Linezolid Metabolism Is Catalyzed by Cytochrome P450 2J2, 4F2, and 1B1

Abstract

The oxazolidinone antibacterial linezolid has been in clinical use for over twenty years, yet knowledge of the contributions of specific cytochrome P450 enzymes to the metabolic clearance of this drug were mostly unknown. In this investigation it was revealed that three P450 enzymes that had not been previously explored in linezolid metabolism, CYP2J2, CYP4F1, and CYP1B1, catalyzed the 2-hydroxylation and deethyleneation of the morpholine moiety of linezolid. The intrinsic clearance for linezolid metabolism in pooled human liver microsomes was low at 0.51 µL/min/mg protein, consistent with its in vivo clearance in humans, and the K M was high (>200 µM). In recombinant human P450 enzymes, a rank order of intrinsic clearance values for linezolid 2-hydroxylation were CYP2J2>>CYP4F2>CYP2C8>CYP1B1≈CYP2D6≈CYP3A4>CYP1A1>CYP3A5, with nine other P450 enzymesshowing no linezolid metabolism The effect of selective inhibitors for these eight P450 enzymes on linezolid metabolism in pooled human liver microsomes was evaluated to provide estimates of the relative fractional contributions of these enzymes to linezolid metabolism. These experiments suggest that CYP2J2 and CYP4F2 contribute about 50% each to linezolid hepatic metabolism. It is proposed that the oxidative metabolic clearance of linezolid is primarily catalyzed by these two unusual P450 enzymes and that this explains the lack of observation of meaningful effects of common perpetrators of drug interactions on linezolid pharmacokinetics. SIGNIFICANCE STATEMENTLinezolid is an important antibacterial drug, but the enzymes involved in its oxidative metabolism were unknown. In this paper, evidence is shown that supports an important role for two enzymes not frequently associated with the metabolism of drugs: CYP2J2 and CYP4F2. These observations offer insight to understand the results of clinical drug-drug interaction studies conducted on linezolid.

Species Specificity and Selection of Models for Drug Oxidations Mediated by Polymorphic Human Enzymes

“…Information from package inserts for some clinically important medicines, such as the DMD-MR-2021-000742 antibiotic linezolid, indicates that no roles of major human P450 enzymes have been identified in liver microsomal drug oxidations, although those studies did not rule out the role of currently uncharacterized P450s (Wynalda et al, 2000). Similarly, recent findings from in vitro assays for linezolid have shown contribution of P450 2J2 (Obach, 2022), an enzyme not generally included in testing at the preclinical drug development stage.…”

Plasma and synovial fluid concentrations of linezolid in patients with knee osteoarthritis infected with Staphylococcus aureus

“…Plasma-to-synovialfluid ratios of 0.76 ± 0.34 for linezolid have been reported in terms of the knee gap after a single dose of 600 mg of linezolid [15]. Although the package insert information of linezolid suggests the presence of two minor morpholine ring-opening metabolites in human plasma or urine (in-house document), recently reported 2-hydroxylated linezolid (mediated by cytochrome P450 2 J2 [9]) was not considered so far, to our knowledge, in the fluid samples in the clinical setting. In the current study, similar plasma and synovial fluid concentrations of parent compound linezolid in two patients with knee osteoarthritis infected with Staphylococcus aureus were demonstrated after patients were administered multiple doses of 600 mg by twice daily intravenous infusion.…”

“…Samples (10 μL) were infused using an autosampler. The retention times of linezolid and its possible 2-hydroxylated metabolite [9] were 7.4 and 5.9 min respectively. Data are given as means and standard deviations from triplicate determinations.…”

“…The pharmacokinetics of linezolid in healthy subjects [6] and in obese patients with cellulitis [7] have been reported. Linezolid is reportedly hydroxylated by as-yet unidentified cytochrome P450s [8], but recently a role for P450 2 J2 in linezolid 2-hydroxylation was indicated [9].…”

“…Under the current clinical setting, it was focused to find any hydroxylated linezolid metabolite(s) in the plasma or synovial fluid samples. To investigate the possible P450 2 J2-dependent 2-hydroxylation of linezolid [9], an incubation mixture was prepared consisting of 100 pmol/ mL recombinant P450 2 J2 protein (Corning, Woburn, MA, USA), 0.40 mM linezolid, an NADPH-generating system (0.50 mM NADP + , 5.0 mM glucose 6-phosphate, and 0.50 units/mL glucose 6-phosphate dehydrogenase), and 100 mM potassium phosphate buffer (pH 7.4) in a total volume of 0.20 mL. The reaction was carried out at 37 °C for 60 min and was terminated by adding 0.60 mL of acetonitrile.…”