Bioactivation and reactivity research advances - 2022 year in review‡.

With the 50th year mark since the launch of Drug Metabolism and Disposition journal, the field of drug metabolism and bioactivation has advanced exponentially in the past decades (Guengerich 2023).This has, in a major part, been due to the continued advances across the whole spectrum of applied technologies in hardware, software, machine learning (ML), and artificial intelligence (AI). LC-MS platforms continue to evolve to support key applications in the field, and automation is also improving the accuracy, precision, and throughput of these supporting assays. In addition, sample generation and processing is being aided by increased diversity and quality of reagents and bio-matrices so that what is being analyzed is more relevant and translatable. The application of in silico platforms (applied software, ML, and AI) is also making great strides, and in tandem with the more traditional approaches mentioned previously, is significantly advancing our understanding of bioactivation pathways and how these play a role in toxicity. All of this continues to allow the area of bioactivation to evolve in parallel with associated fields to help bring novel or improved medicines to patients with urgent or unmet needs.Shuai Wang and Cyrus Khojasteh, on behalf of the authors.

Biotransformation research advances - 2022 year in review.

This annual review is the eighth of its kind since 2016 (Baillie et al. 2016, Khojasteh et al. 2017, Khojasteh et al. 2018, Khojasteh et al. 2019, Khojasteh et al. 2020, Khojasteh et al. 2021, Khojasteh et al. 2022). Our objective is to explore and share articles which we deem influential and significant in the field of biotransformation.

The Absolute Bioavailability and Absorption, Metabolism, and Excretion of Ipatasertib, a Potent and Highly Selective Protein Kinase B (Akt) Inhibitor.

Ipatasertib (GDC-0068) is a potent, highly selective, small-molecule inhibitor of protein kinase B (Akt) being developed by Genentech/Roche as a single agent and in combination with other therapies for the treatment of cancers. To fully understand the absorption, metabolism, and excretion of ipatasertib in humans, an open-label study using 14C-radiolabeled ipatasertib was completed to characterize the absolute bioavailability (period 1) and mass balance and metabolite profiling (period 2). In period 1, subjects were administered a 200 mg oral dose of ipatasertib followed by an 80 µg (800 nCi) intravenous dose of [14C]-ipatasertib. In period 2, subjects received a single oral dose containing approximately 200 mg (100 µCi) [14C]-ipatasertib. In an integrated analytical strategy, accelerator mass spectrometry was applied to measure the 14C microtracer intravenous pharmacokinetics in period 1 and fully profile plasma radioactivity in period 2. The systemic plasma clearance and steady-state volume of distribution were 98.8 L/h and 2530 L, respectively. The terminal half-lives after oral and intravenous administrations were similar (26.7 and 27.4 hours, respectively) and absolute bioavailability of ipatasertib was 34.0%. After a single oral dose of [14C]-ipatasertib, 88.3% of the administered radioactivity was recovered with approximately 69.0% and 19.3% in feces and urine, respectively. Radioactivity in feces and urine was predominantly metabolites with 24.4% and 8.26% of dose as unchanged parent, respectively; indicating that ipatasertib had been extensively absorbed and hepatic metabolism was the major route of clearance. The major metabolic pathway was N-dealkylation mediated by CYP3A, and minor pathways were oxidative by cytochromes P450 and aldehyde oxidase. SIGNIFICANCE STATEMENT: The study provided definitive information regarding the absolute bioavailability and the absorption, metabolism, and excretion pathways of ipatasertib, a potent, novel, and highly selective small-molecule inhibitor of protein kinase B (Akt). An ultrasensitive radioactive counting method, accelerator mass spectrometry was successfully applied for 14C-microtracer absolute bioavailability determination and plasma metabolite profiling.

Characterization of Hepatic UDP-Glucuronosyltransferase Enzyme Abundance-Activity Correlations and Population Variability Using a Proteomics Approach and Comparison with Cytochrome P450 Enzymes.

The expression of ten major drug-metabolizing UDP-glucuronosyltransferase (UGT) enzymes in a panel of 130 human hepatic microsomal samples was measured using a liquid chromatography-tandem mass spectrometry-based approach. Simultaneously, ten cytochromes P450 and P450 reductase were also measured, and activity-expression relationships were assessed for comparison. The resulting data sets demonstrated that, with the exception of UGT2B17, 10th to 90th percentiles of UGT expression spanned 3- to 8-fold ranges. These ranges were small relative to ranges of reported mean UGT enzyme expression across different laboratories. We tested correlation of UGT expression with enzymatic activities using selective probe substrates. A high degree of abundance-activity correlation (Spearman's rank correlation coefficient > 0.6) was observed for UGT1As (1A1, 3, 4, 6) and cytochromes P450. In contrast, protein abundance and activity did not correlate strongly for UGT1A9 and UGT2B enzymes (2B4, 7, 10, 15, and 17). Protein abundance was strongly correlated for UGTs 2B7, 2B10, and 2B15. We suggest a number of factors may contribute to these differences including incomplete selectivity of probe substrates, correlated expression of these UGT2B isoforms, and the impact of splice and polymorphic variants on the peptides used in proteomics analysis, and exemplify this in the case of UGT2B10. Extensive correlation analyses identified important criteria for validating the fidelity of proteomics and enzymatic activity approaches for assessing UGT variability, population differences, and ontogenetic changes. SIGNIFICANCE STATEMENT: Protein expression data allow detailed assessment of interindividual variability and enzyme ontogeny. This study has observed that expression and enzyme activity are well correlated for hepatic UGT1A enzymes and cytochromes P450. However, for the UGT2B family, caution is advised when assuming correlation of expression and activity as is often done in physiologically based pharmacokinetic modeling. This can be due to incomplete probe substrate specificities, but may also be related to presence of inactive UGT protein materials and the effect of splicing variations.

Advances in the study of drug metabolism - symposium report of the 12th Meeting of the International Society for the Study of Xenobiotics (ISSX).

The 12th International Society for the Study of Xenobiotics (ISSX) meeting, held in Portland, OR, USA from July 28 to 31, 2019, was attended by diverse members of the pharmaceutical sciences community. The ISSX New Investigators Group provides learning and professional growth opportunities for student and early career members of ISSX. To share meeting content with those who were unable to attend, the ISSX New Investigators herein elected to highlight the "Advances in the Study of Drug Metabolism" symposium, as it engaged attendees with diverse backgrounds. This session covered a wide range of current topics in drug metabolism research including predicting sites and routes of metabolism, metabolite identification, ligand docking, and medicinal and natural products chemistry, and highlighted approaches complemented by computational modeling. In silico tools have been increasingly applied in both academic and industrial settings, alongside traditional and evolving in vitro techniques, to strengthen and streamline pharmaceutical research. Approaches such as quantum mechanics simulations facilitate understanding of reaction energetics toward prediction of routes and sites of drug metabolism. Furthermore, in tandem with crystallographic and orthogonal wet lab techniques for structural validation of drug metabolizing enzymes, in silico models can aid understanding of substrate recognition by particular enzymes, identify metabolic soft spots and predict toxic metabolites for improved molecular design. Of note, integration of chemical synthesis and biosynthesis using natural products remains an important approach for identifying new chemical scaffolds in drug discovery. These subjects, compiled by the symposium organizers, presenters, and the ISSX New Investigators Group, are discussed in this review.

Human Cytochrome P450 1A1 Adapts Active Site for Atypical Nonplanar Substrate.

The human cytochrome P450 1A1 (CYP1A1) is well known for chemical activation of procarcinogens and often has a substrate scope of small and highly planar compounds. Substrates deviating from these characteristics are certainly known, but how these larger and nonplanar substrates are accommodated and oriented within the CYP1A1 active site is not understood. Herein a new X-ray structure of CYP1A1 bound to the pan-Pim kinase inhibitor GDC-0339 reveals how the CYP1A1 active site cavity is reconfigured to bind larger and nonplanar compounds. The shape and size of the cavity are controlled by structural elements in the active site roof, with major changes in the conformation of the F helix break and relocation of Phe224 from the active site to the protein surface. This altered CYP1A1 active site architecture is consistent with the proposed mechanism for CYP1A1 generation of an unusual aminoazepane-rearranged metabolite for this substrate. SIGNIFICANCE STATEMENT: Cytochrome P450 1A1 metabolizes drugs, procarcinogens, and toxins and although previous structures have revealed how its stereotypical planar, aromatic compounds are accommodated in the CYP1A1 active site, this is not the case for flexible and nonplanar compounds. The current work determines the X-ray structure of CYP1A1 with such a flexible, nonplanar Pim kinase inhibitor, revealing significant modification of the CYP1A1 roof that accommodate this preclinical candidate and support an unusual intramolecular rearrangement reaction.

Novel Homodimer Metabolites of GDC-0994 via Cytochrome P450-Catalyzed Radical Coupling.

Two novel homodimer metabolites were identified in rat samples collected during the in vivo study of GDC-0994. In this study, we investigated the mechanism of the formation of these metabolites. We generated and isolated the dimer metabolites using a biomimetic oxidation system for NMR structure elucidation to identify a symmetric dimer formed via carbon-carbon bond between two pyrazoles and an asymmetric dimer formed via an aminopyrazole-nitrogen to pyrazole-carbon bond. In vitro experiments demonstrated formation of these dimers was catalyzed by cytochrome P450 enzymes (P450s) with CYP3A4/5 being the most efficient. Using density functional theory, we determined these metabolites share a mechanism of formation, initiated by an N-H hydrogen atom abstraction by the catalytically active iron-oxo of P450s. Molecular modeling studies also show these dimer metabolites fit in the CYP3A4 binding site in low energy conformations with minimal protein rearrangement. Collectively, the results of these experiments suggest that formation of these two homodimer metabolites is mediated by CYP3A, likely involving activation of two GDC-0994 molecules by a single P450 enzyme and proceeding through a radical coupling mechanism. SIGNIFICANCE STATEMENT: These studies identified structures and enzymology for two distinct homodimer metabolites and indicate a novel biotransformation reaction mediated by CYP3A. In it, two molecules may bind within the active site and combine through radical coupling. The mechanism of dimerization was elucidated using density functional theory computations and supported by molecular modeling.

Regional Proteomic Quantification of Clinically Relevant Non-Cytochrome P450 Enzymes along the Human Small Intestine.

Current challenges in accurately predicting intestinal metabolism arise from the complex nature of the intestine, leading to limited applicability of available in vitro tools as well as knowledge deficits in intestinal physiology, including enzyme abundance. In particular, information on regional enzyme abundance along the small intestine is lacking, especially for non-cytochrome P450 enzymes such as carboxylesterases (CESs), UDP-glucuronosyltransferases (UGTs), and sulfotransferases (SULTs). We used cryopreserved human intestinal mucosa samples from nine donors as an in vitro surrogate model for the small intestine and performed liquid chromatography tandem mass spectrometry-based quantitative proteomics for 17 non-cytochrome P450 enzymes using stable isotope-labeled peptides. Relative protein quantification was done by normalization with enterocyte marker proteins, i.e., villin-1, sucrase isomaltase, and fatty acid binding protein 2, and absolute protein quantification is reported as picomoles per milligram of protein. Activity assays in glucuronidations and sequential metabolisms were conducted to validate the proteomics findings. Relative or absolute quantifications are reported for CES1, CES2, five UGTs, and four SULTs along the small intestine: duodenum, jejunum, and ileum for six donors and in 10 segments along the entire small intestine (A-J) for three donors. Relative quantification using marker proteins may be beneficial in further controlling for technical variabilities. Absolute quantification data will allow for scaling factor generation and in vivo extrapolation of intestinal clearance using physiologically based pharmacokinetic modeling. SIGNIFICANCE STATEMENT: Current knowledge gaps exist in intestinal protein abundance of non-cytochrome P450 enzymes. Here, we employ quantitative proteomics to measure non-cytochrome P450 enzymes along the human small intestine in nine donors using cryopreserved human intestinal mucosa samples. Absolute and relative abundances reported here will allow better scaling of intestinal clearance.

Characterization of Differential Tissue Abundance of Major Non-CYP Enzymes in Human.

The availability of assays that predict the contribution of cytochrome P450 (CYP) metabolism allows for the design of new chemical entities (NCEs) with minimal oxidative metabolism. These NCEs are often substrates of non-CYP drug-metabolizing enzymes (DMEs), such as UDP-glucuronosyltransferases (UGTs), sulfotransferases (SULTs), carboxylesterases (CESs), and aldehyde oxidase (AO). Nearly 30% of clinically approved drugs are metabolized by non-CYP enzymes. However, knowledge about the differential hepatic versus extrahepatic abundance of non-CYP DMEs is limited. In this study, we detected and quantified the protein abundance of eighteen non-CYP DMEs (AO, CES1 and 2, ten UGTs, and five SULTs) across five different human tissues. AO was most abundantly expressed in the liver and to a lesser extent in the kidney; however, it was not detected in the intestine, heart, or lung. CESs were ubiquitously expressed with CES1 being predominant in the liver, while CES2 was enriched in the small intestine. Consistent with the literature, UGT1A4, UGT2B4, and UGT2B15 demonstrated liver-specific expression, whereas UGT1A10 expression was specific to the intestine. UGT1A1 and UGT1A3 were expressed in both the liver and intestine; UGT1A9 was expressed in the liver and kidney; and UGT2B17 levels were significantly higher in the intestine than in the liver. All five SULTs were detected in the liver and intestine, and SULT1A1 and 1A3 were detected in the lung. Kidney abundance was the most variable among the studied tissues, and overall, high interindividual variability (>15-fold) was observed for UGT2B17, CES2 (intestine), SULT1A1 (liver), UGT1A9, UGT2B7, and CES1 (kidney). These differential tissue abundance data can be integrated into physiologically based pharmacokinetic (PBPK) models for the prediction of non-CYP drug metabolism and toxicity in hepatic and extrahepatic tissues.

Development of a mass spectrometry-based tryptophan 2, 3-dioxygenase assay using liver cytosol from multiple species.

A novel and rapid method to determine the potency of inhibitors for tryptophan 2, 3-dioxygenase (TDO2) activities in human and preclinical species was successfully developed and validated utilizing LC-MS/MS. Previously reported TDO2 activity assays are resource intensive, requiring cloning and overexpression of TDO2. Here, we demonstrated that liver cytosol contained sufficient active TDO2 for evaluating the potency of TDO2 inhibitors across multiple species. TDO2 expression in human cytosol was estimated by LC-MS/MS to be 41 pmoL/mg cytosolic protein, with similar levels in dogs and monkeys, whereas mice and rats had 9.6 and 5.0-fold greater expression, respectively. Reaction conditions for TDO2-mediated conversion of l-tryptophan to kynurenine were optimized. Marked differences in kinetic parameters and inhibition potency were observed in TDO2 across species, with different Km values in dog (0.055 mM), monkey (0.070 mM), human (0.19 mM), mouse (0.32 mM) and rat (0.36 mM). Subsequently, IC50 values were determined for a series of TDO2 inhibitors in liver cytosol of five species, and good agreement with the literature values was observed for human enzyme. Taken together, these data indicate that TDO2 inhibition can be rapidly determined in readily available hepatic cytosol to assess potential species differences in potency.

Characterizing the in vitro species differences in N-glucuronidation of a potent pan-PIM inhibitor GNE-924 containing a 3,5-substituted 6-azaindazole.

1. Glucuronidation of amines has been shown to exhibit large species differences, where the activity is typically more pronounced in human than in many preclinical species such as rat, mouse, dog and monkey. The purpose of this work was to characterize the in vitro glucuronidation of GNE-924, a potent pan-PIM inhibitor, to form M1 using liver microsomes (LM) and intestinal microsomes (IM). 2. M1 formation kinetics varied highly across species and between liver and intestinal microsomes. In LM incubations, rat exhibited the highest rate of M1 formation (CLint,app) at 140 ± 10 µL/min/mg protein, which was approximately 30-fold higher than human. In IM incubations, mouse exhibited the highest CLint,app at 484 ± 40 µL/min/mg protein, which was >1000-fold higher than human. In addition, CLint,app in LM was markedly higher than IM in human and monkey. In contrast, CLint,app in IM was markedly higher than LM in dog and mouse. 3. Reaction phenotyping indicated that UGT1A1, UGT1A3, UGT1A9, UGT2B4 and the intestine-specific UGT1A10 contributed to the formation of M1. 4. This is one of the first reports showing that N-glucuronidation activity is significantly greater in multiple preclinical species than in humans, and suggests that extensive intestinal N-glucuronidation may limit the oral exposure of GNE-924.

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.

CYP1A1-Mediated Intramolecular Rearrangement of Aminoazepane in GDC-0339.

GDC-0339 is a novel small molecule pan-Pim kinase inhibitor that was discovered as a potential treatment of multiple myeloma. During the in vitro and in vivo metabolite profiling of GDC-0339, a metabolite was detected that had the same elemental composition as the parent but was distinct with respect to its chromatographic separation and mass spectrometric fragmentation pattern. High resolution tandem mass spectrometry data indicated the metabolite was modified at the aminoazepane moiety. The structure was solved by nuclear magnetic resonance analysis of the isolated metabolite and further confirmed by comparing it to a synthetic standard. These results indicated that the metabolite was formed by an intramolecular amine replacement reaction with the primary amine forming a new attachment to pyrazole without any change in stereochemistry. In vitro experiments showed cytochrome P450s catalyzed the reaction and demonstrated high isoform selectivity by CYP1A1. Results from kinetic experiments showed that the CYP1A1-mediated rearrangement of GDC-0339 was an efficient reaction with apparent turnover number (kcat) and Michaelis constant (Km) of 8.4 minutes-1 and 0.6 µM, respectively. The binding of GDC-0339 to the cytochrome P450 active site was examined by characterizing the direct inhibition of CYP1A1-mediated phenacetin O-deethylation, and GDC-0339 was a potent competitive inhibitor with Ki of 0.9 µM. This high affinity binding was unexpected given a narrow active site for CYP1A1 and GDC-0339 does not conform structurally to known CYP1A1 substrates, which are mostly polyaromatic planar molecules. Further, we explored some of the structural requirements for the rearrangement reaction and identified several analogs to GDC-0339 that undergo this biotransformation.

Novel Mechanism of Decyanation of GDC-0425 by Cytochrome P450.

GDC-0425 [5-((1-ethylpiperidin-4-yl)oxy)-9H-pyrrolo[2,3-b:5,4-c']dipyridine-6-carbonitrile] is an orally bioavailable small-molecule inhibitor of checkpoint kinase 1 that was investigated as a novel cotherapy to potentiate chemotherapeutic drugs, such as gemcitabine. In a radiolabeled absorption, distribution, metabolism, and excretion study in Sprague-Dawley rats, trace-level but long-lived 14C-labeled thiocyanate was observed in circulation. This thiocyanate originated from metabolic decyanation of GDC-0425 and rapid conversion of cyanide to thiocyanate. Excretion studies indicated decyanation was a minor metabolic pathway, but placing 14C at nitrile magnified its observation. Cytochrome P450s catalyzed the oxidative decyanation reaction in vitro when tested with liver microsomes, and in the presence of 18O2, one atom of 18O was incorporated into the decyanated product. To translate this finding to a clinical risk assessment, the total circulating levels of thiocyanate (endogenous plus drug-derived) were measured following repeated administration of GDC-0425 to rats and cynomolgus monkeys. No overt increases were observed with thiocyanate concentrations of 121-154 µM in rats and 71-110 µM in monkeys receiving vehicle and all tested doses of GDC-0425. These findings were consistent with results from the radiolabel rat study where decyanation accounted for conversion of <1% of the administered GDC-0425 and contributed less than 1 µM thiocyanate to systemic levels. Further, in vitro studies showed only trace oxidative decyanation for humans. These data indicated that, although cyanide was metabolically released from GDC-0425 and formed low levels of thiocyanate, this pathway was a minor route of metabolism, and GDC-0425-related increases in systemic thiocyanate were unlikely to pose safety concerns for subjects of clinical studies.

Utility of CYP3A4 and PXR-CAR-CYP3A4/3A7 Transgenic Mouse Models To Assess the Magnitude of CYP3A4 Mediated Drug-Drug Interactions.

Species differences in the expression, activity, regulation, and substrate specificity of metabolizing enzymes preclude the use of animal models to predict clinical drug-drug interactions (DDIs). The objective of this work is to determine if the transgenic (Tg) Cyp3a-/-Tg-3A4Hep/Int and Nr1i2/Nr1i3-/--Cyp3a-/-Tg-PXR-CAR-3A4/3A7Hep/Int (PXR-CAR-CYP3A4/3A7) mouse models could be used to predict in vivo DDI of 10 drugs; alprazolam, bosutinib, crizotinib, dasatinib, gefitinib, ibrutinib, regorafenib, sorafenib, triazolam, and vandetinib (as victims); with varying magnitudes of reported CYP3A4 clinical DDI. As an assessment of the effect of CYP3A4 inhibition, these drugs were coadministered to Cyp3a-/-Tg-3A4Hep/Int mice with the CYP3A inhibitor, itraconazole. For crizotinib, regorafenib, sorafenib, and vandetanib, there was no significant increase of AUC observed; with alprazolam, bosutinib, ibrutinib, dasatinib, and triazolam, pretreatment with itraconazole resulted in a 2-, 4-, 17-, 7-, and 15-fold increase in AUC, respectively. With the exception of gefinitib for which the DDI effect was overpredicted (12-fold in Tg-mice vs 2-fold in the clinic), the magnitude of AUC increase observed in this study was consistent (within 2-fold) with the clinical DDI observed following administration with itraconazole/ketoconazole. As an assessment of CYP3A4 induction, following rifampin pretreatment to PXR-CAR-3A4/3A7Hep/Int mice, an 8% decrease in vandetanib mean AUC was observed; 39-52% reduction in AUC were observed for dasatinib, ibrutinib, regorafenib, and sorafenib compared to vehicle treated mice. The greatest effect of rifampin induction was observed with alprazolam, bosutinib, crizotinib, gefitinib, and triazolam where 72-91% decrease in AUC were observed. With the exception of vandetanib for which rifampin induction was under-predicted, the magnitude of induction observed in this study was consistent (within 2-fold) with clinical observations. These data sets suggest that, with two exceptions, these transgenic mice models were able to exclude or capture the magnitude of CYP3A4 clinical inhibition and induction. Data generated in transgenic mice may be used to gain confidence and complement in vitro and in silico methods for assessing DDI potential/liability.

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

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.

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.

Elucidating the Mechanisms of Formation for Two Unusual Cytochrome P450-Mediated Fused Ring Metabolites of GDC-0623, a MAPK/ERK Kinase Inhibitor.

Two isomeric metabolites of GDC-0623 [5-((2-fluoro-4-iodophenyl)amino)-N-(2-hydroxyethoxy)imidazo[1,5-a]pyridine-6-carboxamide], a mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) kinase inhibitor, were identified in radiolabeled mass balance studies in rats and dogs (approximately 5% in excreta) and were also observed in human circulation (nonradiolabeled). Mass spectrometric data indicated that both metabolites had formed a new ring structure fused to the imidazopyridine core. Given their unusual structures, we conducted experiments to elucidate their chemical structures and understand the mechanisms for their formation. For the first metabolite, M14, a pyrazol-3-ol ring was generated by N-N bond formation between the aniline and hydroxamate. For the second metabolite, M13, an imidazol-2-one was generated by a Hofmann-type rearrangement that involved C-C bond cleavage and C-N bond formation. Both reactions were catalyzed by CYP2C9 and CYP2C19. M14 was generated directly from GDC-0623 and we speculate that its formation was via oxidative activation of the hydroxamic ester by cytochrome P450 (P450) and intramolecular nucleophilic displacement of the ester side chain. M13 (the rearranged metabolite) formed from the N-reduced hydroxamate (amide) and not from GDC-0623 directly. We propose for M13 that a P450-mediated reaction formed a cationic amide intermediate, which enabled the molecular rearrangement of the imidazopyridine core migrating from the amide carbon to the nitrogen and subsequent cyclization reaction. Each of these metabolic pathways constitutes a novel biotransformation mediated by P450 enzymes.

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.

Dose-dependent exposure and metabolism of GNE-892, a ß-secretase inhibitor, in monkeys: contributions by P450, AO, and P-gp.

(R)-2-Amino-1,3',3'-trimethyl-7'-(pyrimidin-5-yl)-3',4'-dihydro-2'H-spiro[imidazole-4,1'-naphthalen]-5(1H)-one (GNE-892) is an orally administered inhibitor of ß-secretase 1 (ß-site amyloid precursor protein cleaving enzyme 1, BACE1) that was developed as an intervention therapy against Alzheimer's disease. A clinical microdosing strategy was being considered for de-risking the potential pharmacokinetic liabilities of GNE-892. We tested whether dose-proportionality was observed in cynomolgus monkey as proof-of-concept for a human microdosing study. With cryopreserved monkey hepatocytes, concentration-dependency for substrate turnover and the relative contribution of P450- versus AO-mediated metabolism were observed. Characterization of the kinetics of these metabolic pathways demonstrated differences in the affinities of P450 and AO for GNE-892, which supported the metabolic profiles that had been obtained. To test if this metabolic shift occurred in vivo, mass balance studies in monkeys were conducted at doses of 0.085 and 15 mg/kg. Plasma exposure of GNE-892 following oral administration was more than 20-fold greater than dose proportional at the high-dose. P-gp-mediated efflux was unable to explain the discrepancy. The profiles of metabolites in circulation and excreta were indicative that oxidative metabolism limited the exposure to unchanged GNE-892 at the low dose. Further, the in vivo data supported the concentration-dependent metabolic shift between P450 and AO. In conclusion, microdosing of GNE-892 was not predictive of pharmacokinetics at a more pharmacologically relevant dose due to saturable absorption and metabolism. Therefore, it is important to consider ADME liabilities and their potential concentration-dependency when deciding upon a clinical microdosing strategy.