NO-aspirin: mechanism of action and gastrointestinal safety

doi:10.1016/S1590-8658(03)00047-1

Digestive and Liver Disease

Volume 35, Supplement 2, May 2003, Pages S9-S19

https://doi.org/10.1016/S1590-8658(03)00047-1 Get rights and content

Abstract

Nitric oxide-releasing aspirins are new chemical entities obtained by adding a nitric oxide-releasing moiety to aspirin. NCX-4016 is the prototype of this family of molecules.

NCX-4016 consists of the parent molecule (aspirin) linked to a ‘spacer’ via an ester linkage, which is in turn connected to a nitric oxide-releasing moiety. Both aspirin and nitric oxide moieties of NCX-4016 contribute to its effectiveness, the latter occurring via both cyclic guanosyl monophosphate-dependent and -independent mechanisms

In vitro studies have shown that NCX-4016 inhibits platelet aggregation induced by aspirin-sensitive (arachidonic acid) and aspirin-insensitive (thrombin) agonist. In contrast to aspirin, NCX-4016 exerts a multilevel regulation of inflammatory target, including caspase-1 and NF-κB. This broad spectrum of activities translates to an increased potency of this drug in modulating cardiovascular inflammation.

Human studies have shown, that while nitric oxide-aspirin maintains its anti-thrombotic activity, it spares the gastrointestinal tract. Indeed, a 7-day course of NCX-4016 results in 90% reduction of gastric damage caused by equimolar doses of aspirin.

Further studies are ongoing to define whether this superior anti-inflammatory and anti-thrombotic profile translates in clinical benefits in patients with cardiovascular diseases.

Section snippets

Aspirin: the same mechanism for activity and toxicity

Aspirin exerts its effect primarily by interfering with the biosynthesis of cyclic prostanoids, i.e., thromboxane A₂ (TXA₂), prostacyclin, and other prostaglandins [1]. These prostanoids are generated by the enzymatically catalyzed oxidation of arachidonic acid, which is itself derived from membrane phospholipids (Fig. 1). Arachidonic acid is metabolized by the enzyme prostaglandin (PG) H-synthase, which, through its cyclooxygenase (COX) and peroxidase activities, results in the production of

NO-aspirins

An approach that has been taken to develop a drug that inhibits COX-1 activity that spares the gastrointestinal tract is the coupling of aspirin to a nitric oxide (NO)-releasing moiety (Fig. 2). NO exerts protective effects on the stomach [14] and mediates many components of gastrointestinal mucosal defence (Table 1), thus the addition of the NO-releasing moiety to aspirin will results in a new chemical entity that maintains and possibly expands the pharmacological properties of parent drug,

NCX-4016

NCX-4016 is the prototype of NO-aspirins. The structure of NCX-4016, shown in Fig. 2, consists of the parent molecule (aspirin) linked to a ‘spacer’ via an ester linkage, which is in turn connected to an NO-releasing moiety [22], [23], [24]. Both the aspirin and the NO moieties of NCX-4016 contribute to its effectiveness, the latter occurring via both cyclic guanosyl monophosphate (cGMP)-dependent and -independent mechanisms (Table 2). In contrast to conventional NO donors, NO-aspirin is stable

Gastrointestinal safety and anti-platelet activity of NCX-4016 in humans: the proof of concept

In terms of gastrointestinal safety, NCX-4016 can be clearly distinguished from aspirin. This compound does not produce gastric or intestinal damage in laboratory animals as is seen with aspirin [21], [22], [23]. Preclinical studies have extensively demonstrated that NCX-4016 is considerably well tolerated, even at the dose of 250 mg/kg, despite it completely suppress COX-1 activity in the gastric mucosa [24]. Although several mechanisms have been postulated to explain the gastro-sparing

Conclusion

Human studies have confirmed that NCX-4016 imparts its activity by inhibiting COX-dependent and COX-independent, NO-sensitive, targets. These studies have proven that addition of a NO-releasing moiety to aspirin results in a new chemical entity that exerts a multilevel regulation of the coagulation network. Despite its potent anti-thrombotic activity NO-aspirin spares the gastric mucosa, and is significantly better tolerated than aspirin. Further studies are ongoing to define whether this

Conflict of interest statement

Research described in this paper was partially supported by a grant from Nicox to S. Fiorucci. P. Del Soldato is Scientific Director of Nicox.

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2023, Nitric Oxide in Health and Disease: Therapeutic Applications in Cancer and Inflammatory Disorders
In the last three decades, several experimental evidences pointed out the involvement of chronic inflammation in promoting the development of cancer, supporting all stages of tumorigenesis, tumor growth, and spread. In addition, the importance of the tumor microenvironment, an indispensable participant for tumor establishment and growth, turned out by highlighting the important role that stromal and inflammatory cells have in orchestrating the promotion of tumor cells proliferation, survival, and migration. The relationship between inflammation and cancer, the underlying mechanisms, the cells involved, as well as the corresponding crosstalks are, herein, briefly reviewed.
In particular, the effects mediated by cyclooxygenase-2 (COX-2) and corresponding product prostaglandin (PG) E2 on tumor growth and tumor immune evasion are discussed along with corresponding molecular mechanisms.
Immunotherapy has become a milestone in cancer treatment, strikingly improving life expectancy of patients with a broad variety of malignancies while dramatically reducing the side effects of the therapy. The progresses obtained with immunotherapy (in particular with Programmed cell deth protein 1 (PD-1)/programmed cell death ligand 1 (PD-L1) inhibitors) in cancer treatment are briefly reviewed along with the underlying mechanisms. The effects exerted by the COX-2/PGE₂ axis on PD-1/PD-L1 expression, and more in general on the immune system, are discussed.
The clinical investigations on cancer prevention of nonsteroidal antiinflammatory drugs and corresponding results are herein commented along with the side effects that hampered the clinical trials. The outcome obtained with NO-releasing drugs in overcoming these side effects is commented in comparison with the corresponding results obtained with the NO-donor COX-2 inhibitors that we have developed. Moreover, the effect that NO release may have on tumor initiation, growth, and metastasis as well as in controlling immunosuppression is briefly reviewed.
Taking into account the issues discussed above, we think that the NO-donor COX-2 inhibitor (12e) (VA694) and corresponding backups can be good candidates for further studies as therapeutic agents for cancer treatment, either in combination with immune checkpoint (PD-1/PD-L1) inhibitors or with standard chemotherapy.
Conformational analysis for infrared spectroscopy and theoretical calculations of some 2-bromo-2-propyl 2-aryl-acetates, ibuprofen and naproxen analogs
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Recently, considerable attention has been directed towards the development of reversible derivatives, such as prodrugs and mutual prodrugs, via chemical modifications. Chemical modification or derivatization temporarily masks the acidic group of NSAIDs and appears to be a promising and fruitful method for reducing or preventing the GIT toxicity via the local insult mechanism. [6–9] Most prodrugs from NSAIDs have been synthesized by derivatization of the free carboxylic group (-COOH) of the NSAID. [8,9]
Conformational analysis of new para–substituted 2-bromo-2-propyl 2-aryl-acetates (Y = H, OMe, Cl, and NO₂) (R1), ibuprofen (R2), and naproxen (R3) analogs using infrared (IR) spectroscopy and theoretical calculations was performed to determine the preferential conformers of these compounds in solvents with increasing polarity (CCl₄, CH₃Cl, and CH₃CN). The aryl-bromo-esters were synthesized via the coupling reactions of 2-bromo-2-methylpropan-1-ol and the corresponding carboxylic acids, with good yields (∼36‒70%). The IR spectra showed that these compounds presented only one conformation, and the experimental data were supported by the theoretical results obtained by density functional theory (DFT) calculations using the 6311+G (2df, 2p) basis set. The calculations revealed that all the studied compounds presented two stable geometric conformations, which agrees with the data obtained experimentally in CCl₄. Theses conformers are stabilized by intramolecular hydrogen bonds. However, the orbital interaction calculations using the natural bond orbital (NBO) method showed that the $η$ _O $\to σ$ *_C-C, $η$ _O $\to σ$ *_C-O, $η$ _O $\to σ$ *_C-O and $η$ _O $\to π$ *_C-O hyper-conjugations are the main interactions that stabilize the conformations. The compounds preferentially adopt the anti-conformation because the steric effect between the gauche bromo and oxygen atoms overrides the hyper-conjugative interactions, in addition to the stabilizing σ_C-H→ σ*_C-Br interactions in the conformers.
Conformational study of the electronic interactions and nitric oxide release potential of new S‑nitrosothiols esters derivatives of ibuprofen, naproxen and phenyl acids substituted (SNO-ESTERS): Synthesis, infrared spectroscopy analysis and theoretical calculations
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NO released from NO-derivatives of NSAID, promotes vasodilatation of veins and arteries, inhibits platelet aggregation, and reduces the formation of blood clots [6,7]. In addition, it has been shown that NO exerts beneficial effects to the gastrointestinal (GI) tract, maintaining the integrity of the gastric mucosal barrier [4,5,8]. Different chemical strategies have been used to provide NSAIDs with the capacity to release NO. Nitrooxybutyl‑ester derivatives of NSAID display anti-inflammatory activity comparable to the respective parental NSAID against acute inflammation in rodent models, but with reduced GI toxicity [9].
The conformational study on the new S‑nitrosothiols esters (SNO-ESTERS): para–substituted (X = H, OMe, Cl and NO₂) S‑nitrosothiol derivatives 2‑methyl‑2‑(sulfanyl)propyl phenylacetates (R1), 2‑(4‑isobutylphenyl)propanoate (ibuprofen, R2), and 2‑(4‑isobutylphenyl)propanoate of 2‑methyl‑2‑(nitrososulfanyl)propyl (naproxen, R3) was performed using infrared spectroscopy (IR) in solvents with increasing polarity (CCl₄, CH₃Cl, and CH₃CN), and theoretical calculations, to determine the preferential conformer and the potential of these compounds to release nitric oxide (NO). S‑Nitrosothiols were synthesized by esterification reactions, using chlorides of the corresponding carboxylic acids, with good yields (~60%). IR results showed that these compounds presented only one conformation, and the experimental data were supported by the theoretical results obtained by density functional theory (DFT) calculations using the 6311+G (2df, 2p) basis set. The calculations revealed that all S‑nitrosothiols presented one preferential anticlinal (ac) geometric conformation, which agrees with the data obtained experimentally in CCl₄. These conformers are stabilized by intramolecular hydrogen bonds. Examination of the geometry with regard to the RSNO group revealed that these compounds are preferentially in the trans (anti) conformation. The calculation of the orbital interactions using the Natural Bond Orbital (NBO) method showed that the n_O(NO) → σ_(SN)^∗ hyper-conjugative interaction increases the SN bond length. The strong n_S → π_(NO)^∗ interaction and electronic delocalization induces a partial π character to the SN bond. The weak σ_SN bond indicates strong delocalization of the electron pair in O (NO) by the n_O(NO) → σ_(SN)^∗ interaction, thereby increasing the capacity of NO release from SNO-ESTERS.
Gasotransmitters and the immune system: Mode of action and novel therapeutic targets
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Gasotransmitters are a group of gaseous molecules, with pleiotropic biological functions. These molecules include nitric oxide (NO), hydrogen sulfide (H₂S), and carbon monoxide (CO). Abnormal production and metabolism of these molecules have been observed in several pathological conditions. The understanding of the role of gasotransmitters in the immune system has grown significantly in the past years, and independent studies have shed light on the effect of exogenous and endogenous gasotransmitters on immune responses. Moreover, encouraging results come from the efficacy of NO-, CO- and H₂S -donors in preclinical animal models of autoimmune, acute and chronic inflammatory diseases. To date, data on the influence of gasotransmitters in immunity and immunopathology are often scattered and partial, and the scarcity of clinical trials using NO-, CO- and H₂S -donors, reveals that more effort is warranted. This review focuses on the role of gasotransmitters in the immune system and covers the evidences on the possible use of gasotransmitters for the treatment of inflammatory conditions.
NOSH-aspirin (NBS-1120), a novel nitric oxide- and hydrogen sulfide-releasing hybrid has enhanced chemo-preventive properties compared to aspirin, is gastrointestinal safe with all the classic therapeutic indications
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Aspirin is chemopreventive; however, side effects preclude its long-term use. NOSH-aspirin (NBS-1120), a novel hybrid that releases nitric oxide and hydrogen sulfide, was designed to be a safer alternative. Here we compare the gastrointestinal safety, anti-inflammatory, analgesic, anti-pyretic, anti-platelet, and chemopreventive properties of aspirin and NBS-1120 administered orally to rats at equimolar doses. Gastrointestinal safety: 6 h post-administration, the number and size of hemorrhagic lesions in stomachs were counted; tissue samples were frozen for PGE₂, SOD, and MDA determination. Anti-inflammatory: 1 h after drug administration, the volume of carrageenan-induced rat paw edemas was measured for 5 h. Anti-pyretic: fever was induced by LPS (ip) an hour before administration of the test drugs, core body temperature was measured hourly for 5 h. Analgesic: time-dependent analgesic effects were evaluated by carrageenan-induced hyperalgesia. Antiplatelet: anti-aggregatory effects were studied on collagen-induced platelet aggregation of human platelet-rich plasma. Chemoprevention: nude mice were gavaged daily for 25 days with vehicle, aspirin or NBS-1120. After one week, each mouse was inoculated subcutaneously in the right flank with HT-29 human colon cancer cells. Both agents reduced PGE₂ levels in stomach tissue; however, NBS-1120 did not cause any stomach ulcers, whereas aspirin caused significant bleeding. Lipid peroxidation induced by aspirin was higher than that exerted by NBS-1120. SOD activity was significantly inhibited by aspirin but increased by NBS-1120. Both agents showed similar anti-inflammatory, analgesic, anti-pyretic, and anti-platelet activities. Aspirin increased plasma TNFα more than NBS-1120-treated animals. NBS-1120 was better than aspirin as a chemopreventive agent; it dose-dependently inhibited tumor growth and tumor mass.
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VA694, a promising cyclooxigenase-2 (COX-2)-inhibiting hybrid drug endowed with nitric oxide (NO) releasing properties (NO-COXIB), showed COX-2-selective inhibitory effects, associated with interesting anti-inflammatory and anti-nociceptive activities. Therefore, we studied the effects of VA694 on cartilage metabolism, in comparison with Naproxcinod, a COX inhibitor and NO donor (CINOD), and Naproxen, a traditional non-steroidal–anti-inflammatory drug (NSAID) on human osteoarthritic chondrocyte cultures. IL-1β-stimulated chondrocytes showed a significant decrease in cell viability (P < 0.001). VA694, Naproxcinod and Naproxen alone didn't significantly affect cell viability, while it restored cell viability in cultures stimulated by IL-1β. The presence of IL-1β determined a significant increase (P < 0.001) in PGE₂ levels measured by an ELISA assay, and in COX-2 and MMP-3, -9, and -13 gene expression analyzed by RT-PCR. VA694, Naproxcinod and Naproxen, at both concentrations analyzed, significantly counteracted the negative effects induced by IL-1β. VA694, Naproxcinod and Naproxen pre-treatment were able to inhibit IL-1β-induced NF-κB activation, when measured as its nuclear translocation (p50 and p65 subunits). Naproxcinod and Naproxen pre-treatment didn't affect cytoplasmic NF-κB levels; VA694 decreased the cytoplasmic levels of both subunits. Our data suggest that VA694, Naproxcinod and Naproxen, exert anti-inflammatory and chondroprotective effects on OA chondrocytes.

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NO-aspirin: mechanism of action and gastrointestinal safety

Abstract

Section snippets

Aspirin: the same mechanism for activity and toxicity

NO-aspirins

NCX-4016

Gastrointestinal safety and anti-platelet activity of NCX-4016 in humans: the proof of concept

Conclusion

Conflict of interest statement

Lancet

Gastroenterology

Gastroenterology

Eur J Pharmacol

Life Sci

Gastroenterology

Thromb Res

Gastroenterology.

Gastroenterology

J Pharm Biomed Anal

Gastroenterology

Thromb Res

Cell

Curr Biol

Curr Opin Immunol

Trends Immunol

Gastroenterology

Free Radic Biol Med.

Arch. Biochem. Biophys.

Eur. J. Pharmacol

Life Sci

Dig Liver Dis

Aspirin as an antiplatelet drug

New Engl J Med

Cyclo-oxygenase isoenzymes. Structural basis for selective inhibition of cyclo-oxygenases by anti-inflammatory agents

Dig Liver Dis.

Acetylation of prostaglandin synthase by aspirin

Proc Natl Acad Sci USA

Aspirin

Circulation

Unorthodox routes to prostanoid formation: new twists in cyclooxygenase-initiated pathways

J Clin Invest.

Dose effects of aspirin on gastric prostaglandins and stomach mucosal injury

Ann Intern Med

Aspirin for the primary prevention of cardiovascular events: a summary of the evidence for the US Preventive Services Task Force

Ann Intern Med.

Randomised trial of prophylactic daily aspirin in British male doctors

Br Med J