Metabolism and pharmacokinetics of the cyclin-dependent kinase inhibitorR-roscovitine in the mouse

Abstract

R-roscovitine (seliciclib, CYC202) is a cyclin-dependent kinase inhibitor currently in phase II clinical trials in patients with cancer. Here, we describe its mouse metabolism and pharmacokinetics as well as the identification of the principal metabolites in hepatic microsomes, plasma, and urine. Following microsomal incubation of R-roscovitine at 10 μg/mL (28 μmol/L) for 60 minutes, 86.7% of the parent drug was metabolized and 60% of this loss was due to formation of one particular metabolite. This was identified as the carboxylic acid resulting from oxidation of the hydroxymethyl group of the amino alcohol substituent at C2 of the purine core present in R-roscovitine. Identification was confirmed by chemical synthesis and comparison of an authentic sample of the R-roscovitine-derived carboxylate metabolite (COOH-R-roscovitine). Other minor metabolites were identified as C8-oxo-R-roscovitine and N9-desisopropyl-R-roscovitine; these accounted for 4.9% and 2.6% of the parent, respectively. The same metabolic pattern was observed in vivo, with a 4.5-fold lower AUC∞ for R-roscovitine (38μmol/L/h) than for COOH-R-roscovitine (174 μmol/L/h). Excretion of R-roscovitine in the urine up to 24 hours post-dosing accounted for an average of only 0.02% of the administered dose of 50 mg/kg, whereas COOH-R-roscovitine represented 65% to 68% of the dose irrespective of the route of administration (i.v., i.p., or p.o.). A partially deuterated derivative (R-roscovitine-d9) was synthesized to investigate if formation of COOH-R-roscovitine could be inhibited by replacement of metabolically labile protons with deuterium. After 60 minutes of incubation of R-roscovitine-d9 or R-roscovitine with mouse liver microsomes, formation of COOH-R-roscovitine-d9 was decreased by ∼24% compared with the production of COOH-R-roscovitine. In addition, the levels of R-roscovitine-d9 remaining were 33% higher than those of R-roscovitine. However, formation of several minor R-roscovitine metabolites was enhanced with R-roscovitine-d9, suggesting that metabolic switching from the major carbinol oxidation pathway had occurred. Synthetic COOH-R-roscovitine and C8-oxo-R-roscovitine were tested in functional cyclin-dependent kinase assays and shown to be less active than R-roscovitine, confirming that these metabolic reactions are deactivation pathways.

Pyridoimidazolones as Novel Potent Inhibitors of v-Raf Murine Sarcoma Viral Oncogene Homologue B1 (BRAF)

“…The reaction was stopped by the addition of methanol (3 volumes), containing roscovitine (500 nM) used as an internal standard. The parent compound and metabolites were measured by liquid chromatography mass spectrometry with electrospray in positive ionization mode on 2 instruments as previously described . The chromatographic separation was achieved on a Synergi polar RP column (5 cm, 4 μm particle, 4.6 mm i.d.…”

Development of Novel, Highly Potent Inhibitors of V-RAF Murine Sarcoma Viral Oncogene Homologue B1 (BRAF): Increasing Cellular Potency through Optimization of a Distal Heteroaromatic Group

“…The reaction was stopped by the addition of methanol (3 volumes), containing roscovitine (500 nM) used as an internal standard. The parent compound and metabolites were measured by liquid chromatography−mass spectrometry with electrospray in positive ionization mode on two instruments as previously described . The chromatographic separation was achieved on a Synergi polar RP column (5 cm, 4 μm particle, 4.6 mm i.d.…”

“…The chromatographic separation was achieved on a Synergi polar RP column (5 cm, 4 μm particle, 4.6 mm i.d. ; Phenomenex, Cheshire, U.K.) with gradient and mass spectrometry conditions similar to those described by Nutley et al…”

“…The parent compound and metabolites were measured by liquid chromatography-mass spectrometry with electrospray in positive ionization mode on two instruments as previously described. 36 The chromatographic separation was achieved on a Synergi polar RP column (5 cm, 4 μm particle, 4.6 mm i.d. ; Phenomenex, Cheshire, U.K.) with gradient and mass spectrometry conditions similar to those described by Nutley et al 36 Pharmacokinetics.…”

“…36 The chromatographic separation was achieved on a Synergi polar RP column (5 cm, 4 μm particle, 4.6 mm i.d. ; Phenomenex, Cheshire, U.K.) with gradient and mass spectrometry conditions similar to those described by Nutley et al 36 Pharmacokinetics. Female BALB/cAnNCrl mice at least 6 weeks of age were used for the PK analyses except for the intravenous administration of 1m, which was carried out with female Crl:CD1-Foxn1nu mice bearing V600E mutant BRAF WM266.4 tumor xenografts.…”