Abstract
Nitric-oxide synthase, the enzyme responsible for mammalian nitric oxide generation, and cytochrome P450, the major enzymes involved in drug metabolism, share striking similarities. Therefore, it makes sense that cytochrome P450 drug mediated biotransformations might play an important role in the pharmacological modulation of nitric oxide synthase. In this work, we have undertaken an integrated in vitro assessment of the hepatic metabolism and nitric oxide modulation of previously described dual inhibitors (imidazoles and macrolides) of these enzymes in order assess the implication of CYP450 activities over production of nitric oxide. In vitro systems based in human liver microsomes and activated mouse macrophages were developed for these purposes. Additionally in vitro production the hepatic metabolites of dual inhibitor, roxithromycin, was investigated achieving the identification and isolation of main hepatic biotransformation products. Our results suggested that for some macrolide compounds, the cytochrome P450 3A4 derived drug metabolites have an important effect on nitric oxide production and might critically contribute to the pharmacological immunomodulatory activity observed.
Highlights
In a broad sense, nitric-oxide synthase (NOS) belongs to the CY450 family since the label cytochrome P450 (CYP450) has come to encompass a huge and widely distributed group of closely related enzymes containing a thiolate-ligated heme
The main objective of this work was to assess in vitro, the implication of the CYP450 activities in the modulation of nitric oxide (NO) production. In this regard we proposed a working hypothesis in which well-known compounds with the ability for inhibiting both activities (CYP450 and NOS) might display differential ability to inhibit NO production depending on the CYP450 functionality of the cell in vitro model considered
The results from the LPS stimulated RAW 264.7 macrophages in vitro model showed that the 3 members of the imidazole derivate family displayed moderate to weak inhibition potential in NO production (IC50 = 7.20, >10 μM and 7.35 μM, respectively, Table 1), in good agreement with previous works using cultured cell line models (Bogle and Vallance, 1996)
Summary
Nitric-oxide synthase (NOS) belongs to the CY450 family since the label cytochrome P450 (CYP450) has come to encompass a huge and widely distributed group of closely related enzymes containing a thiolate-ligated heme. Structurally different, they have a mechanistic resemblance since NOS converts L-arginine to L-citrulline and nitric oxide (NO) through a P450-like process and complementarily CYP450 was found able to transform N-hydroxy-L-arginine into NO and citrulline. The CYP450 system performs this function by oxidizing, hydrolyzing or reducing the chemicals This enables another group of enzymes, conjugation enzymes, to attach polar groups to make the metabolites water soluble so that they can be excreted in the urine (Martin and Fay, 2001). The biological effectiveness and the potential toxicity of NOS inhibitors might be strongly influenced by their CYP450 metabolism
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