Ryan Takahashi

Two allelic variants of aldo-keto reductase 1A1 exhibit reduced in vitro metabolism of daunorubicin.

Aldo-keto reductases (AKRs) are a class of NADPH-dependent oxidoreductases that have been linked to metabolism of the anthracyclines doxorubicin (DOX) and daunorubicin (DAUN). Although widely used, cardiotoxicity continues to be a serious side effect that may be linked to metabolites or reactive intermediates generated in their metabolism. In this study we examine the little known effects of nonsynonymous single nucleotide polymorphisms of human AKR1A1 on the metabolism of these drugs to their alcohol metabolites. Expressed and purified from bacteria using affinity chromatography, the AKR1A1 protein with a single histidine (6x-His) tag exhibited the greatest activity using two test substrates: p-nitrobenzaldehyde (5.09 ± 0.16 μmol/min/mg of purified protein) and dl-glyceraldehyde (1.24 ± 0.17 μmol/min/mg). These activities are in agreement with published literature values of nontagged human AKR1A1. The 6x-His-tagged AKR1A1 wild type and allelic variants, E55D and N52S, were subsequently examined for metabolic activity using DAUN and DOX. The tagged variants showed significantly reduced activities (1.10 ± 0.42 and 0.72 ± 0.47 nmol of daunorubicinol (DAUNol) formed/min/mg of purified protein for E55D and N52S, respectively) compared with the wild type (2.34 ± 0.71 nmol/min/mg). The wild type and E55D variant metabolized DOX to doxorubicinol (DOXol); however, the levels fell below the limit of quantitation (25 nM). The N52S variant yielded no detectable DOXol. A kinetic analysis of the DAUN reductase activities revealed that both amino acid substitutions lead to reduced substrate affinity, measured as significant increases in the measured Km for the reduction reaction by AKR1A1. Hence, it is possible that these allelic variants can act as genetic biomarkers for the clinical development of DAUN-induced cardiotoxicity.

Absorption, Metabolism, Excretion, and the Contribution of Intestinal Metabolism to the Oral Disposition of [14C]Cobimetinib, a MEK Inhibitor, in Humans

The pharmacokinetics, metabolism, and excretion of cobimetinib, a MEK inhibitor, were characterized in healthy male subjects (n = 6) following a single 20 mg (200 μCi) oral dose. Unchanged cobimetinib and M16 (glycine conjugate of hydrolyzed cobimetinib) were the major circulating species, accounting for 20.5% and 18.3% of the drug-related material in plasma up to 48 hours postdose, respectively. Other circulating metabolites were minor, accounting for less than 10% of drug-related material in plasma. The total recovery of the administered radioactivity was 94.3% (±1.6%, S.D.) with 76.5% (±2.3%) in feces and 17.8% (±2.5%) in urine. Metabolite profiling indicated that cobimetinib had been extensively metabolized with only 1.6% and 6.6% of the dose remaining as unchanged drug in urine and feces, respectively. In vitro phenotyping experiments indicated that CYP3A4 was predominantly responsible for metabolizing cobimetinib. From this study, we concluded that cobimetinib had been well absorbed (fraction absorbed, Fa = 0.88). Given this good absorption and the previously determined low hepatic clearance, the systemic exposures were lower than expected (bioavailability, F = 0.28). We hypothesized that intestinal metabolism had strongly attenuated the oral bioavailability of cobimetinib. Supporting this hypothesis, the fraction escaping gut wall elimination (Fg) was estimated to be 0.37 based on F and Fa from this study and the fraction escaping hepatic elimination (Fh) from the absolute bioavailability study (F = Fa × Fh × Fg). Physiologically based pharmacokinetics modeling also showed that intestinal clearance had to be included to adequately describe the oral profile. These collective data suggested that cobimetinib was well absorbed following oral administration and extensively metabolized with intestinal first-pass metabolism contributing to its disposition.

The Effect of Allelic Variation in Aldo-Keto Reductase 1C2 on the in Vitro Metabolism of Dihydrotestosterone

Aldo-keto reductase (AKR) 1C2 is a human, cytosolic enzyme that has an important role in the deactivation of the potent androgen dihydrotestosterone (DHT). AKR1C2 can regulate the extent and duration of activation of the androgen receptor by catalyzing the reduction of DHT to the less potent receptor ligand 3α-diol. In this study, we functionally characterize in vitro the effect of 11 naturally occurring nonsynonymous single nucleotide polymorphisms on the ability of AKR1C2 to reduce DHT to 3α-diol. The wild-type and variant enzymes were expressed using a transfected insect cell system, and their kinetic activities were measured using both a specific fluorogenic probe and DHT as substrates. This functional characterization demonstrates that several variant AKR1C2 proteins have significantly reduced or altered reductase activities as shown by their measured kinetic parameters. Data from our two separate in vitro studies revealed significant reductions in Vmax for two variants (F46Y and L172Q) and significantly lower apparent Km values for three variants (L172Q, K185E, and R258C) compared with the wild type. These results provide evidence that several naturally occurring nonsynonymous single nucleotide polymorphisms in AKR1C2 result in reduced enzyme activities. These variant AKR1C2 alleles may represent one factor involved in the variable degradation of DHT in vivo.

Aldo-Keto Reductase 1C2 Fails to Metabolize Doxorubicin and Daunorubicin in Vitro

The anthracycline drugs are important for the treatment of a number of malignancies; however, their clinical use is associated with dose-dependent severe chronic cardiotoxicity. Although the mechanism for this side effect has not yet been identified, the alcohol metabolites formed during daunorubicin (DAUN) and doxorubicin (DOX) therapies have been implicated. The alcohol metabolites of DAUN and DOX, daunorubicinol (DAUNol) and doxorubicinol (DOXol), respectively, are generated through reduction of the C-13 carbonyl function, which is reportedly mediated by members of the aldo-keto reductase and carbonyl reductase families of proteins. In our search for potential biomarkers for the occurrence of this side effect, we examined the activity of recombinant aldo-keto reductase enzymes, aldo-keto reductase (AKR) 1A1 and AKR1C2, with DAUN and DOX as substrates. Using purified histidine-tagged recombinant proteins and the direct measurement of metabolite formation with a high-performance liquid chromatography-fluorescence assay, we did not observe DAUNol or DOXol generation in vitro by AKR1C2, whereas AKR1A1 did catalyze the reduction reactions. DAUNol was generated by AKR1A1 at a rate of 1.71 ± 0.09 nmol/min/mg protein, and a low level of DOXol was produced by AKR1A1; however, it was below the limits of quantification for the method. These data suggest that the generation of DAUNol or DOXol by AKR1C2 metabolism in vivo is unlikely to occur during anthracycline treatment.

Absorption, Distribution, Metabolism, and Excretion of [14C]GDC-0449 (Vismodegib), an Orally Active Hedgehog Pathway Inhibitor, in Rats and Dogs: A Unique Metabolic Pathway via Pyridine Ring Opening

2-Chloro-N-(4-chloro-3-(pyridin-2-yl)-phenyl)-4-(methylsulfonyl)-benzamide (GDC-0449, vismodegib) is a potent and selective first-in-class small-molecule inhibitor of the Hedgehog signaling pathway and is currently in clinical development. In this study, we investigated the metabolic fate and disposition of GDC-0449 in rats and dogs after a single oral administration of [14C]GDC-0449. An average of 92.4 and 80.4% of the total administered radioactivity was recovered from urine and feces in rats and dogs, respectively. In both species, feces were the major route of excretion, representing 90.0 and 77.4% of the total dose in rats and dogs, respectively. At least 42.1 and 30.8% of the dose was absorbed in rats and dogs, respectively, based on the total excretion of radioactivity in bile and urine. GDC-0449 underwent extensive metabolism in rats and dogs with the major metabolic pathways being oxidation of the 4-chloro-3-(pyridin-2-yl)-phenyl moiety followed by phase II glucuronidation or sulfation. Three other metabolites resulting from an uncommon pyridine ring opening were found, mainly in feces, representing 1.7 to 17.7% of the dose in total in rats and dogs. In plasma, the total radioactivity was absorbed quickly in both rats and dogs, and unchanged GDC-0449 was the predominant circulating radioactive species in both species (>95% of total circulating radioactivity). Quantitative whole-body autoradiography in rats showed that the radioactivity was well distributed in the body, except for the central nervous system, and the majority of radioactivity was eliminated from most tissues by 144 h.

Structure-Based Design of Tricyclic NF-κB Inducing Kinase (NIK) Inhibitors That Have High Selectivity over Phosphoinositide-3-kinase (PI3K)

We report here structure-guided optimization of a novel series of NF-κB inducing kinase (NIK) inhibitors. Starting from a modestly potent, low molecular weight lead, activity was improved by designing a type 11/2 binding mode that accessed a back pocket past the methionine-471 gatekeeper. Divergent binding modes in NIK and PI3K were exploited to dampen PI3K inhibition while maintaining NIK inhibition within these series. Potent compounds were discovered that selectively inhibit the nuclear translocation of NF-κB2 (p52/REL-B) but not canonical NF-κB1 (REL-A/p50).

Use of Transgenic Mouse Models to Understand the Oral Disposition and Drug-Drug Interaction Potential of Cobimetinib, a MEK Inhibitor

Data from the clinical absolute bioavailability (F) study with cobimetinib suggested that F was lower than predicted based on its low hepatic extraction and good absorption. The CYP3A4 transgenic (Tg) mouse model with differential expression of CYP3A4 in the liver (Cyp3a−/−Tg-3A4Hep) or intestine (Cyp3a−/−Tg-3A4Int) and both liver and intestine (Cyp3a−/−Tg-3A4Hep/Int) were used to study the contribution of intestinal metabolism to the F of cobimetinib. In addition, the effect of CYP3A4 inhibition and induction on cobimetinib exposures was tested in the Cyp3a−/−Tg-3A4Hep/Int and PXR-CAR-CYP3A4/CYP3A7 mouse models, respectively. After i.v. administration of 1 mg/kg cobimetinib to wild-type [(WT) FVB], Cyp3a−/−Tg-3A4Hep, Cyp3a−/−Tg-3A4Int, or Cyp3a−/−Tg-3A4Hep/Int mice, clearance (CL) (26-35 ml/min/kg) was similar in the CYP3A4 transgenic and WT mice. After oral administration of 5 mg/kg cobimetinib, the area under the curve (AUC) values of cobimetinib in WT, Cyp3a−/−Tg-3A4Hep, Cyp3a−/−Tg-3A4Int, or Cyp3a−/−Tg-3A4Hep/Int mice were 1.35, 3.39, 1.04, and 0.701 μM⋅h, respectively. The approximately 80% lower AUC of cobimetinib in transgenic mice when intestinal CYP3A4 was present suggested that the intestinal first pass contributed to the oral CL of cobimetinib. Oxidative metabolites observed in human circulation were also observed in the transgenic mice. In drug-drug interaction (DDI) studies using Cyp3a−/−Tg-3A4Hep/Int mice, 8- and 4-fold increases in oral and i.v. cobimetinib exposure, respectively, were observed with itraconazole co-administration. In PXR-CAR-CYP3A4/CYP3A7 mice, rifampin induction decreased cobimetinib oral exposure by approximately 80%. Collectively, these data support the conclusion that CYP3A4 intestinal metabolism contributes to the oral disposition of cobimetinib and suggest that under certain circumstances the transgenic model may be useful in predicting clinical DDIs.

Abstract B160: Assessing human absorption, metabolism, routes of excretion and the contribution of intestinal metabolism to the oral clearance of cobimetinib, a MEK inhibitor.

Cobimetinib (GDC-0973; XL518) is a potent and highly selective inhibitor of MEK1/2, currently in clinical development as an anticancer agent. The purpose of this investigation was to determine the human absorption, metabolism and routes of excretion of cobimetinib after oral administration to healthy human subjects as well as to assess the contribution of intestinal metabolism to the oral clearance (CL) of cobimetinib. A single 20 mg [14C]-cobimetinib (200 μCi) oral dose was administered to six healthy volunteers. Blood, urine and feces were collected for determination of levels of cobimetinib and cobimetinib-related radioactivity in each matrix. In separate healthy subject study, the CL and bioavailability (F) of cobimetinib were determined from an open-label, 2-way crossover absolute bioavailability study after intravenous (IV; 2 mg) and oral (20 mg) administration of cobimetinib to healthy volunteers (n=20; 19 completed). Approximately 94% of the [14C] dose was recovered in excreta. Cobimetinib was predominantly excreted in feces with 77% of radioactivity recovered in the feces vs. 18% of the dose recovered in urine. Cobimetinib was extensively metabolized, as only 1.6% and 6.6% of unchanged cobimetinib was observed in urine and feces, respectively. The % of the cobimetinib dose absorbed was 88% (Fa), based on the sum of % total radioactivity identified in urine and the % of total metabolites in feces. Unchanged cobimetinib and 5 metabolites; oxidative, glucuronide and glycine conjugate metabolites were identified in plasma. After IV and oral administration of cobimetinib, hepatic extraction (EH) of cobimetinib was 13% and the F of cobimetinib was 46%. Based on the % of cobimetinib absorbed and, if only EH contributed to the CL of cobimetinib, the predicted F (F = Fa*Fint*FH) would be 76%. The lower observed F of 46% suggested that after oral administration, 43% of cobimetinib undergoes extra-hepatic CL, likely via intestinal metabolism. Furthermore, comparison of plasma metabolite profiles after IV and oral dosing showed significantly higher (2 to 5-fold) metabolite to parent ratios after oral dosing, consistent with the contribution of intestinal metabolism. Additionally, using physiologically based pharmacokinetic modeling the IV pharmacokinetic profile of cobimetinib was well described using the observed IV CL. However, the oral PK profile was best described by the addition of intestinal CL. The collective data confirm that cobimetinib is extensively metabolized and excreted in feces after oral dosing. There appears to be a greater contribution of intestinal metabolism than hepatic metabolism to the overall oral CL of cobimetinib. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B160. Citation Format: Edna Choo, Ryan Takahashi, Isabelle Rooney, Mary Gates, Alan Deng, Luna Musib. Assessing human absorption, metabolism, routes of excretion and the contribution of intestinal metabolism to the oral clearance of cobimetinib, a MEK inhibitor. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B160.

Absorption, metabolism and excretion of cobimetinib, an oral MEK inhibitor, in rats and dogs

Abstract 1. The absorption, metabolism and excretion of cobimetinib, an allosteric inhibitor of MEK1/2, was characterized in mass balance studies following single oral administration of radiolabeled (14C) cobimetinib to Sprague–Dawley rats (30 mg/kg) and Beagle dogs (5 mg/kg). 2. The oral dose of cobimetinib was well absorbed (81% and 71% in rats and dogs, respectively). The maximal plasma concentrations for cobimetinib and total radioactivity were reached at 2–3 h post-dose. Drug-derived radioactivity was fully recovered (∼90% of the administered dose) with the majority eliminated in feces via biliary excretion (78% of the dose for rats and 65% for dogs). The recoveries were nearly complete after the first 48 h following dosing. 3. The metabolic profiles indicated extensive metabolism of cobimetinib prior to its elimination. For rats, the predominant metabolic pathway was hydroxylation at the aromatic core. Lower exposures for cobimetinib and total radioactivity were observed in male rats compared with female rats, which was consistent to in vitro higher clearance of cobimetinib for male rats. For dogs, sequential oxidative reactions occurred at the aliphatic portion of the molecule. Though rat metabolism was well-predicted in vitro with liver microsomes, dog metabolism was not. 4. Rats and dogs were exposed to the two major human circulating Phase II metabolites, which provided relevant metabolite safety assessment. In general, the extensive sequential oxidative metabolism in dogs, and not the aromatic hydroxylation in rats, was more indicative of the metabolism of cobimetinib in humans.

Applying Stable Isotope Labeled Amino Acids in Micropatterned Hepatocyte Coculture to Directly Determine the Degradation Rate Constant for CYP3A4

The rate of enzyme degradation (kdeg) is an important input parameter for the prediction of clinical drug-drug interactions (DDIs) that result from mechanism-based inactivation or induction of cytochrome P450 (P450). Currently, a large range of reported estimates for CYP3A4 enzyme degradation exists, and consequently extensive uncertainty exists in steady-state predictions for DDIs. In the current investigations, the stable isotope labeled amino acids in culture technique was applied to a long-lived primary human hepatocyte culture, HepatoPac, to directly monitor the degradation of CYP3A4. This approach allowed selective isotope labeling of a population of de novo synthesized CYP3A4 and specific quantification of proteins with mass spectrometry to determine the CYP3A4 degradation within the hepatocytes. The kdeg estimate was 0.026 ± 0.005 hour−1. This value was reproduced by cultures derived across four individual donors. For these cultures, the data indicated that CYP3A4 mRNA and total protein expression (i.e., labeled and unlabeled P450s), and activity were stable over the period where degradation had been determined. This kdeg value for CYP3A4 was in good agreement with recently reported values that used alternate analytical approaches but also employed micropatterned primary human hepatocytes as the in vitro model.