Nitric Oxide Prodrug Research Articles

INDQ/NO, a Bioreductively Activated Nitric Oxide Prodrug

The design, synthesis, and development of INDQ/NO, a novel nitric oxide (NO) prodrug targeted by a bioreductive trigger, are described. INDQ/NO, an indolequinone-diazeniumdiolate is found to be metabolized to produce NO by DT-diaphorase, a bioreductive enzyme that is overexpressed in certain cancers and hypoxic tumors. Cell-based assays revealed that INDQ/NO induces DNA damage and is a potent inhibitor of cancer cell proliferation.

Activity observed in a phase I dose escalation trial of the hypoxia-activated, NO prodrug, RRx001.

241 Background: RRx-001, the first member of a new class of anticancer compounds sourced from the defense industry, is a hypoxia-selective, nitrite reductase-activated prodrug of nitric oxide (NO). The active metabolites selectively enhance oxidative and nitrosative stress in cancer cells through glutathione depletion, ROS formation, and the generation of high local concentrations of NO. The objectives of this Phase 1 dose-escalation trial were to investigate safety, dose-limiting toxicities (DLTs), pharmacodynamics and pharmacokinetics (PK) of RRx-001. Methods: Eligible patients had advanced solid tumor malignancies; ECOG PS 0-2; adequate bone marrow function. In a 3+3 escalation design, RRx-001 was administered intravenously (IV) over 2-3 hours once weekly for 8 weeks to patients with advanced cancer in successive dose-escalating cohorts. PK was collected on days 1 and 50, and tumor blood flow, assessed by CEUS or DCE/DWI-MRI, was performed on days 1 and 8. Tumor response was determined every 8 weeks. Results: To date 12 patients have been dosed across 3 successive cohorts (10, 16.7, and 24.6 mg/m2). Tumor types included pancreas (3), colorectal (5), head and neck (2), melanoma (1), and cholangiocarcinoma (1). No DLTs or treatment-associated SAEs were observed. Transient infusion site pain was reported in 10/12 patients, grade 1 (9/10) or grade 2 (1/10), generally managed by lengthening the infusion duration and using peripheral venous access. No relevant treatment-related changes in laboratory values were observed. Seven patients were evaluable for response, with 1 partial response (parotid tumor). Three patients had stable disease ≥ 2 months (2 pancreas, 1 colorectal with treatment duration 10+ months, exceeding the response to his previous chemo regimen). One pancreas patient with an increase in tumor size due to treatment-related central necrosis was diagnosed with pseudoprogression and remained on study for 4 months. Conclusions: RRx-001, an anticancer agent with a novel structure and mechanism of action, is well tolerated with mild infusion-site pain. PK and PD data will be presented. Escalation is ongoing, but preliminary evidence of anti-tumor activity is promising. Clinical trial information: NCT01359982.

Stereochemical Origins of Chromophore Extension in O2-Substituted Diazeniumdiolates, Prodrugs of Nitric Oxide

Ultraviolet spectral data for nitric oxide prodrugs of structure R2NN(O)=NOR′ tend to fall into two classes: those in which R2N is pyrrolidinyl have λmax near 255 nm when R′ is a simple alkyl substituent, while those in which R2N is not pyrrolidinyl display λmax around 230 nm. To test the hypothesis that this spectral difference might reflect an electronic interaction large enough to affect key bond lengths and the configuration at the R2N nitrogen, we have compared the crystal structures of two compounds in which R′ was β-d-glucopyranosyl tetraacetate and R2N was pyrrolidinyl versus diethylamino. The X-ray studies revealed a pyramidyl R2N nitrogen for the diethylamino derivative while the pyrrolidine derivative’s R2N approached planarity, consistent with substantial electronic interaction between the N(O)=NOR′ chromophore and pyrrolidine’s (but not diethylamine’s) nitrogen atom. Compensatory changes in key bond lengths were also seen. These findings indicate that overlap between the pyrrolidine nitrogen’s p orbital and the N(O)=NOR′ chromophore can be an important determinant of structure and electron distribution in the diazeniumdiolate series. Ultraviolet spectral data for nitric oxide prodrugs of structure R2NN(O)=NOR′ tend to fall into two classes and the wavelength of the ultraviolet absorbance maximum is a convenient and rather sensitive indicator of the tetrahedral versus planar steric arrangement at the R2N3 nitrogen.

Bioreductively-Activated Prodrugs of Nitric Oxide (NO)

Bioreductively-Activated Prodrugs of Nitric Oxide (NO)

Optimization of Cardiovascular Stent against Restenosis: Factorial Design-Based Statistical Analysis of Polymer Coating Conditions

The objective of this study was to optimize the physicodynamic conditions of polymeric system as a coating substrate for drug eluting stents against restenosis. As Nitric Oxide (NO) has multifunctional activities, such as regulating blood flow and pressure, and influencing thrombus formation, a continuous and spatiotemporal delivery of NO loaded in the polymer based nanoparticles could be a viable option to reduce and prevent restenosis. To identify the most suitable carrier for S-Nitrosoglutathione (GSNO), a NO prodrug, stents were coated with various polymers, such as poly (lactic-co-glycolic acid) (PLGA), polyethylene glycol (PEG) and polycaprolactone (PCL), using solvent evaporation technique. Full factorial design was used to evaluate the effects of the formulation variables in polymer-based stent coatings on the GSNO release rate and weight loss rate. The least square regression model was used for data analysis in the optimization process. The polymer-coated stents were further assessed with Differential scanning calorimetry (DSC), Fourier transform infrared spectroscopy analysis (FTIR), Scanning electron microscopy (SEM) images and platelet adhesion studies. Stents coated with PCL matrix displayed more sustained and controlled drug release profiles than those coated with PLGA and PEG. Stents coated with PCL matrix showed the least platelet adhesion rate. Subsequently, stents coated with PCL matrix were subjected to the further optimization processes for improvement of surface morphology and enhancement of the drug release duration. The results of this study demonstrated that PCL matrix containing GSNO is a promising system for stent surface coating against restenosis.

Nitrite anion stimulates ischemic arteriogenesis involving NO metabolism

Nitric oxide (NO) is a potential regulator of ischemic vascular remodeling, and as such therapies augmenting its bioavailability may be useful for the treatment of ischemic tissue diseases. Here we examine the effect of administering the NO prodrug sodium nitrite on arteriogenesis activity during established tissue ischemia. Chronic hindlimb ischemia was induced by permanent unilateral femoral artery and vein ligation. Five days postligation; animals were randomized to control PBS or sodium nitrite (165 μg/kg) therapy twice daily. In situ vascular remodeling was measured longitudinally using SPY angiography and Microfil vascular casting. Delayed sodium nitrite therapy rapidly increased ischemic limb arterial vessel diameter and branching in a NO-dependent manner. SPY imaging angiography over time showed that nitrite therapy enhanced ischemic gracillis collateral vessel formation from the profunda femoris to the saphenous artery. Immunofluorescent staining of smooth muscle cell actin also confirmed that sodium nitrite therapy increased arteriogenesis in a NO-dependent manner. The NO prodrug sodium nitrite significantly increases arteriogenesis and reperfusion of established severe chronic tissue ischemia.

Synthesis and characterization of a multi-arm poly(acrylic acid) star polymer for application in sustained delivery of cisplatin and a nitric oxide prodrug.

Functionalized polymeric nanocarriers have been recognized as drug delivery platforms for delivering therapeutic concentrations of chemotherapies. Of this category, star-shaped multiarm polymers are emerging candidates for targeted delivery of anti-cancer drugs, due to their compact structure, narrow size distribution, large surface area and high water solubility. In this study, we synthesized a multi-arm poly(acrylic acid) star polymer via MADIX/RAFT polymerization and characterized it using NMR and size exclusion chromatography. The poly(acrylic acid) star polymer demonstrated excellent water solubility and extremely low viscosity, making it highly suited for targeted drug delivery. Subsequently, we selected a hydrophilic drug, cisplatin, and a hydrophobic nitric oxide-donating prodrug, O2-(2,4-dinitrophenyl) 1-[4-(2-hydroxy)ethyl]-3-methylpiperazin-1-yl]diazen-1-ium-1,2-diolate, as two model compounds to evaluate the feasibility of using poly(acrylic acid) star polymers for delivery of chemotherapeutics. After synthesizing and characterizing two poly(acrylic acid) star polymer-based nanoconjugates, poly(acrylic acid)-cisplatin (acid-Pt) and poly(acrylic acid)-nitric oxide prodrug (acid-NO), the in vitro drug release kinetics of both acid-Pt and acid-NO were determined at physiological conditions. In summary, we have designed and evaluated a polymeric nanocarrier for sustained-delivery of chemotherapies, either as a single treatment or a combination therapy regimen.

Nitric oxide-releasing nanoparticle combination therapy to overcome drug resistance in platinum-resistant breast cancers.

278 Background: The most successful cancer chemotherapy regimens usually involve the administration of multiple cytotoxic agents simultaneously. Because different chemotherapies may have different mechanisms of action; it is difficult for cancer cells to become resistant to combination therapy. Recent investigations have discovered that nitric oxide (NO) may be involved in the reversal of cisplatin (CDDP) resistance in chemotherapy. Therefore, we sought to develop a combination regimen involving a NO-releasing prodrug (NO1) and cisplatin for the treatment of breast cancer. Methods: The prodrug NO1 was synthesized to be localized in tumor cells and to release nitric oxide over 10 hours. The antiproliferative activity of NO1 was determined in human breast cancer cell line, MDA-MB-468LN. To evaluate the effectiveness of the CDDP/NO combination regimen, platinum-resistant MDA-MB-468LN cells were treated with either cisplatin alone ([CDDP]=0.01, 0.02 or 0.03 mM) or the CDDP/NO combination ([CDDP]=0.01, 0.02 or 0.03 mM; [NO1]=0.02 mM). The cell viability was determined 48 hours post-treatment using a live/dead trypan blue exclusion assay. Results: The toxicity to breast cancer cells was higher than JS-K, a systemic NO agent currently in preclinical development. (NO1: IC50=26µM; JS-K: IC50=42 µM). In addition, NO1 had a long sustained release of nitric oxide, approximately one fold longer than JS-K (NO1: t1/2=6 h; JS-K: t1/2=3 h). The combination treatments resulted in 6.6-, 6.9- and 7.5-fold increase in cell death compared to cisplatin treatment alone at cisplatin concentrations of 0.01, 0.02 and 0.03 mM, respectively. A subsequent study in a murine xenograft of head and neck cancer found NO-1 conjugated to nanoparticles increased survival a mean of 10 weeks compared to controls (p<0.0001). Conclusions: Nitric oxide prodrug NO-1 and nanoparticles of it have high anticancer activity in vivo and they can reverse platinum resistance in vitro. Ongoing studies are examining the safety and efficacy of CDDP/NO combination therapy in mouse xenografts of human breast cancer.

Treatment of blood flow abnormality using mucosal delivery of nitric oxide.

This review focuses on clinical application of intravaginal formulations containing nitric oxide (NO). Poly(D,L-lactic acid-co-glycolic acid)-based microparticles or nanoparticles encapsulated with nitric oxide prodrugs, such as diethylenetriamine diazeniumdiolate and S-nitrosoglutathione, have been developed for the treatment of blood flow abnormality in various diseases including diabetes. Advanced nanotechnology allows for production of novel formulations with the capability of long-term protection, preserving the integrity of the NO donors, and delivering NO in a controlled and sustained release manner at the mucosal sites. The gene expressions of MAPK and PKC in the vaginal mucosa upon exposure to microparticles were evaluated for the mechanistic study involved with blood flow changes. The blood flow changes and protein expression of the vaginal mucosa upon exposure to intravaginal formulations containing NO donors supported that NO therapy would be suitable for the treatment of blood flow abnormality. This review subsequently would help to establish a scientific foundation for clinical trials of intravaginal NO delivery systems in humans.

TAp73 Induction by Nitric Oxide: REGULATION BY CHECKPOINT KINASE 1 (CHK1) AND PROTECTION AGAINST APOPTOSIS

Nitric oxide (NO) is a potent activator of the p53 tumor suppressor protein, thereby inducing cell cycle arrest and apoptosis. However, little is known about the regulation of the two other p53-family members, p63 and p73, by nitrogen oxides. We report here an up-regulation of p73 by NO in p53-null K-562 leukemia cells. Chemical NO prodrugs or macrophage iNOS activity induced an accumulation of the TAp73α isoform in these cells, whereas macrophages from iNOS(-/-) mice did not. NO also up-regulated TAp73 mRNA expression, suggesting a transcriptional regulation. The checkpoint kinases Chk1 and Chk2 can regulate TAp73 induction after DNA damage. We show that these kinases were rapidly phosphorylated upon NO treatment. Genetic silencing or pharmacological inhibition of Chk1 impaired NO-mediated accumulation of TAp73α. Because NO is known to block DNA synthesis through ribonucleotide reductase inhibition, the up-regulation of TAp73α might be caused by DNA damage induced by an arrest of DNA replication forks. In support of this hypothesis, DNA replication inhibitors such as hydroxyurea and aphidicolin similarly enhanced TAp73α expression and Chk1 phosphorylation. Moreover, inhibition of Chk1 also prevented TAp73α accumulation in response to replication inhibitors. The knockdown of TAp73 with siRNA sensitized K-562 cells to apoptosis induced by a nitrosative (NO) or oxidative (H(2)O(2)) injury. Therefore, TAp73α has an unusual cytoprotective role in K-562 cells, contrasting with its pro-apoptotic functions in many other cell models. In conclusion, NO up-regulates several p53 family members displaying pro- and anti-apoptotic effects, suggesting a complex network of interactions and cross-regulations between NO production and p53-related proteins.

JS-K, a nitric oxide-releasing prodrug, induces breast cancer cell death while sparing normal mammary epithelial cells

Targeted therapy with reduced side effects is a major goal in cancer research. We investigated the effects of JS-K, a nitric oxide (NO) prodrug designed to release high levels of NO when suitably activated, on human breast cancer cell lines, on non-transformed human MCF-10A mammary cells, and on normal human mammary epithelial cells (HMECs). Cell viability assay, flow cytometry, electron microscopy, and Western blot analysis were used to study the effects of JS-K on breast cancer and on mammary epithelial cells. After a 3-day incubation, the IC50s of JS-K against the breast cancer cells ranged from 0.8 to 3 µM. However, JS-K decreased the viability of the MCF-10A cells by only 20% at 10-µM concentration, and HMECs were unaffected by 10 µM JS-K. Flow cytometry indicated that JS-K increased the percentages of breast cancer cells under-going apoptosis. Interestingly, flow cytometry indicated that JS-K increased acidic vesicle organelle formation in breast cancer cells, suggesting that JS-K induced autophagy in breast cancer cells. Electron microscopy confirmed that JS-K-treated breast cancer cells underwent autophagic cell death. Western blot analysis showed that JS-K induced the expression of microtubule light chain 3-II, another autophagy marker, in breast cancer cells. However, JS-K did not induce apoptosis or autophagy in normal human mammary epithelial cells. These data indicate that JS-K selectively induces programmed cell death in breast cancer cells while sparing normal mammary epithelial cells under the same conditions. The selective anti-tumor activity of JS-K warrants its further investigation in breast tumors.

The nitric oxide prodrug JS-K is effective against non-small-cell lung cancer cells in vitro and in vivo: involvement of reactive oxygen species.

Non-small-cell lung cancer is among the most common and deadly forms of human malignancies. Early detection is unusual, and there are no curative therapies in most cases. Diazeniumdiolate-based nitric oxide (NO)-releasing prodrugs are a growing class of promising NO-based therapeutics. Here, we show that O(2)-(2,4-dinitrophenyl)-1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate (JS-K) is a potent cytotoxic agent against a subset of human non-small-cell lung cancer cell lines both in vitro and as xenografts in mice. JS-K treatment led to 75% reduction in the growth of H1703 lung adenocarcinoma cells in vivo. Differences in sensitivity to JS-K in different lung cancer cell lines seem to be related to their endogenous levels of reactive oxygen species (ROS)/reactive nitrogen species (RNS). Other related factors, levels of peroxiredoxin 1 (PRX1) and 8-oxo-deoxyguanosine glycosylase (OGG1), also correlated with drug sensitivity. Treatment of the lung adenocarcinoma cells with JS-K resulted in oxidative/nitrosative stress in cells with high basal levels of ROS/RNS, which, combined with the arylating properties of the compound, was reflected in glutathione depletion and alteration in cellular redox potential, mitochondrial membrane permeabilization, and cytochrome c release. Inactivation of manganese superoxide dismutase by nitration was associated with increased superoxide and significant DNA damage. Apoptosis followed these events. Taken together, the data suggest that diazeniumdiolate-based NO-releasing prodrugs may have application as a personalized therapy for lung cancers characterized by high levels of ROS/RNS. PRX1 and OGG1 proteins, which can be easily measured, could function as biomarkers for identifying tumors sensitive to the therapy.

Inorganic nitrite and chronic tissue ischaemia: a novel therapeutic modality for peripheral vascular diseases

Ischaemic tissue damage represents the ultimate form of tissue pathophysiology due to cardiovascular disease, which is the leading cause of morbidity and mortality across the globe. A significant amount of basic research and clinical investigation has been focused on identifying cellular and molecular pathways to alleviate tissue damage and dysfunction due to ischaemia and subsequent reperfusion. Over many years, the gaseous molecule nitric oxide (NO) has emerged as an important regulator of cardiovascular health as well as protector against tissue ischaemia and reperfusion injury. However, clinical translation of NO therapy for these pathophysiological conditions has not been realized for various reasons. Work from our laboratory and several others suggests that a new form of NO-associated therapy may be possible through the use of nitrite anion (sodium nitrite), a prodrug which can be reduced to NO in ischaemic tissues. In this manner, nitrite anion serves as a highly selective NO donor in ischaemic tissues without substantially altering otherwise normal tissue. This surprising and novel discovery has reinvigorated hopes for effectively restoring NO bioavailability in vulnerable tissues while continuing to reveal the complexity of NO biology and metabolism within the cardiovascular system. However, some concerns may exist regarding the effect of nitrite on carcinogenesis. This review highlights the emergence of nitrite anion as a selective NO prodrug for ischaemic tissue disorders and discusses the potential therapeutic utility of this agent for peripheral vascular disease.

The Nitric Oxide Prodrug V-PROLI/NO Inhibits Cellular Uptake of Proline

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.

Abstract 2651: Cellular uptake and stability studies of the nitric oxide-generating pro-drug JS-K

Abstract The nitric oxide (NO) prodrug O2-(2,4-dinitrophenyl)1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate, or JS-K, has potent antineoplastic activity in vitro and in vivo. Potential targets of JS-K include acute myeloid leukemia, multiple myeloma, hepatocellular carcinoma, prostate cancer and non-small cell lung cancer. JS-K is sparingly soluble in aqueous media. Preliminary pharmacokinetic studies have shown that JS-K has a short half-life in vivo. We have developed a nanoscale micelle formulation for JS-K using Pluronic® P123 polymers. Pluronics® are polyethylene oxide / polypropylene oxide copolymers that are non-toxic and non-irritant. This formulation prolongs the half-life of JS-K in biologic media in vitro. The purpose of this study was to determine the effect of the P123 Pluronic® formulation on the cellular distribution of JS-K. We compared micelle-solubilized JS-K to free JS-K using the HL-60 human myeloid leukemia cell line. JS-K was added at a concentration of 50 μM to HL-60 cells incubated at 37°C in phosphate buffered saline (PBS), RPMI-1640 with 10% fetal bovine serum (FBS), or PBS with 10% FBS. JS-K recovery from the nuclei and cytoplasm was measured using High Pressure Liquid Chromatography (HPLC). In all experiments, cellular uptake of P123-formulated JS-K was greater than that of free JS-K. The highest levels of recovery of P123-formulated JS-K were from nuclei. Indeed, recovery of JS-K from the cytoplasm was minimal. When cells were incubated in PBS, the percent recovery of free JS-K from nuclei at 15, 30, 60 and 120 minutes was 5.03+2.41; 5.00+2.77, 10.83+5.32 and 0.58+0.22, respectively. Under the same conditions and at the same time points, the percent recovery of P123-formulated JS-K was 9.05+3.5, 20.00+7.07, 21.15+3.14 and 27.73+4.30, respectively (average of two different experiments; P<0.05). When cells were incubated in PBS/10%FBS, the percent recovery of free JS-K from nuclei at 30, 60, 120 and 240 minutes was 3.55+1.92, 3.15+ 1.37, 2.13+1.08, and 1.83+1.55, respectively. Under the same conditions and at the same time points, the percent recovery of P123-formulated JS-K was 20.74+6.57, 12.70+3.41, 16.03+3.59, and 13.22+1.55, respectively (average of three different experiments; P<0.05). When cells were incubated in RPMI/10%FBS, the percent recovery of free JS-K from nuclei at 15, 30, 60, and 120 minutes was 0.38+0.15, 0.45+0.20, 0.43+0.22, and 0.2+0.09, respectively. Under the same conditions and at the same time points, the percent recovery of P123-formulated JS-K was 2.93+1.43, 4.68+1.40, 9.30+1.37 and 11.90+3.81, respectively (average of two different experiments; P<0.05). We conclude that a Pluronic® P123 formulation increases the stability of JS-K in biologic media and facilitates penetration of the drug into the nucleus. This clinically useful formulation could therefore enhance the cytotoxic effects of JS-K and increase its therapeutic efficacy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2651.

JS-K, a Nitric Oxide Prodrug, Has Enhanced Cytotoxicity in Colon Cancer Cells with Knockdown of Thioredoxin Reductase 1

BackgroundThe selenoenzyme thioredoxin reductase 1 has a complex role relating to cell growth. It is induced as a component of the cellular response to potentially mutagenic oxidants, but also appears to provide growth advantages to transformed cells by inhibiting apoptosis. In addition, selenocysteine-deficient or alkylated forms of thioredoxin reductase 1 have also demonstrated oxidative, pro-apoptotic activity. Therefore, a greater understanding of the role of thioredoxin reductase in redox initiated apoptotic processes is warranted.MethodologyThe role of thioredoxin reductase 1 in RKO cells was evaluated by attenuating endogenous thioredoxin reductase 1 expression with siRNA and then either inducing a selenium-deficient thioredoxin reductase or treatment with distinct redox challenges including, hydrogen peroxide, an oxidized lipid, 4-hydroxy-2-nonenol, and a nitric oxide donating prodrug. Thioredoxin redox status, cellular viability, and effector caspase activity were measured.Conclusions/SignificanceIn cells with attenuated endogenous thioredoxin reductase 1, a stably integrated selenocysteine-deficient form of the enzyme was induced but did not alter either the thioredoxin redox status or the cellular growth kinetics. The oxidized lipid and the nitric oxide donor demonstrated enhanced cytotoxicity when thioredoxin reductase 1 was knocked-down; however, the effect was more pronounced with the nitric oxide prodrug. These results are consistent with the hypothesis that attenuation of the thioredoxin-system can promote apoptosis in a nitric oxide-dependent manner.

Nitrogen-bound diazeniumdiolated amidines.

In contrast to amidines bearing ionizable alpha-CH bonds, which react with nitric oxide (NO) to add diazeniumdiolate groups at their alpha-carbons, benzamidine forms an N-bound diazeniumdiolate that can be further derivatized at the other amidine nitrogen and/or the terminal oxygen to form caged NO compounds as potential NO prodrugs.

Stabilization of the nitric oxide (NO) prodrugs and anticancer leads, PABA/NO and Double JS-K, through incorporation into PEG-protected nanoparticles.

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.

Glycosylated PROLI/NO derivatives as nitric oxide prodrugs.

GlcNAc-PROLI/NO prodrugs that are activated by N-acetylglucosaminidase to release nitric oxide (NO) are described. A classical acid-amine coupling is used to bifunctionalize these PROLI/NO prodrugs, which on activation generate up to 4 mol of NO, a peptide residue, and an N-acetylglucosamine residue. Many of the prodrugs synthesized are efficient sources of intracellular NO.

The Nitric Oxide Prodrug JS-K and Its Structural Analogues as Cancer Therapeutic Agents

Nitric oxide (NO) prodrugs of the diazeniumdiolate class are routinely used as reliable sources of nitric oxide in chemical and biological laboratory settings. O(2)-(2,4-dinitrophenyl) diazeniumdiolates, which are derivatized forms of ionic diazeniumdiolates, have been found to show potent anti-proliferative activity in a variety of cancer cells, presumably through the effects of NO. One important member of this class of diazeniumdiolates, O(2)-(2,4-dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate (JS-K), has shown promise as a novel cancer therapeutic agent in a number of animal models. This review describes the developments in chemical and biochemical characterization and structure-activity relationship of JS-K and its analogues. In addition, some molecular mechanistic insights into the observed anti-proliferative activity of JS-K are discussed. Finally, a structural motif is presented for O(2)-(aryl) diazeniumdiolate nitric oxide prodrugs that show potency comparable with that of JS-K.