2.3. Human ADME study

The radiolabeled drug [¹⁴C]‐ribociclib succinate salt was synthesized by the Isotope Laboratory of Novartis, Basel, Switzerland. The synthetic route is described in Table S4. The final drug product was analyzed by the Isotope Laboratory and Pharmaceutical and Analytical Development department of Novartis and was released for human use according to predefined specifications. The chemical and radiochemical purity of the drug was 99.7%, with individual impurities accounting for ≤ 0.15%. The nominal specific radioactivity of [¹⁴C]‐ribociclib was 0.62 kBq/mg, referring to free base. The study drug was provided as capsules of 200 mg ¹⁴C‐ribociclib. Three capsules were packed per bottle, which provided the dose of 600 mg.

This single‐center, open‐label, single oral dose study enrolled six healthy, non‐smoking, male Caucasian volunteers who were determined as being in good health according to their medical history, physical examination, vital signs, electrocardiogram, laboratory tests, and urinalysis. Healthy male volunteers were selected as the foundation of the extensive human ADME data should be based on a small cohort (6) of young healthy male volunteers with subsequent extension/bridging to actual patients as needed for the investigation of variables such as age, gender, ethnicity, and health on the metabolic profile. Subjects with relevant radiation exposure of > 0.2 mSv in the 12 months prior to the initiation of the study were excluded. The subjects were exposed to a radiation dose of 1.77 mSv maximally, which was calculated according to the guidelines of the International Commission on Radiological Protection. The clinical study was performed at PRA Health Sciences, Zuidlaren, the Netherlands, in accordance with Good Clinical Practice guidelines and the 1964 Declaration of Helsinki and subsequent revisions. The study protocol and dosimetry calculations were reviewed by the Independent Ethics Committee for the center, and written informed consent was obtained from all subjects before entering the study.

Safety analysis included monitoring and recording of all adverse events, laboratory tests (ophthalmologic exam, hematology, blood chemistry, and urinalysis), vital signs, electrocardiogram, and physical examination.

After an overnight fasting of approximately 10 hours, each subject received a single oral dose of [¹⁴C]‐ribociclib 600 mg in three capsules of 200 mg each, which were taken consecutively with 1 glass of noncarbonated water. Subjects continued to fast for 4 hours after drug administration. After dosing, blood (plasma), urine, and feces samples were collected for 21 days at 0, 0.25, 0.5, 1, 1.5, 2, 3, 6, 12, 24, 36, 48, 72, 96, 120, 144, 168, 192, 216, 240, 264, 312, 360, 408, 456, and 504 h post dose. Urine was collected from 0‐6, 6‐12, and 12‐24 h post dose, then over 24‐hour intervals until 504 hours post dose. Feces was collected over 24‐h intervals until 504 hours post dose, during which time the volunteers were confined to the clinic. Vomitus was also scheduled to be collected for 12 hours post dosing. However, no volunteers vomited during the study.

Ribociclib and LEQ803 were measured in plasma and urine using a validated bioanalytical assay. Plasma aliquots at each time point were subjected to protein precipitation with three volumes of acetonitrile containing 0.1% (v:v) formic acid, followed by dilution and analysis by liquid chromatography‐tandem mass spectrometry in selected reaction monitor‐positive ion mode using heated electrospray ionization as the ionization technique. For urine aliquots, six volumes of acetonitrile containing 0.1% (v:v) formic acid were added, followed by dilution and analysis in the same way as for plasma. Components were separated using a YMC‐Triart C18 (2.0 x 30 mm, 1.9 µm) column (YMC Co. Ltd.). Mobile phase A was held at 95% for 0.5 min, then reduced to 80% at 0.8 min, 60% at 2 min, and 5% at 2.5 min, where it was held until 4 min. Finally, it was increased to 95% at 4.1 min. Mobile phase A was 0.1% formic acid in H₂O while mobile phase B was 0.1% formic acid in ACN:Isopropanol 8:2 v:v. The validated range for ribociclib and LEQ803 in plasma and urine was 1.0 (LLOQ) and 1000 ng/mL (ULOQ), using 50 µL of sample per analysis.

Total radioactivity in blood and plasma was analyzed by accelerator mass spectrometry (AMS) on a National 5.3.2. Electrostatics Corporation 1.5SDH Compact AMS System (Middleton) in the bioanalytical laboratory of Accium BioSciences, Seattle, WA, USA. A known aliquot of each blood and plasma specimen was transferred to a prebaked quartz tube. Sample volumes were selected to achieve approximately 1–2 mg total carbon. An AMS batch consisted of unknown specimens and chemical blank(s) to monitor for any in‐process contamination. The samples were then dried using vacuum centrifugation and submitted to graphitization. Graphitization consisted of combustion of samples followed by reduction to graphite according to published methods. 12 Briefly, approximately 200‐mg copper oxide was added to each combustion tube containing the dried sample residue. The combustion tubes were flame‐sealed under vacuum and combusted at approximately 900°C to form carbon dioxide (CO₂). Combusted samples were then attached to a disposable transfer system connected to a septa‐sealed vial. This vial contained a minimum of 100 mg of zinc powder, several 3‐mm glass beads, and a smaller vial containing 2‐ to 6‐mg iron powder. The transfer system was evacuated with a vacuum pump and the tip of the combustion tube was broken to allow release of gases into the septa‐sealed vial, which was submerged slightly in liquid nitrogen. Gases, such as CO₂ and H₂O, condensed at the bottom of the vial while noncondensing gases were purged using the vacuum pump. To avoid cross‐contamination, all parts that came into contact with the sample were disposed of and replaced with each use. The septa‐sealed vials were then placed in a heat block maintained between 515°C and 525°C for approximately 5 hours. During this stage, carbon from CO₂ reduced to solid graphite, adhering to the surface of the iron powder. The resulting iron‐graphite mixture was pressed into individual cathodes and submitted for AMS measurement. Pressed, individual cathodes were loaded on to the AMS sample wheel for measurement. A typical AMS measurement batch contained unknown samples, certified standards to normalize all measurements, machine blanks ([¹⁴C]‐free graphite of natural origin) to assess the sensitivity of the spectrometer, and chemical blanks (blanks prepared with a [¹⁴C]‐free substance) to characterize the extraneous carbon introduced during graphite batch preparation.

Radioactivity contents in urine and feces homogenates were determined at the bioanalytical laboratory of PRA International, Zuidlaren, the Netherlands. For urine, duplicate (1000 µL) aliquots were placed into 7‐mL glass vials (Perkin Elmer), after which 5 mL of scintillation cocktail (Ultima Gold™, Perkin Elmer) was added. After vortex mixing for 5 seconds, each sample was placed in a Tri‐Carb™ 3100 TR liquid scintillation analyzer (Perkin Elmer, Waltham, MA, USA) 30 minutes before counting. The total [¹⁴C]‐radioactivity of the samples was determined by counting until a statistical error (two standard deviations) of 0.5% was obtained with a counting time of 10 or 30 minutes, depending on the level of radioactivity.

For feces homogenates, quadruplicate, accurately weighed (500 mg) aliquots were dried in a stove at 50ºC for 3 hours. After the addition of 100‐µL combustion aid (Perkin Elmer) to the dry homogenates, the samples were combusted in a sample oxidizer model 307 (Perkin Elmer). The absorber agent for CO₂ was 7‐mL Carbo‐Sorb® E (Perkin Elmer). At the end of the combustion cycle, the absorber was mixed with 13 mL of the scintillant Permafluor® E (Perkin Elmer). The samples were placed in the liquid scintillation analyzer for 60 minutes before counting. The total [¹⁴C]‐radioactivity of the samples was determined by counting until a statistical error (2 s) of 0.5% was obtained with a counting time of 10 or 30 minutes, depending on the level of radioactivity.

A plasma pool across the six healthy volunteers included in the study was prepared at the time points of 1 hour, 3 hours, 24 hours, and 48 hours post dose. For each time point, an equal volume of plasma was taken from each subject and combined. Plasma pool samples were processed by protein precipitation with acetonitrile. The protein pellet that resulted from precipitation with acetonitrile was washed further with additional aliquots of acetonitrile. All acetonitrile washes were combined and evaporated under nitrogen. For the 3‐ and 24‐hour plasma pools, the extraction procedure was slightly modified, including precipitation of the plasma proteins by addition of a 1:1 mixture of acetonitrile:methanol, followed by washing of the protein pellet with the same solvent mixture. All extracts were reconstituted with a solution of 15‐mM ammonium formate and acetonitrile (95:5 v/v). Extraction and reconstitution recoveries were measured in all four plasma samples in order to calculate and overall recovery.

Quantitative metabolite profiling in plasma was accomplished using a Shimadzu Prominence high‐performance liquid chromatography (HPLC) system (Shimadzu, Columbia, MD, USA), coupled with fraction collection and analysis of total radioactivity in individual fractions using AMS (as described above). A second injection of each sample, conducted immediately after the first, was used to provide HPLC fractions for metabolite identification. The fractions identified as containing radioactivity from the AMS analysis were analyzed by HPLC coupled with high‐resolution mass spectrometry to identify the metabolites. The HPLC‐MS/MS methodology used is described below.

For each subject, urine and feces pools were created by combining identical percentages of the volumes of the different excreta fractions, with the objective of representing> 95% of the radioactivity eliminated via each route. Average pools across the six subjects were also created by combining equal percentages (of total urine and feces excreted over the time interval) of the individual subject pools. Urine pools were analyzed directly for metabolite profiling (column recovery of a representative sample was measured), whereas feces pools were subjected to extraction. Specifically, to each aliquot of feces homogenate, two volumes of acetonitrile were added. After centrifugation, the pellet was washed further with an additional two volumes of acetonitrile. Additional pellet wash steps were done with methanol, dimethyl sulfoxide, acetone, and dichloromethane. All washes were combined, evaporated under nitrogen, and reconstituted with a solution of 15‐mM ammonium formate (pH 3.5). This feces extract reconstitute was analyzed for metabolite profiling. Combined extraction and reconstitution (overall recovery) and column recovery were measured.

Metabolite profiling in urine and feces was accomplished using an Agilent model 1200 HPLC (Agilent Technologies, Basel, Switzerland) coupled with a Waters Synapt‐G2‐S HDMS (Waters, Milford, MA, USA) and a Gilson fraction collector GX‐271 (Gilson Inc, Middleton, WI, USA). The HPLC column used was a Phenomenex Kinetex, C18, 250 × 4.6 mm, equipped with a pre‐column of the same type (2.1 × 4.6 mm), which was placed in a column oven at 40°C. Separation of components was achieved using 15‐mM ammonium formate solution in water adjusted at pH 3.5 with formic acid as mobile phase A and acetonitrile:methanol (9:1, v/v) as mobile phase B. Flow rate was 1.25 mL/min. The HPLC gradient was as follows: initial conditions were 5% mobile phase B for 1 minute, then increased to 20% at 10 minutes post injection where it was held (isocratic) up to 20 minutes. From there, mobile phase B was increased to 35% at 30 minutes, and to 100% to 35 minutes where it was held (isocratic) up to 45 minutes. The post column flow was directed to the fraction collector operated in a time‐slice mode and containing 96‐well Lumaplates (Perkin Elmer). The plates were dried at room temperature and the radioactivity was measured using a microplate scintillation counter model TopCount NXT (Perkin Elmer) instrument (up to 3 × 100‐minute counting time). Chromatograms were evaluated using Microsoft Excel 2010. Metabolites were identified during the same injections using data generated by the high‐resolution mass spectrometer. The molecular ions and key fragments of each drug‐related component were determined. Key fragments for each metabolite were derived from their product ion mass spectra and were used to determine the metabolite structures. Where possible, structural assignments were supported by exact mass measurement, comparison with synthetic standards and/or Hydrogen/Deuterium (H/D) exchange LC‐MS(/MS).