In vitro metabolism of a nitroderivative of acetylsalicylic acid (NCX4016) by rat liver: LC and LC–MS studies

Abstract

The metabolism of a nitroderivative of acetylsalicylic acid, benzoic acid, 2-(acetyloxy)-3-[(nitrooxy)methyl]phenyl ester (NCX4016), the lead compound of a new class of NO-releasing non steroidal-antiinflammatory drugs has been studied in vitro in rat liver subcellular fractions (S 9000 × g, microsomes, cytosol). Samples were extracted with CH 3 CN (2 vol.) containing 1% H 3 PO 4 (2 M), vortexed for 3 min and then centrifuged for 5 min at 5000 rpm. Supernatants were diluted with 0.02 M phosphoric acid and analysed by reverse-phase LC. Linearity of calibration for NCX4016 and metabolites was observed over the range 0.25-50 mg/ml with coefficients of determination greater than 0.9996. Extraction efficiency from spiked liver samples ranged from 85 to 95% for all the analytes. In the S 9000×g fraction, NCX4016 undergoes rapid metabolization, with the formation of salicylic acid (SA) and [3-(nitrooxymethyl)phenol] (HBN). HBN is then rapidly metabolised to 3-hydroxybenzylalcohol (HBA), and mainly to a new metabolic species, whose formation takes place specifically in the liver cell cytosol. LC-MS analysis (electrospray ionisation) of the cytosol extract in negative and positive-ion modes furnished deprotonated [M − H] − and protonated [M +H] + molecular ions at m/z 412 and 414, respectively, accompanied by the typical clusters with sodium. MS/MS analysis in negative-ion mode, by selection and collision of the ion at m/z 412, gave a fragmentation pattern characterized by the ions at m/z 272 and 254, which allowed to assign the structure of 1-(glutathion-Syl)methylene-3-hydroxy-benzene, a conjugated product between GSH and the benzyl carbon atom of HBN. In rat liver cytosol HBN is completely metabolised to this thioether adduct within 30 min incubation; the process is enzymatically mediated by GSH transferase and strictly dependent on GSH availability. The relevance of this new metabolic pathway in NCX4016 detoxification by rat liver is discussed.

Nitric oxide–releasing aspirin and indomethacin are potent inhibitors against colon cancer in azoxymethane-treated rats: effects on molecular targets

“…In vitro studies have shown that NO-aspirin inhibits COX-1 and COX-2, and that the presence of the spacer and NO-donating moiety in the molecule slows the kinetics of COX-1 inhibition by NCX 4016 compared with aspirin (34). The positional isomer of NO-aspirin used in this study (meta) is converted essentially into salicylic acid, 3-hydroxybenzylalcohol, and a conjugated product between glutathione and the aromatic spacer linking the aspirin moiety to the NOreleasing group (35,36). In rats, following a single administration of NO-aspirin, the main metabolites in plasma were NO x , salicylic acid, and nitrosothiols, with no intact drug observed.…”

Reversal to cisplatin sensitivity in recurrent human ovarian cancer cells by NCX-4016, a nitro derivative of aspirin

“…Although the use of NSAIDs is limited by their gastrointestinal (GI) and renal toxicity, NOreleasing NSAIDs were shown to exert antiinflammatory and analgesic effects that were at least as potent as those of the parent drug, without causing GI tract toxicity (16)(17)(18)(19). The in vivo metabolism of these drugs has been established reasonably well in a rat model (14,20), except that the precise mechanism by which NO is released from the nitro moiety has yet to be understood. NCX-4016 is metabolized by esterases in the liver cells and in plasma to salicylic acid and 3-(nitrooxymethyl)phenol, which is rapidly metabolized to 3-hydroxybenzylalcohol and NO (20).…”

“…The in vivo metabolism of these drugs has been established reasonably well in a rat model (14,20), except that the precise mechanism by which NO is released from the nitro moiety has yet to be understood. NCX-4016 is metabolized by esterases in the liver cells and in plasma to salicylic acid and 3-(nitrooxymethyl)phenol, which is rapidly metabolized to 3-hydroxybenzylalcohol and NO (20). The carbon atom of 3-(nitrooxymethyl)phenol can react with GSH to form 1-(glutathion-S-yl)methylene-3-hydroxybenzene.…”

“…The carbon atom of 3-(nitrooxymethyl)phenol can react with GSH to form 1-(glutathion-S-yl)methylene-3-hydroxybenzene. This process is enzymatically mediated by GSH transferase and is dependent on the availability of GSH (20). Napoli et al (21,22) showed that NCX-4016 reduced experimental restenosis, an effect associated with lower vascular smooth muscle cell proliferation.…”

Searching for New NO-Donor Aspirin-like Molecules: A New Class of Nitrooxy-acyl Derivatives of Salicylic Acid

“…Unlike the previous NO-donor aspirins that seem to require enzymatic metabolism for NO release, [10][11][12] these products proved to release NO under the action of thiols. 13 Intracellular release induced by glutathione is potentiated by ascorbic acid.…”