2.1. Non‐clinical ADME studies

ADME studies (including QWBA in pigmented and non‐pigmented rats) using radiolabeled ribociclib were conducted in rat and dog. Relevant institutional and national guides for the care and use of laboratory animals were followed. Exposure of organs to total radioactivity was measured by QWBA of tissue sections. 11 Placental transfer in rat and rabbit was assessed by comparison of ribociclib concentrations in maternal and fetal plasma. Details of the dosing routes, formulations used, and the sampling schedules are provided in Table S1. The synthetic route of [³H] ‐ and [¹⁴C]‐ribociclib, which were used in selected ADME studies, is described in Table S4.

In these studies, PK of ribociclib in plasma, and total radioactivity analysis in blood, plasma, urine, and feces were measured by a combination of validated bioanalytical assays and LSC measurements. Metabolite profiling was conducted on plasma, urine, bile, and feces samples in the rat and dog ADME studies, and further metabolite profiling on plasma and milk was conducted in a dedicated rat milk excretion study.

Plasma samples obtained from these studies were extracted with equal volumes of acetonitrile. Additional extractions of the protein pellet with acetonitrile and/or acetone were conducted as needed to maximize radioactivity recovery. Feces samples were initially extracted with two to four volumes of acetonitrile. Additional extractions of the pellet following centrifugation and removal of supernatant were conducted with acetonitrile and/or acetone as needed to maximize radioactivity recovery. Urine and bile samples were analyzed directly following centrifugation. Radioactivity recoveries following the centrifugation step were measured by LSC. Milk samples were extracted with two volumes of acetonitrile, following centrifugation and removal of supernatant, the pellets were re‐suspended in 100 µL of water and re‐extracted with 200 µL acetonitrile.

QWBA studies were conducted with the tritium label in Hanover Wistar and partially pigmented Long‐Evans rats. A further QWBA study was conducted with the carbon‐14 label in male Long Evans rats in order to support the dosimetry calculation for the human ADME study. Briefly, 40‐µm thick lengthwise dehydrated whole body sections were exposed for 1 day to Fuji BAS III imaging plates (Fuji Photo Film Co., Ltd., J‐Tokyo) in a lead‐shielded box and room temperature, and scanned in a Fuji BAS 5000 phosphor imager at a 50‐µm scanning step. Concentrations of total radiolabeled components in the tissues were determined by comparative densitometry and digital analysis of the autoradiogram; blood samples of known radioactivity concentrations processed under the same conditions as the samples to analyze were used as calibrators.

Metabolite profiling in the ADME studies was conducted using liquid chromatography‐mass spectrometry. Radioactivity profiles were generated by diversion of the majority of the post‐column flow into 96‐well yttrium silicate scintillation‐coated plates (Deepwell Lumaplates; Perkin Elmer Life and Analytical Sciences Inc), by means of Gilson FC204 fraction collectors (Gilson). Metabolites were identified by mass spectrometry (Waters QTOF operating under MassLynx 4.1) and, where feasible, by comparison with authentic reference standards.

Placental transfer of ribociclib was assessed in rat and rabbit embryofetal development toxicology studies. A single plasma sample on the last day of dosing in both studies, at 3 hours post dose, was taken from the fetuses and ribociclib was measured using a validated bioanalytical assay. Resulting concentrations were compared with maternal plasma concentrations. In vitro blood distribution of [³H]‐ribociclib was investigated at the nominal blood concentrations of 100, 1000, and 10 000 ng/mL (rat and dog) and at 10, 100, 1000, and 10 000 ng/mL (human). Heparinized blood was spiked with [³H]‐ribociclib and incubated for 1 h at 37°C with constant agitation. After the incubation, blood cells and plasma were separated by centrifugation (1500 g, 10 min, 37°C). Total radioactivity was measured by LSC on triplicate aliquots, taken before (blood) and after (plasma) centrifugation. The hematocrit of the whole blood was determined in triplicate after centrifugation in micro hematocrit capillaries (13 000 g, 5 min). Calculation of f_p and C_bc/C_p is described in Table S5.

In vitro plasma protein binding of [³H]‐ribociclib was investigated at nominal plasma concentrations of 100 and 1000 ng/mL (rat and dog) and at 10, 100, 1000, and 10 000 ng/mL (human). Stock solutions were spiked into plasma to achieve the intended concentrations. After incubation for 1 hour at 37°C under constant gentle agitation, the spiked plasma samples (n = 3) were centrifuged (2000 g, 10 min, 37°C) in pre‐warmed Centrifree devices. Total radioactivity was determined in the ultrafiltrate (C_u, concentration of unbound compound) and in the sample introduced into the reservoir before ultrafiltration (C_p). The unbound (f_u) and the bound (f_b) fraction in plasma were calculated as follows: f_u (%) = C_u/C_p × 100; f_b (%) = 100‐F_u (%).