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

doi:10.1016/S0731-7085(02)00147-4

Journal of Pharmaceutical and Biomedical Analysis

Volume 29, Issue 6, 1 August 2002, Pages 1061-1071

https://doi.org/10.1016/S0731-7085(02)00147-4 Get rights and content

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 CH₃CN (2 vol.) containing 1% H₃PO₄ (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 μg/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-S-yl)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.

Introduction

Benzoic acid, 2-(acetyloxy)-3-[(nitrooxy)methyl]phenyl ester (NCX4016) (Fig. 1), the nitroderivative of aspirin, is the lead compound of a new class of NO-releasing non steroidal anti-inflammatory drugs (NO-NSAIDs). In pre-clinical studies the drug has been shown to display antiaggregatory and antithrombotic activity by a dual mechanism of action involving inhibition of cyclooxygenase (COX) and release of nitric oxide (NO), the latter acting on guanylate cyclase in both platelets and vascular smooth muscle cells [1], [2]. The drug was also found to be devoid of typical toxic effects of COX-1 inhibitors (NSAIDs) on gastric mucosa also following repeated oral administration. This through inhibition of neutrophil adherence to vascular endothelium [3]. In addition, recent results indicated that NCX4016 is a powerful agent in improving the post-ischemic ventricular dysfunction in the rabbit heart [4] and in reducing the infarct size in rat heart induced by ischemia-reperfusion [5].

While the pharmacokinetic profile of the drug following single or repeated oral doses in the rat is known (no NCX4016 has been detected in plasma, but only salicylic acid) [2], [6], no studies have been performed on its metabolism in vitro or in vivo in the animal.

As a first step, to elucidate the metabolic picture of the drug, in this work we have studied the in vitro metabolism of NCX4016 by rat liver, using the combined approach of LC–UV–DAD and LC–UV–DAD–MS/MS for the identification and quantitative determination of metabolites in different hepatic subcellular fractions: S 9000×g supernatant fraction, cytosol (105 000×g supernatant fraction) and microsomes, with the final aim to define the key metabolic steps of biotransformation of the drug and the kinetics of its metabolization by the liver.

Section snippets

Chemicals

HPLC-grade and analytical-grade organic solvents were purchased from Merck (Bracco, Milan, Italy). HPLC-grade water was prepared with a Milli-Q water purification system. Benzoic acid, 2-(acetyloxy)-3-[(nitrooxy)methyl]phenyl ester (NO-ASA; NCX4016) and the NO-ASA metabolites: benzoic acid, 2-(hydroxy)-3-[(nitrooxy)methyl]phenyl ester (NCX4023), benzoic acid, 2-(acetyloxy)-3-(hydroxymethyl)phenyl ester (NCX4017), benzoic acid, 2-(hydroxy)-3-(hydroxymethyl)phenyl ester (NCX4019) and

In vitro studies

In Fig. 1 are reported the structures of NCX4016 and of some postulated metabolites of the drug which could arise from different biotransformation pathways involving single or multiple hydrolytic cleavage of the different ester functions in the molecule (acetate, benzoate and nitrate esters). Firstly we developed a reverse-phase HPLC method for analysis of all the metabolites in liver samples. The chromatographic profile reported in Fig. 2b is obtained from an extract of the native S 9000×g

Conclusions

A reliable HPLC assay for the simultaneous determination of NCX 4016 and its metabolites in liver tissue has been developed, which provides satisfactory accuracy and precision for all the analytes. The method has been successfully applied to the study of the in vitro metabolism of the drug by rat liver, to get an insight into the key metabolic steps of its hepatic biotransformation and detoxification and seems to be applicable also to the study of the in vivo metabolism of the drug. Although

Acknowledgements

Financial support from MURST (Cofinanziamento Programma Nazionale 2000) is gratefully acknowledged.

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

Abstract

Introduction

Section snippets

Chemicals

In vitro studies

Conclusions

Acknowledgements

Thromb. Res.

Biochem. Pharmacol.

Biochem. Biophys. Res. Commun.

J. Pharm. Biomed. Anal.

Cardiovasc. Drug Rev.