. However, during immunotherapy (high NO), NOS2 inhibition or macrophage depletion reversed the ability of IL-2/α-CD40 treatment to reduce lung metastases but had no effect on primary tumor burden. Furthermore, IL-2/α-CD40 induced the IFN-γ– and NO-dependent decrease in matrix metalloproteinase (MMP) expression and activity, concomitant with increases in tissue inhibitor of metalloproteinase (TIMP) 1 and E-cadherin expression within tumors. Finally, treatment of tumor-bearing mice with the NO donor JS-K significantly reduced metastases. These data differentiate the mechanism for primary anti-tumor effects of IL-2/α-CD40 immunotherapy, which are independent of NO, from the NO-dependent inhibition of metastases. Furthermore, reduced MMP9 activity implicates M1-polarized macrophages within the tumor microenvironment as critical components of therapeutic response. Our data demonstrate the mechanistic basis for IL-2/α-CD40–mediated control of metastases and suggest that the context-dependent application of NO donors may hold promise for prevention of metastatic disease.

O(2)-(2,4-Dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate (JS-K) and O(2)-{2,4-dinitro-5-[4-(N-methylamino)be nzoyloxy]phenyl} 1-(N,N-dimethylamino)diazen-1-ium-1,2-diolate (PABA/NO) are O(2)-arylated diazeniumdiolates that have shown promising in vivo activity in a variety of rodent cancer models, including prostate cancer, leukemia, liver cancer, multiple myeloma, and ovarian cancer. This compound class was designed to be activated for anti-cancer effects by glutathione-S-transferase (GST)-induced release of cytotoxic nitric oxide (NO), but mechanistic studies have implicated a variety of pathways, some GST/NO-related, some not. Current work is focused on improving formulations and other drug development activities, as well as exploring possible new applications of these agents and their analogs. The selectivity of these drugs for attacking tumors while exhibiting little toxicity toward normal tissues suggests considerable promise for the treatment of various tumor types.

We report the stabilization of the nitric oxide (NO) prodrugs and anticancer lead compounds, PABA/NO (O(2)-{2,4-dinitro-5-[4-(N-methylamino)benzoyloxy]phenyl} 1-(N,N-dimethylamino)diazen-1-ium-1,2-diolate) and "Double JS-K" 1,5-bis-{1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diol-2-ato}-2,4-dinitrobenzene, through their incorporation into polymer-protected nanoparticles. The prodrugs were formulated in block copolymer-stabilized nanoparticles with sizes from 220 to 450 nm by a novel rapid precipitation process. The block copolymers, with polyethylene glycol (PEG) soluble blocks, provide a steric barrier against NO prodrug activation by glutathione. Too rapid activation and NO release has been a major barrier to effective administration of this class of compounds. The nanoparticle stabilized PABA/NO are protected from attack by glutathione as evidenced by a significant increase in time taken for 50% decomposition from 15 min (unformulated) to 5 h (formulated); in the case of Double JS-K, the 50% decomposition time was extended from 4.5 min (unformulated) to 40 min (formulated). The more hydrophobic PABA/NO produced more stable nanoparticles and correspondingly more extended release times in comparison with Double JS-K. The hydrophobic blocks of the polymer were either polystyrene or polylactide. Both blocks produced nanoparticles of approximately the same size and release kinetics. This combination of PEG-protected nanoparticles with sizes appropriate for cancer targeting by enhanced permeation and retention (EPR) and delayed release of NO may afford enhanced therapeutic benefit.

An improved synthesis of V-PROLI/NO, a cytochrome P450-activated nitric oxide (NO) prodrug, in an overall yield of 26% in four steps from prolinol is reported; the previously published yield of this transformation was 1%. Using this revised strategy, the sarcosine analogue (14) of V-PROLI/NO was prepared. Finally, the methyl ester of V-PROLI/NO (15) was found to be an esterase-activated prodrug form of V-PROLI/NO.

The nitric oxide (NO) prodrug JS-K is shown to have anticancer activity. To profile the molecular events associated with anticancer effects of JS-K, HL-60 leukemia cells were treated with JS-K and subjected to microarray and real-time RT-PCR analysis. JS-K induced concentration- and time-dependent gene expression changes in HL-60 cells corresponding to the cytolethality effects. The apoptotic genes (caspases, Bax, and TNF-α) were induced, and differentiation-related genes (CD14, CD11b, and vimentin) were increased. For acute phase protein genes, some were increased (p53, c-jun) while others were suppressed (c-myc, cyclin E). The expression of anti-angiogenesis genes thrombospondin-1 and CD36 and genes involved in tumor cell migration such as tissue inhibitors of metalloproteinases, were also increased by JS-K. Confocal analysis confirmed key gene changes at the protein levels. Thus, multiple molecular events are associated with JS-K effects in killing HL-60, which could be molecular targets for this novel anticancer NO prodrug.

Inorganic arsenic shows great promise in human cancer chemotherapy, although hepatotoxicity is a major limiting side-effect. O(2)-Vinyl 1-[2-(Carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate (V-PROLI/NO) [Correction added after publication 19 December 2008: 1-[2-(Carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate (V-PROLI/NO) was corrected to O(2)-Vinyl 1-[2-(Carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate (V-PROLI/NO)] is a nitric oxide (NO) donor prodrug that is metabolized by liver cytochromes P450 to release NO. Other NO-releasing agents have been shown to mitigate arsenic toxicity. Thus, the effects of V-PROLI/NO pretreatment on the toxicity of inorganic arsenic (as NaAsO(2)) were studied in vitro in a human liver (HepG2) cell line. HepG2 cells acted upon the prodrug to release NO, as assessed by nitrite levels, in a dose- and time-dependent fashion to maximal levels of 57-fold above control levels. In cells pretreated with V-PROLI/NO (200 microM, 24 h) then exposed to arsenic for an additional 24 h, arsenic was much less toxic (LC(50) = 151.9 +/- 5.9 microM) than in control cells (LC(50) = 90.5 +/- 6.5 microM) and the reduced cytolethality was directly related to the level of NO produced. V-PROLI/NO also increased CYP2E1 transcriptional expression in a dose-dependent manner and CYP2E1 expression was directly related to the level of NO produced and the reduction in arsenic cytotoxicity. V-PROLI/NO pretreatment markedly reduced arsenic-induced apoptosis as measured by DNA fragmentation. Pretreatment with V-PROLI/NO suppressed phosphorylation of JNK1/2 after arsenic exposure. Arsenic increased metallothionein, a metal-binding protein important in arsenic tolerance, and V-PROLI/NO pretreatment caused additional increases in metallothionein levels. Thus, the prodrug, V-PROLI/NO, protects against arsenic toxicity in cultured human liver cells, reducing cytolethality, apoptosis and dysregulation of mitogen-activated protein kinases, through generation of NO formed after metabolism by liver cell enzymes, possibly including CYP2E1.

Type II diabetes mellitus (DM) and metabolic syndrome are associated with accelerated restenosis following vascular interventions due to neointimal hyperplasia. The efficacy of nitric oxide (NO)-based therapies is unknown in these environments. Therefore, the aim of this study is to examine the efficacy of NO in preventing neointimal hyperplasia in animal models of type II DM and metabolic syndrome and examine possible mechanisms for differences in outcomes. Aortic vascular smooth muscle cells (VSMC) were harvested from rodent models of type II DM (Zucker diabetic fatty), metabolic syndrome (obese Zucker), and their genetic control (lean Zucker). Interestingly, NO inhibited proliferation and induced G0/G1 cell cycle arrest to the greatest extent in VSMC from rodent models of metabolic syndrome and type II DM compared with controls. This heightened efficacy was associated with increased expression of cyclin-dependent kinase inhibitor p21, but not p27. Using the rat carotid artery injury model to assess the efficacy of NO in vivo, we found that the NO donor PROLI/NO inhibited neointimal hyperplasia to the greatest extent in type II DM rodents, followed by metabolic syndrome, then controls. Increased neointimal hyperplasia correlated with increased reactive oxygen species (ROS) production, as demonstrated by dihydroethidium staining, and NO inhibited this increase most in metabolic syndrome and DM. In conclusion, NO was surprisingly a more effective inhibitor of neointimal hyperplasia following arterial injury in type II DM and metabolic syndrome vs. control. This heightened efficacy may be secondary to greater inhibition of VSMC proliferation through cell cycle arrest and regulation of ROS expression, in addition to other possible unidentified mechanisms that deserve further exploration.

Nitric oxide (NO) prodrugs such as O(2)-(2,4-dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate (JS-K) are a growing class of promising NO-based therapeutics. Nitric oxide release from the anti-cancer lead compound, JS-K, is proposed to occur through a nucleophilic aromatic substitution by glutathione (GSH) catalyzed by glutathione S-transferase (GST) to form a diazeniumdiolate anion that spontaneously releases NO. In this study, a number of structural analogues of JS-K were synthesized and their chemical and biological properties were compared with those of JS-K. The homopiperazine analogue of JS-K showed anti-cancer activity that is comparable with that of JS-K but with a diminished reactivity towards both GSH and GSH/GST; both the aforementioned compounds displayed no cytotoxic activity towards normal renal epithelial cell line at concentrations where they significantly diminished the proliferation of a panel of renal cancer cell lines. These properties may prove advantageous in the further development of this class of nitric oxide prodrugs as cancer therapeutic agents.

Glycosylated diazeniumdiolates of structure R 2NN(O)NO-R' (R' = a saccharide residue) are potential prodrugs of the nitric oxide (NO)-releasing but acid-sensitive R 2NN(O)NO (-) ion. Moreover, cleaving the acid-stable glycosides under alkaline conditions provides a convenient protecting group strategy for diazeniumdiolate ions. Here, we report comparative hydrolysis rate data for five representative glycosylated diazeniumdiolates at pH 14, 7.4, and 3.8-4.6 as background for further developing both the protecting group application and the ability to target NO pharmacologically to macrophages harboring intracellular pathogens. Confirming the potential in the latter application, adding R 2NN(O)NO-GlcNAc (where R 2N = diethylamino or pyrrolidin-l-yl and GlcNAc = N-acetylglucosamin-l-yl) to cultures of infected mouse macrophages that were deficient in inducible NO synthase caused rapid death of the intracellular protozoan parasite Leishmania major with no host cell toxicity.

Diazeniumdiolate anions and their prodrug forms are reliable sources of nitric oxide (NO) that have generated interest as promising therapeutic agents. A number of structural analogues of O(2)-(2,4-dinitro-5-(4-(N-methylamino)benzoyloxy)phenyl) 1-(N,N-dimethylamino)diazen-1-ium-1,2-diolate (PABA/NO), an anti-cancer lead compound that is designed to release NO upon activation by glutathione, were prepared. The nitric oxide release patterns of these O(2)-(2,4-dinitrophenyl) diazeniumdiolates in the presence of glutathione were tested and it was found that in the absence of competing pathways, these compounds release nearly quantitative amounts of NO. The ability of PABA/NO and its structural analogues to inhibit human leukemia cell proliferation was determined and it was found that compounds releasing elevated amounts of NO displayed superior cytotoxic effects.

Nitric oxide (NO)-based therapies effectively inhibit neointimal hyperplasia in animal models of arterial injury and bypass grafting, but are not available clinically. We created a simple, effective, locally-applied NO-eluting therapy to prevent restenosis following vascular procedures. We investigated the efficacy of perivascular delivery of two different distinctly different diazeniumdiolate NO donors, 1-[2-(carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate (PROLI/NO), (short half-life) and diazeniumdiolated poly(acrylonitrile) (PAN/NO), (long half-life), in powder or gel form (30% poloxamer 407), at inhibiting neointimal hyperplasia using the rat carotid artery injury model. Two weeks post-injury, all of the NO-eluting therapies successfully reduced neointimal hyperplasia. However, most dramatically, PROLI/NO powder reduced intimal area by 91.2% (P

Nitric oxide prodrugs derived from hindered secondary amines were prepared. The decomposition patterns of these prodrugs indicate that alpha-methyl groups around the nitrogen bearing the diazeniumdiolate group prolong their half-life in aqueous buffer.

Arsenite is an important cancer chemotherapeutic. The liver is a major target tissue of arsenic toxicity and hepatotoxicity may limit its chemotherapeutic efficacy. O(2)-vinyl 1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate (V-PYRRO/NO) is a liver-selective nitric oxide (NO)-producing prodrug metabolized by hepatic P450 enzymes to release NO locally. V-PYRRO/NO protects against various organic or inorganic hepatotoxicants but any role in arsenic hepatotoxicity is undefined. Thus, we studied the effects of V-PYRRO/NO (0-1000muM) pretreatment on inorganic arsenic-induced toxicity in cultured rat liver (TRL 1215) cells. These cells metabolized the prodrug to release NO, producing extracellular nitrite levels to 41.7-fold above control levels (7.50+/-0.38 microM) after 24h V-PYRRO/NO (1000 microM) exposure. The effect of pretreatment with V-PYRRO/NO (24h) on the cytolethality of arsenic (as NaAsO(2)) exposure (24h) was assessed. Arsenic was markedly less toxic in V-PYRRO/NO pretreated cells (LC(50)=30.3 microM) compared to control (LC(50)=20.1 microM) and the increases in LC(50) showed a direct relationship to the level of NO produced (measured as nitrite). Consistent with the cytolethality data, V-PYRRO/NO pretreatment markedly reduced arsenic-induced apoptosis as assessed by DNA fragmentation. Activation of the c-Jun N-terminal kinase (JNK) pathway can be critical to apoptosis and pretreatment with V-PYRRO/NO suppressed arsenic-induced JNK activation. V-PYRRO/NO pretreatment modestly increased metallothionein (MT), a metal-binding protein, but greatly enhanced arsenic induction of MT. Thus, V-PYRRO/NO pretreatment directly mitigates arsenic toxicity in cultured liver cells, reducing cytolethality, apoptosis and related JNK pathway activation, apparently through generation of NO. The role of NO in reducing the hepatotoxicity of arsenical chemotherapeutics in vivo deserves additional study.

A novel group of O2-acetoxymethyl-protected diazeniumdiolate-based non-steroidal anti-inflammatory prodrugs (NONO-NSAIDs) were synthesized by esterifying the carboxylate group of aspirin, ibuprofen, or indomethacin with O2-acetoxymethyl 1-[N-(2-hydroxyethyl)-N-methylamino]diazeniumdiolate. The resulting nitric oxide (•NO)-releasing prodrugs (7–9) did not exhibit in vitro cyclooxygenase (COX) inhibitory activity against the COX-1 and COX-2 isozymes (IC50s > 100 μM). In contrast, prodrugs 7 and 8 significantly decreased carrageenan-induced rat paw edema showing enhanced in vivo anti-inflammatory activities (ID50’s = 552 and 174 μmol/kg, respectively) relative to those of the parent NSAIDs aspirin (ID50 = 714 μmol/kg), and ibuprofen (ID50 = 326 μmol/kg).. The rate of porcine liver esterase-mediated •NO release from prodrugs 7–9 (2 moles of •NO/mol of test compound in 0.6–6.5 min) was substantially higher compared to that observed without enzymatic catalysis (about 1 mol of •NO/mol of test compound in 40–48 h). These incubation studies suggest that both •NO and the parent NSAID would be released upon in vivo activation (hydrolysis) by esterases. Data acquired in an in vivo ulcer index (UI) assay showed that NONO-aspirin (UI = 0.8), NONO-indomethacin (UI = 1.3), and particularly NONO-ibuprofen (UI = 0), were significantly less ulcerogenic compared to the parent drugs aspirin (UI = 57), ibuprofen (UI = 46) or indomethacin (UI = 34) at equimolar doses. The release of aspirin and •NO from the NONO-aspirin (7) prodrug constitutes a potentially beneficial property for the prophylactic prevention of thrombus formation and adverse cardiovascular events such as stroke and myocardial infarction.

Experimental evidence suggests that nitric oxide (NO) and hydrogen sulfide (H2S) signaling pathways are intimately intertwined, with mutual attenuation or potentiation of biological responses in the cardiovascular system and elsewhere. The chemical basis of this interaction is elusive. Moreover, polysulfides recently emerged as potential mediators of H2S/sulfide signaling, but their biosynthesis and relationship to NO remain enigmatic. We sought to characterize the nature, chemical biology, and bioactivity of key reaction products formed in the NO/sulfide system. At physiological pH, we find that NO and sulfide form a network of cascading chemical reactions that generate radical intermediates as well as anionic and uncharged solutes, with accumulation of three major products: nitrosopersulfide (SSNO(-)), polysulfides, and dinitrososulfite [N-nitrosohydroxylamine-N-sulfonate (SULFI/NO)], each with a distinct chemical biology and in vitro and in vivo bioactivity. SSNO(-) is resistant to thiols and cyanolysis, efficiently donates both sulfane sulfur and NO, and potently lowers blood pressure. Polysulfides are both intermediates and products of SSNO(-) synthesis/decomposition, and they also decrease blood pressure and enhance arterial compliance. SULFI/NO is a weak combined NO/nitroxyl donor that releases mainly N2O on decomposition; although it affects blood pressure only mildly, it markedly increases cardiac contractility, and formation of its precursor sulfite likely contributes to NO scavenging. Our results unveil an unexpectedly rich network of coupled chemical reactions between NO and H2S/sulfide, suggesting that the bioactivity of either transmitter is governed by concomitant formation of polysulfides and anionic S/N-hybrid species. This conceptual framework would seem to offer ample opportunities for the modulation of fundamental biological processes governed by redox switching and sulfur trafficking.

PABA/NO [O(2)-{2,4-dinitro-5-[4-(N-methylamino)benzoyloxy]phenyl} 1-(N,N-dimethylamino) diazen-1-ium-1,2-diolate] is a nitric oxide (NO)-releasing arylating agent designed to be selectively activated by reaction with glutathione (GSH) on catalysis by glutathione S-transferase P1 (GSTP1), an enzyme frequently overexpressed in cancer cells. PABA/NO has proven active in several cancer models in vitro and in vivo, but its tendency to be metabolized via a variety of pathways, some that generate inactive metabolites and hydrolysis products, limits its potential as a drug. Here we show that a simple replacement of cyano for nitro at the 4 position to give compound 4b ('p-cyano-PABA/NO') has the dual effect of slowing the undesired side reactions while enhancing the proportion of NO release and arylating activity on catalysis by GSTP1. Compound 4b showed increased resistance to hydrolysis and uncatalyzed reaction with GSH, along with a more favorable product distribution in the presence of GSTP1. It also showed significant proapoptotic activity. The data suggest p-cyano-PABA/NO to be a more promising prodrug than PABA/NO, with better selectivity toward cancer cells.

The literature provides evidence that metabolic nitric oxide (NO) release mediates the cytotoxic activities (against human leukemia and prostate cancer xenografts in mice) of JS-K, a compound of structure R(2)N-N(O)=NO-Ar for which R(2)N is 4-(ethoxycarbonyl)piperazin-1-yl and Ar is 2,4-dinitrophenyl. Here we present comparative data on the potencies of JS-K and 41 other O(2)-arylated diazeniumdiolates as inhibitors of HL-60 human leukemia cell proliferation, as well as in the NCI 51-cell-line screen for six of them. The data show JS-K to be the most potent of the 42 in both screens and suggest that other features of its structure and metabolism besides NO release may contribute importantly to its activity. Results with control compounds implicate JS-K's arylating ability, and the surprisingly low IC(50) value of the N-(ethoxycarbonyl)piperazine byproduct of NO release suggests a role for the R(2)N moiety. In addition to the above-mentioned in vivo activities, JS-K is shown here to be carcinostatic in a rat liver cancer model.

Hydrolytic deamidation of asparagine (Asn) and glutamine (Gln) residues to aspartate (Asp) and glutamate (Glu), respectively, can have significant biological consequences. We hypothesize that a wholly different mechanism of deamidation might occur in the presence of aerobic nitric oxide (NO). Accordingly, we examined the deamidating ability of aerobic NO toward three model peptides, 2,4-dinitrophenyl (DNP)-Pro-Gln-Gly, Lys-Trp-Asp-Asn-Gln, and Ser-Glu-Asn-Tyr-Pro-Ile-Val, incubating them with the NO-generating compound, Et(2)N[N(O)NO]Na (DEA/NO, 30-48 mM), in aerobic, pH 7.4, buffer at 37 degrees C. DNP-Pro-Glu-Gly was detected after 2 h, while Lys-Trp-Asp-Asp-Gln, Lys-Trp-Asp-Asn-Glu, and Ser-Glu-Asp-Tyr-Pro-Ile-Val were detected within 10 min, accumulating in apparent yields of up to approximately 10%. In the latter case, tyrosine nitration was also observed, producing the expected nitrotyrosine residue. DEA/NO solutions preincubated to exhaust the NO before the peptides were added did not induce detectable deamidation. The data demonstrate that aerobic NO exposures can lead to nitrosative deamidation of peptides, a pathway that differs from the established hydrolytic deamidation mechanism in several key respects: the by-products of the former are molecular nitrogen and an acid (HONO) while that of the latter is a base (NH(3)); the nitrosative path can in principle proceed in the absence of water molecules; Asn is much more easily deamidated than Gln in the hydrolytic pathway, while the two amino acid residues were deamidated to a similar extent by exposure to NO in the presence of oxygen.

Nitric oxide (NO) is an endogenous, diffusible, transcellular messenger shown to affect regulatory and signaling pathways with impact on cell survival. Exposure to NO can impart direct post-translational modifications on target proteins such as nitration and/or nitrosylation. As an alternative, after interaction with oxygen, superoxide, glutathione, or certain metals, NO can lead to S-glutathionylation, a post-translational modification potentially critical to signaling pathways. A novel glutathione S-transferase pi (GSTpi)-activated pro-drug, O(2)-[2,4-dinitro-5-[4-(N-methylamino)benzoyloxy]phenyl]1-(N,N-dimethylamino)diazen-1-ium-1,2-diolate (PABA/NO), liberates NO and elicits toxicity in vitro and in vivo. We now show that PABA/NO induces nitrosative stress, resulting in undetectable nitrosylation, limited nitration, and high levels of S-glutathionylation. After a single pharmacologically relevant dose of PABA/NO, S-glutathionylation occurs rapidly (5 min) and is sustained for approximately 7 h, implying a half-life for the deglutathionylation process of approximately 3 h. Two-dimensional SDS-polyacrylamide gel electrophoresis and immunoblotting with a monoclonal antibody to S-glutathionylated residues indicated that numerous proteins were S-glutathionylated. Subsequent matrix-assisted laser desorption ionization/time of flight analysis identified 10 proteins, including beta-lactate dehydrogenase, Rho GDP dissociation inhibitor beta, ATP synthase, elongation factor 2, protein disulfide isomerase, nucleophosmin-1, chaperonin, actin, protein tyrosine phosphatase 1B (PTP1B), and glucosidase II. In addition, we showed that sustained S-glutathionylation was temporally concurrent with drug-induced activation of the stress kinases, known to be linked with cell death pathways. This is consistent with the fact that PABA/NO induces S-glutathionylation and inactivation of PTP1B, one phosphatase that can participate in deactivation of kinases. These effects were consistent with the presence of intracellular PABA/NO or metabolites, because cells overexpressing MRP1 were less sensitive to the drug and had reduced levels of S-glutathionylated proteins.

[reaction: see text] The enediamine tautomer of a variety of substituted amidine free bases reacts with nitric oxide (NO) to produce compounds containing a carbon-bound diazeniumdiolate [R1R2R3C-N(O)=NO-] functional group (previously called "nitrosohydroxylamines"). The new reaction has been shown to be quite general, although the nature of the products does vary. Amidines containing more than one replaceable hydrogen produce polydiazeniumdiolates as intermolecular salts, while those in which only one diazeniumdiolation can occur provide zwitterionic salts. These diazeniumdiolated amidines are shown to be useful NO donor compounds which undergo very slow spontaneous dissociation on dissolution in pH 7.4 phosphate buffer to produce mixtures of NO and nitrous oxide containing mostly NO. The most advantageous manifestation of the new discovery is the preparation of the monodiazeniumdiolated amidine zwitterions. Reaction of the medically relevant alpha-adrenergic agonists tetrahydrozoline and idazoxan produced monodiazeniumdiolated amidine zwitterions from which NO release was observed for up to 28 days and showed little sign of ending. The reaction should be applicable to a variety of pharmaceutical agents, including NO synthase inhibitors, antitumor agents, and antibacterials.

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 pi isoform of glutathione S-transferase, an enzyme expressed at high levels in many tumors. We have used mouse embryo fibroblasts (MEFs) null for GSTpi (GSTpi(-/-)) to show that the absence of GSTpi 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 GSTpi and/or various combinations of gamma-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 GSTpi-activated metabolite of PABA/NO was effluxed by MRP1 in a GSH-dependent 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.

The nitric oxide (NO) donor, O(2)-vinyl 1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate (V-PYRRO/NO), is metabolized by P450 enzymes to release NO in the liver and possibly other tissues. V-PYRRO/NO has been shown to be hepatoprotective, but little is known about its effect in the kidney, another organ rich in P450s. Thus, mice were given V-PYRRO/NO (0.4-5.4 mg/ml, 8 microl/h) before and/or after a nephrotoxic dose of acetaminophen (APAP; 600 mg/kg, i.p.) to examine its nephroprotective effects. V-PYRRO/NO administration significantly reduced APAP-induced nephrotoxicity in a dose- and time-dependent manner, as evidenced by mitigation of increased blood urea nitrogen levels and by amelioration of renal pathology, specifically interstitial congestion, proximal tubular cell degeneration and necrosis. The best protection was observed at the highest dose (5.4 mg/ml) and with V-PYRRO/NO pretreatment (4-16 h). Implanting V-PYRRO/NO pumps simultaneously with APAP also attenuated APAP nephrotoxicity. The protection is probably not due to a decreased APAP toxication metabolism, as similar depletion of renal glutathione levels was observed regardless of V-PYRRO/NO treatment. APAP-induced renal lipid peroxidation was reduced by V-PYRRO/NO, as determined by the concentrations of hydroxynonenals and malondialdehyde. In summary, this study demonstrates that the NO donor V-PYRRO/NO is effective in blocking APAP-induced nephrotoxicity in mice. The protection is probably due to multiple mechanisms involving attenuation of APAP-induced congestion and lipid peroxidation in the kidney.

Diazeniumdiolates, compounds of structure R1R2NN(O)=NOR3, which have also been called NONOates, have proven useful for treating an increasing diversity of medical disorders in relevant animal models. Here, I review the chemical features that make them such excellent starting points for designing materials capable of targeting reliable and controllable fluxes of bioactive NO for in vitro and in vivo applications. This is followed by a consideration of recent proof-of-concept studies that underscore what I believe to be the substantial clinical promise of such materials. Examples covered include progress toward inhibiting restenosis after angioplasty, preparing thromboresistant medical devices, reversing vasospasm, and relieving pulmonary hypertension. Together with a very recent report describing the beneficial effects of diazeniumdiolate therapy in a patient with acute respiratory distress syndrome, the results of the animal experiments support the prediction that a broad selection of problems in clinical medicine can be solved by judiciously mining the enormous variety of possible R1R2NN(O)=NOR3 structures.

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 γ-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.

O 2 -Vinyl 1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate (V-PYRRO/NO), a liver-selective nitric oxide (NO)-donating prodrug, is metabolized by hepatic enzymes to release NO within the liver. This study was undertaken to examine the effects of V-PYRRO/NO ond-galactosamine/lipopolysaccharide (GlaN/LPS)-induced liver injury in mice. Mice were given injections of V-PYRRO/NO (10 mg/kg, s.c. at 2-h intervals) before and after GlaN/LPS (700 mg/30 μg/kg, i.p.). V-PYRRO/NO administration dramatically reduced GlaN/LPS-induced hepatotoxicity, as evidenced by reduced serum alanine aminotransferase activity and improved pathology. To examine the mechanisms of the protection, cDNA microarray was performed to profile the gene expression pattern in livers of mice treated with GlaN/LPS, GlaN/LPS plus V-PYRRO/NO, or controls. V-PYRRO/NO administration greatly ameliorated GlaN/LPS-induced alterations in the expression of genes encoding the stress response, DNA damage/repair response, and drug-metabolizing enzymes in accordance with hepatoprotection. Gel shift assay and Western blot analysis supported microarray results, showing that V-PYRRO/NO suppressed GlaN/LPS-induced activation of nuclear factor-κB and GlaN/LPS-induced increases in caspase-1, caspase-8, tumor necrosis factor receptor 1 (TNFR1)-associated death domain, and TNF-related apoptosis-inducing ligand. Immunohistochemical analysis further revealed that GlaN/LPS-induced activation of TNFR1, caspase-3, and hepatocellular apoptosis was ameliorated by V-PYRRO/NO treatment. GlaN/LPS-induced elevation of hepatic caspase-3 activity was diminished by V-PYRRO/NO treatment. In addition, V-PYRRO/NO alone suppressed the basal expression of genes encoding inducible NO synthase and TNF-α-related components, as revealed by mouse 1.2 array. In summary, this study demonstrates that the liver-selective NO donor, V-PYRRO/NO, is effective in blocking GlaN/LPS-induced hepatotoxicity in mice, and that this protection appears to involve, at least in part, the suppression of the TNF-α-mediated cell death pathways. * V-PYRRO/NO : O 2 -vinyl 1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate NO : nitric oxide LPS : lipopolysaccharide GlaN/LPS : galactosamine plus LPS ALT : alanine aminotransferase TNF-α : tumor necrosis factor-α TNFR : tumor necrosis factor receptor NF-κB : nuclear factor-κB TRADD : TNFR1-associated death domain TRAIL : TNF-related apoptosis inducing ligand TBST : Tris-buffered saline/Tween 20 TUNEL : terminal deoxynucleotidyl transferase dUTP nick-end labeling G3PDH, glyceraldehyde-3-phosphate dehydrogenase

Background and purpose There is an unmet medical need for novel NAFLD treatments. Here we have examined the effects of liver-selective NO donor (V-PYRRO/NO) as compared with metformin on hepatic steatosis and glucose tolerance in mice fed high fat diet. Material and methods Effects of V-PYRRO/NO (5 mg kg −1 ) or metformin (616 mg kg −1 ) were examined in C57BL/6J mice fed high fat diet (HF, 60 kcal% fat). Quantitative determination of steatosis, liver fatty acid composition and western blot analysis of selected proteins involved in mitochondrial biogenesis, fatty acid de novo synthesis and oxidation, triacylglycerols and cholesterol transport from the liver were performed. Liver NO x and nitrate concentration and blood biochemistry were also analyzed. Results V-PYRRO/NO and metformin reduced liver steatosis with simultaneous reduction of total liver triacylglycerols, diacylglycerols and ceramides fraction and reversed HF-induced decrease in UFA/SFA ratio. V-PYRRO/NO substantially improved postprandial glucose tolerance, while the effect of metformin was modest and more pronounced on HOMA IR index. The anti-steatotic mechanism of V-PYRRO/NO was dependent on NO release, differed from that of metformin and involved improved glucose tolerance and inhibition of de novo fatty acid synthesis by Akt activation and ACC phosphorylation. In turn, major mechanism of metformin action involved increased expression of proteins implicated in mitochondrial biogenesis and metabolism (PGC-1α, PPARα, COX IV, cytochrome c, HADHSC). Conclusions V-PYRRO/NO acts as a liver-specific NO donor prodrug affording pronounced anti-steatotic effects and may represent an efficient, mechanistically novel approach to prevent liver steatosis and insulin resistance.

Controlled release of nitric oxide (NO*) may be useful in the treatment of a variety of vascular disorders. NO* donors of the diazeniumdiolate family with different rates of spontaneous NO* release have been synthesized. In the current study responses to seven diazeniumdiolate NO* donors (DEA/NO*, DETA/NO*, OXI/NO*, PIPERAZI/NO*, PROLI/NO*, SPER/NO*, and SULFI/NO*) were investigated in the hindquarters vascular bed of the cat. Intravenous injections of all NO* donors caused dose-dependent decreases in systemic arterial pressure and the rank order of potency was SNP > DEA/NO* > PIPERAZI/NO* > SPER/NO* > PROLI/NO* > OXI/NO*. Injections of all NO* donors into the hindlimb perfusion circuit caused dose-related decreases in hindquarters perfusion pressure that were similar to the order of potency in decreasing systemic arterial pressure. The rank order of the time required for the response to return to 50% of the maximal decrease in pressure (T(1/2)) and total duration of action of the NO* donors was SPER/NO* > PIPERAZI/NO* > DEA/NO* > OXI/NO* > DETA/NO* > PROLI/NO* > SULFI/NO*. After treatment with the NO* synthase inhibitor, N(omega)-nitro-L-arginine methyl ester (100 mg/kg, i.v.), hindlimb vasodilator responses to the NO* donors were not significantly different, but vasodilator responses to acetylcholine were significantly reduced. After treatment with zaprinast (2 mg/kg, i.v.), a type V cyclic 3',5'-guanosine monophosphate-specific phosphodiesterase inhibitor, the duration of vasodilator responses to the NO* donors, as measured by T(1/2), was increased significantly, whereas the duration of the response to the beta2-adrenergic receptor agonist albuterol was unchanged. These data suggest that diazeniumdiolate NO* donors are endothelium-independent, directly stimulate soluble guanylate cyclase, and decrease vascular resistance by increasing cyclic 3',5'-guanosine monophosphate levels in the hindquarters vascular bed of the cat.

Proatherogenic oxidized low-density lipoprotein (oxLDL) induces endothelial apoptosis. We investigated the anti-apoptotic effects of intracellular and extracellular nitric oxide (*NO) donors, iron chelators, cell-permeable superoxide dismutase (SOD), glutathione peroxidase mimetics, and nitrone spin traps. Peroxynitrite (ONOO-)-modified oxLDL induced endothelial apoptosis was measured by DNA fragmentation, TUNEL assay, and caspase-3 activation. Results indicated the following: (i) the lipid fraction of oxLDL was primarily responsible for endothelial apoptosis. (ii) Endothelial apoptosis was potently inhibited by *NO donors and lipophilic phenolic antioxidants. OxLDL severely depleted Bcl-2 levels in endothelial cells and *NO donors restored Bcl-2 protein in oxLDL-treated cells. (iii) The pretreatment of a lipid fraction derived from oxLDL with sodium borohydride or potassium iodide completely abrogated apoptosis in endothelial cells, suggesting that lipid hydroperoxides induce apoptosis. (iv) Metalloporphyrins dramatically inhibited oxLDL-induced apoptosis in endothelial cells. Neither S-nitrosation of caspase-3 nor induction of Hsp70 appeared to play a significant role in the antiapoptotic mechanism of *NO in oxLDL-induced endothelial apoptosis. We propose that cellular lipid peroxyl radicals or lipid hydroperoxides induce an apoptotic signaling cascade in endothelial cells exposed to oxLDL, and that *NO inhibits apoptosis by scavenging cellular lipid peroxyl radicals.

Diazeniumdiolate ions of structure R(2)N[N(O)NO](-) (1) are of pharmacological interest because they spontaneously generate the natural bioregulatory species, nitric oxide (NO), when dissolved in aqueous media. Here we report the kinetic details for four representative reactivity patterns: (a) straightforward dissociation of the otherwise unfunctionalized diethylamine derivative 2 (anion 1, where R = Et) to diethylamine and NO; (b) results for the zwitterionic piperazin-1-yl analogue 4, for which the protonation state of the neighboring basic amine site is an important determinant of dissociation rate; (c) data for 5, a diazeniumdiolate derived from the polyamine spermine, whose complex rate equation can include terms for a variety of medium effects; and (d) the outcome for triamine 6 (R = CH(2)CH(2)NH(3)(+)), the most stable structure 1 ion identified to date. All of these dissociations are acid-catalyzed, with equilibrium protonation of the substrate preceding release of NO. Specific rate constants and pK(a) values for 2-6 have been determined from pH/rate profiles. Additionally, a hypsochromic shift (from approximately 250 to approximately 230 nm) was observed on acidifying these ions, allowing determination of a separate pK(a) for each substrate. For 6, the pK(a) value obtained kinetically was 2-3 pK(a) units higher than the value obtained from the spectral shift. Comparison of the ultraviolet spectra for 6 at various pH values with those for O- and N-alkylated diazeniumdiolates suggests that protonation at the R(2)N nitrogen initiates dissociation to NO at physiological pH, with a second protonation (at oxygen) accounting for both the spectral change and the enhanced dissociation rate at pH4. Our results help to explain the previously noted variability in dissociation rate of 5, whose half-life we found to increase by an order of magnitude when its concentration was raised from near-zero to 1 mM, and provide mechanistic insight into the factors that govern dissociation rates among diazeniumdiolates of importance as pharmacologic progenitors of NO.

The metal binding protein metallothionein (MT) is involved in zinc homeostasis since it typically binds large amounts of zinc. Free zinc can control MT gene expression by interacting with metal-sensitive transcription factors. However, the precise factors governing intracellular release of metal ions from MT remain unknown. Aerobic nitric oxide (NO) can nitrosate thiol groups in proteins, and MT-bound cadmium is released by NO exposure. Thus, we hypothesized that NO may also be effective at displacing zinc from MT in cultured cells and that this could be an important physiological control mechanism in zinc homeostasis and utilization. In this study, DETA/NO, an agent that spontaneously generates NO with a 20-h half life in physiological media, was used to study the release of zinc from MT and the induction of MT in TRL1215 cells (a normal rat liver cell line). Zinc or cadmium was given at levels inducing MT production, followed by DETA/NO (20-200 microM) to produce controlled NO exposure in both cell lines. Although both metals activated MT gene expression, MT-I mRNA and MT protein were further increased when DETA/NO was given after zinc or cadmium treatment. Additionally, NO from DETA/NO clearly displaced MT-bound zinc, as evidenced by G-75 gel-filtration chromatography. The released zinc or cadmium probably then stimulates further MT gene expression. These results suggest that NO may play an important role in regulation of cellular zinc homeostasis by providing a controlled release mechanism for metal ions stored in MT, and NO-mediated release of MT-bound zinc could in turn activate gene expression, such as with the MT gene.