Metabolism and pathways for denitration of organic nitrates in the human liver.

Liver first-pass metabolism differs considerably among organic nitrates, but little information exists on the mechanism of denitration of these compounds in hepatic tissue. The metabolism of nitrooxybutyl-esters of flurbiprofen and ferulic-acid, a class of organic nitrates with potential therapeutic implication in variety of different conditions, was investigated in comparison with glyceryl trinitrate (GTN) in human liver by a multiple approach, using a spontaneous metabolism-independent nitric oxide (NO) donor [3-(aminopropyl)-1-hydroxy-3-isopropyl-2-oxo-1-triazene (NOC-5)] as a reference tool. Nitrooxybutyl-esters were rapidly and quantitatively metabolized to their respective parent compounds and the organic nitrate moiety nitrooxybutyl-alcohol (NOBA). Differently from GTN, which was rapidly and completely metabolized to nitrite, NOBA was slowly metabolized to nitrate. In contrast to the spontaneous NO donor NOC-5, NOBA and GTN did not generate detectable NO and failed to suppress the activity of cytochrome P450, an enzyme known to be inhibited by NO. The direct identification of NOBA after liver metabolism targets this compound as the functional organic nitrate metabolite of nitrooxybutyl-esters. Moreover, the investigation of the pathways for denitration of NOBA and GTN suggests that organic nitrates are not primarily metabolized to NO in the liver but to different extents of nitrite or nitrate depending in their different chemical structure. Therefore, cytochrome P450-dependent metabolism of concomitant drugs is not likely to be affected by oral coadministration of organic nitrates. However, the first pass may differently affect the pharmacological profile of organic nitrates in connection with the different extent of denitration and the distinct bioactive species generated and exported from the liver (nitrate or nitrite).

Pharmacokinetic profile of defibrotide in patients with renal impairment.

Hepatic veno-occlusive disease, also called sinusoidal obstruction syndrome (VOD/SOS), is an unpredictable, potentially life-threatening complication of hematopoietic stem cell transplant conditioning. Severe VOD/SOS, generally associated with multiorgan dysfunction (pulmonary or renal dysfunction), may be associated with >80% mortality. Defibrotide, recently approved in the US, has demonstrated efficacy treating hepatic VOD/SOS with multiorgan dysfunction. Because renal impairment is prevalent in patients with VOD/SOS, this Phase I, open-label, two-part study in adults examined the effects of hemodialysis and severe or end-stage renal disease (ESRD) on defibrotide pharmacokinetics (PK). Part 1 compared defibrotide PK during single 6.25 mg/kg doses infused with and without dialysis. Part 2 assessed defibrotide plasma PK after multiple 6.25 mg/kg doses in nondialysis-dependent subjects with severe/ESRD versus healthy matching subjects. Among six subjects enrolled in Part 1, percent ratios of least-squares mean and 90% confidence intervals (CIs) on dialysis and nondialysis days were 109.71 (CI: 97.23, 123.78) for maximum observed plasma concentration (Cmax); 108.39 (CI: 97.85, 120.07) for area under the concentration-time curve to the time of the last quantifiable plasma concentration (AUC0-t); and 109.98 (CI: 99.39, 121.70) for AUC extrapolated to infinity (AUC0-∞). These ranges were within 80%-125%, indicating no significant effect of dialysis on defibrotide exposure/clearance. In Part 2, defibrotide exposure parameters in six subjects with severe/ESRD after multiple doses (AUC0-t, 113 µg·h/mL; AUC over dosing interval, 113 µg·h/mL; Cmax, 53.8 µg/mL) were within 5%-8% of parameters after the first dose (AUC0-t, 117 µg·h/mL; AUC0-∞, 118 µg·h/mL; Cmax, 54.9 µg/mL), indicating no accumulation. Defibrotide peak and extent of exposures in those with severe/ESRD were ~35%-37% and 50%-60% higher, respectively, versus controls, following single and multiple doses. One adverse event (vomiting, possibly drug-related) was reported. These findings support defibrotide prescribing guidance stating no dose adjustment is necessary for hemodialysis or severe/ESRD.

Chemiluminescence and LC-MS/MS analyses for the study of nitric oxide release and distribution following oral administration of nitroaspirin (NCX 4016) in healthy volunteers.

The metabolic fate of nitric oxide (NO) released from nitroaspirin, benzoic acid, 2-(acetyloxy)-3-[(nitrooxy)methyl]phenyl ester (NCX 4016), the lead compound of a new class of NO-releasing non-steroidal anti-inflammatory drugs (NO-NSAIDs) has been studied in eight healthy male Caucasian subjects following p.o. administration of 1600 mg (single dose), by monitoring at different times in plasma the bioactive storage forms of NO, S-nitrosothiols (RSNO) and its oxidation products (NOx). Plasma levels of NOx and RSNO and urinary levels of NOx were determined by an ozone-based chemiluminescent assay using a sensitive Nitric Oxide Analyzer (LOQ: 10 pmol NO injected). In parallel plasma samples were analyzed by a newly developed LC-MS/MS method for analysis of NCX 4015, the metabolite bearing the nitrate ester function. Using MS/MS with multiple reaction monitoring (MRM) in negative ion mode for NCX 4015 and the internal standard (NCX 4015- 13C-D2) it was possible to detect with sufficient accuracy and precision the metabolite in plasma with a quantification limit of 78.1 ng ml(-1). Concentration versus time profile of plasma NCX 4015 gave a Cmax value of 161.94 +/- 47.4 ng ml(-1) and a tmax 4.5 +/- 1 h. The results indicate that both NOx and RSNO (these last for the first time determined in vivo in man following oral administration of a NO-donor drug) are effective plasma markers of NO release in vivo, the latter being an earlier indicator of NO distribution (tmax 2.0 +/- 0.6 h versus 5.4 +/- 1.2 h).

In vitro metabolism of (nitrooxy)butyl ester nitric oxide-releasing compounds: comparison with glyceryl trinitrate.

We investigated the in vitro metabolism of two (nitrooxy)butyl ester nitric oxide (NO) donor derivatives of flurbiprofen and ferulic acid, [1,1'-biphenyl]-4-acetic acid-2-fluoro-alpha-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.

Treatment of portal hypertension with NCX-1000, a liver-specific NO donor. A review of its current status.

Portal hypertension, a life threatening complication of liver cirrhosis, results from increased intrahepatic resistance and increased portal blood inflow through a hyperdynamic splanchnic system. The increased intrahepatic vascular tone is the result of an enhanced activity of endogenous vasoconstrictors and a deficiency of nitric oxide (NO) release by sinusoidal endothelial cells. These pathophysiological events provide the rational basis for using NO-based therapies for the treatment of portal hypertension. Clinical studies have demonstrated that nitrate therapy results in a significant reduction of portal pressure as assessed by hepatic venous portal gradient but causes vasodilation in both systemic arterial and venous vascular beds, aggravating the progression of the vasodilatory syndrome of cirrhotic patients. For this reason, the ideal drug for the treatment of portal hypertension should act by decreasing intrahepatic vascular resistance, without worsening the splanchnic/systemic vasodilatation. NCX-1000 is the prototype of a family of NO-releasing derivatives of ursodeoxycholic acid (UDCA). These compounds are releasing selectively, from parenchymal and non-parenchymal hepatic cells, biologically active NO into the liver microcirculation with no detectable effect on systemic circulation. Preclinical studies have shown that long- and short-term administration of NCX-1000 to rodents with chronic liver injury protects against the development of portal hypertension and reduces the intrahepatic hyperreactivity to alpha1-adrenoceptor agonists. The finding of increased liver nitrite/nitrate content in NCX-1000-treated animals together with an increase in cGMP levels in their liver homogenates suggests that this nitro-compound behaves as a liver-selective NO donor. In contrast to conventional NO-donors such as isosorbide mono- and di-nitrate, which are also used for primary and secondary prevention of gastrointestinal bleeding, NCX-1000 has no effect on mean arterial pressure in either normal or cirrhotic animals indicating the absence of adverse systemic effect. In summary, these data suggest that NCX-1000 may provide a novel therapy for the treatment of patients with portal hypertension.