Dual Nitric Oxide Research Articles

Ru(II) based dual nitric oxide donors: electrochemical and photochemical reactivities and vasorelaxant effect with no cytotoxicity.

The synthesized complexes, cis-[Ru(NO)(NO2)(phen)2](PF6)2 (NONO2P) and cis-[Ru(NO)(NO2)(bpy)2](PF6)2 (NONO2B), were characterized by using elemental analysis, voltammetry and electronic and vibrational spectroscopy. Under electrochemical and photochemical stimulation in an aqueous medium, there are indications of the formation of complexes, which suggests that the nitro and nitrosyl groups are converted into nitric oxide. Both compounds do not show cytotoxic activity against human umbilical vein endothelial cells (HUVECs). The cis-[Ru(NO)(NO2)(phen)2](PF6)2 complex presented vasorelaxation activity in superior mesenteric arteries from Wistar rats: the biphasic concentration-response curve indicates two sites of action. In the presence of NO scavengers, we observed an impaired relaxing effect induced by NONO2P, suggesting that the vasorelaxant effect is due to NO production from this compound.

Abstract 4841: Molecular targets of NOSH-naproxen (AVT-219), a dual nitric oxide and hydrogen sulfide-releasing hybrid, in a xenograft mouse model of colon cancer

Abstract Introduction: Long-term use of non-steroidal anti-inflammatory drugs (NSAIDs) may prevent colon cancer, but can cause serious gastrointestinal (GI), cardiovascular (CV), and renal side effects. Naproxen has a relatively favorable CV profile, but is notorious for its GI toxicity. To improve its safety profile, we developed NOSH-naproxen, where we linked nitric oxide (NO) and hydrogen sulfide (H2S) releasing moieties to the native molecule, the rational being the observations that NO and H2S may modulate some components of the local mucosal defense systems which should lead to reduced GI toxicity. Indeed, we recently reported that NOSH-naproxen displayed enhanced GI safety, with potent anti-inflammatory, analgesic, anti-pyretic, and anti-platelet properties. Here we describe the effects of NOSH-naproxen on the growth properties of several human colon cancer cell lines, and on its molecular targets in a xenograft mouse model of colon cancer. Methods: NOSH-naproxen was synthesized by us with 1H-NMR verification. Colon cancer cell lines: HT-29, HCT 15, and SW480; Cell growth: MTT. Xenografts: Male athymic nude (NU/NU) mice (N = 15) were implanted s.c. in the right flank with SW480 cells, when the tumors reached an average sizes of ∼100 mm3, the mice were randomly divided into 3 groups and gavaged daily with either vehicle (0.5% CMC) or equimolar concentrations of NOSH-naproxen (100 mg/kg) or naproxen (45 mg/kg). Tumor volume was measured every three days; after 30 days the mice were sacrificed, tumors collected, weighed, photographed and stored in formalin for IHC studies. Results: NOSH-naproxen inhibited the growth of HT-29, HCT 15, and SW480 cells with IC50 values of 90 ± 10, 100 ± 8, and 98 ± 7 nM at 24 hr, respectively. The corresponding IC50s for naproxen were 2800 ± 165, 2950 ± 215, and 3110 ± 185 nM. This represents an enhanced potency of ∼30,000-fold in the 3 cell lines at 24 h. In xenografts, all the mice treated with naproxen died within 2 weeks from what appeared to be GI bleeding. However, the NOSH-naproxen treated mice did not show any overt signs of toxicity. At sacrifice, NOSH-naproxen-treated mice had a mean reduction in tumor volume of 82%, and a mean reduction in tumor mass of 55%. NOSH-naproxen inhibited growth of these cancer cell xenografts as a result of reduced proliferation (decreased PCNA expression), and induction of apoptosis (increased number of TUNEL positive cells). NF-êB, activated in untreated xenografts was significantly inhibited by NOSH-naproxen. Other molecular targets affected by NOSH-naproxen were: induction of iNOS, significant reduction in FoxM1, and increases in p53. Conclusions: NOSH-naproxen is more potent and efficacious than naproxen and appears to be significantly safer. It targets parameters important in determining cellular kinetics, inflammation, and signaling pathways important in the carcinogenic process. Citation Format: Pascale L. Duvalsaint, Mitali Chattopadhyay, Ravinder Kodela, Khosrow Kashfi. Molecular targets of NOSH-naproxen (AVT-219), a dual nitric oxide and hydrogen sulfide-releasing hybrid, in a xenograft mouse model of colon cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4841.

Synthesis and anti-cancer potential of the positional isomers of NOSH-aspirin (NBS-1120) a dual nitric oxide and hydrogen sulfide releasing hybrid.

We recently reported the synthesis of NOSH-aspirin, a novel hybrid compound capable of releasing both nitric oxide (NO) and hydrogen sulfide (H2S). In NOSH-aspirin, the two moieties that release NO and H2S are covalently linked at the 1, 2 positions of acetyl salicylic acid, i.e., ortho-NOSH-aspirin. Here we report on the synthesis of meta- and para-NOSH-aspirins. We also made a head-to-head evaluation of the effects of these three positional isomers of NOSH-aspirin on colon cancer cell kinetics and induction of reactive oxygen species, which in recent years has emerged as a key event in causing cancer cell regression. Electron donating/withdrawing groups incorporated about the benzoate moiety significantly affected the potency of these compounds with respect to colon cancer cell growth inhibition.

NOSH-aspirin (NBS-1120), a dual nitric oxide and hydrogen sulfide-releasing hybrid, reduces inflammatory pain.

The development of nitric oxide (NO)- and hydrogen sulfide (H2S)-releasing nonsteroidal anti-inflammatory drugs (NSAIDs) has generated more potent anti-inflammatory drugs with increased safety profiles. A new hybrid molecule incorporating both NO and H2S donors into aspirin (NOSH-aspirin) was recently developed. In the present study, the antinociceptive activity of this novel molecule was compared with aspirin in different models of inflammatory pain. It was found that NOSH-aspirin inhibits acetic acid-induced writhing response and carrageenan (Cg)-induced inflammatory hyperalgesia in a dose-dependent (5–150 μmol/kg, v.o.) manner, which was superior to the effect of the same doses of aspirin. NOSH-aspirin’s antinociceptive effect was also greater and longer compared to aspirin upon complete Freund’s adjuvant (CFA)-induced inflammatory hyperalgesia. Mechanistically, NOSH-aspirin, but not aspirin, was able to reduce the production/release of interleukin-1 beta (IL-1β) during Cg-induced paw inflammation. Furthermore, NOSH-aspirin, but not aspirin, reduced prostaglandin E2-induced hyperalgesia, which was prevented by treatment with a ATP-sensitive potassium channel (KATP) blocker (glibenclamide; glib.). Noteworthy, the antinociceptive effect of NOSH-aspirin was not associated with motor impairment. The present results indicate that NOSH-aspirin seems to present greater potency than aspirin to reduce inflammatory pain in several models. The enhanced effects of NOSH-aspirin seems to be due to its ability to reduce the production of pronociceptive cytokines such as IL-1 β and directly block hyperalgesia caused by a directly acting hyperalgesic mediator in a mechanism dependent on modulation of KATP channels. In conclusion, we would like to suggest that NOSH-aspirin represents a prototype of a new class of analgesic drugs with more potent effects than the traditional NSAID, aspirin.

P09 Therapeutic potential of NOSH-aspirin, a dual nitric oxide- and hydrogen sulfide-donating hybrid in colon cancer

Colorectal cancer is one of the major causes of cancer deaths worldwide. In patients with advanced disease, the efficacy of currently available treatments is limited. Nonsteroidal anti-inflammatory drugs (NSAIDs) in general and aspirin (ASA) in particular are prototypical chemopreventive agents. However, the use of NSAIDs is limited by their significant toxicity (gastrointestinal, renal, and cardiovascular). In our search for a “better NSAID”, we have developed NOSH-aspirin for the treatment of colon cancer. The notion that a modified NSAID maybe used to treat cancer is radical. NOSH-aspirin (NBS-1120) is a hybrid molecule that releases nitric oxide (NO) and hydrogen sulfide (H 2 S), two gasotransmitters of physiological relevance. NOSH-aspirin is the first NSAID-based agent that is active against colon cancer in the nano-molar range, is devoid of gastrointestinal and cellular toxicity, preferentially affects cancer cells compared to normal cells, and has a pleotropic mechanism of action. In vitro, NOSH-aspirin inhibited the growth of HT-29, HCT 15, and SW480 human colon cancer cell lines with IC 50 values of 48 ± 3, 50 ± 5, and 60 ± 4 nM at 24 h, respectively. The corresponding IC 50 for aspirin in all 3 cell lines was > 5,000,000 nM. This represents an enhanced potency of > 80,000-fold in the 3 cell lines at 24 h. Using HT-29 colon cancer cells, NOSH-aspirin had IC 50 s for cell growth inhibition of 20 nM at 48 h and 8 nM at 72 h. The corresponding values for aspirin were, 2,500,000 nM and 2,000,000 nM, respectively. This represents potency enhancements of ∼125–000-fold and ∼250,000-fold. Although highly potent, NOSH-aspirin was without any cytotoxicity as determined by lactate dehydrogenase release. Inhibiting NO and H 2 S release from NOSH-aspirin, increased the IC 50 for cell growth inhibition to ∼800,000 nM at 24 h. NOSH-aspirin had a synergistic effect with 5-flurouracil (5-FU), in HT-29 cells its IC 50 for cell growth inhibition at 24 h went down from 50 nM to 4 nM in presence of sub-toxic levels of 5-FU. Mechanistically, NOSH-aspirin dose-dependently induced apoptosis; inhibited proliferation; caused a G 0 /G 1 cell cycle block; increased reactive oxygen species (ROS); inhibited NF-#kappa B, regulating it by reciprocal S-nitrosylation and S-sulfhydration; and inhibited cytokine-induced NOS2 expression. In vivo, using a xenograft model of colon cancer, NOSH-aspirin dose-dependently reduced the growth of established tumors, reduced proliferation, induced apoptosis, reduced NF-#kappa B, and induced ROS. NOSH-aspirin also exhibited potent anti-inflammatory properties, inhibiting both cyclo-oxygense (COX)-1 and COX-2, thus reducing prostaglandin (PG) E 2 levels in a carrageenan-induced rat paw edema model. In conclusion, NOSH-aspirin is a promising candidate against cancer modulating key parameters that regulate cellular mass.

A diazen-1-ium-1,2-diolate analog of 7-azabenzobicyclo[2.2.1]heptane: Synthesis, nitric oxide and nitroxyl release, in vitro hemodynamic, and anti-hypertensive studies

1-(7-Azabenzobicyclo[2.2.1]heptane)diazen-1-ium-1,2-diolate (16) was designed with the expectation that it would act as a dual nitric oxide (NO) and nitroxyl (HNO) donor that is not carcinogenic or genotoxic. Compound 16, with a suitable half-life (17.8min) in PBS at pH 7, released NO (19%) and HNO (22%) during a 2h incubation in PBS at pH 7. In addition, compound 16 exhibited a significant in vitro positive inotropic effect, increased the rates of contraction and relaxation, and increased coronary flow rate, but did not induce a chronotropic effect. Furthermore, compound 16 (13.7mgkg−1, po dose) provided a significant reduction in the blood pressure of mice up to 3h post-drug administration. All these data suggest that compound 16 constitutes an attractive ‘lead-compound’ that could have potential applications to treat cardiovascular disease(s) such as congestive heart failure.

Abstract B34: NOSH-aspirin (NBS-1120) a dual nitric oxide and hydrogen sulfide releasing hybrid for treatment of cancer

Abstract Introduction: Regular uses of non-steroidal anti-inflammatory drugs (NSAIDs) in general and aspirin in particular have been associated with reductions in a number of cancers including, colon, breast, pancreas and others. However, NSAIDs have significant side effects, which precludes their wide spread use. In our search for a “better NSAID”, we have synthesized a series of nitric oxide-, and hydrogen sulfide-releasing hybrid compounds that have IC50s for cell growth inhibition in the low nano-molar range. We have termed these NOSH compounds. Our lead agent, NOSH-aspirin is in the order of 100,000 to 250,000-fold more potent than aspirin in inhibiting colon cancer cell growth over a 24-72h-time period. NOSH-aspirin although highly potent, is essentially devoid of any cyto-toxicity as judged by lactate dehydrogenase (LDH) release. Here we report our results on the effects of NOSH-aspirin treatment on established tumors in a xenograft model of human colon, breast (both ER- and ER+), and pancreatic cancer cells. Methods: Compound: NOSH-aspirin was synthesized and purified by us with 1H-NMR verification. Cell lines: Colon, HT-29, HCT 15, and SW 480; breast, MCF-7 (ER+), MDA-MB-231 (ER-, PR-, which does not over express Her2), and HMEpC, normal human mammary epithelial; pancreas, MIA PaCa2 and BxPC3. Xenografts: Male athymic nude (NU/NU) mice (N=10 per cell line) were implanted s.c. in the right flank with HT-29, MCF-7, MDA-MB-231, MIA PaCa2 cells (2 x 106) suspended in 50% Matrigel, after 10 days the animals from each implanted cell line were randomly divided into 2 groups (N = 5/group) and gavaged daily with NOSH-aspirin (100 mg/kg body weight) or vehicle. Tumor volume and animal weight were recorded every 3 days. After 3 weeks of treatment, mice were sacrificed, tumors excised, weighed, and fixed in 10% buffered formalin for immunohistochemical studies. Results: NOSH-aspirin's IC50 in nM at 24h for cell growth inhibition were HT-29 (50 ± 2), HCT 15 (52 ± 3), SW480 (55 ± 5), MCF-7 (250 ± 10), MDA-MB-231 (95 ± 7), HMEpC (20,000 ± 3,000), MIAPaCa2 (52 ± 4), and BxPC3 (62 ± 5). Aspirin's IC50 in all cell lines was greater than 5,000,000 nM at 24h. In all 4 xenografts, NOSH-aspirin had no effect on the weight of the mice and there were no overt signs of toxicity. Tumor volume was reduced as a function of treatment time in all 4 xenografts. Interestingly, the growth rate of ER(−) xenografts was significantly greater than that of ER(+) ones and NOSH-aspirin had a greater effect on the ER(−) vs ER(+) xenografts. Tumor mass for each group were, colon: 2.8 ± 0.51g in untreated and 0.32 ± 0.067g in treated (89% reduction, P=0.005); breast, ER(+): 0.46 ± 0.056g untreated and 0.21 ± 0.035g treated, (55% reduction, P=0.022), ER(−): 1.2 ± 0.33g untreated and 0.11 ± 0.058g treated (91% reduction, P=0.006); pancreas: 2.47 ± 0.24g untreated and 0.62 ± 0.25g treated, (75% reduction, P=0.0031). NOSH-aspirin inhibited growth of these cancer cell xenografts as a result of reduced proliferation (decreased PCNA expression), and induction apoptosis (increased number of TUNEL positive cells). NF-κB, activated in untreated xenografts was significantly inhibited by NOSH-aspirin. Conclusions: NOSH-aspirin preferentially affects cancer cells and has potent anti-cancer properties. It targets parameters important in determining cellular mass, and inflammation. Citation Format: Mitali Chattopadhyay, Ravinder Kodela, Diana H. Lee, Thuy-Tien C. Le, Khosrow Kashfi. NOSH-aspirin (NBS-1120), a dual nitric oxide and hydrogen sulfide-releasing hybrid for treatment of cancer. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr B34.

Asymmetric dimethylarginine: A molecule responsible for the coexistence of insulin resistance and atherosclerosis via dual nitric oxide synthase inhibition

Asymmetric dimethylarginine (ADMA) has been recently identified as the major endogenous inhibitor of soluble nitric oxide synthase. Its systemic accumulation was observed in conjunction with atherosclerosis and several cardiovascular and metabolic diseases. Here, we propose that ADMA causes insulin resistance by the inhibition of the neuronal isoform of nitric oxide synthase, while the simultaneously observed atherosclerosis is the consequence of endothelial nitric oxide synthase (NOS) inhibition. Our hypothesis rests on animal models in which experimental insulin resistance was induced by intraportal administration of non-selective and selective neuronal nitric oxide synthase inhibitors, N-methyl-L-arginine (L-NMMA) or 7-nitroindazole. In these models, loss of hepatic nitric oxide productions is presumed to hinder a very potent insulin sensitizing mechanism referred to as meal induced sensitization that is anatomically linked to the nitrergic fibers of the anterior hepatic plexus. Cause and effect relationship between ADMA and insulin resistance has been proposed previously by others however the nature of this relationship has not been elucidated in detail. In our hypothesis, we suggest that ADMA by inhibiting both the neuronal and the endothelial forms of NOS, results both in insulin resistance and in accelerated atherosclerosis, therefore ADMA is the molecule responsible for the coexistence of these two conditions. We also suggest animal models and human studies to test our hypothesis, the results of which may offer novel approaches in the prevention of insulin resistance and atherosclerosis.