RESUMO
Camonsertib is a novel ATR kinase inhibitor in clinical development for advanced cancers targeting sensitizing mutations. This article describes the identification and biosynthesis of an N-glucuronide metabolite of camonsertib. This metabolite was first observed in human hepatocyte incubations and was subsequently isolated to determine the structure, evaluate its stability as part of bioanalytical method development and for use as a standard for estimating its concentration in Phase I samples. The N-glucuronide was scaled-up using a purified bacterial culture preparation and was subsequently isolated using preparative chromatography. The bacterial culture generated sufficient material of the glucuronide to allow for one- and two-dimensional 1H and 13C NMR structural elucidation and further bioanalytical characterization. The NOE data combined with the gradient HMBC experiment and molecular modeling, strongly suggests that the point of attachment of the glucuronide results in the formation of (2S,3S,4S,5R,6R)-3,4,5-trihydroxy-6-(5-(4-((1R,3r,5S)-3-hydroxy-8-oxabicyclo[3.2.1]octan-3-yl)-6-((R)-3-methylmorpholino)-1H-pyrazolo[3,4-b]pyridin-1-yl)-1H-pyrazol-1-yl)tetrahydro-2H-pyran-2-carboxylic acid. Significance Statement This is the first report of a glucuronide metabolite of camonsertib formed by human hepatocyte incubations. This study reveals the structure of an N-glucuronide metabolite of camonsertib using detailed elucidation by one- and two-dimensional NMR after scale-up using a novel bacterial culture approach yielding significant quantities of a purified metabolite.
RESUMO
6ß-Hydroxy-21-desacetyl deflazacort (6ß-OH-21-desDFZ) is a major circulating but not biologically active metabolite of deflazacort (DFZ). In vitro studies were performed to evaluate cytochrome P450 (CYP)- and transporter-mediated drug interaction potentials of 6ß-OH-21-desDFZ. Up to 50 µM, the highest soluble concentration in the test system, 6ß-OH-21-desDFZ weakly inhibited (IC50 > 50 µM) the enzyme activity of CYPs 1A2, 2B6, 2C8, 2C9, and 2D6, while moderately inhibiting CYP2C19 and CYP3A4 with IC50 values of approximately 50 and 35 µM, respectively. The inhibition was neither time-dependent nor metabolism-based. Incubation of up to 50 µM 6ß-OH-21-desDFZ with plated cryopreserved human hepatocytes for 48 h resulted in no meaningful concentration-dependent induction of either mRNA levels or enzyme activity of CYP1A2, CYP2B6, or CYP3A4. In transporter inhibition assays, 6ß-OH-21-desDFZ, up to 50 µM, did not show interaction with human OAT1, OAT3, and OCT2 transporters. It weakly inhibited (IC50 > 50 µM) human MATE1, MATE2-K, and OCT1 transporter activity, and moderately inhibited human MDR1, OATP1B1, and OATP1B3 transporter activity with IC50 values of 19.81 µM, 37.62 µM, and 42.22 µM, respectively. 14 C-6ß-OH-21-desDFZ was biosynthesized using bacterial biotransformation and the subsequent study showed that 6ß-OH-21-desDFZ was not a substrate for human BCRP, MDR1, MATE1, MATE2-K, OAT1, OATP1B1, OATP1B3, and OCT2 transporters, but appeared to be an in vitro substrate for the human OAT3 uptake transporter. At plasma concentrations of 6ß-OH-21-desDFZ seen in the clinic, CYP- and transporter-mediated drug-drug interactions are not expected following administration of a therapeutic dose of DFZ in Duchenne muscular dystrophy (DMD) patients.
