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CARDIOVASCULAR
Division of Internal and Cardiovascular Medicine (T.C., M.L., P.G.), Section of Biochemistry (L.C.), Department of Internal Medicine, University of Perugia, Perugia, Italy; and NicOx Research Institute, Milan, Italy (R.M.)
Received May 23, 2005; accepted September 1, 2005.
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Abstract |
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). Two isoforms of the enzyme have been identified, COX-1 and COX-2, that are the product of two distinct genes (Appleby et al., 1994), and both isoforms are inhibited by nonsteroidal anti-inflammatory drugs (NSAIDs) (Smith et al., 1997; Marnett et al., 1999).
Aspirin is unique among NSAIDs because it covalently modifies COX by acetylating a serine residue positioned in the arachidonic acid-binding channel of the enzyme (Ser529 of COX-1 and Ser516 of COX-2), thus irreversibly inactivating it and preventing the generation of prostaglandins and thromboxane A2 (Loll et al., 1995). Ser529 is not directly involved in catalysis; in fact, mutation of this residue has no effect on the enzyme activity, but it is strategically located, and its acetylation prevents the access of the substrate to the catalytic site of the enzyme (Loll et al., 1995). Other NSAIDs, such as ibuprofen and indomethacin, inhibit competitively and reversibly COX and prevent the irreversible acetylation of COX-1 by aspirin by occupying the active site channel of the enzyme, thereby preventing aspirin from reaching its target (Catella-Lawson et al., 2001; Ouellet et al., 2001). Mature normal human platelets express only COX-1, and because they are anucleate cells unable to synthesize enzyme de novo, the effect of aspirin on them is permanent and cumulative (Patrono, 1994). Thus, the cardioprotective effect of aspirin is exerted through the irreversible and permanent impairment of thromboxane A2-dependent platelet function, which reduces the development of acute arterial thrombosis (Patrono, 1994). One of the limitations of NSAIDs is the occurrence of side effects in the gastrointestinal tract (e.g., gastritis and ulceration), resulting from the inhibition of vasodilator and cytoprotective prostanoids in the gastric mucosa, which limits the use of these compounds (Wallace, 1997).
In the past few years, nitric oxide (NO)-releasing NSAIDs have been developed (Burgaud et al., 2002) with the aim of reducing gastrointestinal toxicity by exploiting the protective activity of locally released NO on gastric mucosa (Wallace, 1997; Lanas et al., 2000). They are synthesized by the ester linkage of a NO-releasing moiety to conventional NSAIDs, such as aspirin (NCX 4016), flurbiprofen (HCT 1026), or naproxen (HCT 3012). For NCX 4016, the acetylsalicylic acid molecule is linked to a chemical spacer (hydroxybenzylalcohol) bearing the NO-donating moiety benzenemethanol-3-hydroxy-
-nitrate (NCX 4015), and the resulting molecule has a molecular mass 1.8-fold greater than that of aspirin (Del Soldato et al., 1999).
Nitroaspirin, besides its better gastric tolerability (Fiorucci et al., 2003), seems to be superior to aspirin in terms of platelet inhibition (Lechi et al., 1996; Mezzasoma et al., 1999) and antithrombotic activity (Momi et al., 2000, 2005), and this is probably due to a combination of the antiplatelet effects of aspirin and the broad-range cardiovascular activities of NO (Mezzasoma et al., 1999; Gresele et al., 2003). Moreover, differently from aspirin, NCX 4016 inhibits the activity of COX-2 of stimulated monocytes in human whole blood through a NO-mediated mechanism (Corazzi et al., 2003).
However, although NCX 4016 inhibits COX-1 with a potency similar to that of aspirin (Lechi et al., 1996; Mezzasoma et al., 1999; Corazzi et al., 2003), it is not clear whether the inhibitory activity is exerted directly by the whole molecule or whether it is the consequence of its enzymatic transformation, in whole blood or in other organs, into acetylsalicylic acid. It is indeed conceivable that the steric hindrance created by the spacer and the NO-releasing moiety of NCX 4016 may hamper it from entering the COX-1 enzymatic channel and thus from irreversibly blocking the enzyme. In fact, some reports have questioned the effectiveness of nitroaspirin in blocking platelet COX-1 (Wallace et al., 1999; Wainwright et al., 2002), and it has been hypothesized that NCX 4016 may either block only reversibly COX-1 or it may act as a reservoir of aspirin that is then slowly released in blood to exert its pharmacologic effects.
The understanding of the mechanism of COX blockade by NCX 4016 is relevant to the complete unraveling of the pharmacokinetic/pharmacodynamic profile of the drug required for further clinical studies. Aims of the present study were to test whether NCX 4016 and/or its analog/metabolites inhibit directly the activities of COXs, to assess whether the inhibition of COX is reversible, and to evaluate whether other NSAIDs may compete with this inhibition.
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Materials and Methods |
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; Gresele et al., 1987). Serum was separated by centrifugation (10 min; 2000g) and frozen at –80°C for later assay of TxB2 (Gresele et al., 1988). To assess the effect of the prolongation of preincubation time on the COX-1-suppressing activity, the tested compounds were incubated at room temperature for 1, 4, and 24 h, with 1-ml aliquots of citrated [3.8%; 1/10 (v/v)] whole blood; 0.2 mM CaCl2 and 1 U/ml thrombin were then added, and the samples were allowed to clot at 37°C for 60 min. Serum was then separated, as above described, for later assay of TxB2.
COX-2 Activity in Human Whole Blood. The effects of the drugs under study on whole blood monocyte COX-2 activity were assessed by incubating each compound, or its vehicle, with 1-ml aliquots of heparinized (10 IU/ml) human whole blood for 15 min before the addition of 10 µg/ml LPS (derived from Escherichia coli 026:B6; Sigma-Aldrich, St. Louis, MO). Samples were incubated for 24 h at 37°C in a 5% CO2 atmosphere, and then plasma was separated by centrifugation (10 min; 2000g at room temperature) and immediately frozen at –80°C for later assay of PGE2 (Patrignani et al., 1994; Corazzi et al., 2003). Given that the above-mentioned assay, although useful for the testing of the effects of NSAIDs and cyclooxygenase-2 inhibitors (Patrignani et al., 1994; Chan et al., 1999), is largely insensitive to aspirin due to rapid deacetylation of the molecule by plasmatic esterases, we also used a modified whole blood assay for the measurement of monocyte COX-2 activity in which aspirin was previously proven to be effective (Warner et al., 1999). Human whole blood monocytes were isolated from buffy coat, as described previously (Corazzi et al., 2003), seeded into plastic Petri dishes, and exposed to LPS (10 µg/ml) for 24 h to induce COX-2 expression. The medium was then removed, and human heparin-anticoagulated whole blood (100 µl) was added together with the test agents or vehicle. Sixty minutes later, A23187
[GenBank]
(calcimycin; 50 µM) was added, and the samples were further incubated for 30 min. The culture plates were then centrifuged, and supernatant plasma was removed for later assay of PGE2, as a measure of the activity of COX-2 in human monocytes.
Measurement of TxB2 and Prostaglandin E2. Immunoreactive serum TxB2 was measured by a specific radioimmunoassay, as described previously (Gresele et al., 1987; Vezza et al., 1995). Briefly, unextracted serum samples were diluted with the assay buffer (Tris-HCl-buffered isotonic saline; pH 7.5). The final volume of the assay mixture was 200 µl, and the mixture comprised 
4000 cpm of radiolabeled [3H]TxB2 (specific activity 104 Ci/mM; GE Healthcare, Little Chalfont, Buckinghamshire, UK) and a highly specific rabbit anti-TxB2 antiserum diluted 1:35,000 in assay buffer.
The IC50 for unlabeled TxB2 was 170 pg/ml, and the least detectable amount was 25 pg/ml TxB2. Cross-reactivities of the antiserum are as follows: PGD2, 1.6%; PGF2, 0.04%; PGE2, <0.01%; PGF1, <0.01%; PGE1, <0.01%; 6-keto-PGF1, <0.01%; and arachidonic acid, <0.01%. Radioimmunoassay of PGE2 was performed on nonextracted, highly diluted serum samples using a highly specific antiserum and tritiated tracers as described previously (Vezza et al., 1993).
Purified Ovine COX (oCOX)-1 and oCOX-2 Assay. COX activity was determined by a spectrophotometric assay (optical density 610 nm) using 200 units of oCOX-1 or oCOX-2 (Cayman Chemical, Ann Arbor, MI) in 600 µl of enzyme reaction buffer (100 mM sodium phosphate, pH 6.5, 0.5 µM hematin, and 1 mg/ml gelatin), and 0.6 µl of the tested compound dissolved in dimethyl sulfoxide.
The enzymatic reaction was initiated by adding 100 µM TMPD (Sigma-Aldrich, Milan, Italy) and 100 µM arachidonic acid (saturating condition; Sigma-Aldrich) in assay buffer. Inhibitors were added to the incubation reaction at different time intervals before the addition of TMPD and arachidonic acid. The velocities observed in the presence of different inhibitor concentrations were divided by the velocity observed for enzyme samples preincubated with vehicle, and this ratio was multiplied by 100 to yield percentage of control activity. The enzyme activity was measured by the estimation of the initial velocity of TMPD oxidation as indicated by the increase in absorbance at 610 nm, as described previously (Ouellet et al., 2001). In our experimental conditions, 1 unit of enzyme activity produced an increase in TMPD absorbance of 0.001 per minute, as also reported by Copeland et al. (1994).
The irreversibility of the inactivation of oCOX-1 by the tested inhibitors was assessed by repeating the COX activity assay after extensive dialysis. Enzyme (2000 units) was incubated with inhibitors in a volume of 1 ml of enzyme reaction buffer for 15 min at 4°C. After incubation, the samples were dialyzed against 500 ml of inhibitor-free reaction buffer for 5 h. After dialysis, the remaining enzymatic activity, assessed as described above, was measured in comparison with samples of oCOX-1 incubated with vehicle before dialysis.
Competition experiments were carried out by adding the potential competing inhibitor 15 min before the addition of the tested inhibitor (aspirin or NCX 4016). After an additional 15 min, the reaction mixture was extensively dialyzed for 4 h, and then COX-1 activity was determined as described above.
Measurement of NCX 4016 and Its Derivatives by HPLC. After each oCOX-1 assay, the reaction mixture was collected and assessed by HPLC for NCX 4016, its metabolites, or aspirin to exclude degradation in vitro of NCX 4016. The samples (0.2-ml aliquots) were extracted with ice-cooled CH3CN (2 volumes) containing 1% orthophosphoric acid (50%) and centrifuged at 20,000g at 4°C for 10 min. Supernatants were immediately submitted to liquid chromatography.
Liquid chromatography analyses were carried out on an Agilent 1100 series system (Agilent Technologies, Palo Alto, CA) equipped with a binary pump; a UV-visible diode array programmable detector operating at 230-, 240-, 280-, and 300-nm wavelengths; an autosampler; an in-line degasser; and Agilent Chemstation software. Separations were achieved by reverse-phase elution with a Synergi Polar-RP 80A column (Phenomenex, Torrance, CA) (150 x 4.60 mm i.d., particle size 4 µm), equipped with a Polar-RP precolumn (4 x 3 mm i.d.) and maintained at 25°C. The mobile phase, 0.1 M KH2PO4, pH 2/CH3CN (9:1) (solvent A), and CH3CN (solvent B), was delivered at a flow rate of 1 ml/min with a gradient elution: from 100% solvent A to 60% solvent B in 30 min. Under these conditions, the retention times (minutes) were as follows: NCX 4023, 30.7; NCX 4016, 29.5; NCX 4215, 27.1; NCX 4019, 22.3; NCX 4017, 20.8; NCX 4015, 19.0; salicylic acid, 13.6; acetylsalicylic acid, 11.9; and 3-hydroxybenzoic acid, 5.3 min.
Statistical Analysis. Results are expressed as means ± S.E.M. Differences between controls and treatments were analyzed by using the two-way analysis of variance followed by Tukey's test for multiple comparisons or by the Student's t test. A p value of less than 0.05 was considered as statistically significant.
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Results |
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, 2004).
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), only the acetylated compounds NCX 4017 and NCX 4215 strongly inhibited TxB2 production. On the contrary, the deacetylated metabolites (NCX 4023 and NCX 4015) did not affect TxB2 formation or only marginally inhibited it (NCX 4019) at the highest concentration tested (Fig. 2).
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Effect of NCX 4016, Analog, and Metabolites on Monocyte COX-2 Activity. Incubation of heparinized whole blood with 10 µg/ml LPS resulted in an approximately 20-fold increased production of PGE2 at 24 h compared with saline-incubated samples.
Besides NCX 4016 (Corazzi et al., 2003), only the derivatives containing the NO-donating moiety (NCX 4023 and NCX 4215) inhibited human monocyte COX-2 in whole blood (more than 70% inhibition at 300 µM for both); the NO-donor moiety itself (NCX 4015; 300 µM) slightly but significantly inhibited PGE2 production (48.8 ± 2.3% inhibition; p < 0.05 versus LPS-stimulated control), whereas the NCX 4016 derivatives devoid of the nitrooxy group (NCX 4017) and of the acetyl and nitrooxy group (NCX 4019) were totally inactive (data not shown).
When similar experiments were carried out in a modified human whole blood COX-2 assay, which does not involve extensive aspirin deacetylation, we found that 300 µM aspirin strongly inhibited LPS-induced COX-2 activity (98% of inhibition; p < 0.01) as did NCX 4016 (87% inhibition; p < 0.05) and the acetylated derivatives NCX 4017 and NCX 4215 (60 and 70% inhibition, respectively; p < 0.05). The nonacetylated NCX 4016 metabolites were instead almost inactive (NCX 4019, 30% inhibition; NCX 4023, 23% inhibition; NCX 4015, 10% inhibition; p = N.S.) (Table 1). In this assay, which uses large doses of the calcium ionophore A23187 [GenBank] as a trigger of PGE2 formation, NO-donors only marginally inhibit monocyte COX-2 activity (data not shown).
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Effect of NCX 4016, Analog, and Metabolites on Isolated, Purified oCOX-1 and oCOX-2. Among NCX 4016 analog and derivatives, only its acetyl-containing metabolites and its analog NCX 4215 suppressed the activity of isolated, purified oCOX-1 and oCOX-2, whereas all of the deacetylated derivatives were ineffective (Table 2). NCX 4016 was somewhat less potent at inhibiting oCOX-1 compared with aspirin, with IC50 values of 68 and 27 µM, respectively. Concerning the NCX 4016 analog/metabolites, relative potency was as follows: for oCOX-1, NCX 4016 > NCX 4215 > NCX 4017; and for oCOX-2, NCX 4016 > NCX 4017 > NCX 4215. HPLC analysis of the incubation mixtures collected after each reaction showed that NCX 4016 acts directly on oCOXs without undergoing further metabolization (Table 3).
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Concerning the time taken to inhibit isolated oCOX-1, aspirin inhibited enzyme activity already by 56.5 ± 1.5% 30 s after the addition, whereas NCX 4016 at this time point gave almost no inhibition (0.86 ± 0.1% inhibition). The prolongation of the preincubation time produced a progressive increase of the degree of inhibition of oCOX-1 with both aspirin and NCX 4016; however, NCX 4016 took longer to reach its maximal effect, with a time to half-maximal inhibition significantly longer than that of aspirin (9 ± 1.05 min versus 26.8 ± 1.5 s, respectively; p < 0.05) (Fig. 4).
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), it prevented the inhibition of the enzyme by both aspirin and NCX 4016. In fact, salicylic acid (10.8 µM) preincubated for 15 min before the addition of aspirin (22.2 µM) produced a decrease in the inhibition observed with aspirin alone (35 ± 0.89% inhibition without salicylic aid; 16 ± 0.57% inhibition with salicylic acid). The same was observed by preincubating salicylic acid (25.3 µM) for 15 min before the addition of nitroaspirin (1.66 µM) (16 ± 0.7% inhibition without salicylic aid; 7.5 ± 0.8% inhibition with salicylic acid). Similarly, the deacetylated NCX 4016 metabolites (NCX 4023, NCX 4019, and NCX 4015) competed with NCX 4016 or with aspirin for oCOX-1 inhibition (data not shown).
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Discussion |
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). So far, in spite of the ability of a large series of other compounds to inhibit COX-1, only aspirin has entered in the therapeutic armamentarium for thrombosis prevention.
Aspirin, however, has several limitations, including side effects at the gastrointestinal level, an antiplatelet activity limited to thromboxane-mediated activation, and little or no activity on atheroma formation. For these reasons, in the search of novel antithrombotic agents with wider activity, a NO-releasing aspirin (NCX 4016) has been developed by linking aspirin, through a spacer, to a NO-donating moiety (Del Soldato et al., 1999; Gresele et al., 2003). Nitroaspirin was found to exert anti-inflammatory and antithrombotic effects through multiple mechanisms, extending beyond the COX-1 pathway.
Our data show for the first time that NCX 4016 is able to directly and irreversibly inhibit COX-1, without a requirement for metabolic breakdown. NCX 4016, which is stable in buffer, is rapidly enzymatically degraded in a series of metabolites in plasma or in whole blood (Carini et al., 2004), and so far, it is still unknown whether the inhibition of COX-1 by NCX 4016 is exerted by the whole molecule or whether it requires metabolism with release of acetylsalicylic acid.
We have worked with an in vitro system with isolated, purified ovine COX-1 resuspended in a reaction buffer not containing plasma or cell fractions, thus excluding degradation of the drug by other enzymatic systems (glutathione S-transferase and aldehyde dehydrogenase), which could have accounted for the COX-inhibitory activity previously detected in vitro or in vivo in whole blood or in platelet suspensions (Lechi et al., 1996; Mezzasoma et al., 1999; Momi et al., 2000; Corazzi et al., 2003). In such a condition, it has previously been demonstrated that NCX 4016 is stable, without any degradation into aspirin and/or deacetylated or denitrated fragments (NicOx Research Institute, data on file; Carini et al., 2002). A further confirmation that NCX 4016 acts as a whole molecule derives from HPLC assays of the reaction mixtures, which showed that NCX 4016 was active on isolated, purified COXs without undergoing metabolism.
The inhibition of isolated COX-1 that we observed is compatible with acetylation of the enzyme, as demonstrated by dialysis experiments in which oCOX-1 that had been incubated with aspirin or NCX 4016 was still largely inhibited after removal of the drug by dialysis, similarly to aspirin. On the contrary, when the purified enzyme was incubated with indomethacin, a reversible, competitive inhibitor of COX-1, the suppression of enzymatic activity was no longer evident after dialysis, indicating that there had been no covalent modification of COX by indomethacin, as reported previously (Kulmacz and Lands, 1985). Furthermore, we observed that the inhibitory effect of NCX 4016 on COX is to a large extent antagonized by the preincubation of the enzyme with a competitive inhibitor (indomethacin) or with salicylic acid. This result is consistent with competitive inhibition by indomethacin of the access of NCX 4016 to the acetylation site of platelet COX-1 (Loll et al., 1995; Catella-Lawson et al., 2001) and represents one further suggestion that nitroaspirin acts on COX-1 with the identical mechanism of aspirin.
In agreement with this conclusion is the observation that among NCX 4016 analog/metabolites, only those containing the acetylsalicylic acid moiety suppressed the activity of platelet COX-1 and of monocyte COX-2, the latter under conditions preventing the degradation of the acetylated compounds by plasmatic esterases. The derivatives containing the NO-donating moiety also inhibited the activity of human monocyte COX-2 stimulated by LPS challenge of whole blood, an activity in part regulated by NO (Corazzi et al., 2003).
Therefore, it seems that in the NCX 4016 molecule, the hydroxybenzylalcohol spacer and the NO-donating moiety do not create a steric hindrance preventing NCX 4016 from reaching its target within the enzymatic channel of COX-1. However, some data suggest that they may delay it. In fact, in the experiments with isolated, purified oCOX-1, 50% inhibition of the enzyme activity was reached only after 9 min of incubation with NCX 4016 as opposed to 26 s with aspirin, suggesting a slower penetration of the molecule in the enzymatic channel of COX. Penetration of NCX 4016 through the cell membrane may to some extent be delayed, too, compared with aspirin, because experiments in whole blood showed an even longer time to maximal inhibition of platelet COX-1 with NCX 4016 than with aspirin.
In vivo studies in animals (Cuzzolin et al., 1996; Momi et al., 2005) as well as data in humans (Fiorucci et al., 2003) demonstrated that inhibition of serum TxB2 by NCX 4016 after oral administration is somewhat lower than with equimolar aspirin. This may be the consequence of a large preabsorptional deacetylation of the drug, with only relatively little NCX 4016 absorbed as such; of the longer time taken by NCX 4016 for platelet COX-1 inhibition; and of partial competition by larger amounts of deacetylated metabolites formed after NCX 4016 than of salicylate formed after aspirin administration (NicOx Research Institute, data on file; Fiorucci et al., 2003).
Indeed, the potential competition exerted by in vivo-generated salicylic acid or by deacetylated NCX 4016 derivatives (Carini et al., 2004) versus NCX 4016 for COX inhibition (de Gaetano et al., 1985) is a point worth considering. In our in vitro system, we observed that both salicylic acid and deacetylated NCX 4016 derivatives compete with NCX 4016 or aspirin, preventing, in part, the inhibition of COX-1. Although this competition may be of concern, it has previously been demonstrated that the combination of salicylate with aspirin, at therapeutically relevant dose ratios, may result in an almost complete dissociation of the effect of the drug on platelets and blood vessels by protecting primarily endothelial COX from aspirin inhibition (de Gaetano et al., 1985; Gambino et al., 1988). Considering that NCX 4016 seems to act on COX-1 with the identical mechanism of aspirin, it is conceivable that the effects of salicylate previously described with aspirin will be observed also with NCX 4016.
Although no unmetabolized NCX 4016 has been detected so far in peripheral blood after oral administration of the drug (Carini et al., 2004), the detection limits of the analytical methods used (approximately 1 µM) (Carini et al., 2004) are still compatible with the presence of amounts of NCX 4016 sufficient to inhibit COX-1 (Corazzi et al., 2003). Moreover, it is conceivable that some unmodified NCX 4016 may enter the mesenteric and preportal circulation and then undergo complete deacetylation in the liver. Thus, the demonstration that the whole NCX 4016 molecule affords irreversible inhibition of COX-1 is therapeutically relevant because the largest part of platelet COX-1 inhibition observed with low-dose aspirin takes place in the prehepatic circulation (Pedersen and FitzGerald, 1984; Cerletti et al., 1985; Giuliano and Warner, 1999). Further studies with blood sampling in different circulatory districts after oral administration of NCX 4016 need to address whether the COX-1 inhibitory profile of NCX 4016 found with isolated cells or purified enzymes is reproduced in vivo in the portal or systemic circulation.
In conclusion, NCX 4016 may act in vitro on COX-1 as a direct and irreversible inhibitor, similarly to aspirin, and simultaneously display a range of other actions related to NO.
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Acknowledgements |
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Footnotes |
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ABBREVIATIONS: COX, cyclooxygenase; NSAID, nonsteroidal anti-inflammatory drug; NO, nitric oxide; TxB2, thromboxane B2; LPS, lipopolysaccharide; PG, prostaglandin; oCOX, ovine cyclooxygenase; TMPD, N,N,N',N'-tetramethyl-p-phenylenediamine; HPLC, high-performance liquid chromatography; NCX 4016, benzoic acid, 2-(acetyl-oxy)-3-[(nitrooxy)methyl]phenyl ester; NCX 4019, 2-(hydroxy)-3-(hydroxymetyl)phenyl ester; NCX 4017, 2-(acetyloxy)-3-(hydroxymetyl) phenyl ester; NCX 4023, 2-(hydroxy)-3-[(nitrooxy)methyl]phenyl ester; NCX 4215, acetylsalicylic acid 4-(nitroxy)butyl ester; NCX 4015, benzenemethano-3 hydroxy--nitrate.
Address correspondence to: Dr. Paolo Gresele, Department of Internal Medicine, Division of Internal and Cardiovascular Medicine, University of Perugia, Via Enrico dal Pozzo, 06126, Perugia, Italy. E-mail: grespa{at}unipg.it
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), whereas it had only a minor effect on the inhibitory potency of aspirin (
) (*, p < 0.05 versus aspirin). Data represent mean ± S.E.M. (n = 5).
