For intravenous (IV) administration, LY was formulated in 10 % N methyl pyrrolidine / 18 %

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1 Supplementary Material and Methods Drug formulation For intravenous (IV) administration, LY was formulated in 10 % N methyl pyrrolidine / 18 % hydroxypropyl β cyclodextrin in 22.5 mm phosphate buffer at ph 3.0. For oral administration, LY was suspended in 1 % w/v hydroxyethylcellulose / 0.25 % v/v polysorbate 80 / 0.05 % v/v antifoam in purified water. While all PK experiments within this study were performed with the methanesulfonate salt, the disposition of the free base is comparable (data not shown). All preclinical characterization in mice bearing colo 205 tumors was performed with the methanesulfonate salt of LY Mice bearing A375 xenograft tumors received the free base form of LY Tumor implantation Colo 205 human colorectal and A375 human melanoma tumor cell lines were obtained directly from the ATCC cell bank and authenticated by IDEXX RADIL (Columbia, MO) using CellCheck 9, an STR based DNA profiling and multiplex PCR technique, to detect both contamination and misidentification. All cells were passaged for fewer than 2 months in the author s laboratory. Upon implantation, cells were harvested at sub confluence with trypsin and rinsed twice with growth medium without serum. Tumor growth was initiated by subcutaneous injection of 5 x 10 6 colo 205 cells or 10 x 10 6 A375 cells in a 1:1 mixture of serum free growth medium and Matrigel (BD Bioscience, Franklin Lakes, NJ) in the rear flank of each female immunodeficient mouse (5 6 weeks old, Harlan Laboratories, Indianapolis, IN). When mean tumor volume reached approximately 150 mm 3, the animals were randomized by tumor size and body weight and placed into their respective treatment groups. All in vivo studies were performed according to the Institutional Animal Care and Use Protocols. Studies to assess CDK4/6 and cell cycle inhibition in colo 205 xenograft tumors 1

2 First, a dose response study was performed after a single oral dose of 3.125, 6.25, 12.5, 25, and 50 mg/kg (n=5 per dose level); animals were sacrificed at 24 hours post dose. Then, 4 time course studies were carried out after a single oral dose of 25 (two studies) and 50 mg/kg (one study), wherein animals were sacrificed at 1, 3, 6, 12, 24, 36, and 48 hours post dose (n=5 per time point). An additional timecourse experiment was performed after a single oral dose of 25 mg/kg, wherein animals were sacrificed at 1, 3, 6, 12, and 24 hours post dose (n=5 per time point). Finally, 3 multiple dose time course studies were carried out to assess CDK4/6 and cell cycle inhibition at steady state; LY was administered once a day for 21 (100 mg/kg) or 56 days (50 mg/kg), or for 56 days with a 7 day break after each 21 day dosing period (50 mg/kg). Animals were sacrificed at 336 and 504 hours post dose for the 100 mg/kg regimen, and at 672 and 1344 hours post dose for the 50 mg/kg regimens (n=5 per dose and time point). Blood and tumor tissue were collected from each animal at the point of sacrifice to obtain plasma drug concentration and biomarker measurements. The levels of p Rb, TopoIIα, and phh3 were measured in every tumor tissue sample collected in every study. A summary of the available experimental data is provided in Supplementary Table 1. Determination of LY in plasma An internal standard compound in acetonitrile/methanol (1:1 v/v) was added to samples of plasma to precipitate protein and the samples were centrifuged prior to analysis. The supernatants were analyzed by injection and rapid gradient elution on a Javelin Betasil C18 column (20 x 2.1 mm cartridge, mobile phase A: water/1 M NH 4 HCO 3, 2000:10 v/v, mobile phase B: MeOH/1 M NH 4 HCO 3, 2000:10 v/v). The eluted analytes were detected by LC MS/MS analysis using a Sciex API 4000 triple quadrupole mass spectrometer. The concentration of LY in plasma was determined from standards prepared 2

3 under identical conditions, with a limit of quantification of 1 ng/ml and assay imprecision of less than 20 %. Determination of p Rb, TopoIIα and phh3 in tumor lysates Tumors were processed by Western blot to determine p Rb, TopoIIα, and phh3 expression. Briefly, the tumor samples were pulverized and lysed in 1 % SDS buffer containing both protease (Roche Applied Science, Indianapolis, IN) and phosphatase inhibitors (Sigma Aldrich, St Louis, MO). The crude lysates were heated to C and then clarified by centrifugation. The clarified lysates were analyzed for expression by immunoblotting following electrophoresis on 4 20 % gradient gels. For the experiments performed in colo 205 xenograft tumors, biomarker levels were quantified using mouse anti p Rb serine 780 (BD Pharmingen, San Diego, CA), rabbit anti TopoIIα (Santa Cruz Biotechnology, Dallas, TX), and rabbit anti phh3 serine 10 (Millipore Upstate Biotechnology, Lake Placid, NY), normalized for mouse anti Glyceraldehyde 3 phosphate dehydrogenase (GAPDH, Meridian Life Science, Biodesign, Saco, ME). For the experiments performed in A375 xenograft tumors, an alternative source of the TopoIIα antibody was used (Abcam, Cambridge, UK), and data were not normalized for GAPDH. All biomarker data are presented as a percentage proportion of control levels, determined as an average of 8 control mice. Measurement of tumor size The largest diameter of the tumor (L) and the perpendicular diameter (W) were measured with a caliper. Tumor volume was estimated using the following equation: Tumor Volume L W (1) It was assumed that tumor density is equal to 1 mg/mm 3. 3

4 Supplementary Table 1. Summary of available PK and biomarker data Doses (mg/kg) Dosing Regimen Time Points Mice Per Time Point PK experiments 1 QDx1, IV 0.08, 0.25, 0.5, 1, 2, 4, 8, 12, 24 h 2 3 Yes 3, 12.5, 25, 50 QDx1 0.25, 0.5, 1, 2, 4, 8, 12, 24 h 2 3 Yes 3.125, 6.25, 12.5, 25, 50 QDx1 24 h 5 No 25 QDx1 1, 3, 6, 12, 24 h 5 No 25, 50 QDx1 1, 6, 24, 36, 48 h 5 No 50 QDx1 24 h 5 No 50 QDx21 14, 21 days 10 No 100 QDx21 14, 21 days 5 No Biomarker experiments in colo 205 xenograft tumors 3.125, 6.25, 12.5, 25, 50 QDx1 24 h 5 Yes 25, 50 QDx1 1, 6, 24, 36, 48 h 5 Yes 25 QDx1 1, 3, 6, 12, 24 h 5 Yes 50 QDx56 28, 56 days 5 No 50 Two cycles of QDx21, 7 days rest 28, 56 days 5 No 100 QDx21 14, 21 days 5 Yes Biomarker experiments in A375 xenograft tumors 22.5, 45, 90 QDx3 72 h 5 No LY was administered orally unless otherwise stated Used in Parameter Estimation? 4

5 Supplementary Table 2. Definitions of PK, biomarker and tumor model parameters Acronym Definition A gut Amount in the gut compartment A transit,i Amount in transit compartment, i C 50 Concentration at which half maximal tumor shrinkage occurs C Cen Concentration in the central compartment C Per Concentration in the peripheral compartment E [LY] Effect of LY concentration on tumor death E Drug Inhibitory effect of the drug on the production of p Rb E phh3 Effect of phh3 inhibition on tumor growth IC 50 Plasma concentration at which p Rb is inhibited by 50 % I max Maximum inhibitory potential of LY on p Rb k a First order absorption rate constant k death Rate of tumor shrinkage k el Rate of elimination from the precursor compartment k G1S Rate of transition from late G1 to S phase k in Zero order input into the precursor compartment K m,abs Michaelis Menten constant for drug absorption into the central compartment K m,elim Michaelis Menten constant for drug elimination from the central compartment k MG1 Rate of transition from M to G1 phase k R Rate of transition from early to late G1 phase k SG2 Rate of transition from S to G2 phase k stasis Rate of transition between growing and non growing tumor volume Q Bi directional flow between the central and peripheral compartments T g Growing tumor volume T ng Non growing tumor volume V Cen Volume of the central compartment V max,abs Maximum velocity of drug absorption into the central compartment V max,elim Maximum velocity of drug elimination from the central compartment V Per Volume of the peripheral compartment γ 1 Sigmoidicity of the phh3 dependent effect on tumor growth inhibition γ 2 Sigmoidicity of concentration dependent effect on tumor shrinkage λ 0 Exponential tumor growth parameter λ 1 Linear tumor growth parameter ψ Sigmoidicity between exponential and linear growth at threshold tumor size Estimated tumor weight at time zero ω 0 5

6 Supplementary Figure Legends Figure 1. External validation of the PK model by VPC in colo 205 xenograft bearing mice receiving a single 6.25, 12.5, 25 and 50 mg/kg oral dose of LY , or with oral doses of 50 and 100 mg/kg administered daily for 21 days. The circles denote observed concentration time data. The solid line represents the median of 1000 individuals simulated by the model and the shaded area represents the 5 th and 95 th percentiles of the 90% confidence interval around the median prediction. Figure 2. VPC of the biomarker model in colo 205 xenograft bearing mice following a single 3.125, 6.25 or 12.5 mg/kg oral dose of LY The circles denote observed p Rb, TopoIIα and phh3 data in treated tumors, expressed in percent of the control value observed in the vehicle group. The solid line represents the median of 1000 individuals simulated by the model and the shaded area represents the 5 th and 95 th percentiles of the 90% confidence interval around the median prediction. Figure 3. External validation of the biomarker model by VPC with data collected in colo 205 following daily oral dosing of 50 mg/kg of LY for 56 days or two cycles of 50 mg/kg for 21 days with a 7 day rest period. The circles denote observed p Rb, TopoIIα and phh3 data in treated tumors, expressed in percent of the control value observed in the vehicle group. The solid line represents the median of 1000 individuals simulated by the model and the shaded area represents the 5 th and 95 th percentiles of the 90% confidence interval around the median prediction. Figure 4. Model simulations of the plasma concentration, the biomarker levels of p Rb, TopoIIα and phh3, and the effect parameters driving tumor growth inhibition for a 21 day daily dosing regimen of 25 (red line), 50 (blue line) and 100 mg/kg (green line) LY using the mean parameter estimates. Figure 5. Model simulated dose response curve for the average (red line), minimum (green line) and maximum (blue line) levels of p Rb and phh3 over a 24 period at steady state following daily dosing. 6

7 Figure 6. Model simulations of tumor size dynamics for 17 days of growth followed by a 21 day daily dosing regimen of 25, 50 and 100 mg/kg of LY for the full model (green line) or for the model incorporating tumor growth inhibition by cell cycle arrest alone (blue line), compared to control tumor growth (red line). 7