Organic nitrates metabolism in the liver

biological effect of NO3-. In fact, nitrate can be converted to nitrite in vivo resulting in increased NO2- plasmatic levels. Nitrite in turn is liable of biological effect trough serial reduction to bioactive NO in blood and tissues. This newly described nitrate-nitrite-NO pathway represents a fundamentally different mechanism for the generation of NO in mammals that complement the L-arginine/NOS pathway along the physiological and pathological oxygen and proton gradients (Fig. 24).

responsible for the deactivation of organic nitrates, acting, at least in liver, as an acute scavenger of NO bioavailability.

The “high hepatic clearance” organic nitrate GTN undergoes extensive liver first-pass metabolism¹¹⁹ at one of the three nitrate moieties leaving a dinitrate (GDN) which, having a longer half life circulates systemically¹¹⁷. Despite its extensive first pass metabolism GTN can still be administered by oral route for prophylactic purposes. In our experimental setting GTN was rapidly denitrated in the liver to inorganic nitrite by a clearance based GST-mediated mechanism. This implies that both the combination of GDN and the inorganic nitrite that would be generated during first passage could account for the effect of this compound when taken by oral route for prophylaxis purposes. As already stressed in the first part of the thesis nitrite is in fact emerging as a stable circulating NO pool and a variety of pathways have been described for the reduction of nitrite to NO in blood and tissues³⁸. Such NO formation is expected to be slower and more prolonged than direct release of NO from most NO donors. In addition, NO formation from nitrite is heavily dependent on oxygenation, pH and redox milieu meaning that generation is greatly enhanced in hypoxic or ischaemic areas²¹⁴. Inorganic nitrite is strongly protective in animal models of ischaemia-reperfusion injury and therefore the potential use of nitrite as a therapeutic agent for the treatment of cardiovascular diseases is being considered. A limitation of nitrite therapy is however that NO2- supplementation by the oral route may cause loss of bioavailability due to considerable conversion of nitrite to NO and reactive nitrogen intermediates under the acidic gastric conditions⁶⁴. Our results suggest the interesting possibility of using high hepatic clearance organic nitrates, by oral route to deliver nitrite distal to the liver in different pathological conditions like hypertension, stroke and myocardial infarction.

Nitrooxybutyl-ester compounds are organic nitrates structurally different from GTN. In our attempt to investigate their metabolism we found that in the liver the two derivatives of flurbiprofen and ferulic acid are rapidly cleaved to the respective parent compounds and the NO-bearing organic nitrate moiety NOBA by the action of hepatic esterases (Fig. 25). This latter is therefore the compound retaining the “NO like”

activity of this class of compounds. Interestingly, NOBA shows an intermediate degree of metabolism (GTS mediated denitration to NOx) between the “high hepatic clearance” organic nitrate GTN and the “low hepatic clearance” compound ISMN. The slower metabolism of NOBA in the liver compared with that of GTN might allow this compound to overcome the liver first passage and reach (at least in part) the systemic circulation and being bioactivated to NO in the vascular compartment, red blood cells or elsewhere where a different enzymatic pool than in the liver exists. Moreover the NOx species generated during first passage could also contribute to “NO like” effect of these compounds.

Interestingly, in a recent work it was demonstrated that inorganic nitrate given to rats for 1 week in the drinking water, dose dependently inhibited the acute gastric injury and inflammatory response caused by administration of the non-steroidal anti-inflammatory drug diclofenac²¹⁵. This effect can be attributed to conversion of nitrate to nitrite and then NO in the GI track. It follows that the NOx generated from nitrooxybutyl-ester derivatives of flurbiprofen or ferulic acid during first passage could protect the stomach and greatly contribute to their final biological effect.

The absence of intrahepatic generation of NO from organic nitrates as suggested by the results of the present thesis provides potentially important information for the development of new compounds and especially for those undergoing liver first pass metabolism or for molecules specifically designed to deliver NO to this organ. NO is involved in the inactivation of CYP^144,145, therefore generation of NO during first

passage from organic nitrates given in association with other CYP metabolism-dependent drugs could lead to severe drug interactions. Our results suggest that CYP-dependent drug metabolism of concomitant drugs should not be affected by oral co-administration of organic nitrates. On another hand in particular conditions NO production in the liver might be beneficial. Physiologically the liver plays a central role in the metabolism and immune processes, and is a major target organ influenced by NO. NO production may be decreased during chronic liver diseases. A variety of pharmacological direct NO releasing prodrugs have been developed, and, when used appropriately, most of them have shown beneficial effects in the liver in a variety of pathological settings¹⁴⁶. Due to their partially established safety profile the possibility to develop organic nitrates as therapeutic agents in the liver may come obvious from these observations. However the result of our metabolic studies cast doubts on the usefulness of organic nitrates as possible therapeutic NO donor agents in the liver.

In conclusion, the biological effect of different organic nitrates will certainly depend from the location in which the metabolism occurs and the concomitant production of nitrite and nitrate can contribute significantly to their final pharmacological profile. It follows that the therapeutic potential of organic nitrates will not be unlocked until a clear identification of the mechanism of denitration in the different tissues is fully established.

Fig. 25. Metabolism of nitrooxybutyl ester derivatives of flurbiprofen and ferulic acid in the liver. HCT 1026 and NCX 2057 are rapidly cleaved to the respective parent compounds and NOBA by the action of hepatic esterases.

The slower metabolism of NOBA to NOx species might allow this compound to reach the systemic circulation and be bioactivated to NO in vascular tissue, red blood cells or elsewhere. The NOx generated during first pass could be recycled to bioactive NO through the nitrate/nitrite/NO pathway contributing to the biological effect of these molecules.

6 CONCLUSIONS

PART I (PAPERS I, II, II)

 Inorganic nitrate is a substrate for systemic generation of nitrite and eventually nitric oxide in humans.

 Salivary ingestion following the enterosalivary recirculation of inorganic nitrate is fully responsible for the systemic nitrite generation after nitrate intake in humans.

 In the mouth, inorganic nitrate taken up by salivary glands is converted to nitrite by bacterial nitrate reductases and swallowed with saliva. This pathway is the major source for systemic nitrite increase after an oral nitrate intake in humans (prokaryotic pathway).

 Mammalian cells possess nitrate reductase activity.

 The enterosalivary recirculation of inorganic nitrate and saliva ingestion maximize exposure of inorganic nitrate in the gut and liver where the mammalian nitrate reductase activity is maximal. This pathway contributes to systemic generation of nitrite after nitrate intake in humans (eukaryotic pathway)

 Under basal conditions ingestion of saliva following the enterosalivary recirculation of inorganic nitrate in humans could be a major source of endogenous circulating nitrite.

 Mechanistically the findings of the present thesis can explain the strong biological effect of inorganic nitrates and suggest a role for this anion in the protective effect of vegetables.

PART II (PAPERS IV, V)

 Organic nitrates are metabolized in the liver exclusively by the cytosolic enzyme GST through a clearance based mechanism leading acutely to the formation of NOx but not NO.

 In the liver nitrooxybutyl-ester derivatives of flurbiprofen and ferulic acid are rapidly cleaved by esterases to the respective parent compounds and the organic nitrate moiety NOBA.

 NOBA is therefore the compound retaining the “NO like” activity of nitrooxybutyl-ester derivatives of flurbiprofen and ferulic acid.

 Differently from GTN, NOBA is only slowly denitrated by GST in the liver and therefore it has the potential to survive (at last in part) liver first pass metabolism and be bioactivated in other tissues.

 The generation of NOx species from organic nitrates during first pass metabolism could greatly contribute to the “NO like” effect of these compounds when taken by oral route.

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