Comparison between 3-Nitrooxyphenyl acetylsalicylate (NO-ASA) and O2-(acetylsalicyloxymethyl)-1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate (NONO-ASA) as safe anti-inflammatory, analgesic, antipyretic, antioxidant prodrugs

Abstract

Chronic inflammation is an underlying etiological factor in carcinogenesis; nonsteroidal anti-inflammatory drugs (NSAIDs) and their chemically modified NO-releasing prodrugs (NO-NSAIDs) are promising chemopreventive agents. The aim of this study was to conduct a head-to-head comparison between two NO-ASAs possessing different NO donor groups, an organic nitrate [3-nitrooxyphenyl acetylsalicylate (NO-ASA; NCX-4016)] and an N-diazeniumdiolate [NONO-ASA, O(2)- (acetylsalicyloxymethyl)-1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate (NONO-ASA; CVM-01)], as antiulcerogenic, analgesic, anti-inflammatory, and antipyretic agents. All drugs were administered orally at equimolar doses. For antiulcerogenic study, 6 h after administration, the number and size of hemorrhagic lesions in stomachs from euthanized animals were counted. Tissue samples were frozen for prostaglandin E(2) (PGE(2)), superoxide dismutase (SOD), and malondialdehyde determination. For anti-inflammatory study, 1 h after drug administration, the volume of carrageenan-induced rat paw edemas was measured for 6 h. For antipyretic study, 1 h after dosing, fever was induced by intraperitoneal LPS, and body core temperatures measured for 5 h. For analgesic study, time-dependent analgesic effect of prodrugs was evaluated by carrageenan-induced hyperalgesia. Drugs were administered 30 min after carrageenan. NO-ASA and NONO-ASA were equipotent as analgesic and anti-inflammatory agents but were better than aspirin. Despite a drastic reduction of PGE(2) in stomach tissue, both prodrugs were devoid of gastric side effects. Lipid peroxidation induced by aspirin was higher than that observed by prodrugs. SOD activity induced by both prodrugs was similar, but approximately 2-fold higher than that induced by aspirin. CVM-01 is as effective as NCX-4016 in anti-inflammatory, analgesic, and antipyretic assays in vivo, and it showed an equivalent safety profile in the stomach. These results underscore the use of N-diazeniumdiolate moieties in drug design.

Fig. 1.

Chemical structures of aspirin and NO-releasing aspirins, NCX 4016 (NO-ASA) and CVM-01 (NONO-ASA).

Fig. 2.

NO-ASA and NONO-ASA do not cause gastric damage. Drugs were administered orally at equimolar doses (1 mmol/kg), and effects on the stomach were evaluated as indicated under Materials and Methods. A, ASA caused severe gastric damage (UI = 50 ± 7 mm), whereas both NO-ASA and NONO-ASA were gastric damage-sparing (UI = 2 ± 0.2 and 3 ± 0.3 mm, respectively). B, all three drugs also caused erosions of the gastric mucosa, but the damage was significantly less in the NO-ASA and NONO-ASA groups compared with the ASA group. Results are mean ± S.E.M. for 9 to 12 rats in each group, ∗, P < 0.01 compared with the ASA group.

Fig. 3.

Effects of ASA, NO-ASA, and NONO-ASA on gastric PGE₂ level, lipid peroxidation (MDA), and SOD. Four groups of rats were treated with vehicle, ASA, NO-ASA, and NONO-ASA, and their stomachs were removed and processed as described under Materials and Methods. A, all three drugs caused a significant reduction in gastric mucosal PGE₂ levels (A). Results are mean ± S.E.M. of 9 to 12 rats in each group, ∗, P < 0.05 versus vehicle group. B, ASA caused an almost 6-fold increase in MDA levels for NO-ASA- and NONO-ASA-treated rats, and MDA levels were comparable with those in the vehicle-treated rats. Results are mean ± S.E.M. for five to seven rats in each group, †, P < 0.01 versus ASA group. C, ASA caused a significant reduction in SOD activity, whereas NO-ASA and NONO-ASA did not have an effect. Results are mean ± S.E.M. of five to seven rats, ∗, P < 0.05 versus vehicle group.

Fig. 4.

Anti-inflammatory properties of ASA, NO-ASA, and NONO-ASA. Rat paw edema was induced by carrageenan injection, as described under Materials and Methods. A, all three drugs caused a significant reduction in paw volume at all time points. Results are mean ± S.E.M. of five rats in each group, ∗, P < 0.05 versus vehicle-treated rats at all time points. B, all three drugs also caused a significant reduction in PGE₂ levels in the paw exudate. Results are mean ± S.E.M. for five rats in each group, †, P < 0.01 versus vehicle; ∗, P < 0.05 versus NO-ASA and NONO-ASA.

Fig. 5.

Effect of ASA and its NO-releasing prodrugs on plasma TNF-α. Rats were treated with equimolar amounts (1 mmol/kg) of each drug, and plasma TNF-α was measured as described under Materials and Methods. ASA caused a significant rise in plasma TNF-α; however, this rise was significantly lower in the NO-ASA- and NONO-ASA-treated rats. Results are mean ± S.E.M. for three rats in each group: ∗, P < 0.01 versus vehicle; †, P < 0.01 versus ASA; §, P < 0.05 versus NO-ASA.

Fig. 6.

ASA, NO-ASA, and NONO-ASA reduce LPS-induced fever and raise the threshold for hyperalgesia. A, LPS (50 μg/kg i.p.) was administered to the animals 1 h before administration of the test drugs. Core body temperature was recorded at 30 min and hourly thereafter for 5 h. Results are mean ± S.E.M. for five rats in each group. ∗, P < 0.05 versus vehicle for all three drugs from 1 to 5 h. B, mechanical pain threshold was increased in a time-dependent manner by all three drugs; however, both NO-ASA and NONO-ASA were better than ASA, especially during the last 2 h of the experiment. Results are mean ± S.E.M. for five rats in each group. §, P < 0.05 versus vehicle for all three drugs from 1 to 5 h; †, P < 0.05 for NO-ASA versus ASA and NONO-ASA from 2 to 5 h; ∗, P < 0.05 for NONO-ASA versus ASA from 4 to 5 h.

Fig. 7.

Time course of NO_x levels in plasma after NO-ASA and NONO-ASA administration. Plasma concentration of NO was quantified indirectly as the concentration of NO₃⁻/NO₂⁻ using the Griess method. Results are mean ± S.E.M. of three rats at each time point, P < 0.01 versus zero time at all time points.