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Asthma is one of the most prevalent diseases in the world, for which the mainstay treatment has been inhaled glucocorticoids (GCs). Despite the widespread use of these drugs, approximately 30% of asthma sufferers exhibit some degree of steroid insensitivity or are refractory to inhaled GCs. One hypothesis to explain this phenomenon is interpatient variability in the clearance of these compounds. The objective of this research is to determine how metabolism of GCs by the CYP3A family of enzymes could affect their effectiveness in asthmatic patients. In this work, the metabolism of four frequently prescribed inhaled GCs, triamcinolone acetonide, flunisolide, budesonide, and fluticasone propionate, by the CYP3A family of enzymes was studied to identify differences in their rates of clearance and to identify their metabolites. Both interenzyme and interdrug variability in rates of metabolism and metabolic fate were observed. CYP3A4 was the most efficient metabolic catalyst for all the compounds, and CYP3A7 had the slowest rates. CYP3A5, which is particularly relevant to GC metabolism in the lungs, was also shown to efficiently metabolize triamcinolone acetonide, budesonide, and fluticasone propionate. In contrast, flunisolide was only metabolized via CYP3A4, with no significant turnover by CYP3A5 or CYP3A7. Common metabolites included 6β-hydroxylation and 6-dehydrogenation for triamcinolone acetonide, budesonide, and flunisolide. The structure of 6-flunisolide was unambiguously established by NMR analysis. Metabolism also occurred on the D-ring substituents, including the 21-carboxy metabolites for triamcinolone acetonide and flunisolide. The novel metabolite 21-nortriamcinolone acetonide was also identified by liquid chromatography–mass spectrometry and NMR analysis.

Pactimibe sulfate is a novel acyl coenzyme A:cholesterol acyltransferase inhibitor. We conducted metabolic studies of pactimibe and its plasma metabolite, R-125528. Pactimibe had multiple metabolic pathways including indolin oxidation to form R-125528, ω-1 oxidation, N-dealkylation, and glucuronidation. Among them, the indolin oxidation and the ω-1 oxidation were dominant and were mainly catalyzed by CYP3A4 and CYP2D6, respectively. The intrinsic clearance (CLint) values for these pathways in human hepatic microsomes were 0.63 and 0.76 µl/min/mg-protein, respectively. On the other hand, the metabolic reaction for R-125528 was restricted. It was demonstrated that ω-1 oxidation was the only pathway that could eliminate R-125528 from the systemic circulation. To our surprise, only CYP2D6-expressing microsomes could catalyze the reaction, and ω-1 oxidation was strongly correlated with the CYP2D6 marker reaction, dextromethorphan O-demethylation (r2= 0.90), in human hepatic microsomes. Although R-125528 is an atypical substrate for CYP2D6 because of its acidity, the Kmvalue was 1.8 µM for the reaction in human hepatic microsomes and the CLintvalue was as high as 75.0 µl/min/mg-protein. These results suggested that the systemic clearance of R-125528 was highly dependent on CYP2D6 activity and that several studies with CYP2D6 including drug-drug interaction and polymorphism sensitivity should be performed during development from the viewpoint of metabolite safety assessment. The finding that R-125528, an acidic compound devoid of basic nitrogen, was a good substrate for CYP2D6 raised a question about previously reported CYP2D6 models based on a critical electrostatic interaction with Asp301and/or Glu216.

Diglucuronidation is a novel glucuronidation reaction where the second glucuronosyl moiety is attached at the C2' position of the first glucuronosyl moiety. To examine whether diglucuronidation takes place in endogenous substrates in vivo, control urine and bile samples were collected from male Crl:CD(SD) IGS rats, beagle dogs, and cynomolgus monkeys and analyzed by liquid chromatography-mass spectrometry (LC-MS) after solid phase extraction. Several diglucuronides of C19steroids, including M1 (C31H46O14) and M2 (C31H44O14), were detected in the urine and bile of the dogs but not in the excreta of the rats and monkeys. A milligram quantity of M1 was successfully isolated from the pooled dog urine and analyzed by nuclear magnetic resonance (NMR) spectroscopy. M1 was unambiguously identified as epiandrosterone 3-O-diglucuronide by comparing the LC-MS and two-dimensional NMR data of M1 with those of the biosynthesized epiandrosterone 3-O-diglucuronide. M2 was identified as dehydroepiandrosterone 3-O-diglucuronide. According to these findings, the diglucuronidation reaction was proven to be occurring on steroid hormones in vivo in dogs.