The glucuronidation of all UGT1A1 substrates is likely to be impaired in subjects carrying the UGT1A1*6 and UGT1A1*62 alleles, although the reduction in metabolic clearance might vary with the substrate. The Y486D mutation appears to greatly reduce most, but not all, UGT1A activities.

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Studies were conducted to evaluate the potential mechanismbased inactivation of recombinant and human liver microsomal CYP2C8 by clinically used drugs. Several tricyclic antidepressants, calcium channel blockers, monoamine oxidase inhibitors, and various other known CYP3A4 inhibitors exhibited greater inhibition of CYP2C8 (paclitaxel 6␣-hydroxylation) following preincubation, consistent with mechanism-based inactivation. Inactivation of recombinant CYP2C8 by phenelzine, amiodarone, verapamil, nortriptyline, fluoxetine, and isoniazid was of the pseudo-first order type and was characterized by respective inactivation kinetic constants (K I and k inact ) of 1. etine, and isoniazid, but not amiodarone. In contrast, inactivation by phenelzine resulted from heme destruction by free radicals. Studies with human liver microsomes (HLMs) revealed that nortriptyline, verapamil, and fluoxetine were not mechanism-based inactivators (MBIs) of CYP2C8. Simultaneous inactivation of CYP2C8 and CYP3A4 (paclitaxel 3Ј-phenyl-hydroxylation) was observed using amiodarone, isoniazid, and phenelzine with the efficiency of inactivation greater for the CYP3A4 pathway. With the exception of phenelzine, glutathione and superoxide dismutase failed to protect CYP2C8 (recombinant and HLMs) or CYP3A4 from inactivation by MBIs. However, the alternate CYP2C8 substrate, torsemide, prevented CYP2C8 inactivation in all cases. These data are consistent with mechanism-based inactivation of CYP2C8 by a range of commonly prescribed drugs, several of which have been implicated in clinically important drug-drug interactions.

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Dimethylarginine dimethylaminohydrolase 1 (DDAH1) is responsible for metabolism of an endogenous inhibitor of nitric oxide synthase (NOS), asymmetric dimethylarginine (ADMA), which plays a key role in modulating angiogenesis. In addition to angiogenesis, tumours can establish a vascular network by forming vessel-like structures from tumour cells; a process termed vasculogenic mimicry (VM). Here, we identified over-expression of DDAH1 in aggressive MDA-MB-231, MDA-MB-453 and BT549 breast cancer cell lines when compared to normal mammary epithelial cells. DDAH1 expression was inversely correlated with the microRNA miR-193b. In DDAH1+ MDA-MB-231 cells, ectopic expression of miR-193b reduced DDAH1 expression and the conversion of ADMA to citrulline. In DDAH1− MCF7 cells, inhibition of miR-193b elevated DDAH1 expression. Luciferase reporter assays demonstrated DDAH1 as a direct target of miR-193b. MDA-MB-231 cells organised into tube structures in an in vitro assay of VM, which was significantly inhibited by DDAH1 knockdown or miR-193b expression. Mechanistically, we found miR-193b regulates cell proliferation and migration of MDA-MB-231 cells, whilst DDAH1 knockdown inhibited cell migration. These studies represent the first evidence for DDAH1 expression, regulation and function in breast cancer cells, and highlights that targeting DDAH1 expression and/or enzymatic activity may be a valid option in the treatment of aggressive breast cancers.

AimsTo characterize potential mechanism-based inactivation (MBI) of major human drugmetabolizing cytochromes P450 (CYP) by monoamine oxidase (MAO) inhibitors, including the antitubercular drug isoniazid. MethodsHuman liver microsomal CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A activities were investigated following co-and preincubation with MAO inhibitors. Inactivation kinetic constants ( K I and k inact ) were determined where a significant preincubation effect was observed. Spectral studies were conducted to elucidate the mechanisms of inactivation. ResultsHydrazine MAO inhibitors generally exhibited greater inhibition of CYP following preincubation, whereas this was less frequent for the proparg ylamines, and tranylcypromine and moclobemide. Phenelzine and isoniazid inactivated all CYP but were most potent toward CYP3A and CYP2C19. Respective inactivation kinetic constants ( K I and k inact ) for isoniazid were 48.6 µ M and 0.042 min − 1 and 79.3 µ M and 0.039 min − 1 . Clorgyline was a selective inactivator of CYP1A2 (6.8 µ M and 0.15 min − 1 ). Inactivation of CYP was irreversible, consistent with metabolite-intermediate complexation for isoniazid and clorgyline, and haeme destruction for phenelzine. With the exception of phenelzine-mediated CYP3A inactivation, glutathione and superoxide dismutase failed to protect CYP from inactivation by isoniazid and phenelzine. Glutathione partially slowed (17%) the inactivation of CYP1A2 by clorgyline. Alternate substrates or inhibitors generally protected against CYP inactivation. ConclusionsThese data are consistent with mechanism-based inactivation of human drugmetabolizing CYP enzymes and suggest that impaired metabolic clearance may contribute to clinical drug-drug interactions with some MAO inhibitors. CYP inactivation by MAO inhibitorsBr J Clin Pharmacol 61 :5 571

What is already known about this subject • Gliclazide is a widely used oral hypoglycaemic agent. • The major metabolites of gliclazide formed in vivo have been identified. • However, the cytochrome P450 enzymes catalysing the rate‐limiting pathways of gliclazide elimination are unknown. What this study adds • CYP2C9 is the major enzyme involved in the various hydroxylation pathways of gliclazide, although a contribution of CYP2C19 to tolymethylhydroxylation, the major metabolic route, cannot be discounted. • Factors known to influence CYP2C9 activity will provide the main source of variability in gliclazide pharmacokinetics. Aims To identify the human cytochrome P450 (CYP) enzymes responsible for the formation of the 6β‐hydroxy (6β‐OHGz), 7β‐hydroxy (7β‐OHGz) and hydroxymethyl (MeOH‐Gz) metabolites of gliclizide (Gz). Methods 6β‐OHGz, 7β‐OHGz and MeOH‐Gz formation by human liver microsomes and a panel of recombinant human P450s was measured using a high‐performance liquid chromatography procedure, and the kinetics of metabolite formation was determined for each pathway. Effects of prototypic CYP enzyme selective inhibitors were characterized for each of the microsomal metabolic pathways. Results Microsomes from six human livers converted Gz to its 6β‐OHGz, 7β‐OHGz, and MeOH‐Gz metabolites, with respective mean (± SD) Km values of 461 ± 139, 404 ± 143 and 334 ± 75 µm and mean Vmax values of 130 ± 55, 82 ± 31 and 268 ± 115 pmol min−1 mg−1, respectively. Vmax/Km ratios for the microsomal reactions parallelled relative metabolite formation in vivo. Sulfaphenazole inhibited microsomal 6β‐OHGz, 7β‐OHGz and MeOH‐Gz formation by 87, 83 and 64%, respectively, whereas S‐mephenytoin caused significant inhibition (48%) of only MeOH‐Gz formation. Recombinant CYP2C9, CYP2C18 and CYP2C19 catalysed all hydroxylation pathways, whereas CYP2C8 formed only 6β‐OHGz and 7β‐OHGz. Conclusion Taken together, the results indicate that CYP2C9 is the major contributor to Gz metabolic clearance, although CYP2C19 may also be involved in MeOH‐Gz formation (the major metabolic pathway). Factors known to influence CYP2C9 activity will provide the main source of variability in Gz pharmacokinetics.

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