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Supplemental material to this article can be found at: http://dmd.aspetjournals.org/content/suppl/2015/10/08/dmd.115.066282.DC1

1521-009X/44/1/28–39$25.00 http://dx.doi.org/10.1124/dmd.115.066282 AND DISPOSITION Drug Metab Dispos 44:28–39, January 2016 Copyright ª 2015 by The American Society for and Experimental Therapeutics Absorption, Metabolism, , and the Contribution of Intestinal Metabolism to the Oral Disposition of [14C], a MEK Inhibitor, in s

Ryan H. Takahashi, Edna F. Choo, Shuguang Ma, Susan Wong, Jason Halladay,1 Yuzhong Deng, Isabelle Rooney, Mary Gates, Cornelis E.C.A. Hop, S. Cyrus Khojasteh, Mark J. Dresser,2 and Luna Musib

Departments of and (R.H.T., E.F.C., S.M., S.W., J.H., Y.D., C.E.C.A.H., S.C.K.,), Product Development Oncology (I.R.), Early Clinical Development (M.G.), and (M.J.D, L.M.), Genentech, South San Francisco, California

Received July 14, 2015; accepted October 7, 2015 Downloaded from

ABSTRACT

The pharmacokinetics, metabolism, and excretion of cobimetinib, cobimetinib had been well absorbed (fraction absorbed, Fa =0.88). a MEK inhibitor, were characterized in healthy male subjects (n =6) Given this good absorption and the previously determined low following a single 20 mg (200 mCi) oral dose. Unchanged cobime- hepatic , the systemic exposures were lower than tinib and M16 ( conjugate of hydrolyzed cobimetinib) were expected (, F = 0.28). We hypothesized that intestinal dmd.aspetjournals.org the major circulating species, accounting for 20.5% and 18.3% of metabolism had strongly attenuated the oral bioavailability of the drug-related material in plasma up to 48 hours postdose, cobimetinib. Supporting this hypothesis, the fraction escaping respectively. Other circulating were minor, accounting gut wall elimination (Fg) was estimated to be 0.37 based on F and for less than 10% of drug-related material in plasma. The total Fa from this study and the fraction escaping hepatic elimination (Fh) recovery of the administered radioactivity was 94.3% (61.6%, S.D.) from the absolute bioavailability study (F =Fa 3 Fh 3 Fg). with 76.5% (62.3%) in and 17.8% (62.5%) in . Physiologically based pharmacokinetics modeling also showed that

profiling indicated that cobimetinib had been extensively metabo- intestinal clearance had to be included to adequately describe the at ASPET Journals on September 27, 2021 lized with only 1.6% and 6.6% of the dose remaining as unchanged oral profile. These collective data suggested that cobimetinib was drug in urine and feces, respectively. In vitro phenotyping exper- well absorbed following and extensively me- iments indicated that CYP3A4 was predominantly responsible for tabolized with intestinal first-pass metabolism contributing to its metabolizing cobimetinib. From this study, we concluded that disposition.

Introduction phase III coBRIM study, cobimetinib plus vemurafenib reduced the risk Cobimetinib [GDC-0973/XL518, chemically identified as (S)- of disease worsening or death by half in patients with BRAF V600- (3,4-difluoro-2-(2-fluoro-4-iodophenylamino)phenyl)(3-hydroxy- mutated metastatic melanoma (hazard ratio = 0.51; 95% confidence – , 3-(piperidin-2-yl)azetidin-1-yl)methanone] (Fig. 1) is a novel interval = 0.39 0.68; P 0.0001). The median progression-free therapeutic being developed by F. Hoffman-La Roche, survival was 9.9 months for cobimetinib plus vemurafenib compared Ltd. (Basel, Switzerland) and Genentech. The molecule is a potent and with 6.2 months with vemurafenib alone (Larkin et al., 2014). In 2015, highly selective inhibitor of MEK1/2, a kinase that activates ERK1/2 in the U.S. and Drug Administration granted Priority Review (http:// the mitogen-activated protein kinase signaling cascade. The mitogen- www.fda.gov/ForPatients/Approvals/Fast/default.htm) for a New Drug activated protein kinase signaling cascade transduces multiple pro- Application for cobimetinib in combination with vemurafenib for the – liferative and differentiating signals within tumor cells and includes treatment of patients with BRAF V600 mutation positive advanced four major mammalian mitogen-activated protein kinase pathway melanoma. modules: ERK1 and ERK2, c-Jun NH2-terminal kinase, p38 kinase, radiolabeled studies are the accepted standard for providing a and ERK5 (Johnson and Lapadat, 2002; Roberts and Der, 2007). In the definitive understanding of the absorption, distribution, metabolism, and excretion properties of a drug since the radiolabel assures that all of the drug-related material can be accounted for. From the radiolabel 1Current affiliation: Anacor Pharmaceuticals, Inc., Palo Alto, CA. study, in addition to obtaining pharmacokinetics (PK) for the drug, the 2Current affiliation: Denali Therapeutics, Inc., South San Francisco, CA. identity and concentrations of circulating metabolites and the pathways dx.doi.org/10.1124/dmd.115.066282. of elimination (metabolism or excretion) are revealed (Beumer et al., s This article has supplemental material available at dmd.aspetjournals.org. 2006; Penner et al., 2009). Prior to the human mass balance study,

ABBREVIATIONS: ACN, ; AE, adverse event; AUC, area under the curve; CL, clearance; DDI, drug-; F, bioavailability; Fa, fraction absorbed; Fg, fraction escaping gut wall elimination; Fh, fraction escaping hepatic elimination; HLM, human microsome; LC, liquid chromatography; MS, mass spectrometry; PBPK, physiologically based pharmacokinetics; PK, pharmacokinetics; SRM, selected reaction monitoring; t1/2, half-life; UDPGA, UDP ; UGT, UDP-. 28 Absorption, Metabolism, and Excretion of Cobimetinib In Humans 29

Materials and Methods Radiolabeled Drug and Reference Compounds. Cobimetinib and [14C] cobimetinib (radiochemical purity .98%) were synthesized by F. Hoffman-La Roche. For [14C]cobimetinib, the radiolabel was evenly distributed in the fluoro- iodoaniline ring (55 mCi/mmol specific activity) (Fig. 1). Synthetic standards for metabolites M12, M16, and M19 were synthesized at Genentech and Roche 13 (Basel, Switzerland). C6-cobimetinib (used as an internal standard for bioanalysis) was synthesized at Ricerca Biosciences (Concord, OH). Materials. 1-Aminobenzotriazole was purchased from Spectrum Chemical Corporation (Gardena, CA); CYP3cide was purchased from Toronto Research Chemicals (Toronto, Canada); and other chemical inhibitors (furafylline, , , , sulfaphenazole, , ketocona- zole, troleandomycin, ), reduced b-nicotinamide adenine dinucleo- tide phosphate tetrasodium salt (NADPH), UDP glucuronic acid (UDPGA) Fig. 1. Chemical structure of [14C]cobimetinib. The asterisk denotes the location of trisodium salt, and alamethicin were purchased from Sigma-Aldrich (St. Louis, the 14C radiolabel, which was uniformly distributed throughout the fluoro- MO). All other chemicals and solvents were of analytic grade and were obtained iodoaniline ring. from commercial sources. Human liver microsomes [(HLMs), pool of 150 donors, mixed sex)], CYP Supersomes (CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C18,

14 2C19, 2D6, 2E1, 3A4, and 3A5), and UDP-glucuronosyltransferase (UGT) Downloaded from cobimetinib was characterized in the preclinical species with C- Supersomes (UGT1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, radiolabeled mass balance studies (Choo et al., 2012). In rats and 2B15, and 2B17) were purchased from BD Biosciences (San Jose, CA). dogs, the administered dose was well absorbed, with 70%–80% of the CYP3A5 genotyped single donor HLMs were purchased from Xenotech radioactivity recovered in urine and bile. Metabolism was extensive (Lenexa, KS) and Corning Discovery Labware (Tewksbury, MA). with biliary excretion of metabolites as the primary pathway for Study Design. This was a single-center, open-label, nonrandomized study elimination. The major metabolic pathways were oxidative, although with the oral administration of radiolabeled cobimetinib (20 mg with approxi- some species differences in metabolism existed with rats primarily mately 200 mCi of radioactivity) to six healthy male subjects to determine the PK dmd.aspetjournals.org hydroxylating the aromatic core and dogs sequentially oxidizing the of the parent drug and to characterize metabolites in circulation and excreta. The piperidine-azetidine moieties (Takahashi, RH et al., manuscript in study was conducted at the Covance Clinical Research Unit (Madison, WI). The study followed the guidelines of the World Medical Association Declaration of preparation). Helsinki in its revised edition (http://www.wma.net/en/30publications/10policies/ The PK of cobimetinib following oral administration in patients with b3/), the current guidelines for Good Clinical Practice (http://www.fda.gov/ solid tumors have also been previously described (Musib et al., 2011). ScienceResearch/SpecialTopics/RunningClinicalTrials/), and other applicable Cobimetinib exhibited dose-proportional kinetics (;3.5–100 mg) with the regulatory requirements. All subjects provided written informed consent. coefficient of variability in exposure [area under the curve (AUC)] ranging Subjects. Volunteers that were eligible for inclusion in this study were male at ASPET Journals on September 27, 2021 from 21% to 120%. It has a low apparent clearance (CL) with a terminal (18–55 years of age; body mass index, 18.5–29.9 kg/m2) in good health, as half-life (t1/2) of approximately 50 hours, supporting once-daily dosing. determined from medical history, vital signs, 12-lead electrocardiogram, and The PK of cobimetinib following oral and i.v. administration have also clinical laboratory evaluations. Exclusion criteria included any clinically been characterized in healthy subjects and the absolute bioavailability of significant allergic disease or clinical manifestation of any significant metabolic, cobimetinib was determined to be 46.2% (Musib et al., 2013). The human dermatologic, hepatic, renal, hematologic, pulmonary, cardiovascular, gastro- intestinal, neurologic, or psychiatric disorder, or having received any other mass balance study described here was conducted in healthy subjects; investigational drug within 5 half-lives or 30 days prior (whichever was longer) therefore, the administered dose (20 mg) was lower than the clinical or a radiolabeled investigational drug within 6 months prior. Subjects with therapeutic dose for patients (60 mg). Given its dose-proportional history of glaucoma or retinal vein occlusion, neurosensory retinal detachment, PK, extrapolation of data from the 20-mg dose group in healthy subjects or predisposing factors to retinal vein occlusion were also excluded. was expected to adequately assess the fate of cobimetinib at its clinical Dose Preparation and Administration. The final dose of cobimetinib was therapeutic dose in patients. However, a caveat is that differences in prepared by the Covance Clinical Research Unit by dissolving 20 mg (200 mCi) of exposures between healthy subjects and cancer patients have been reported [14C]cobimetinib in Crystal Light (Kraft , Northfield, IL) solution. The (Cheeti et al., 2013; Coutant et al., 2015). dosed radioactivity was not expected to represent a significant radiation Physiologically based PK (PBPK) modeling is a powerful tool that exposure risk in man based on dosimetry from quantitative whole-body allows for predictions and investigations, for example, in drug-drug autoradiography using rats (Choo et al., 2012). Each subject was admitted on day -1, and on day 1 after at least a 10-hour fast each subject received the single interactions (DDIs), special populations, formulation changes, regional 20-mg dose as an oral solution followed by 240 ml of to rinse the dosing absorption, etc. (Agoram et al., 2001; Zhao et al., 2011; Heikkinen container. Subjects remained ambulatory (seated or standing) for 1 hour et al., 2012; Huang and Rowland, 2012). For cobimetinib, a base PBPK following dose administration. Subjects were restricted from caffeinated model was developed that described the i.v. PK profile. This model beverages, grapefruit juice, , and concomitant during the enabled simulations where the effects of food, permeability, particle entire duration of the study. Subjects were confined to the clinic for a minimum size, and on the absorption of cobimetinib were tested with of 13 days and a maximum of 28 days. Subjects were discharged when and sensitivity analysis and these data have been previously reported plasma radioactivity levels were below the limit of quantitation in two (Musib et al., 2013). The mass balance study in humans provided data consecutive samples and $90% of the administered dose had been recovered , that refined and verified the inputs for the cobimetinib PBPK model, or there was minimal excretion of radioactivity in urine and feces ( 1% of dose) and thereby increased confidence in the model for simulating the effects in consecutive collections. Sample Collection. Blood samples for PK analysis of cobimetinib and of other clinical scenarios, such as DDIs. metabolite profiling and identification were collected predose and at 0.5, 1, 2, The objectives of the current study were to determine the routes of 4, 6, 8, 12, 24, 48, 72, 96, 120, 144, 192, 264, 336, and 408 hours postdose. excretion, characterize the metabolites, and understand the factors Urine was collected predose and at 0–6, 6–12, and 12–24 hours postdose, and affecting the /disposition of cobimetinib. In addition, then at 24-hour intervals until the subject was discharged from the clinic. For PBPK simulations were used to test the hypothesis that intestinal each period, all urine produced by a subject was collected and stored in dark metabolism plays a role in the oral disposition of cobimetinib. containers and refrigerated. At the end of the collection period, the bulk urine 30 Takahashi et al. was mixed thoroughly, the total weight recorded, and aliquots transferred to provide additional chromatographic separation of late-eluting metabolites. This polypropylene containers and stored frozen at approximately 270C until gradient was the same as method A up to reaching 42% B, but then it was analysis. Fecal samples were collected at predose and 24-hour intervals until increased to 58% B over 22 minutes, increased to 100% B over 4 minutes, and the subject was discharged from the clinic. Each fecal collection was then the column was flushed and re-equilibrated as in method A. For detection, transferred to a tared container and homogenized with approximately 2 to the flow from the column was split 10:1 with the minor component directed to the 3 volumes of water and the weight of the fecal homogenate sample was mass spectrometer and the major component directed to a fraction collector for recorded. For each collection interval, 10 g of each fecal homogenate sample collection to DeepWell LumaPlate 96-well plates (Perkin Elmer, Waltham, MA) was transferred to a separate container for measurement of total radioactivity based on time (15 seconds/fraction). Following fraction collection, the plates by liquid scintillation counting and the remaining bulk fecal homogenate were dried under vacuum using a Savant (Thermo Scientific) with low or samples were stored at 220C or below until they were used for preparing medium heat setting for up to 8 hours. The radioactivity in each fraction was pooled samples for biotransformation analyses. measured using a TopCount Scintillation and Luminescence Counter (Perkin Determination of Radioactivity and Cobimetinib Plasma Concentrations. Elmer) for 5 minutes. Radiochromatograms were reconstructed using Laura Radioactivity and cobimetinib plasma concentrations were determined at Evaluation software (LabLogic, Sheffield, United Kingdom) and all radiopeaks Covance Laboratories, Inc. (Madison, WI). The radioactivity levels in plasma that were discernable over background (signal at least 3Â) were integrated to and urine were determined by liquid scintillation counting, and in blood and determine the distribution of radioactivity in each sample. feces they were determined by combustion followed by liquid scintillation Metabolite Identification. The proposed metabolites structures were de- 14 counting of the trapped CO2. Cobimetinib was quantified in plasma using a termined from MS data that corresponded to the radiodetection of drug-related validated liquid chromatography (LC)–tandem mass spectrometry (MS/MS) analytes. All radiopeaks in plasma and the radiopeaks that accounted for greater 13 method (Deng et al., 2014). The plasma samples were mixed with C6- than 0.5% of the dose in urine or 1% of the dose in feces were targeted for Downloaded from cobimetinib and then prepared using supported liquid extraction. The plasma identification. Mass spectra were obtained with an LTQ Orbitrap high-resolution extracts were concentrated under , and reconstituted for analysis. The mass spectrometer equipped with an electrospray ionization source from Thermo LC-MS/MS system consisted of a CTC HTS PAL autosampler (LEAP Scientific. The electrospray ion source voltage was 5.0 kV. The heated capillary Technologies, Carrboro, NC), LC-10AD pumps (Shimadzu, Columbia, MD), temperature was 350C. The scan-event cycle consisted of a full-scan mass and an API4000 triple quadrupole mass spectrometer (AB Sciex, Foster City, spectrum at a mass resolving power of 30,000 (at m/z 400) and the corresponding CA). The samples were injected onto a Luna PFP (2 Â 50 mm, 3 mm; data-dependent MS/MS and MSn scans were acquired at a resolving power of

Phenomenex, Torrance, CA) column and eluted with a gradient method that 7500. Accurate mass measurements were performed using external calibration. dmd.aspetjournals.org used mobile phases A (water with 0.1% formic acid) and B [acetonitrile Pharmacokinetic Evaluation. The PK parameters were calculated by (ACN) with 0.1% formic acid]. The total run time was 3 minutes and the flow noncompartmental methods (Gibaldi and Perrier, 1982) using WinNonlin rate was 0.5 ml/min. The ionization was conducted in the positive ion mode version 5.1.1 (Pharsight, Mountain View, CA). The AUC0–t values were 13 for cobimetinib and its internal standard, C6-cobimetinib, using the calculated for hour 0 to the last measurable concentration (t), which were 264 selected reaction monitoring (SRM) transitions m/z 532.1 → 249.1 and hours for total radioactivity in plasma and 408 hours for cobimetinib in plasma.

538.1 → 255.1, respectively. The standard curve ranged from 0.200 to The AUC0–‘ value was calculated as AUC0–t plus the extrapolated area from the 100 ng/ml using 50 ml of plasma, and the lower limit of quantification last measurable concentration to infinity. The AUCs were calculated using the of cobimetinib was 0.200 ng/ml. linear trapezoidal rule and t1/2 was calculated from the apparent terminal at ASPET Journals on September 27, 2021 Extraction of Metabolites from Biologic Samples. Plasma samples at elimination phase rate constant. The PK parameter estimates are presented as predose and at 1, 2, 4, 8, 24, and 48 hours postdose were pooled by combining mean 6 S.D. To estimate the plasma concentrations of metabolites, the percent equal volumes of plasma from each subject, and the subject-pooled samples at contributions from radioprofiling were multiplied by the concentrations of total these selected time points were profiled. Time-based pooling of plasma was not radioactivity (ng-Eq/g) for the 1, 2, 4, 6, 24, and 48 hours postdose plasma pursued since the recovery of radioactivity from plasma was different across time samples. The plasma exposures for the parent compound and metabolites points and biased the metabolite profile to earlier time points. Plasma samples (AUC0–48) were calculated from the concentration-time data with the trapezoidal (2 ml) were extracted twice with two volumes of ACN. The extracts from each method using Excel 2003 (Microsoft, Redmond, WA). step were combined and concentrated under vacuum, then redissolved with In Vitro Metabolism. Cobimetinib was incubated with HLMs (0.5 mg/ water:methanol (2:1, v/v, ;0.2 ml for approximately 10Â concentration of ml) in potassium phosphate buffer (100 mM, pH 7.4) with the necessary radioactivity) for radiometric analysis. Time-pooled urine and feces samples cofactors. Incubations for P450-mediated CLs were supplemented with were prepared for each subject by mixing an equal percentage of the excreted NADPH (1 mM). Incubations for UGT-mediated CLs were supplemented volume or weight of each collection. The pooled urine and feces samples with MgCl2 (10 mM) and UDPGA (5 mM) and the microsomes were represented .90% of the radioactivity excreted in that route (pooled urine activated with alamethicin (50 mg/mg microsomal protein, preincubated samples were for periods of 0–144 to 0–192 hours; pooled feces samples were for on ice for 5 minutes). For CYP phenotyping experiments, mechanism- periods of 0–96 to 0–312 hours postdose, depending on the rate that radioactivity based CYP inactivators (1-aminobenzotriazole, P450, 1 mM; furafylline, was excreted by the individual subject). Urine samples were concentrated under CYP1A2, 10 mM; or troleandomycin, CYP3A, 20 mM) were preincubated vacuum, the radioactivity components were redissolved with the addition of a for 15 minutes with HLMs that were supplemented with NADPH. Other small volume of ACN, and the supernatant was injected onto the LC column. inhibitors (tranylcypromine, CYP2A6, 1 mM; ticlopidine, CYP2B6/2C19, Fecal homogenates were extracted twice with ACN and the supernatants were 10 mM; quercetin, CYP2C8/2C9, 10 mM; sulfaphenazole, CYP2C9, 10 mM; separated and pooled together. The combined supernatants were evaporated to quinidine, CYP2D6, 1 mM; and fluconazole, UGT2B7, 2.5 mM) were added dryness under vacuum and the residues were reconstituted with water:methanol immediately before cobimetinib was added. The contribution of CYP3A5 to (2:1, v/v) before injection onto the LC column. HLM CL of cobimetinib was determined following similar experimental Metabolite Profiling. Chromatographic separations were completed using an conditions as have been previously described (Tseng et al., 2014). For Accela LC system (Thermo Scientific, San Jose, CA) and CTC HTS PAL additional phenotyping experiments, cobimetinib was incubated with P450 autosampler (LEAP Technologies) using a Luna C18(2) column (3 mm, 4.6 Â Supersomes (40 pmol/ml) or UGT Supersomes (0.5 mg/ml). Reactions were 250 mm, Phenomenex) and mobile phases A (0.4% formic acid in water adjusted at 1 mM (except for the M15 generation experiments, when it was 20 mM) to pH 3.2 with hydroxide) and B (methanol). Gradient method A and conducted at 37C. At 0 and 60 minutes, aliquots of the reaction mixture (total run time, 113 minutes) was used for urine and feces samples, where the were transferred to ACN containing internal standard. The samples were initial conditions were 0% B for 3 minutes; increase to 15% B over 5 minutes; centrifuged at 2000 Â g for 10 minutes, and the supernatants were diluted increase to 30% over 15 minutes and maintain for 12 minutes; increase to 42% B with water and analyzed by LC-MS/MS. Cobimetinib was monitored with over 45 minutes; increase to 70% over 7 minutes; and increased to 100% over 4 the SRM transition 532.1 → 249.1 and the percentage drug remaining in the minutes. The column was flushed at 100% B for 5 minutes, returned to 0% B reaction was determined by comparing it with t = 0 samples. For the UGT over 2 minutes, and then re-equilibrated to these conditions for 15 minutes. experiments, M15 (glucuronide conjugate) formation was monitored by Gradient method B (total run time, 128 minutes) was used for plasma samples to SRM with the neutral loss of 176 Da (708.1 → 532.1). For in vitro CL Absorption, Metabolism, and Excretion of Cobimetinib In Humans 31 estimates, the in vitro t1/2 was calculated as 20.693/k with k from the slope of the linear regression of log percentage remaining versus incubation time. The in vitro t1/2 was scaled to hepatic CL with the incubation conditions (concentration, time, and microsomal protein); microsomal content for liver, taken to be 45 mg/g liver; and liver weight, taken to be 20 g/kg body weight (Obach et al., 1997).

Estimating Oral Bioavailability (F), Fraction Absorbed (Fa), Fraction Escaping Gut Wall Elimination (Fg), and PBPK Modeling and Simula- tions. The hepatic CL of cobimetinib (assumed as CLTotal, since renal CL was negligible; 1.6% of dose unchanged in urine) in healthy volunteers was taken from the i.v. dosing arm of the previously reported absolute bioavailability study (Musib et al., 2013). The mean dose normalized AUC from the current study was compared with that from the i.v. dosing study to estimate the oral bioavailability for the current study. The fraction of the oral dose absorbed (Fa) was estimated as the sum of radioactivity recovered in urine and radioactivity in feces that was characterized as metabolites. This assumed that during transit, unabsorbed drug did not degrade and metabolites did not revert to unchanged cobimetinib. The fraction of dose that escaped gut   metabolism (Fg) was then calculated from the relationship of F =Fa Fg Fh Fig. 2. Concentration-time profiles of total radioactivity (solid squares) and Downloaded from 14 (where Fh denotes the fraction escaping hepatic elimination), with F and Fa cobimetinib (open diamonds) in plasma following a single oral dose of [ C] cobimetinib (20 mg, 200 mCi) given to healthy male subjects. Cobimetinib from the current study and Fh from the i.v. dosing study. The PBPKPlus module of GastroPlus simulation software (SimulationsPlus concentrations were determined by LC-MS/MS analysis. Data points are mean values and error bars are S.D. for n = 6 subjects. Inc., Lancaster, CA) was used to simulate the oral PK profile of cobimetinib from this 14C study using inputs as previously described (Musib et al., 2013). The enterocyte binding was set as 5.8% unbound (based on ). mean total radioactivity AUC0–‘ in plasma (4621 ng·Eq·h/g). The mean For all other parameters, default settings of GastroPlus were used. blood/plasma ratios for total radioactivity were 0.805 and 0.979 for dmd.aspetjournals.org Cmax and AUC0–‘, respectively. Mass Balance/Excretion in Urine and Feces. The cumulative Results recoveries of radioactivity from the six subjects are shown graphically Demographic, Safety, and Data. Six healthy male in Fig. 3. An average of 94.3% 6 1.6% of the administered 20 mg dose volunteer subjects (four Caucasians and two African Americans) were of [14C]cobimetinib was recovered over the 408-hour study. The enrolled. Five of the six enrolled subjects completed the study in majority of the administered radioactivity was recovered in feces with a accordance with the protocol. One subject withdrew 17 days after mean cumulative percentage of 76.5% 6 2.4% of the administered at ASPET Journals on September 27, 2021 dosing, but was not replaced and was included in the overall data radioactivity. The radioactivity recovered in urine cumulatively analysis. This withdrawn subject received a full dose of cobimetinib on accounted for 17.8% 6 2 .5% of the administered radioactivity. The day 1; had all plasma, whole blood, urine, and feces samples collected majority of the radioactivity (.80% of the dose) was recovered within through 408 hours postdose; and the cumulative percent of radioactive the first 168 hours postdose. dose recovered in urine and feces for this subject was .90%. The mean Metabolite Profiles in Plasma and Excreta. Radioprofiles were age, weight, and body mass index for the subjects were 32 years (range determined for plasma samples at 1, 2, 4, 6, 24, and 48 hours postdose 21–47 years), 82.2 kg (range 74.7–92.7 kg), and 25.5 kg/m2 (range pooled across all six subjects. Representative profiles are shown in Fig. 20.5–27.6 kg/m2), respectively. 4 and the calculated concentrations of analytes determined for all Overall, the oral dose of cobimetinib was well tolerated. There were plasma profiles are presented in Table 2. The recoveries of radioactivity no serious adverse events (AEs) and no subject was withdrawn from the from plasma by protein precipitation decreased as time increased study as a result of an AE. Five subjects (83.3%) experienced a total of postdose. From the 1-hour sample 93% of the radioactivity was nine AEs, which were all Grade 1. All AEs occurred greater than 24 recovered, whereas from the 48-hour sample 56% of the radioactivity hours after dosing, were mild in severity, and were resolved by the end was recovered. The most abundant analytes at time points up to 24 of the study. The majority (seven of nine events) were considered drug hours were unchanged cobimetinib and M16 (glycine conjugate of related by the investigator. The most common AE was categorized as hydrolyzed cobimetinib), which accounted for 20.5% and 18.3% of the gastrointestinal disorders, which included change in bowel movement, exposures to total drug-related material up to 48 hours, respectively. hard feces, nausea, and diarrhea. To assist/improve their bowel M15 (direct ) accounted for 7.4% of the exposure up to movements, all of the subjects received concomitant single doses of 48 hours. At 24 and 48 hours postdose, M16 and unchanged drug docusate sodium (100 mg). There were no clinically important changes accounted for approximately equal percentages of total plasma in clinical laboratory values, vital signs, physical examinations, radioactivity. Other minor circulating metabolites (accounting for less electrocardiograms, or visual disturbances. than 5% of the total circulating drug-related material) were oxidative Pharmacokinetics. The mean concentration-time profiles for products (M12, M18, M19, M21, and M40) and their glucuronide plasma cobimetinib and total radioactivity are presented in Fig. 2 with conjugates (M6, M20, M44, M45, M57, M59, and M60). Plasma the PK parameter estimates summarized in Table 1. The cobimetinib samples from after 48 hours postdose did not have enough radioactivity and total radioactivity median times to reach Cmax (tmax) were observed in their extracts for profiling. Extracts for plasma from 24 to 192 hours at 4.0 and 2.0 hours, respectively. The mean Cmax for cobimetinib in postdose were analyzed by MS using SRM for cobimetinib (532.1 → plasma was 10.0 ng/ml, while the mean Cmax for total radioactivity in 249.1) and M16 (negative ion mode electrospray, 449.1 → 127.1), plasma was 68.2 ng·Eq/g. After reaching Cmax, cobimetinib and total and M16 levels declined with a slope that was similar to cobimetinib radioactivity concentrations in plasma declined with mean terminal t1/2 (unpublished in-house data). values of 75.5 and 141 hours, respectively. The mean AUC0–‘ for Urine and feces pools from each subject were analyzed for cobimetinib in plasma (495 ng·h/ml) was approximately 10.7% of the metabolite profiles. Representative metabolic profiles are shown in 32 Takahashi et al.

TABLE 1 Pharmacokinetic parameters for cobimetinib and total drug-related radioactivity following a single oral dose of [14C] cobimetinib (20 mg, 200 mCi) given to healthy male subjects

Data for all parameters are mean (S.D.) except for the time to reach Cmax (tmax), which is the median (minimum, maximum); n =6.

Parameter Cobimetinib1 Total Plasma Radioactivity Total Blood Radioactivity

Cmax (ng-Eq/ml) 10.0 (2.70) 68.2 (9.67) 54.9 (5.20) tmax (h) 4.02 (1.00, 8.00) 2.00 (2.00, 2.05) 2.00 (2.00, 2.05) AUC0–t (ng-Eq·h/ml) 455 (179) 3533 (812) 2925 (497) 2 AUC0–‘ (ng-Eq·h/ml) 495 (183) 4621 (1256) 4012 (759) 2 t1/2 75.5 (21.9) 141 (56.9) 97.9 (32.1)

1 Units of ng/ml for Cmax or ng·h/ml for AUC. 2Data for n =4.

Fig. 5 and the distributions of metabolites are presented in Table 3. absorbed (Fa) was likely underestimated and represented the minimum The radioactivity was widely distributed with few major metabolites. value for Fa. Across subjects, the same profile of metabolites was observed with Metabolite Identification. Twenty-five metabolites were identi- reasonable subject-to-subject variability in metabolite abundances fied in the current study that were either in plasma samples or Downloaded from [mean variability in abundances (CV) were 25% and 27% for the accounted for greater than 2% of the administered dose in urine and identified urinary and fecal metabolites, respectively]. Unchanged feces. The proposed structures of these metabolites were derived from cobimetinib in urine accounted for 1.6% of the administered dose. elemental compositions calculated based on the accurate masses M15 was the major metabolite in urine, which accounted for 2.1% of observed for their protonated molecular ions [MH]+ for all metabolites the dose, and all other metabolites were trace level (,1% of the except M16, which was analyzed in negative ion mode, and structural

dose). In feces, unchanged cobimetinib accounted for 6.6% of the elucidation of the product ions observed from collision-induced dmd.aspetjournals.org administered dose. The major metabolites in feces were M5 (dioxi- dissociation experiments. The human metabolites were compared with dation, +30 Da); M10 (mono-oxidation); M29 (trioxidation, +46 Da); those reported in the preclinical species (manuscript in preparation) M56 (trioxidation, +48 Da); and M62 (mono-oxidation of M40), based on their LC retention times, and MS/MS and MSn fragmentation which individually accounted for 5.2%–10.3% of the administered patterns to confirm the same metabolite numbers were assigned to dose. Six other minor metabolites (M21, M52, M53, M55, M28, and common metabolites. For M12, M16, and M19, synthetic standards M40) individually accounted for 2.1%–3.3% of the dose in feces, and were available to confirm the structures of the human metabolites. The the remaining radioactivity was accounted for by trace level proposed metabolic pathways for cobimetinib in humans are summa- metabolites (,2% of the dose). Assuming no degradation of un- rized in Fig. 6 with the structural elucidation data for metabolites at ASPET Journals on September 27, 2021 absorbed drug and no metabolites reverted to unchanged cobimetinib provided in Supplemental Table 2. during transit in the gastrointestinal tract, or enterohepatic recircula- In Vitro Metabolism of Cobimetinib. The CL (hepatic) values of tion (supported by observations in preclinical species), the fraction of 18.7, 2.9, and 17.8 ml/min/kg were observed for cobimetinib with the dose absorbed (Fa) following oral administration in healthy HLMs supplemented with NADPH, UDPGA, and both cofactors, subjects was estimated to be 0.88 using the sum of radioactivity respectively. In preliminary experiments, activation of microsomes recovered in urine (17.8%) and radioactivity in feces that was with alamethicin did not impact the NADPH-dependent metabolism characterized as metabolites (69.9%). It was noted that M15 (glucuronide of cobimetinib and addition of 2% bovine serum albumin to activate conjugate) was absent in feces, presumably due to b-glucuronidase UGT2B7 did not impact the UDPGA-dependent CLs of cobimetinib. activities in the gastrointestinal tract; therefore, the estimated fraction The metabolism of cobimetinib by HLMs was NADPH dependent

Fig. 3. Cumulative excretion of total radioactivity following a single oral dose of [14C]cobimetinib (20 mg, 200 mCi) given to healthy male subjects. Open diamonds are radioactivity in urine, open squares are radioactivity in feces, and solid squares are the sum of urine and feces. Data points are mean values and error bars are S.D. for n = 6 subjects. Absorption, Metabolism, and Excretion of Cobimetinib In Humans 33 Downloaded from dmd.aspetjournals.org at ASPET Journals on September 27, 2021

Fig. 4. Representative metabolite radiochromatograms for plasma pooled from six subjects following a single oral dose of [14C]cobimetinib at 1 hour (top), 4 hours (middle), and 24 hours (bottom) postdose. Signals that were assigned to metabolite structures have been labeled. The signal at ;100 minutes was judged to be an artifact because it was also observed in the radioprofile for predose plasma. and fully inhibited by 1-aminobenzotriazole (a broad-spectrum which suggested that cobimetinib was mostly or exclusively metab- inactivator of P450), indicating that it was primarily P450 mediated. olized by CYP3A4 with minimal contribution by CYP3A5 (un- and troleandomycin effectively inhibited the published in-house data). metabolism, which identified CYP3A as the major P450s involved in Determining the Contribution of First-Pass Intestinal Metabo- metabolizing cobimetinib (Fig. 7A). This was consistent with the lism (Fg) to the Oral Bioavailability of Cobimetinib. The oral findings from experiments using individual expressed recombinant bioavailability for the current study was estimated to be 0.28 6 0.10 P450 Supersomes, where the greatest turnover was measured with from the ratio of the mean dose normalized AUC compared with that rCYP3A4 and rCYP3A5 (Fig. 7B). In the UGT reaction phenotyping following i.v. dosing study (Musib et al., 2013). The relatively low study using Supersomes, M15 formation was mediated mainly by intersubject variability (CV = 28.2%, n = 13) in the i.v. dosing study UGT2B7, and this was confirmed by inhibition by fluconazole (Fig. suggested that hepatic CL did not differ greatly (i.e., CV less than 50%) 7C) (Uchaipichat et al., 2006). The CL values were low with little between healthy volunteer subjects in separate studies. This supported difference between ketoconazole and CYP3cide inhibition with assuming hepatic CL and Fh from a separate study [Fh = 0.87, where genotyped CYP3A5 HLM lots (CYP3A5*1/*1, *1/*3, or *3/*3), FH =12 Eh;Eh =CLs/Qh,blood; and Qh = 20.7 ml/min/kg (87 l/h) for a 34 Takahashi et al.

TABLE 2 14 Plasma concentrations and percentage of total radioactivity AUC0–48 h for cobimetinib and its metabolites following a single oral dose of [ C] cobimetinib (20 mg, 200 mCi) given to healthy male subjects Plasma concentrations were pooled across subjects (n = 6) at individual time points for metabolite profiling.

Plasma Concentration (ng-Eq/ml) Analyte Percentage of Total Radioactivity, AUC0–48 h 1 Hour 2 Hours 4 Hours 6 Hours 24 Hours 48 Hours Cobimetinib 7.92 10.51 7.37 8.03 6.02 2.14 20.5 M6 3.76 4.65 3.83 2.19 ND ND 3.0 M12 4.80 5.96 3.39 2.55 0.85 ND 4.7 M15 5.32 7.46 6.34 4.84 0.66 0.64 7.4 M16 3.63 6.60 6.64 6.50 5.40 2.86 18.3 M18 2.34 3.15 3.09 2.31 ND ND 2.7 M19 1.43 2.12 2.36 1.08 ND ND 1.5 M20 0.39 1.23 1.62 0.63 ND ND 0.9 M21 1.43 1.20 1.03 0.42 ND ND 0.7 M40 2.34 1.16 2.50 1.11 ND ND 1.5 M44 1.82 2.22 1.03 0.87 ND ND 1.2 M45 1.82 2.29 0.74 1.26 ND ND 1.4 M57 1.04 1.37 1.62 1.19 ND ND 1.4 Downloaded from M59 2.86 1.74 1.48 0.97 ND ND 1.3 M60 1.56 0.92 1.18 0.71 ND ND 0.9 Not extracted 3.48 7.33 8.40 10.8 8.37 7.96 31.5 Total radioactivity 48.1 61.7 54.1 46.6 21.3 13.6 100

ND, not detected. dmd.aspetjournals.org 70-kg person] (Davies and Morris, 1993); because the blood-to-plasma 0.02 (mean 6 S.D.) (described previously), Fg was estimated to be ratio of cobimetinib was 0.98, hepatic extraction was directly 0.37 6 0.14. determined from total systemic plasma CL for subjects in the current While the PBPK model was successful in describing the PK study. Taking the fraction of the oral dose absorbed (Fa) to be 0.88 6 profile of cobimetinib after i.v. administration in the absolute at ASPET Journals on September 27, 2021

Fig. 5. Representative metabolite radiochromatograms for urine (upper) and feces (lower) from an individual subject following a single oral dose of [14C]cobimetinib. Absorption, Metabolism, and Excretion of Cobimetinib In Humans 35

TABLE 3 Percentages of administered radioactive dose for cobimetinib and its metabolites in urine and feces following a single oral dose of [14C]cobimetinib (20 mg, 200 mCi) given to healthy male subjects Metabolites that accounted for less than 2% of the administered dose are not individually listed. Values are mean (S.D.) for six subjects.

Percentage Excreted Analyte Urine Feces Total

% of Dose Cobimetinib 1.6 (0.4) 6.6 (1.7) 8.2 (1.9) M5 D 5.2 (0.6) 5.2 (0.6) M10 0.3 (0.1) 10.3 (2.1) 10.7 (2.1) M15 2.1 (0.9) ND 2.1 (0.9) M21 0.9 (0.1) 2.7 (0.6) 3.6 (0.6) M28 ND 2.7 (0.7) 2.7 (0.7) M29 0.3 (0.1) 6.9 (2.4)1 7.9 (2.4) M62 0.7 (0.1) 6.9 (2.4)1 7.9 (2.4) M37 0.3 (0.1) 1.9 (0.6) 2.1 (0.6) M40 0.2 (0.1) 3.3 (0.6) 3.5 (0.5) M49 0.7 (0.1) 1.8 (0.3) 2.5 (0.3)

M52 D 2.4 (0.6) 2.4 (0.6) Downloaded from M53 D 3.1 (0.4) 3.1 (0.4) M55 D 2.1 (0.6) 2.1 (0.6) M56 ND 5.3 (2.6) 5.3 (2.6) Minor metabolites with oxidative deiodination 2.5 (0.5) 4.9 (0.7) 7.3 (1.1) Minor metabolites with oxidation at aromatic 1.3 (0.2) 4.0 (0.9) 5.3 (0.7) Other minor metabolites 3.2 (0.7) 7.1 (0.9) 10.3 (1.1) Total cobimetinib and identified metabolites 14.0 (2.0) 70.2 (2.2) 84.1 (2.6)

Total radioactivity 17.8 (2.5) 76.5 (2.3) 94.3 (1.6) dmd.aspetjournals.org

D, detected only by MS; ND, not detected. 1M29 and M62 co-eluted, and the sum of the two metabolites is presented. bioavailability study described by Musib et al. (2013) (Fig. 8A), (testing ;10-fold lower and higher than the observed/experimental simulation of the oral PK profile with the same parameters value) indicated that permeability (0.1–10 Â 104 cm/s), particle size (predicted Fa = 1.0) overestimated the observed Cmax and AUC (2.5–250 mM), and solubility (0.079–7.9 mg/ml) had little impact by 6-fold (59.2 versus 10.0 ng/ml) and 3-fold (1450 versus 495 mg·h/ml), on the PK profile of cobimetinib (Musib et al., 2013). We did note at ASPET Journals on September 27, 2021 respectively (Fig. 8B). Previously described sensitivity analysis that the observed mean AUC in the current study was ;1.5-fold

Fig. 6. Proposed metabolic pathways of cobimetinib in healthy male subjects following a single 20 mg oral dose. Excreted metabolites that accounted for greater than 5% of the dose and all circulating metabolites are described. M17, M32, and the piperidine monooxidation metabolites were not observed in the current study but are included to describe the likely sequential metabolism of cobimetinib. Samples where a metabolite was observed are indicated in brackets for plasma (P), urine (U), and feces (F). 36 Takahashi et al. Downloaded from dmd.aspetjournals.org at ASPET Journals on September 27, 2021

Fig. 7. Reaction phenotyping for cobimetinib. (A) Effect of selective P450 chemical inhibitors on cobimetinib metabolism in pooled HLMs. (B) Cobimetinib metabolism by expressed recombinant P450 Supersomes. (C) Formation of M15 by alamethicin-activated HLMs and expressed recombinant UGT (solid bars) and effect of fluconazole (UGT2B7 inhibitor, light bars). Data bars are mean values and error bars are S.D. from triplicate incubations.

lower than in the absolute bioavailability study, which was likely due to bile (76.5% of the administered dose in rats and 60.3% of the intersubject variability; however, the base PBPK model overestimated administered dose in dogs), which in humans was consistent with the exposures by 2- to 3-fold in both cases. In order to better describe the majority of radioactivity being recovered as metabolites in feces. PK data (to within 1.5-fold of the observed AUC), addition of intestinal Metabolite profiling of human samples indicated unchanged cobime- CL (to best fit) was necessary (Fig. 8C). Using the refined model, the tinib was the most abundant drug-derived species in plasma. Prior to simulations described the elimination half-life (61 hours predicted elimination, cobimetinib was extensively metabolized, and un- versus 75.5 hours observed) and AUC0–‘ (420 ng·h/ml predicted versus changed drug in urine and feces accounted for 1.6% and 6.6% of 495 ng·h/ml observed) with good success. the dose, respectively. Given the low intersubject variability in total radioactivity PK profiles (CV values for AUC and Cmax were 24% and 28%, Discussion respectively), plasma samples were pooled across subjects at 1, 2, 4, 6, The objective of the current study was to characterize the metab- 24, and 48 hours postdose to characterize the circulating radioactivity olism and excretion of cobimetinib in humans following a single and estimate metabolite exposures. Total radioactivity in plasma 20-mg oral dose. The radioactive dose was fully recovered with the declined more slowly than unchanged drug, which indicated that one majority being eliminated in feces (76.5% of the dose) and lesser or more components of the circulating radioactivity were not un- amounts eliminated in urine (17.8% of the dose). Most of the changed drug. Unchanged cobimetinib and M16 were the major radioactivity (.80% of the dose) was recovered in the first 7 days circulating components up to 48 hours postdose (AUC0–48), accounting following dosing. Cobimetinib was well absorbed with the extent for 20.5% and 18.3% of the circulating drug-related material, re- of absorption estimated to be 88%. This was consistent with findings spectively. No other metabolite in plasma approached 10% of total from nonclinical radiolabeled studies, where 70%–80% of drug-related material. After 48 hours postdose, cobimetinib and M16 the administered radioactivity was recovered in urine and bile declined in parallel, indicating that M16 CL was formation-rate limited. from bile-duct cannulated rats and dogs (Choo et al., 2012). In The total radioactivity in plasma declined to 22% of Cmax at 48 hours the preclinical species, metabolites were predominantly excreted in (61.7 ng·Eq/ml at 2 hours and 13.6 ng·Eq/ml at 48 hours), but the Absorption, Metabolism, and Excretion of Cobimetinib In Humans 37

Fig. 8. The observed PK profile of cobimetinib (symbols, mean 6 S.D.) and PBPK fit (line). (A) 2 mg i.v. fit; (B) single 20 mg oral dose, using parameters from i.v. fit; and (C) single 20 mg oral dose with addition of intestinal CL. Downloaded from dmd.aspetjournals.org

unextractable radioactivity showed little decline after 2 hours and was described by predominantly oxidative that oc- approximately the same concentration at 48 hours (7.33 ng·Eq/ml at curred in successive steps. Oxidation reactions modified the aromatic at ASPET Journals on September 27, 2021 2 hours and 7.96 ng·Eq/ml at 48 hours). Thus, the unextractable portion portion, including deiodination reactions, and at the aliphatic portion, was an increasing percentage of the plasma radioactivity at later time which lead to piperidine ring opening and further modification. points. These observations were consistent with radioactivity becoming Conjugative reactions, mostly glucuronidation, were also evident for associated with plasma protein(s), and once it was associated its cobimetinib and its oxidative metabolites. In mass balance studies in elimination was determined by the catabolism cycle for the protein, rats and dogs, cobimetinib was well absorbed following oral which was long. This became the predominant determinant for the administration and extensively metabolized, and the metabolic path- observed long elimination phase for total radioactivity. Over the first 48 ways were largely overlapping with humans (manuscript in prepara- hours, approximately 31.5% of the radioactivity in plasma was tion). Furthermore, the abundant circulating metabolites in humans unextractable. Converting this to an amount, based on a total plasma were observed in one or more tested preclinical species, which validated volume of 3000 ml for humans (Davies and Morris, 1993) less than the choice of these nonclinical species for toxicological studies of 100 mg or less than 0.1% of the administered dose was in plasma and cobimetinib. In vitro CYP reaction phenotyping described CYP3A4 unextracted. This was consistent with fully recovering the administered as the predominant for cobimetinib metabolism. For the radioactive dose in urine and feces over the 17-day study period. To test direct glucuronidation of cobimetinib (to form M15), UGT2B7 the hypothesis that the long-lived radioactivity was associated with catalyzed the conjugation reaction. With HLMs, glucuronidation was plasma proteins, we employed targeted MS analyses to identify a minor contribution to the complete metabolism. This was cobimetinib-related adducts of proteins or amino acid residues, but consistent with excreta and plasma profiles, which showed at least were unsuccessful, presumably due to the low percentage of modified 63.5% of the dose was transformed by oxidative pathways. M15 was a protein and the limitations of detection of the analytical methods. minor analyte in excreta (2.1% of the dose in urine) and plasma (7.4% Similar observations of incomplete recoveries of radioactivity from of drug-related material). Thus, it appeared that the predominant plasma have been reported for in humans, although in that case pathway for cobimetinib in vivo CL was oxidative metabolism that only unchanged drug was a detectable radiopeak for plasma samples was catalyzed by CYP3A4. (Castellino et al., 2012). In the current study, the absolute bioavailability was determined to be The metabolic profiles for human samples indicated extensive 28%. Since drug absorption did not seem to be a barrier (Fa = 0.88 from metabolism of cobimetinib and there were only minor quantitative this study), the leading hypothesis was that extraction of the cobime- differences observed for the six subjects. There were a few metabolites tinib by the liver, and particularly by the gut, had limited oral exposures. that were identified for the first time in this study, but they were found This hypothesis was consistent with the extensive metabolism observed at trace levels (#2% of the administered dose) except for M56 and the enzymology data that indicated the major metabolic pathways (trioxidation), which accounted for 5% of the dose and was in feces. for cobimetinib were CYP3A4-mediated oxidation, and to a lesser The proposed structures for these metabolites also suggested they extent UGT2B7-mediated glucuronidation. These are formed from primary metabolites that were common between humans expressed in the intestine and liver, and have been implicated as major and the preclinical species. Overall, the metabolism in humans was barriers to oral bioavailability by extracting drug by first-pass 38 Takahashi et al. metabolism (Cubitt et al., 2009; Gertz et al., 2010). Given that the humans. Unchanged cobimetinib was the main circulating species and metabolites generated by the intestine could not be distinguished there were no human unique metabolites observed in circulation. The from those generated by hepatic metabolism, we examined the majority of the drug-related radioactivity was excreted as metabolites in current data set to estimate the extent of gut metabolism that attenuated the feces with minor renal elimination. Cobimetinib metabolism was the systemic oral exposures. First, taking F and Fa from the current study mediated primarily by CYP3A4, which catalyzed multiple oxidative and applying the hepatic CL determined from the i.v. dosing study (Fh = pathways with extensive sequential metabolism. The oxidative modi- 0.87) (Musib et al., 2013), Fg was calculated to be 0.37, which was fications occurred most extensively at the piperidine ring, which indicative that intestinal metabolism contributed significantly to the oral resulted in several ring-opened metabolites, and to a lesser extent at bioavailability of cobimetinib. Consistent with this premise, there was the aromatic core of cobimetinib. In addition to characterizing the 2 good correlation between F versus Fg (r = 0.996), whereas a correlation metabolic fate for cobimetinib, this study allowed the estimation of would not be expected for a compound in which F is only limited by Fa, and in turn supported that intestinal metabolism contributes to hepatic extraction/CLsystemic/total (Supplemental Fig. 2). The interindi- limiting oral bioavailability. These findings increased our understand- vidual and study-to-study variability in mean exposure observed for the ing of the disposition and total metabolism of cobimetinib and have current study and the absolute bioavailability study may be a conse- implications for interpreting and predicting DDIs. quence of the variability in the intestinal gut expression of CYP3A4 in individuals. A 6-fold range in exposures is in keeping with observations Acknowledgments for other that are CYP3A4 substrates and that are metabolized by The authors thank the study volunteers, without whom this study would not Downloaded from the gut (Masica et al., 2004). have been completed. Given the added knowledge of factors that impact cobimetinib disposition, we tested the reliability of a transgenic mouse model with Authorship Contributions differential expression of CYP3A4 in the gut and/or liver for predicting Participated in research design: Takahashi, Choo, Ma, Wong, Halladay, the relative contribution of hepatic and intestinal metabolism (Choo et al., Rooney, Gates, Dresser, Musib. 2015). The results showed good concurrence with the inference from Conducted experiments: Takahashi, Choo, Wong. the human radiolabeled study that cobimetinib was extensively Contributed new reagents or analytic tools: Takahashi, Deng. dmd.aspetjournals.org extracted by metabolism at the gut compared with at the liver. Across Performed data analysis: Takahashi, Choo, Ma, Wong, Halladay, Musib. structurally diverse and highly related species, the relationship Wrote or contributed to the writing of the manuscript: Takahashi, Choo, Ma, between extraction due to metabolism at the gut and liver has not Rooney, Gates, Hop, Khojasteh, Musib. been clear. With benzodiazepines, Fg estimates for , , and are 0.05, 0.6, and 0.5, respectively despite References only a single atom difference for triazolam and alprazolam (Masica Agoram B, Woltosz WS, and Bolger MB (2001) Predicting the impact of physiological and biochemical processes on oral drug bioavailability. Adv Drug Deliv Rev 50 (Suppl 1):S41–S67. at ASPET Journals on September 27, 2021 et al., 2004). The challenges in predicting intestinal metabolism are Beumer JH, Beijnen JH, and Schellens JH (2006) Mass balance studies, with a focus on anti- further exemplified by several drugs that are predominantly cleared by cancer drugs. Clin Pharmacokinet 45:33–58. CYP3A4 (e.g., , , , and ), Castellino S, O’Mara M, Koch K, Borts DJ, Bowers GD, and MacLauchlin C (2012) Human metabolism of lapatinib, a dual kinase inhibitor: implications for . Drug Metab which have low-to-moderate hepatic CL (as determined by liver Dispos 40:139–150. microsomes), but are extracted by the gut very extensively (Yang Cheeti S, Budha NR, Rajan S, Dresser MJ, and Jin JY (2013) A physiologically based phar- macokinetic (PBPK) approach to evaluate pharmacokinetics in patients with cancer. Biopharm et al., 2007). Clearly, developing reliable methods for predicting the Drug Dispos 34:141–154. contribution of intestinal extraction to determining bioavailability is Choo EF, Belvin M, Boggs J, Deng Y, Hoeflich KP, Ly J, Merchant M, Orr C, Plise E, and Robarge K, et al. (2012) Preclinical disposition of GDC-0973 and prospective and an area requiring further study. retrospective analysis of human dose and predictions. Drug Metab Dispos 40: With increased understanding of the absorption, metabolism, and 919–927. 14 — Choo EF, Woolsey S, DeMent K, Ly J, Messick K, Qin A, and Takahashi R (2015) Use of excretion properties of cobimetinib from the Cstudy specifically, transgenic mouse models to understand the oral disposition and drug-drug interaction potential – the estimate for Fa and key insight/confirmation on the involvement of of cobimetinib, a MEK inhibitor. Drug Metab Dispos 43:864 869. — Coutant DE, Kulanthaivel P, Turner PK, Bell RL, Baldwin J, Wijayawardana SR, Pitou C, intestinal metabolism the PBPK model for cobimetinib was refined and Hall SD (2015) Understanding disease-drug interactions in cancer patients: implications for and used to describe the observed PK. With the hepatic CL, the i.v. dosing within the therapeutic window. Clin Pharmacol Ther 98:76–86. Cubitt HE, Houston JB, and Galetin A (2009) Relative importance of intestinal and hepatic profile of cobimetinib was well described (Fig. 8A). However, this glucuronidation—impact on the prediction of drug clearance. Pharm Res 26:1073–1083. base (i.v.) model did not adequately describe the oral concentration Davies B and Morris T (1993) Physiological parameters in laboratory and humans. ; Pharm Res 10:1093–1095. profile of cobimetinib ( 3-fold discrepancy in AUC between Deng Y, Musib L, Choo E, Chapple M, Burke S, Johnson J, Eppler S, and Dean B (2014) observed versus simulated) (Fig. 8B). Therefore, parameters that Determination of cobimetinib in human plasma using protein precipitation extraction and high- influenced the oral disposition of cobimetinib were evaluated. While performance liquid chromatography coupled to mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci 972:117–123. various scenarios were simulated (see Supplemental Table 1), the Gertz M, Harrison A, Houston JB, and Galetin A (2010) Prediction of human intestinal first-pass addition of gut metabolism (to best fit; entered in the model as K and metabolism of 25 CYP3A substrates from in vitro clearance and permeability data. Drug Metab m Dispos 38:1147–1158. Vmax) best described the oral profile of cobimetinib (Fig. 8C), which Gibaldi M and Perrier D (1982) Pharmacokinetics, 2nd ed. Marcel Dekker, New York. nicely corroborated its relatively large (;40%) role in the oral Heikkinen AT, Baneyx G, Caruso A, and Parrott N (2012) Application of PBPK modeling to predict human intestinal metabolism of CYP3A substrates—an evaluation and case study using disposition of cobimetinib. Furthermore, in the clinical interaction GastroPlus. Eur J Pharm Sci 47:375–386. study of cobimetinib with , 6.7- and 3.2-fold increases in Huang SM and Rowland M (2012) The role of physiologically based pharmacokinetic modeling in regulatory review. Clin Pharmacol Ther 91:542–549. cobimetinib AUC and Cmax respectively were observed. To describe the Johnson GL and Lapadat R (2002) Mitogen-activated protein kinase pathways mediated by ERK, – itraconazole DDI, fm CYP3A of 0.78 and Fg of 0.45 were estimated. JNK, and p38 protein kinases. Science 298:1911 1912. Larkin J, Ascierto PA, Dréno B, Atkinson V, Liszkay G, Maio M, Mandalà M, Demidov L, Subsequently, PBPK simulations were used to describe the magnitude Stroyakovskiy D, and Thomas L, et al. (2014) Combined vemurafenib and cobimetinib in of interactions from moderate and weak CYP3A4 inhibitors and BRAF-mutated melanoma. N Engl J Med 371:1867–1876. Masica AL, Mayo G, and Wilkinson GR (2004) In vivo comparisons of constitutive cytochrome inducers (American Society of Clinical Pharmacology and Therapeu- P450 3A activity assessed by alprazolam, triazolam, and midazolam. Clin Pharmacol Ther 76: tics Annual Conference, New Orleans, 2015). 341–349. Musib L, Choo E, Deng Y, Eppler S, Rooney I, Chan IT, and Dresser MJ (2013) Absolute In summary, data from the mass balance study indicated that the oral bioavailability and effect of formulation change, food, or elevated pH with on dose of cobimetinib was well absorbed and extensively metabolized in cobimetinib absorption in healthy subjects. Mol Pharm 10:4046–4054. Absorption, Metabolism, and Excretion of Cobimetinib In Humans 39

Musib L, Eppler S, Choo E, Deng A, Miles D, Hsu B, Rosen L, Sikic B, LoRusso P, Uchaipichat V, Winner LK, Mackenzie PI, Elliot DJ, Williams JA, and Miners JO (2006) Quan- and Ma W, et al. (2011) Abstract 1304: Clinical pharmacokinetics of GDC-0973, an oral titative prediction of in vivo inhibitory interactions involving glucuronidated drugs from in vitro MEK inhibitor, in cancer patients: data from a Phase 1 study. Cancer Res 71 (Suppl 8): data: the effect of fluconazole on glucuronidation. Br J Clin Pharmacol 61:427–439. 1304. Yang J, Jamei M, Yeo KR, Tucker GT, and Rostami-Hodjegan A (2007) Prediction of intestinal Obach RS, Baxter JG, Liston TE, Silber BM, Jones BC, MacIntyre F, Rance DJ, and Wastall P first-pass drug metabolism. Curr Drug Metab 8:676–684. (1997) The prediction of human pharmacokinetic parameters from preclinical and in vitro Zhao P, Zhang L, Grillo JA, Liu Q, Bullock JM, Moon YJ, Song P, Brar SS, Madabushi R, metabolism data. J Pharmacol Exp Ther 283:46–58. and Wu TC, et al. (2011) Applications of physiologically based pharmacokinetic (PBPK) Penner N, Klunk LJ, and Prakash C (2009) Human radiolabeled mass balance studies: objectives, modeling and simulation during regulatory review. Clin Pharmacol Ther 89:259–267. utilities and limitations. Biopharm Drug Dispos 30:185–203. Roberts PJ and Der CJ (2007) Targeting the Raf-MEK-ERK mitogen-activated protein kinase cascade for the treatment of cancer. Oncogene 26:3291–3310. Address correspondence to: Dr. Ryan Takahashi, Drug Metabolism and Tseng E, Walsky RL, Luzietti RA, Jr, Harris JJ, Kosa RE, Goosen TC, Zientek MA, and Obach Pharmacokinetics, Genentech, Inc., 1 DNA Way, MS 412a, South San Francisco, RS (2014) Relative contributions of cytochrome CYP3A4 versus CYP3A5 for CYP3A-cleared drugs assessed in vitro using a CYP3A4-selective inactivator (CYP3cide). Drug Metab Dispos CA 94080. E-mail: [email protected] 42:1163–1173. Downloaded from dmd.aspetjournals.org at ASPET Journals on September 27, 2021 DMD # 66282 Supplemental Data

Supplemental Data

Absorption, Metabolism, Excretion, and the Contribution of Intestinal Metabolism to the

Oral Disposition of [14C]Cobimetinib, a MEK Inhibitor, in Humans

Ryan H. Takahashi, Edna F. Choo, Shuguang Ma, Susan Wong, Jason Halladay, Yuzhong Deng,

Isabelle Rooney, Mary Gates, Cornelis E.C.A. Hop, S. Cyrus Khojasteh, Mark J. Dresser, Luna

Musib

Departments of Drug Metabolism and Pharmacokinetics (R.H.T., E.F.C., S.M., S.W., J.H., Y.D.,

C.E.H., S.C.K.,), Product Development Oncology (I.R.), Early Clinical Development (M.G.), and Clinical Pharmacology (M.J.D, L.M.), Genentech, Inc., 1 DNA Way, South San Francisco,

CA, 94080

Drug Metabolism and Disposition

DMD # 66282 Supplemental Data

Supplemental Methods

Absolute bioavailability study PBPK (Musib et.al, Mol Pharmaceutics, 2013) The partitioning (Kp) were determined by GastroPlus default methods (perfusion limited tissues and permeability limited tissues. The following compound dependent inputs were used: solubility ranging from 23.3 mg/mL at pH 1 to 0.79 mg/mL at pH 6.8; cLogP (3.7), pKa (1.98); protein binding (5.8%); MDCK permeability converted to human Peff (14 × 10-4 cm/s). For all other

parameters, default GastroPlus parameters/settings were used. Fa was predicted using the default

GastroPlus ACAT model.

Various scenarios were tested using the PBPK model by changing hepatic CL, permeability and

addition/inclusion of intestinal first pass metabolism to best capture the oral PK profile of

cobimetinib were as follows (Figure S1 A-F):

a) Increase hepatic CL to the observed CL/F of 25.2 L/h (Musib et al., 2013) (Figure S1-C,

Table S1)

b) Decrease permeability by 10-fold (decreasing %Fa from 99 to 83%) to account for

cobimetinib being a P-gp substrate (Choo et al., 2012) (Figure S1-D, Table S1). This is

an unlikely scenario since dose linearity in exposure was observed from approximately

~3.5 mg to 100 mg

c) Addition/inclusion of intestinal first pass metabolism (value that resulted in “best fit”), to

test the hypothesis that the lower than predicted F observed was due to intestinal first

pass metabolism (Figure S1-E, Table S1)

d) Addition of intestinal first pass metabolism and increased hepatic CL (from 11.7 to 13

L/h) to account for the higher range of CL observed in subjects (Figure S1-F, Table S1) DMD # 66282 Supplemental Data

Supplemental Tables

Table S1. PK parameters from observed profiles of cobimetinib from the absolute bioavailability

study and following PBPK simulations and optimizations

Simulation/O Peff CLG* CLh or %F %Fa Cmax /C0 AUC0-inf T1/2 (hr)

bserved PK (cm/s) (L/h) CL/F (ng/mL) (ng.h/mL)

x104 (L/h)

Observed (Musib et al., 2013)

IVa NA NA 11.7 NA NA 19.5 171 68.8

(28.2) (34.7) (28.2) (24.8)

POb NA NA 25.2 46.2 NA 13.8 716 59.5

(38.0) (24.2) (35.6) (38) (32.5)

Simulations/Optimizations

IVc 1.4 No 11.7 NA 99 25.3 171 61

POd 1.4 No 11.7 84 99 31.2 1430 61

A 1.4 No 25.2 67 99 21.9 526 28

B 0.14 No 11.7 71 84 16.0 1210 61

C 1.4 Yes 11.7 44 99 25.1 753 61

D 1.4 Yes 13 43 99 24.3 664 55

Observed data reported as geometric mean (geometric CV%); a – IV parameters from the absolute bioavailability study; observed, 1080 L (Figure 2A, symbols) b – PO parameters from oral arm of the absolute bioavailability study (Figure 2B, symbols) c – IV simulations using observed CL (Figure 2A, solid line) d – PO simulated using parameters from IV simulation (Figure 2B, solid line)

A – increase hepatic CL to CL/F (Figure 2C) B – decrease permeability (Figure 2D) C - inclusion of intestinal metabolism (Figure 2E) D- inclusion of intestinal CL and increasing hepatic CL (Figure 2F) * Yes/No; inclusion of intestinal first pass metabolism

DMD # 66282 Supplemental Data

Table S2. Proposed metabolites of cobimetinib identified in human samples Observed Ret Characteristic Chemical MH+ Analyte Time Product Ions Structure and MS/MS Product Ions Formula (Error in (min) (m/z) ppm) 376 - I, 249 OH F O 497.0310, H N 532.0694 N Cobimetinib 63.4 C21H22O2N3F3I 375.9439, HN (-1.7) - I H O, 497 249.0402 (100) F 2

F 403

- , 435 I 338 - I, 266 435.1409 (100), OH F O 562.0438 338.0890, H N - N O 2H M5 69.2 C21H20O4N3F3I HN (-1.2) 294.0625, I - 266.0440 O F I, 294 F 376 - - , I, I 221 249 84 OH F O Gluc 530.0516 (100) H N 724.0972 N M6 52.2 C H O N F I HN 27 30 9 3 3 (-0.1) IT: 706, 678, 530, I F 375, 302, 249 O F - Gluc=548

, - - 157 H2O NH3 122 , - 392 I 265 84 512.0444, OH F O 500.0440, H N N 548.0646 418.9501, HN M10 52.7 C21H22O3N3F3I I (-1.1) 403.1511, O F - = H2O 530 391.9397, - = F 2H2O 512 - 265.0352 (100) 419, I 293

- , - , 419 I 293 I 321 - I, 250 OH F O 546.0487 465.2853, H N M12 84.3 C H O N F I N 21 20 3 3 3 (-1.6) 419.1457 (100) HN I - F I, 277 O F 403 387 - , I 249 Gluc OH 84 532.0709 (100), F O H N 708.1012 514.0603, N M15 53.5 C H O N F I HN 27 30 8 3 3 (-1.7) 484.0505, I 249.0401 F F - Gluc=532 - - = Gluc H2O 514

DMD # 66282 Supplemental Data

376 - I, 249 O F H O N [M-H] 450.9749 433, 376, 249 H OH M16 88.9 N C15H9O3N2F3I (-2.7) (100) - I H O, 433 F 2 F 376 171 - I, 249 - , 471.0171, OH H2O 471 F O 546.0488 375.9452, H N - N O 2H M18 58.9 C21H20O3N3F3I HN (-1.5) 249.0400 (100), I 171.1129 F F , - 516.0374, 376 I 249 487 OH - F O H2O 465 403.2329 (100), H N N 546.0478 375.9446, N M19 85.0 C21H20O3N3F3I I (-3.3) 335.2588, O F 291.2326, , - 249.0403 F 221 HF 201 266 OH uc F O Gl H N 422.1696 (100), N 598.2000 HN M20 20.3 C27H31O9N3F3 404.1598, - (-1.2) F Gluc=422 266.0427 - - O Gluc H O=404 F 2

266 - , 374.1485, HF 218 - - , ; , 293.0547, H2O 139 NH3 122 238 - = 266.0431 (100) OH H2O 404 422.1681 F O M21 26.3 C21H23O3N3F3 H N (-1.2) N IT: 386 (100), HN

374, 293, 266, F 293 O - , 139, 122 H2O 374 F - I, 249 OH - F O H O, 517 546.0515 (100), H N 2 564.0591 N M28 55.6 C21H22O4N3F3I 517.0236, HN (-2.0) I 249.0400 2O F - = H2O 546 F , - 451 H2O 433 - 392, I 265 OH 543.0021, F O 578.0387 H N - N 2O 2H M29 65.4 C21H20F3IN3O5 415.1360 (100), HN (-1.2) I - 433.1253 H O=560 O F - 2 2H O=542 - 2 - F = H2O CO 532 438 - , H2O 420 376 190 - 438.1401, I, 249 OH 420.1298, F O 565.0436 H N M37 72.5 C21H21F3IN2O5 375.9448, N (-1.1) OH OH 249.0402 (100), I 190.1075 F O F DMD # 66282 Supplemental Data

438 547.0355, - - , 190 I, 321 H2O 420 376 - 438.1420, I, 249 OH 420.1309, F O 565.0433 H N M40 80.3 C21H21O5N2F3I 375.9467, N (-1.6) OH OH 321.0867, I F O 249.0412 (100), - H O, 547 190.1082 F 2 266 OH F O Gluc H N 598.2011 422.1683 (100), N M44 18.3 C H F N O HN 27 31 3 3 9 (0.7) 404.1580 F -Gluc=422 O -Gluc-H2O=404 F OH No product ion F O Gluc H N N 612.1791 spectra obtained HN 2O-2H M45 22.5 C27H29F3N3O10 (-1.5) due to low ion F abundance O F , - 190 H2O 172 - , - 266 2H2O 154 3H2O 136 OH F O 455.1426 266.0439, H N M49 28.6 C21H22F3N3O6 N (0.4) 190.1079 (100) OH O

F OH O F 190

266 437.3021, OH F O 455.1419 N M52 31.1 C21H22F3N3O6 266.0429 (100), H (-1.1) N O 190.1073 OH F OH O F , - , - 171 NH3 154 H2O 136 266 OH 416.1436, F O 436.1492 H N - M53 34.0 C21H20F3N3O4 266.0440, N O 2H (3.2) HN 171.1135 (100) - F F=416 O F 392 - I, 265 OH F O 548.0638 391.9365, H N N M55 50.2 C21H22F3IN3O3 HN (-2.6) 265.0349 (100) I O F F - - - I H O, 331 I, 265 2 - - - , 2O 2H I H O 345 453 2 - O 453.1527 (100), 2H2O 417 OH F O 417.1310, H N N 580.0541 HN M56 54.2 C21H22F3IN3O5 345.0868, (-1.7) I 296.0781, F

265.0370 F - I, 296

DMD # 66282 Supplemental Data

- I, 249 OH uc F O Gl 546.0499 (100) H N - N O 2H 722.0801 HN I - M57 55.3 C27H28F3IN3O9 OH=705 (-2.2) IT: 705, 676, 546 F - = CO2 676 (100), 249, 221 - - = F I, 221 Gluc 546

- 376, I 249 OH uc F O Gl H N - 546.0505 N O 2H HN 722.0811 (100),528.0394, I - H O=704 M59 58.2 C27H28F3IN3O9 F - 2 (-0.8) 516.0381, CO =676 - 2 249.0398 F Gluc=546 - - = Gluc H2O 528

- I, 249 H O Gluc 595.1805 (100), F O H N N 722.0814 546.0521, HN - I=5 5 M60 61.7 C27H28F3IN3O9 I - 9 (-0.4) 419.1471, - Gluc=546 F O 2H - - 249.0412 I Gluc=419 F

454 - , H2O 436 - , 563.0311, 2H2O 418 391 190 - 454.1363 I, 265 OH 581.0383 (100),436.1256, F O M62 66.1 C H F IN O H N 21 21 3 2 6 N (-1.4) 391.9405, OH OH 265.0360, I O F O - 190.1080 H O, 563 F - , 2 I 294

DMD # 66282 Supplemental Data

Supplemental Figures

Figure S1. The observed concentration-time profile of cobimetinib (symbols) and PBPK simulation (line) from the absolute bioavailability study: A) IV simulation, B) PO simulations; using parameters from IV simulation. Optimization of PBPK inputs: C) increased CL to CL/F,

D) decrease permeability, E) addition of intestinal metabolism, F) inclusion of intestinal first pass and hepatic CL

DMD # 66282 Supplemental Data

Figure S2. Relationship between observed oral bioavailability (F) and estimated intestinal bioavailability (Fg; blue) or hepatic bioavailability (Fh; red), data from the absolute bioavailability study (n=6)*