Estimation of Circulating Drug Metabolite Exposure in Human Using In Vitro Data and Physiologically Based Pharmacokinetic Modeling: Example of a High Metabolite/Parent Drug Ratio.

(R)-4-((4-(((4-((tetrahydrofuran-3-yl)oxy)benzo[d]isoxazol-3-yl)oxy)methyl)piperidin-1-yl)methyl)tetrahydro-2H-pyran-4-ol (TBPT), a serotonin-4 receptor partial agonist, is metabolized to two metabolites: an N-dealkylation product [(R)-3-(piperidin-4-ylmethoxy)-4-((tetrahydrofuran-3-yl)oxy)benzo[d]isoxazole (M1)] and a cyclized oxazolidine structure [7-(((4-(((R)-tetrahydrofuran-3-yl)oxy)benzo[d]isoxazol-3-yl)oxy)methyl)octahydro-3H (M2)]. After administration of TBPT to humans the exposure to M1 was low and the exposure to M2 was high, relative to the parent drug, despite this being the opposite in vitro. In this study, projection of the plasma metabolite/parent (M/P) ratios for M1 and M2 was attempted using in vitro metabolism, binding, and permeability data in static and dynamic physiologically based pharmacokinetic (PBPK) models. In the static model, the fraction of parent clearance yielding the metabolite (which also required taking into account secondary metabolites of M1 and M2), the clearance of the metabolites and parent, and an estimate of the availability of the metabolites from the liver were combined to yield estimated parent/metabolite ratios of 0.32 and 23 for M1 and M2, respectively. PBPK modeling that used in vitro and physicochemical data input yielded estimates of 0.26 and 20, respectively. The actual values were 0.12 for M1/TBPT and 58 for M2/TBPT. Thus, the ratio for M1 was overpredicted, albeit at values less than unity. The ratio for M2/TBPT was underpredicted, and the high ratio of 58 may exceed a limiting ceiling of the approach. Nevertheless, when considered in the context of determining whether a potential circulating metabolite may be quantitatively important prior to administration of a drug for the first time to humans, the approaches succeeded in highlighting the importance of M2 (M/P ratio >> 1) relative to M1, despite M1 being much greater than M2 in vitro.

Metabolite profiling of bendamustine in urine of cancer patients after administration of [14C]bendamustine.

Bendamustine is an alkylating agent consisting of a mechlorethamine derivative, a benzimidazole group, and a butyric acid substituent. A human mass balance study showed that bendamustine is extensively metabolized and subsequently excreted in urine. However, limited information is available on the metabolite profile of bendamustine in human urine. The objective of this study was to elucidate the metabolic pathways of bendamustine in humans by identification of its metabolites excreted in urine. Human urine samples were collected up to 168 h after an intravenous infusion of 120 mg/m(2) (80-95 µCi) [(14)C]bendamustine. Metabolites of [(14)C]bendamustine were identified using liquid chromatography (high-resolution)-tandem mass spectrometry with off-line radioactivity detection. Bendamustine and a total of 25 bendamustine-related compounds were detected. Observed metabolic conversions at the benzimidazole and butyric acid moiety were N-demethylation and γ-hydroxylation. In addition, various other combinations of these conversions with modifications at the mechlorethamine moiety were observed, including hydrolysis (the primary metabolic pathway), cysteine conjugation, and subsequent biotransformation to mercapturic acid and thiol derivatives, N-dealkylation, oxidation, and conjugation with phosphate, creatinine, and uric acid. Bendamustine-derived products containing phosphate, creatinine, and uric acid conjugates were also detected in control urine incubated with bendamustine. Metabolites that were excreted up to 168 h after the infusion included products of dihydrolysis and cysteine conjugation of bendamustine and γ-hydroxybendamustine. The range of metabolic reactions is generally consistent with those reported for rat urine and bile, suggesting that the overall processes involved in metabolic elimination are qualitatively the same in rats and humans.

The molecular basis of CYP2D6-mediated N-dealkylation: balance between metabolic clearance routes and enzyme inhibition.

N-dealkylation is a commonly observed metabolic reaction for drugs containing secondary and tertiary amines. On searching the literature, it is obvious that this reaction is far less common among cytochrome P450 2D6 catalyzed reactions compared with other cytochromes P450. The CYP2D6 pharmacophore and characteristic features in the active site cavity suggest a favored substrate orientation that prevents N-dealkylation from occurring. In this study, the literature was searched for N-dealkylated and non-N-dealkylated CYP2D6 substrates. The hypothesis that was suggested and confirmed demonstrated that N-dealkylation occurs by this enzyme when the preferred site of metabolism is blocked toward other oxidative metabolic pathways. An interesting observation was also that addition of stable groups at preferred sites of metabolism generally improved the metabolic stability but also resulted in retained or increased inhibition of the enzyme. In addition, the effect of pH on N- and O-dealkylation of dextromethorphan was shown to be consistent with the hypothesis that an ionized amino function favored substrate dockings resulting in O-dealkylation.