In Vitro Metabolism of Nitric Oxide-Donating Aspirin: The Effect of Positional Isomerism

Abstract

NO-donating aspirin (NO-ASA) is a potentially important chemopreventive agent against cancer. Since positional isomerism affects strongly its potency in inhibiting colon cancer cell growth, we studied the metabolic transformations of its ortho-, meta-, and para-isomers in rat liver and colon cytosolic, microsomal, and mitochondrial fractions as well as in intact HT-29 human colon cancer cells. NO-ASA and metabolites were determined by high-performance liquid chromatography and products identified by mass spectroscopy, as required. For all three isomers, the acetyl group on the ASA moiety was hydrolyzed rapidly. This was followed by hydrolysis of the ester bond linking the salicylate anion to the spacer. The ortho-and paraisomers produced salicylic acid and a putative intermediate consisting of the remainder of the molecule, which via a rapid step generated nitrate, (hydroxymethyl)phenol, and a conjugate of spacer with glutathione. The meta-isomer, in contrast, generated salicylic acid and (nitroxymethyl)phenol, the latter leading to (hydroxymethyl)phenol and the glutathione-spacer conjugate. This metabolic pathway takes place in its entirety only in the cytosolic fraction of the tissues tested and in intact human colon cancer cells, perhaps reflecting exposure to the cytosolic glutathione S-transferase, which catalyzes the formation of the spacer-glutathione conjugate. Thus, the three positional isomers of NO-ASA differ in their metabolism and these differences correlate with their differential effects on cancer cell growth, underscoring the importance of positional isomerism in modulating drug effects.NO-donating nonsteroidal anti-inflammatory drugs (NONSAIDs) are emerging as an important novel pharmacological class that has already entered the phase of clinical testing. NO-NSAIDs consist of a traditional NSAID to which the -ONO 2 group is covalently bound via a spacer (Fig. 1). Their three main features appear to be enhanced potency, efficacy, and greater safety compared with their NSAID counterparts (reviewed in Rigas et al., 2003;Rigas and Kashfi, 2004). A large body of basic work in recent years indicates that they display novel properties that set them apart from their parent traditional NSAIDs. In view of their envisioned clinical applications, it is becoming increasingly important to know their metabolic transformations by various tissues, especially those that may be targets of their pharmacological action.We have been interested in the use of NO-NSAIDs, in particularly NO-aspirin (NO-ASA), as a safe and effective chemopreventive agent against colon cancer (Williams et al., 2001;Kashfi et al., 2002). In vitro studies using cultured colon and other cell lines indicate that NO-ASA is the most potent among several NO-NSAIDs in inhibiting cell growth (preceding article), which is taken to portend its efficacy in intact organisms. Using an animal model of colon cancer, we and others have demonstrated that NO-NSAIDs, including NO-ASA, are effective in inhibiting aberrant crypt foci, which represent t...

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

“…Some of them are rapidly metabolized, with little or no formation of aspirin, when incubated in serum, plasma, and rat liver subcellular fractions. 9,12 This behavior is in keeping with the knowledge that the loss of the negative charge by the aspirin molecule, because of the esterification of the COOH group (pK a ) 3.5), makes the acetoxy moiety extremely susceptible to enzymatic cleavage. 21 Interestingly, it was recently found that † A portion of this work was presented as an invited lecture at the 41st International Union of Pure and Applied Chemistry (IUPAC) World Chemistry Congress, August 5-11, 2007 neither aspirin nor NO contributes to the antitumor effect of 2, 4, which, in contrast, is due to the quinone methides formed after carboxylic ester hydrolysis.…”

“…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.…”

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.…”

The differential cell signaling effects of two positional isomers of the anticancer NO-donating aspirin

“…NO-ASA, both para [2-(acetyloxy) benzoic acid 4-(nitrooxy methl) phenyl ester] and meta [2-(acetyloxy) benzoic acid 3-(nitrooxy methl) phenyl ester], was synthesized as previously described (22). Antibodies against the phosphorylated forms of cJun, ATF-2, JNK, p38, ERK1/2, Akt and total p21 were purchased from Cell Signaling Technology, Beverly, MA.…”

“…1B, the time-response curves of both isomers follow a similar course: after an initial strong decline during the first 4 h, there is a shallow decrease in the number of viable cells. Although the reason for this biphasic response is unclear, it is conceivable that it reflects the rate of metabolic transformation of NO-ASA (22).…”