Absorption, Distribution, Metabolism, and Excretion (ADME) of Capmatinib (INC280) in Healthy Male Volunteers and In Vitro Aldehyde Oxidase Phenotyping of the Major Metabolite.

Capmatinib (INC280), a highly selective and potent inhibitor of the MET receptor tyrosine kinase, has demonstrated clinically meaningful efficacy and a manageable safety profile in patients with advanced NSCLC harboring MET exon 14 skipping mutations. We investigated the absorption, distribution, metabolism, and excretion of capmatinib in six healthy male volunteers after a single peroral dose of 600 mg 14C-labeled capmatinib. The mass balance, blood and plasma radioactivity, and plasma capmatinib concentrations were determined along with metabolite profiles in plasma, urine, and feces. The metabolite structures were elucidated using mass spectrometry and comparing with reference compounds. The parent compound accounted for most of the radioactivity in plasma (42.9 ± 2.9%). The extent of oral absorption was estimated to be 49.6%; the maximum concentration (Cmax) of capmatinib in plasma was reached at 2 h (median Tmax). The apparent mean elimination half-life of capmatinib in plasma was 7.84 h. Apparent distribution volume (Vz/F) of capmatinib during the terminal phase was moderate to high (geometric mean 473 L). Metabolic reactions involved lactam formation, hydroxylation, N-dealkylation, formation of a carboxylic acid, hydrogenation, N oxygenation, glucuronidation, and combinations thereof. The most abundant metabolite, M16 was formed by imidazo-triazinone formation (lactam formation). Absorbed capmatinib was eliminated mainly by metabolism and subsequent biliary/fecal and renal excretion. Excretion of radioactivity was complete after 7 days. In vitro studies demonstrated that CYP3A was the major P450 enzyme family involved in hepatic microsomal metabolism, and M16 formation was mainly catalyzed by aldehyde oxidase. SIGNIFICANCE STATEMENT: The absorption, distribution, metabolism, and excretion of capmatinib revealed that capmatinib had substantial systemic availability after oral administration. It was also extensively metabolized and largely distributed to the peripheral tissue. Mean elimination half-life was 7.84 h. The most abundant metabolite, M16 was formed by imidazo-triazinone formation and was catalyzed by aldehyde oxidase. Correlation analysis suggested that CYP3A is the major enzyme family involved in the hepatic microsomal metabolism of [14C]capmatinib.


Introduction
The MET proto-oncogene encodes a receptor tyrosine kinase that plays a role in the activation of signaling pathways involved in cell proliferation, migration, and survival (Sierra and Tsao, 2011;Feng et al., 2014;Smyth et al., 2014;Garajova et al., 2015). In normal physiological signaling, the MET pathway is activated by the binding of hepatocyte growth factor (Smyth et al., 2014). Dysregulation of the MET pathway can occur through several mechanisms and may lead to upregulated cell proliferation, survival, and metastasis (Feng et al., 2014;Garajova et al., 2015;Schrock et al., 2016;Tong et al., 2016). One such mechanism is through genomic mutations, including point mutations or insertions/deletions, leading to exon 14 skipping (Kong-Beltran et al., 2006;Feng et al., 2012;Garajova et al., 2015;Schrock et al., 2016;Salgia, 2017). The pathway can also be activated via MET gene amplification, which occurs in about 1-4% of newly diagnosed cases with epidermal growth factor receptor (EGFR)-wild-type non-small-cell lung cancer (NSCLC) (Cappuzzo et al., 2009;Kawakami et al., 2014;Schildhaus et al., 2015).
MET exon 14 skipping (METex14) mutations have been reported in 3-4% of patients with NSCLC (The Cancer Genome Atlas Research et al., 2014;Frampton et al., 2015;Awad et al., 2016;Descarpentries et al., 2018). Patients with METex14 mutations were found to have worse survival outcomes compared with patients without the MET mutation (Ludovini et al., 2012;Tong et al., 2016;Vuong et al., 2018). Capmatinib (INC280) is an orally bioavailable, ATP-competitive, reversible inhibitor of MET, with an half maximal inhibitory concentration (IC 50 ) of 0.13 nmol/L as demonstrated in an in vitro MET kinase assay (Liu et al., 2011;Lara et al., 2017). Capmatinib has also been demonstrated to be highly selective versus other kinases, with more than 1,000-fold selectivity among 442 kinases when tested using the KINOMEscan selectivity screening platform (Baltschukat et al., 2019). It is highly potent as demonstrated in biochemical (IC 50 0.13 nM) and cellular (IC 50 0.3-1.1 nM) assays across a range of tumor types, including lung, gastric cancer, and glioblastoma, and caused regression of MET-dependent (amplified) tumors

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in animal models at well-tolerated doses (Liu et al., 2011;Lara et al., 2017). In biochemical and vitro assays, it was found to be 30 times more potent than crizotinib (IC 50 values of 0.13 and 4 nM, respectively), (Zou et al., 2007;Liu et al., 2011). In Phase I and II clinical studies, single-agent capmatinib has demonstrated strong activity in patients with NSCLC with a high MET gene copy number (GCN), with an overall response rate (ORR) of 53% in patients with MET GCN ≥6 (Schuler et al., 2018) and 68% in treatment-naïve patients with METex14 advanced NSCLC (Wolf, 2019). In addition, the combination of capmatinib with gefitinib has been shown to be a promising treatment for patients with EGFR-mutated, MET-dysregulated NSCLC, particularly for MET-amplified disease, with an ORR of 47% in patients with MET GCN ≥6 (Wu et al., 2018). Capmatinib is now approved for adult patients with metastatic NSCLC with METex14 mutation.
This single-center, open-label study, conducted as per the International Conference on Harmonisation (ICH) M3 guidelines, investigated the absorption, distribution, metabolism, and excretion (ADME) of capmatinib after a single oral dose of 600 mg [ 14 C]capmatinib (5.55 megabecquerel [MBq]) in healthy male volunteers.
In vitro investigations were conducted to identify the role of aldehyde oxidase (AO), xanthine oxidase (XO), and CYP P450 enzymes in the metabolism of capmatinib. The study results provide data to characterize the main elimination pathways.

Study drug
Radiolabeled [ 14 C]capmatinib was prepared by Novartis Isotope Laboratory (IL) of Novartis, Basel, Switzerland. Capmatinib was labeled with 14 C in a metabolically stable position as shown in Fig. 1.
The radiolabeled study drug was provided as individually packaged doses of 12 × 50 mg [ 14 C]capmatinib free base in hard gelatin capsules with a nominal specific activity of 9.25 kilobecquerel (kBq)/mg (total dose of 5.55 MBq). The 12 hand-weighed capsules per bottle corresponded to a nominal dose of 600 mg of capmatinib. The analyses of chemical and DMD # 90324 radiochemical identity, purity, and stability were performed according to predefined and quality assurance-approved specifications, and the drug was released for human use.
The actual specific radioactivity of the solid drug substance, measured at Novartis Isotope Laboratory, was 9.185 kBq/mg. The deviation between the actual specific radioactivity and nominal specific radioactivity was 0.7%. The actual specific radioactivity was used for all further calculations. The radiochemical purity of the radiolabeled drug product was 99.7%, with individual impurities accounting for ≤0.2%. Hence, no impurity is expected to confound the metabolite investigations. Key exclusion criteria included a history of hypersensitivity to the study drug or to drugs of similar chemical classes or excipients; past medical history of or current clinically significant ECG abnormalities/arrhythmias or a family history of a prolonged QT-interval syndrome; relevant radiation exposure (>0.2 millisievert) within 12 months prior to scheduled dosing with [ 14 C]capmatinib; smokers (use of tobacco products in the previous 3 months); any surgical or medical condition that could significantly alter the ADME of drugs (e.g., history of inflammatory bowel syndrome, ulcers, gastrointestinal or rectal bleeding, gastritis, or major gastrointestinal surgery).
The primary objectives of the study were to determine the pharmacokinetics of total radioactivity in blood and plasma, to determine the rates and routes of excretion of [ 14 C]capmatinib-related radioactivity (including mass balance of total drug-related radioactivity in urine and feces), and to characterize the plasma pharmacokinetics of capmatinib. The secondary objective was to assess the safety and tolerability of a single 600 mg oral dose of [ 14 C]capmatinib administered to healthy male volunteers. Exploratory objectives included identification and (semi-) quantification of the metabolites of capmatinib in plasma, urine, and feces in order to elucidate the key biotransformation pathways and clearance mechanisms of capmatinib in humans, and characterization of plasma exposure and pharmacokinetics of metabolites based on radiometry data. This study was performed in accordance with the ethical principles of the Declaration of Helsinki and the principles of Good Clinical Practice. The protocol was approved by an Institutional Review Board at each DMD # 90324 hospital or site, and all volunteers provided written informed consent before any study procedures were performed.

Drug Administration
On Day 1, following a fasting period of at least 10 h, volunteers received a single, oral, nominal dose of 600 mg (5.55 MBq or 150 µCi) [ 14 C]capmatinib free base in 12 capsules of 50 mg each, taken consecutively, together with one glass of non-carbonated water (~240-480 mL). The dose was based on the recommended phase 2 dose for single-agent capmatinib determined in clinical studies (Ma et al., 2015). The expected radiation exposure was estimated according to the recommendations of the International Commission on Radiological Protection.
All blood samples were taken by either direct venipuncture or an indwelling cannula inserted in a forearm vein into K 2 -EDTA (pharmacokinetics) or K 3 -EDTA (hematology) tubes. Three aliquots of 0.3 mL were removed from each sample for radioactivity determination The remaining blood was then centrifuged at 4°C to obtain plasma. From the total plasma, three weighed aliquots of 0.25 mL each were removed for radioactivity determination and two aliquots of 0.5 mL was reserved for the analysis of capmatinib. The remaining plasma was then used for metabolite analysis.
After aliquoting for the different assays, the blood and plasma samples were immediately frozen and stored at <−60°C until analysis.
• Urine and fecal samples This article has not been copyedited and formatted. The final version may differ from this version.

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For each volunteer, a pre-dose (blank) urine sample was collected on Day −1. After administration of the radiolabeled dose, all urine was collected for the time intervals of 0−6, 6−12, 12−24 hours and thereafter in 24-hour fractions up to 168 hours. Aliquot(s) were separated for radioactivity determination. The urine samples were frozen and stored at <−60°C until analysis.
For each volunteer, a pre-dose (blank) fecal sample was also collected on Day -1.
Following the radiolabeled dose on Day 1, all fecal samples were collected during the postdose sample collection period of 168 h. The fecal samples were quantitatively transferred into a container for each volunteer per 24-hour interval. A minimum amount (one to two weight equivalents) of water-containing carboxymethyl cellulose (Sigma Aldrich, NL) was added.
The total amount of carboxymethyl cellulose (by weight) did not exceed 1.5%. The samples were thoroughly homogenized for at least 10 min using an Ultra Turrax mixer. After homogenization, aliquot(s) were separated for radioactivity determination. All remaining homogenate samples were stored at −20°C. Total radioactivity measurement

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Radioactivity in blood, plasma, urine, and feces was measured using liquid scintillation counting, with typical counting times of 10 or 30 min. Detailed summary of the procedure used for detection of radioactivity is included in the supplementary materials.

Determination of capmatinib concentrations in plasma
Concentration of unchanged capmatinib in plasma was measured by a validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay. The detailed procedure used for the determination of capmatinib concentration in plasma is described in the supplementary materials.

Determination of metabolite profiles in plasma and excreta
For metabolite profiling in plasma, the individual samples of volunteers taken at 0.5, 1, 2, 3, 6 and 12 hours after the [ 14 C]capmatinib dose were analyzed. Each plasma sample was extracted 3 times with mixtures of ACN, methanol, and/or water. The final extracts were evaporated, reconstituted in mixtures of ACN and water and analyzed using HPLC. The total recovery of the radioactivity in the plasma samples after extraction, reconstitution and HPLC yielded in average 94.1% (range 92.5%-97.5%) of the total radioactivity AUC 0-12h .
Urine samples from each volunteer were pooled across the collection period of 0-96 hours. On average, the six pools represented 99.3% of the radioactivity excreted with urine.
After centrifugation, a volume of 250 μL from each urine pool was injected into the HPLC-MS system with offline radioactivity detection. The recovery of the radioactivity after centrifugation and HPLC analysis was found to be complete (100.0%).
Feces samples from each volunteer were pooled across the collection period of 0-96 hours. On average, the six pools represented 97.9% of the radioactivity excreted with feces. Aliquots of the feces homogenate (~5 g) were extracted 4 times with ACN, methanol, aqueous ACN acidified with FA, and acetone by sonication and centrifugation. The combined extracts were evaporated and reconstituted in mixtures of ACN and water for further analysis.
The extractions yielded between 91.7% and 98.5% (mean 95.1%) of radioactivity. This article has not been copyedited and formatted. The final version may differ from this version. DMD Fast Forward. Published on July 14, 2020 as DOI: 10.1124/dmd.119.090324 at ASPET Journals on November 5, 2020 dmd.aspetjournals.org

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The stability of capmatinib and its metabolites in the biological samples was demonstrated by repeated extractions and analyses during the study. The stability of capmatinib during sample preparation and HPLC analysis was investigated using blank plasma, urine, and feces spiked with [ 14 C]capmatinib. No degradations were observed.

HPLC instrumentation for metabolite pattern analysis
The chromatography was performed on an Agilent 1100 (Agilent Technologies, Waldbronn, Germany) HPLC system, equipped with a binary capillary pump, a column oven, a degasser, and an ultraviolet-visible spectroscopy (UV/VIS) diode array detector with a standard 13 μL flow cell. The operating software for the HPLC system was Agilent

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smaller portion was directed into the electrospray LC-MS interface. The larger portion was used for UV detection followed by offline radioactivity detection.
Single stage or product ion spectra with exact mass determination were obtained on a time-of-flight mass spectrometer (Synapt G2-S, Waters Corporation, Manchester, UK) in positive ion mode.
Structure assignment was achieved based on molecular ions (M+H + ), exact mass measurements, hydrogen/deuterium exchange experiments and several key fragment characteristic for specific moieties of the molecule; comparison of HPLC or LC-MS data with synthetic reference compounds; or comparison with metabolites from previous metabolism studies. NMR spectroscopy data are provided in the supplemental materials.

In vitro experiments
The metabolite profiles of capmatinib in liver fractions (homogenate, S9, microsomes, and cytosol) were studied in the absence and presence of NADPH. In order to determine the enzymes involved in the catalysis of the formation of major hepatic metabolites, enzyme phenotyping studies were carried out using CYP and AO/XO enzymes.  Table 1). The apparent distribution volume (Vz/F) of capmatinib during the terminal phase was moderate to high (geometric mean: 473 L). The apparent plasma clearance (CL/F) of capmatinib was moderate to high (geometric mean: 45.5 L/h).
The apparent mean terminal half-lives of total radiolabeled components and capmatinib in plasma were 10.6 and 7.84 h, respectively.
The inter-individual variability in systemic exposure (Area under the plasma analyte concentration-time curve from time zero to infinity, AUC inf ) to total radiolabeled components and capmatinib was moderate. The coefficient of variation (CV) for C max was 48.9% for total radiolabeled components and 46.7% for capmatinib. The CV for AUC inf was 34.9% for total radiolabeled components and 36.9% for capmatinib. The shape of the concentration-time curves for radioactivity and capmatinib was comparable in individual volunteers. About 31.5% of plasma radioactivity was due to capmatinib in the period 0-infinity (33.0% of the 14 C-AUC last ), indicating substantial exposure to metabolites. In blood, radioactivity was This article has not been copyedited and formatted. The final version may differ from this version.

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detected for up to 48 h post-dose after which radioactivity levels were near or below LOQ.
The mean blood/plasma AUC-ratio of radioactivity was 1.14 (range 0.96-1.43), indicating that capmatinib-related components were slightly more confined within the blood than the plasma compartment. These data are in line with the in vitro capmatinib blood-to-plasma ratios which were close to unity with ratios of 0.9 to 1.4.
Excretion and mass balance of radioactivity in urine and feces Excretion of radioactivity was complete after 7 days (mean, 99.7%; range 94.8-104%). Fig. 3 depicts the cumulative excretion of radioactivity in urine and feces. A major proportion of radioactivity (97.9%) was excreted within 4 days. Unchanged capmatinib amounted to 42.1 ± 23.0% of the dose and ranged between 9.3% and 72.64%. Differences in the dissolution of the twelve capsules might be a possible explanation for the large differences between individuals. A large proportion of the administered radioactivity was excreted within the feces and amounted to 77.9 ± 10.3% of the dose (range 66.59−92.72). Only 21.8 ± 8.5% of the administered radioactivity was excreted in urine (range 8.88−31.51).

Metabolite profiles in plasma, urine, and feces
Metabolite profiles in plasma for each volunteer were analyzed by HPLC, with radioactivity detection at 0.5, 1, 2, 3, 6, and 12 h time points. No major relative difference was observed between volunteers in the plasma metabolite profiles. Overall, more than 90% of detected radioactive components of the plasma AUC0-12h and extrapolated AUC inf could be covered by the parent drug and structurally characterized metabolites. The parent compound capmatinib contributed to a large proportion of radioactivity in plasma (42.9 ± 2.9% of the plasma AUC 0-12h ). Metabolite M16 (formed by imidazo-triazinone formation) was the most prominent metabolite in plasma and amounted to 21.5 ± 2.1% of the plasma AUC 0-12h (Table   2). Minor proportions of other metabolite peaks were detected, and were attributed to the metabolites M8 and M28, each accounting on average for 5.4-5.9% of the plasma AUC 0-12h.
The metabolites M8 and M28 were formed by hydroxylation of the methylene group and N-dealkylation/hydroxylation (or lactam formation), respectively. The metabolites M18, This article has not been copyedited and formatted. The final version may differ from this version.
Supplementary table 1 provides the plasma AUC 0-12h for capmatinib and its metabolites.
Numerous other metabolites (in total 15) individually contributed to less than 2% of the plasma AUC 0-12h . The metabolites were formed by hydroxylation/lactam formation, hydrolysis of the amide bond, N-dealkylation, N-oxygenation, oxygenation, hydrogenation, and/or glucuronidation.
Unchanged capmatinib in urine was only detected in traces, indicating extensive metabolism of the systemically available capmatinib. The most abundant plasma metabolite M16 (imidazo-triazinone formation) was also found in urine and accounted for 2.9 ± 1.2% of the dose. In addition, numerous other minor metabolites were detected (Supplementary table 2) each less than 2.0% of the dose. No major relative difference was observed between volunteers in the urinary metabolite profiles. More than 19.2% of dose (89% of total radioactivity in the 0-96 h urine pool) could be covered by capmatinib and structurally characterized metabolites.
Two additional metabolites accounted for more than 2% of the radioactivity dose in feces; i.e., the hydrogenated metabolite M26 amounted for 3.3 ± 1.1% and the oxygenated metabolite M20 for 2.3 ± 1.1%. Fifteen other metabolites were identified, with each metabolite contributing less than 2% of the dose each in feces (Supplementary table 2

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cytosol (0.4 mg protein/mL) was investigated after 120 min incubation time. Individual and mean values for 2 incubations are provided in supplementary tables 4A and 4B. In the incubation with human liver S9 fraction (HLS9) without NADPH, the oxidative metabolites M16 and M19 were detected with M8, which might be due to the presence of trace amounts of native NADPH in the S9 preparation (Fig. 8). The catalysis of metabolites M16 and M19 does not require NADPH as enzyme cofactor. The metabolite patterns in HLS9 with NADPH was qualitatively comparable with the pattern obtained in human hepatocytes. The metabolites formed in HLM (M8 and minor ones) and in the HLC (M16 and M19) were both found in HLS9.
2) AO phenotyping and XO involvement in the metabolic formation of M16 and M19.
The apparent Km values for biotransformation to metabolite M16 and M19 in HLC were 8.84 μM and 18.5 μM, respectively; V max was 21.5 pmol/min/mg and 11.2 pmol/min/mg, respectively. The derived intrinsic clearances were low (2.43 μL/mg/min and 0.605 μL/mg/min for M16 and M19 formation, respectively). The enzyme kinetic parameters for the formation of M16 and M19 by AO, XO, and HLC estimated by simulation assuming Michaelis-Menten type behavior are provided in supplementary table 5.
Among the six recombinant or purified molybdenum hydroxylases investigated, significant amount of M16 and M19 was found with recombinant AO expressed in E. coli cells, while XO was able to catalyze the production of M19 (Fig. 9, Supplementary tables 6A and 6B). Allopurinol (XO inhibitor) showed lower or partial inhibition for the formation of M16 and M19 while all four AO inhibitors (raloxifene, menadione, isovanillin, and hydralazine) investigated showed strong or total inhibition in HLC ( Fig. 10 and Supplementary Fig. 2) Figures 3 and 4) In line with these observations, in vitro metabolic profiles obtained from incubations with 14 C-capmatinib using mouse, rat, dog, monkey, and human hepatocytes were similar across species except for dog.  10. Strong inhibition was observed with ketoconazole and azamulin (CYP3A inhibitors, up to 80% and 92 % inhibition, respectively). Other chemical inhibitors tested did not show significant inhibitory effects (Supplementary table 11, supplementary figure 7). Correlation analysis suggested that CYP3A is the major enzyme family involved in the hepatic microsomal metabolism of [ 14 C]capmatinib (Supplementary table 12, supplementary figure   8).

Safety Assessments
There were no discontinuations, deaths, SAEs, or other significant AEs during the study.

Discussion
Assuming that the drug is stable against intestinal bacterial enzymes, the mean oral absorption of capmatinib (based on urinary and fecal [as metabolites] excreted radioactivity, Supplementary table 2) was estimated to be 49.6 ± 20.9% and ranged between 21.0% and 81.7%. The inter-individual differences in exposure are likely caused by the difference in dissolution and differences in absorption. This is supported by the very similar patterns of relative metabolites in plasma, urine, and feces and the large absolute differences in total drug-related exposure in plasma, differences in total radioactivity excreted in feces and urine, as well as large differences in capmatinib (likely not absorbed) in feces. Absorbed capmatinib was eliminated mainly due to metabolism and subsequent biliary/fecal and renal excretion.
The study showed that the CL/F of capmatinib was moderate to high (30.0 to 121 L/h). Mean apparent elimination half-lives of total radiolabeled components (radioactivity) and capmatinib in plasma were 10.6 and 7.84 h, respectively. The Vz/F associated with the terminal phase calculated from plasma concentrations was 473 L. Thus, capmatinib was largely distributed to the peripheral tissues. The ratio of compound-related radioactivity between blood and plasma showed substantial variations, but no special affinity of capmatinib and/or its metabolites for erythrocytes could be concluded. Metabolism mainly occurred by This article has not been copyedited and formatted. The final version may differ from this version. In vitro enzyme phenotyping studies were carried out to determine the enzymes involved in the catalysis of the formation of major hepatic metabolites. The presence of M16 and M19 in HLC and cytosol containing liver fractions and the catalysis of M16 and M19 formation even in absence of NADPH suggested that aldehyde oxidases (which were typically located in the cytosol) were involved in the metabolism of capmatinib and that HLC is the best hepatic cell fraction suitable for the phenotypic investigations of M16 and M19 formation.
AO is a cytosolic molybdenum-containing hydroxylase able to oxidize aldehydes and azaheterocyclic-containing molecules. It is known to catalyze the oxidation of N-heterocyclic drugs such as famciclovir and zaleplon. Formation of an oxidative metabolite in an incubation with cytosolic or S9 fraction in the absence of NADPH reveals the contribution of a non-CYP450 enzyme in the oxidative metabolism. Consequently, the involvement of AO is confirmed by carrying out the same incubation in the presence of an AO inhibitor and observing a reduction in metabolite production. (Pryde et al., 2010). AO is homologous with xanthine oxidase (XO), another mammalian molybdoflavoprotein. In general, AO has the ability to oxidize a broader range of substrates than XO (Kitamura et al., 2006). This article has not been copyedited and formatted. The final version may differ from this version. DMD Fast Forward. Published on July 14, 2020as DOI: 10.1124 at ASPET Journals on November 5, 2020 dmd.aspetjournals.org

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No safety concerns were identified in this study. A single 600 mg oral dose of [ 14 C]capmatinib was safe and well tolerated in healthy male volunteers.

Acknowledgements:
We would like to thank the participants of this study from whom data were taken for analysis. We would also like to thank Albrecht Glänzel and Thomas Mönius (Isotope   Electrospray mass spectrum of capmatinib Positive ion mode, sample cone voltage 40 V, trap collision energy ramp 20-55 eV was used for electrospray ionization. The accurate mass measurements were in agreement with the proposed fragmentation. The differences between calculated and measured masses were ≤1.1 ppm for all indicated fragment ions.