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ABSORPTION, DISTRIBUTION, METABOLISM, AND EXCRETION
Departments of Drug Metabolism and Pharmacokinetics (M.G., S.C., R.M., P.T.) and Medicinal Chemistry (V.C.), NicOx Research Institute, Milan, Italy
Received October 21, 2005; accepted January 18, 2006.
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Abstract |
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-methyl-4-(nitrooxy)butyl ester (HCT 1026) and 3-(4-hydroxy-3-methoxyphenyl)-2-propenoic acid 4-(nitrooxy)butyl ester (NCX 2057), respectively, in rat blood plasma and liver subcellular fractions compared with (nitrooxy)butyl alcohol (NOBA) and glyceryl trinitrate (GTN). HCT 1026 and NCX 2057 undergo rapid ubiquitous carboxyl ester hydrolysis to their respective parent compounds and NOBA. The nitrate moiety of this latter is subsequently metabolized to inorganic nitrogen oxides (NOx), predominantly in liver cytosol by glutathione S-transferase (GST) and to a lesser extent in liver mitochondria. If, however, in liver cytosol, the carboxyl ester hydrolysis is prevented by an esterase inhibitor, the metabolism at the nitrate moiety level does not occur. In blood plasma, HCT 1026 and NCX 2057 are not metabolized to NOx, whereas a slow but sustained NO generation in deoxygenated whole blood as detected by electron paramagnetic resonance indicates the involvement of erythrocytes in the bioactivation of these compounds. Differently from NOBA, GTN is also metabolized in blood plasma and more quickly metabolized by different GST isoforms in liver cytosol. The cytosolic GST-mediated denitration of these organic nitrates in liver limits their interaction with other intracellular compartments to possible generation of NO and/or their subsequent availability and bioactivation in the systemic circulation and extrahepatic tissues. We show the possibility of modulating the activity of hepatic cytosolic enzymes involved in the metabolism of (nitrooxy)butyl ester compounds, thus increasing the therapeutic potential of this class of compounds.
). However, there has been an explosion of activity in the area of hybrid nitrates over the past decade, stimulated by a growing realization that nitrates may represent new therapeutic agents in different areas (Keeble and Moore, 2002). Despite this, the metabolism of several organic nitrates is still to be elucidated. The exact mechanism whereby nitric oxide (NO) is generated from organic nitrates is still unknown, and several mechanisms have been proposed and rejected. Although nonenzymatic pathways involving endogenous sulfhydryl groups (Ignarro et al., 1980) or hemoglobin (Bennett et al., 1986; Cosby et al., 2003) have been suggested to mediate the biotransformation to NO, the attention has also shifted to an enzyme-catalyzed mechanism (Needleman, 1976). Several enzymes have been proposed to be directly or indirectly involved in the generation of NO from organic nitrates, as the cytosolic glutathione S-transferase (GST) (Keen et al., 1976; Taylor et al., 1989; Kurz et al., 1993) and xanthine oxidoreductase (XO) (Millar et al., 1998; Doel et al., 2001), the mitochondrial aldehyde dehydrogenase (Chen et al., 2002; DiFabio et al., 2003; Kollau et al., 2005) and cytochrome bc1 (Nohl et al., 2000), or the microsomal cytochrome P-450 (P450) (Servent et al., 1989; McDonald and Bennett, 1990; Schroder, 1992). Most likely this mechanism is not identical in all tissues or biological mediums, and a cooperation between different enzymes and intracellular compartments is likely to occur (Kozlov et al., 2003). The first metabolic step of organic nitrates was generally thought to be the release of NO, which is consequently oxidized to nitrite (NO2-) and nitrate (NO3-). On these bases, the main metabolic products of NO (NO2- + NO3-) were used to quantify the NO coming from organic nitrates. However, recent findings suggest that biotransformation of organic nitrates should proceed via two steps, where NO2- is an intermediate and precursor of NO rather than a product of NO degradation (Millar et al., 1998; Chen et al., 2002; Kozlov et al., 2003). Cyclooxygenase-inhibiting nitric oxide donators (CINODs) are a novel group of compounds with potential therapeutic applications in a variety of clinical conditions. The general structure of such molecules is a NO-releasing moiety (-ONO2) connected via a linker to the parent compound by a carboxyl ester bond. CINODs with different linkers but the same parent compound have also been developed to modulate the extent of NO release within the same pharmacological target. Although an extensive exploration of pharmacological activities of these compounds in different pharmacological models has been carried out (Keeble and Moore, 2002), a structure-activity relationship in association with the metabolism and NO-releasing characteristics has still not been completely elucidated.
In this article, we investigated the metabolism and associated enzymes of two NO-releasing compounds currently under development for the treatment of Alzheimer's disease, [1,1'-biphenyl]-4-acetic acid 2-fluoro--methyl-4-(nitrooxy-)butyl ester (HCT 1026) and 3-(4-hydroxy-3-methoxyphenyl)-2-propenoic acid 4-(nitrooxy)butyl ester (NCX 2057), (nitrooxy)butyl ester derivatives of flurbiprofen and ferulic acid, respectively (Wenk et al., 2002, 2004; Prosperi et al., 2004). These compounds bear the same aliphatic butyl linker between the parent compound and the NO-releasing group. The role of the aliphatic linker has been studied using (nitrooxy-)butyl alcohol (NOBA), and its metabolic properties were compared with those of the classic NO donor GTN.
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Materials and Methods |
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-methyl,4-hydroxybutyl ester (HCT 1027) and 3-(4-hydroxy-3-methoxyphenyl)-2-propenoic acid 4-hydroxybutyl ester (NCX 2059) (Fig. 1) were synthesized at NicOx Laboratories, Department of Medicinal Chemistry (Milan, Italy). HPLC-grade organic solvents were purchased from Carlo Erba Reagents (Milan, Italy). HPLC-grade water was prepared with a Milli-Q water purification system. GTN and NOBA were kindly provided by Dipharma SpA (Milan, Italy). All other chemicals were purchased from Sigma-Aldrich (Milan, Italy).
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). Liver fraction incubations were performed using protein concentrations reflecting the protein content of the different subcellular fractions in 1 ml of original liver homogenate (Kozlov et al., 2003) (microsomes, 1 mg · ml-1; mitochondria, 1.3 mg · ml-1; and cytosol, 4.1 mg · ml-1). The following cofactor mixtures were used for each incubation: microsomes, 1 mM NADPH; mitochondria, 1 mM NADPH, 0.5 mM NAD+, and 0.5 mM NADH; cytosol, 1 mM NADPH, 0.5 mM NAD+, 0.5 mM NADH, and 2.5 mM reduced glutathione.
HCT 1026, NCX 2057, NOBA, and GTN as well as the inhibitors ethacrynic acid (EA), bromosulfophthalein (BSP), N-ethylmaleimide (NEM), and tetraisopropyl pyrophosphoramide (isoOMPA) were dissolved in acetonitrile, dimethyl sulfoxide, or water and added to the incubation (final solvent concentration 
1% v/v). Drugs were incubated in 0.1 M phosphate buffer, pH 7.4, with rat liver fractions or in rat blood plasma at 37°C under shaking. Inhibitors were added 15 min before drug incubation. At fixed times, 200 µl of incubation mixture was removed and deproteinized with 400 µl of acidified acetonitrile (acetonitrile + 0.5% phosphoric acid, v/v) for HPLC analysis. Another 300 µl of the incubation mixture was removed at the same time for chemiluminescence analysis.
Gas-Phase Chemiluminescence Assay. The total concentrations of inorganic nitrogen oxides (NO2- + NO3- + nitros(yl) species = NOx) and NO2- were determined by gas-phase chemiluminescence with a nitric oxide analyzer (NOA 280I; Sievers, Boulder, CO) after reductive cleavage and subsequent determination of the NO released into the gas phase. The apparatus was described in detail by Lundberg and Govoni (2004). NO signals were collected with NO analysis software for Windows (version 3.2; Ionic Instrument Business Group, Boulder, CO), further manipulated with Origin for Windows, version 7.0 (Microcal, Northampton, MA), and reported as area under the curve.
NOx were reduced to NO with a solution of vanadium(III) chloride in 1 M hydrochloric acid (saturated solution) at 95°C. In this condition, organic nitrates eventually present in the sample do not convert to NO or, at worst, they slightly convert, increasing the baseline but not affecting the shape and the recovery of NOx. Despite this, samples were extracted before analysis with chloroform to remove organic nitrates potentially present and stabilize the baseline and deproteinized with ice-cold ethanol.
Nitrites were reduced to NO with a solution consisting of 45 mM potassium iodide and 10 mM iodine in glacial acetic acid at 60°C. Samples were directly injected into the reducing solution without pretreatment. The methods for NOx and NO2- determination have been described previously by Lundberg and Govoni (2004).
HPLC Analysis. Liquid chromatography analyses were carried out on an Agilent 1100 system (Agilent Technologies, Palo Alto, CA) equipped with an UV-visible diode array programmable detector. Separations were achieved by reverse phase elution with a Synergi Hydro-RP 80 Å column (150 x 4.6 mm i.d., particle size 4 µm; Phenomenex, Torrance, CA) equipped with an AQ-C18 precolumn (4 x 3 mm i.d.) maintained at 25°C. The mobile phase, acetonitrile (solvent A) and 25 mM sodium phosphate buffer, pH 2 (solvent B), was delivered at a flow rate of 1 ml · min-1. HCT 1026 and its metabolites were detected at 
= 246 nm with the following gradient compositions: 0 min, 20% A; 1 to 8 min, 80% A; and 9 to 11 min, 20% A. Under these conditions the retentions times for HCT 1026, HCT 1027, and flurbiprofen were 6.8, 4.7, and 4.2 min, respectively.
NCX 2057 and its metabolites were detected at = 325 nm with the following gradient composition: 0 min, 20% A; 4 to 8 min, 80% A; and 10 to 12 min, 20% A. Under these conditions, the retentions times for NCX 2057, NCX 2059, and ferulic acid were 6.1, 4.6, and 4.1 min, respectively. NCX 2057, HCT 1026, and their metabolites were chromatographically separated and characterized on the basis of the typical UV fingerprint of the pure compounds. Chromatographic peaks were processed for spectral purity characterization by statistical analysis for automated comparison of spectra (Agilent Chemstation software). Only peaks showing spectral matching factors higher than 99% were considered acceptable. Chromatograms were integrated with Agilent Chemstation software, and the area under the curve was converted into concentration using reference compounds.
In Vitro Incubations in Deoxygenated Whole Blood and Electron Paramagnetic Resonance Analysis. Test drugs were incubated to a final concentration of 100 µM in venous deoxygenated rat blood and maintained at 37°C under anaerobic conditions. In this environment, NO avidly binds to deoxyhemoglobin (K = 2.6 x 107 M-1 · s-1), leading to the formation of a stable high-affinity paramagnetic complex, nitrosyl hemoglobin [HbFe(II)NO], that produces a distinctive EPR spectrum (Stamler et al., 1997; Gladwin et al., 2000). The concentration of HbFe(II)NO in the incubation mixture reflects the quantity of nitric oxide accumulated at different times. The apparatus and method were described in detail by Wenk et al. (2004) and Ongini et al. (2004).
Kinetics and Data Analysis. Values for Michaelis-Menten Vmax and Km determinations were obtained from Lineweaver-Burk double-reciprocal plots of velocities (V0) versus substrate concentrations using three to four concentrations (50, 100, 250, and 500 µM) in triplicates. NOx values in the incubations were subtracted from the basal (time 0) and reported as means ± S.D. One-way analysis of variance followed by a Tukey post hoc test was used to evaluate differences between groups. Differences were considered significant at p = 0.05.
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Results |
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Metabolism of GTN in Rat Blood Plasma and Liver Subcellular Fractions. GTN incubated in blood plasma at a concentration of 250 µM showed a very rapid NOx formation (V0 = 12.8 ± 2.5 nmol · ml-1 · min-1), achieving an almost complete conversion to NOx species in 2 h of incubation (Fig. 3C). In liver microsomes and in liver mitochondria, GTN showed an increase of the NOx levels just in the first minutes of incubation (
16% in relation to one nitrate moiety of GTN) and remained stable afterward. In contrast, in liver cytosol GTN was very quickly and completely metabolized to NOx species (80% within 15 min of incubation, V0 = 4.5 ± 0.15 nmol · mg-1 · min-1). NOx levels reached a complete conversion to NOx species after 1 h of incubation and did not change significantly up to 6 h of incubation (Fig. 3C).
GTN was stable in boiled deactivated cytosol (data not shown). Enzymatic Michaelis-Menten parameters measured in cytosol incubations for the formation of NOx reflected the fast metabolism at the nitrate moiety level of GTN in comparison to NOBA (Vmax GTN >> Vmax NOBA) (Table 1). The kinetics of NOx generation at the different GTN concentrations used for Km and Vmax calculations are depicted in Fig. 3D.
Effect of isoOMPA on the Metabolism of HCT 1026 and NCX 2057 in Liver Cytosol. A selective inhibitor of butyrylcholinesterase, isoOMPA, incubated at the concentration of 2.5 mM in liver cytosol inhibited 1) the metabolism of HCT 1026 and NCX 2057 (incubated at a final concentration of 250 µM) to flurbiprofen and ferulic acid, respectively, and 2) the metabolism of the nitrate moiety to NOx species. Moreover, HCT 1027 and NCX 2059 were never observed during the incubation time. In particular, as shown in Fig. 4A, the concentration of HCT 1026 was less than 20% reduced after 6 h of incubation with the inhibitor, whereas the metabolites flurbiprofen and NOx did not exceed 16% of formation.
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Nitric Oxide Generation from HCT 1026 and NCX 2057 in Whole Deoxygenated Blood. HCT 1026 and NCX 2057 incubated in deoxygenated whole blood at a concentration of 100 µM produced a slow and sustained HbFe(II)NO accumulation (Fig. 5A). HbFe(II)NO formation from HCT 1026 and NCX 2057 was not significantly different, ranging from 0.2 ± 0.1 to 29.8 ± 5.6 µM for HCT 1026 and from 0.7 ± 0.5 to 28.0 ± 6.7 µM for NCX 2057 after 15 min and 4 h of incubation, respectively (Fig. 5B). Analogously, the initial rate (V0) of HbFe(II)NO formation accounted for 29.2 nmol · min-1 and 27.6 nmol · min-1 for HCT 1026 and NCX 2057, respectively. The effects could not be attributed to endogenous NO production, because the vehicle was devoid of any effect under these experimental conditions.
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Discussion |
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; Keeble and Moore, 2002). Moreover, previous observations demonstrated that the fast biotransformation of HCT 1026 in S9 liver fraction incubations was not influenced by the presence or absence of cofactors (S. C. Casagrande, M. G. Govini, R. M. Maucci, and P. T. Tocchetti, unpublished data). This finding indicated that the initial carboxyl ester hydrolysis of the compound is not P450-mediated and might result from the action of carboxyl esterases.
Our results related to the metabolism of HCT 1026, NCX 2057, and NOBA in the presence of a selective inhibitor of butyrylcholinesterase (Fig. 4) allow us to conclude that 1) the first fast and extensive carboxyl ester hydrolysis to the respective parent compound and NOBA is esterase-dependent, 2) the metabolism at the nitrate moiety level is not esterase-mediated, and 3) this latter result does not occur unless carboxyl ester hydrolysis takes place. Justifying these results are the assertions that 1) the first carboxyl ester metabolic step leading to the formation of the parent compound and NOBA is essential for the metabolism of the NO-releasing moiety and 2) NOBA is the active metabolite retaining NO bioactivity. As also supported by similar metabolism at the nitrate moiety level of NOBA (Fig. 3, A and B) in comparison with that of HCT 1026 and NCX 2057 (Fig. 2) and by an analysis of the Michaelis-Menten parameters in the cytosolic fraction (Table 1), NOBA is thus the compound to note when searching for NO-releasing properties and associated enzyme(s) involved with (nitrooxy)butyl ester NO-releasing compounds such as HCT 1026 and NCX 2057.
Direct evidence of NO generation from HCT 1026 and NCX 2057 has been obtained by using EPR. Both HCT 1026 and NCX 2057 exhibited the same profile of HbFe(II)NO formation, suggesting that the final transformation to bioactive NO comes from the same chemical entity. In effect, the fast carboxyl ester hydrolysis metabolism of HCT 1026 and NCX 2057 with subsequent formation of NOBA and the stability of the latter in blood plasma might allow NOBA to reach erythrocytes and be transformed into bioactive NO within this compartment. This is a further confirmation of the key role played by NOBA in the delivery and biotransformation of this class of compounds. Moreover, this finding demonstrates that an active role is played by erythrocytes in the bioactivation of (nitrooxy)butyl ester compounds and suggests that hemoglobin is the possible mediator of this biotransformation (Bennett et al., 1986; Cosby et al., 2003).
Although the chemical structures of the NO-releasing moieties of GTN and NOBA are identical (-ONO2), it is clear that the NO-donating characteristics are different. As also demonstrated by our results, a comparison between the in vitro metabolism of NOBA and GTN showed significant differences both in terms of specificity to the incubation matrices and extent of metabolic products (NOx) formed over the incubation time (Fig. 3, A and C). In particular and differently from NOBA, the rapid metabolism at the nitrate moiety level of GTN in blood plasma might be related to the rapid fall of systemic blood pressure associated to this NO-donor drug.
Recently, the hypothesis that the first metabolic step of organic nitrates is a direct enzymatic bioactivation to NO with a consequent rapid oxidation of this latter to NOx has been replaced by evidence of a direct 1e- reduction to NO2- (Chen et al., 2002; Kozlov et al., 2003). NO2- can then be nonenzymatically or enzymatically converted into bioactive NO (Lundberg and Weitzberg, 2005) and/or can be oxidized to NO3-. In view of this latter mechanism of NO generation, the measurement of NOx species gives information regarding the extent of the first metabolic conversion to species (NO2-) retaining potential NO bioactivity.
In liver, NOBA is metabolized to NOx mainly in the cytosolic fraction and to a minor extent in the mitochondrial fraction. A negligible metabolism to NOx species occurs in microsomes, and this leads to exclusion of the superfamily of P450 as species involved in a direct catalytic activity of the nitrate moiety of (nitrooxy)butyl ester compounds. Instead, a role in the metabolism of NOBA is played by mitochondria as recently reported also for GTN (Chen et al., 2002; DiFabio et al., 2003; Kollau et al., 2005).
Considering the cytosolic enzymes, XO has recently been reported to catalyze the anaerobic reduction of GTN to NO2- and then to NO (Millar et al., 1998; Doel et al., 2001). However, incubations of NOBA or GTN in liver cytosol in the presence of an excess of a selective inhibitor of XO did not change the metabolism of these compounds (Fig. 7). Thus, XO, in oxygenated conditions, seems not to be directly involved in the metabolism of the nitrate moiety of both (nitrooxy)butyl ester compounds and GTN.
Because GTN metabolism is mediated by a cytosolic glutathione-dependent organic nitrate reductase (Needleman, 1976), our evidence that an (-SH)-alkylating agent such as NEM in liver cytosol inhibited the metabolism to NOx species of NOBA and GTN (Fig. 6, C and D) and that the metabolism of both compounds was inhibited in a concentration-dependent manner by BSP (Fig. 8, A and C) is a clear demonstration of the involvement of GST in the direct metabolism of the nitrate moiety. Interestingly though, another widely used inhibitor of GST, EA, did not affect the metabolism of NOBA but still inhibited the metabolism of GTN (Fig. 8, B and D).
Rat liver is a very complex tissue containing at least 14 GST isoenzymes belonging to the alpha, pi, mu, and theta classes. However, the alpha and mu GST classes are widely represented (56 and 43%, respectively) (Turella et al., 2003).
BSP and EA exhibit different inhibition values (Ki) on the alpha and mu GST isoforms of rat liver (Singhal et al., 1996). Differently from that of GTN, the negligible inhibition of EA on liver cytosolic NOBA metabolism suggests a prevalent metabolic activity of one of these two isoforms rather then a wider involvement of different GST isoforms.
The major rat hepatic cytosolic GST isoforms are not normally present in blood plasma and are released in blood only after liver damage (Igarashi et al., 1988). Differently from NOBA, the evidence of an extensive metabolism at the nitrate moiety level of GTN in blood plasma suggests the involvement of other enzymes (in addition to GST) in the direct denitration of this drug.
Kozlov et al. (2003) demonstrated that in liver GTN is directly metabolized to NO2- in the cytoplasm, and NO2- can be subsequently metabolized to NO in the mitochondria or endoplasmic reticulum by different enzymes. The lack of physiological response of GTN in liver could be explained by the difficulty of NO2- to reach other subcellular compartments also because of its rapid oxidation to NO3- (Ignarro et al., 1993) as, in fact, is evidenced by the analysis of the ratio NO2-/NO3- in our cytosolic incubations. We identified the cytosolic enzyme involved in the transformation of GTN and NOBA to NOx species as GST. GST had already been shown to be involved in the metabolism of GTN in vascular tissue. However, this enzyme was demonstrated to be capable of reduction to NO2- but not to NO (Kurz et al., 1993). Thus, the cytosolic GST-mediated denitration of these organic nitrates in liver might partially prevent the compounds from reaching other subcellular compartments (such as mitochondria) or tissues intact and being transformed into bioactive NO. For these reasons, GST might be seen as an enzyme responsible for the deactivation of organic nitrates in cytosol, acting, at least in liver, as a scavenger of NO bioavailability.
We demonstrated that, differently from GTN, NOBA seems to be more specifically metabolized by GST (alpha or mu isoform). Moreover, although GTN is completely and rapidly converted to NOx species by cytosolic GST, NOBA is consistently more slowly metabolized in a linearly dependent manner. The slower liver cytosolic metabolism of NOBA, compared with that of GTN, might allow this compound to reach (at least in part) the subcellular compartment (such as mitochondria) capable of bioactivation to NO or diffuse back into blood, being slowly converted to nitrosyl hemoglobin within erythrocytes and/or reach the vascular system and exert the NO action. Moreover, selective inhibition of GST might decrease the scavenging effect on these organic nitrates in liver, suggesting the possibility of increasing the bioavailability of NO in other tissues.
In conclusion, it is likely that the mechanism of NO generation from organic nitrates depends on the location in which the metabolism occurs, from pathological conditions and varies in relation to the chemical nature of the organic nitrate. Although these aspects complicate the search for the mechanisms of NO generation, the therapeutic potential of these drugs will not be unlocked until a clear identification of the metabolic steps by which they finally provide NO is established.
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Footnotes |
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ABBREVIATIONS: GTN, glyceryl trinitrate; NO, nitric oxide; NO2-, nitrite; NO3-, nitrate; GST, glutathione S-transferase; XO, xanthine oxidoreductase; P450, cytochrome P-450; CINOD, cyclooxygenase-inhibiting nitric oxide donator; HCT 1026, [1,1'-biphenyl]-4-acetic acid 2-fluoro--methyl,4-(nitrooxy)butyl ester; NCX 2057, 3-(4-hydroxy-3-methoxyphenyl)-2-propenoic acid 4-(nitrooxy)butyl ester; NOBA, (nitrooxy)butyl alcohol; HCT 1027, [1,1'-biphenyl]-4-acetic acid 2-fluoro--methyl,4-hydroxybutyl ester; NCX 2059, 3-(4-hydroxy-3-methoxyphenyl)-2-propenoic acid 4-hydroxybutyl ester; HPLC, high-performance liquid chromatography; EA, ethacrynic acid; BSP, bromosulfophthalein; NEM, N-ethylmaleimide; isoOMPA, tetraisopropylpyrophosphoramide; EPR, electron paramagnetic resonance; NOx, inorganic nitrogen oxide(s); HbFe(II)NO, nitrosyl hemoglobin.
Address correspondence to: Dr. Mirco Govoni, Department of Drug Metabolism and Pharmacokinetics, NicOx Research Institute, Via Ariosto 21, 20091, Bresso, Milan, Italy. E-mail: govoni{at}nicox.it
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). 
, kinetics of flurbiprofen and ferulic acid formation from HCT 1026 and NCX 2057, respectively, whereas the direct denitrated metabolites HCT 1029 and NCX 2059 were not observed over the incubation time either in blood plasma or in liver subcellular fractions. 
, time course of NOx species generated over the incubation time. NOx values are subtracted from basal (time 0). Data are presented as means ± S.D., n 
3.
), and liver cytosol (
). B and D, kinetics of NOx generation at different NOBA (B) and GTN (D) concentrations (50, 100, 250, and 500 µM) in liver cytosol. Values are subtracted from basal (time 0). Data are presented as means ± S.D., n 
) or absence (
