Intracranial Evaluation of the In Vivo Pharmacokinetics, Brain Distribution, and Efficacy of Rucaparib in Poster 3888 BRCA-Mutant, Triple-Negative Breast Cancer Minh Nguyen,1 Liliane Robillard,1 Thomas C. Harding,1 Jim J. Xiao,1 Andrew D. Simmons,1 Hartmut Kristeleit,2 Michelle (Mingxiang) Liao1 1Clovis Oncology, Inc., Boulder, CO; 2Guy’s and St Thomas’ NHS Foundation Trust, London, UK

INTRODUCTION In Vitro Validation of BRCA1mut MDA-MB-436 as a Suitable Free Fractions of PARP Inhibitors Were Determined in Rucaparib Demonstrates In Vivo Efficacy in the Rucaparib Shows Resolution of Neurological • Poly(ADP-ribose) polymerase (PARP) inhibitors, including Model for PARP Inhibitor Testing In Vitro Plasma and Brain Homogenate Binding Assays Intracranial BRCA1mut MDA-MB-436 Model Symptoms in a Patient with TNBC rucaparib, niraparib, olaparib, talazoparib, and veliparib, are Fraction unbound of PARP inhibitors, mean ± SD, % approved or in clinical development for many cancers. There Cytotoxicity of PARP inhibitors IC50, mean ± SD, µM • An intracranial MDA-MB-436 efficacy study was performed to evaluate rucaparib • Case study of a 51-year-old patient with BRCA2 c.6225dupA activity in the CNS based on prior reports of PARP inhibitor activity in this model2 (p.Val2076fs) mutation. Patient experienced progressive CNS disease is very limited clinical data on the effects of PARP inhibitors in Cell line BRCA status Rucaparib Niraparib Olaparib Talazoparib Veliparib Rucaparib Niraparib Olaparib Talazoparib Veliparib mut mut 18 months after whole brain radiation therapy, with headaches, slurred the central nervous system (CNS) MDA-MB-436 (BRCA1 ) MDA-MB-436 (BRCA1 ) MDA-MB-231 Wild type 6.71 ± 1.89 7.15 ± 1.03 19.69 ± 0.59 2.98 ± 0.01 >20 Plasma (1 µM) 30.7 ± 1.94 16.3 ± 0.93 11.6 ± 0.80 3.47 ± 0.31a 60.1 ± 7.53 Tumor bioluminescence Body weight speech, facial pain, and numbness in right side of face and right hand • Preclinical studies have suggested that veliparib and niraparib 1011 10 1,2 BRCA1 Plasma (10 µM) 28.4 ± 0.79 16.7± 0.85 12.2 ± 1.18 3.75 ± 0.24b 58.5 ± 4.61 • Following 1 cycle of rucaparib, complete resolution of neurological can cross the blood-brain barrier (BBB), whereas 1010 MDA-MB-436 0.09 ± 0.01 0.17 ± 0.09 0.11 ± 0.05 0.004 ± 0.001 1.86 ± 0.45 M 5 mutated E

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i Baseline 2 months 5 months -10 understand the distribution and efficacy of PARP inhibitors in orthotopic and intracranial studies B 106 • PARP inhibitors displayed concentration-independent free fraction in mouse plasma and the CNS 105 -15 • Talazoparib and veliparib had the lowest and highest IC50, respectively, in MDA-MB-436 20 30 40 50 60 20 30 40 50 60 brain homogenate, except for talazoparib, which had a higher free fraction in brain Dosing period Dosing period Days post tumor implant Days post tumor implant – In vitro plasma and brain protein binding and in vivo cells, whereas the IC50 for rucaparib, niraparib, and olaparib were comparable homogenate at the higher concentration evaluated pharmacokinetic (PK) studies were conducted using Vehicle Rucaparib 150 mg/kg BID Niraparib 50 mg/kg QD rucaparib, niraparib, olaparib, talazoparib, and veliparib • The free fractions were used to calculate PK parameters described below QD, once daily. – Antitumor activities of rucaparib and niraparib were Rucaparib In Vivo Efficacy in MDA-MB-436 Orthotopic • Rucaparib and niraparib demonstrated statistically significant (P<0.05 2-way analysis of variance; day 57) tumor suppression relative to control tumor evaluated in a BRCA1-mutant, intracranial, triple-negative Model Correlates with Drug Exposure In Vivo PK Study to Evaluate Brain Penetration of PARP breast cancer (TNBC) model volumes in the MDA-MB-436 intracranial model, with rucaparib showing greater MDA-MB-436 (BRCA1mut) Inhibitors in Male CD-1 Mice inhibition than niraparib (P=0.0017) – A case report demonstrating rucaparib clinical activity in TNBC orthotopic model 2500 • Mice treated with rucaparib and niraparib showed maximum body weight loss of the CNS of a patient with BRCA2-mutant breast cancer is Rucaparib - 150 mg/kg Niraparib - 50 mg/kg Olaparib - 150 mg/kg

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Reagents: Cell lines were obtained from ATCC and cultured according to their e T 0 4 8 12 16 20 24 0 4 8 12 16 20 24 0 4 8 12 16 20 24 m 500 instructions. Compounds were obtained from MedChemExpress. Time (hours) Time (hours) Time (hours) Cytotoxicity assays: Cells were seeded in 384-well plates and treated with PARP Vehicle 0 Talazoparib - 1 mg/kg Veliparib - 150 mg/kg

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by oral gavage (PO) BID for 28 days at doses shown in the graph (n=10 mice/group). e all PARP inhibitors evaluated in a murine model with an m MDA-MB-436 orthotopic model. Tumor regressions of 105% and 112% TGI were 1 m 1 CSF 2 0.6 Tumor volumes were calculated as V = 0.5 a × b , where a and b are the long and 0.1 0.1 observed at 50 and 150 mg/kg BID, respectively 0 4 8 12 16 20 24 0 4 8 12 16 20 24 intact BBB. However, rucaparib and niraparib demonstrated short diameters of the tumor, respectively. Time (hours) Time (hours) photon/second) 0.4 10

10 antitumor efficacy in a BRCA1-mutant, intracranial, TNBC Rucaparib In vivo PK and PD study: Nontumor-bearing NOD/SCID female mice were given a Dose, exposure, and response relationship in the MDA-MB-436 model • For all PARP inhibitors, the C and AUC were higher in the plasma than in the brain × single dose of rucaparib for PK assessment (n=3 mice/group), whereas NOD/SCID max 0-inf 0.2 mouse model a a ( BLI female mice bearing MDA-MB-436 tumors were given 5 doses of rucaparib BID to Dose PK PK/PD Efficacy and CSF • Clinical activity has been observed in a patient with derive tumor to plasma ratios (n=4 mice/group). Rucaparib was given PO at the Plasma Plasma PAR Tumor Plasma TGI Pharmacokinetic parameters of PARP inhibitors • BLI of mice from an intracranial efficacy study on day 57 confirmed the presence doses shown in the table. Drug levels were assessed by LC-MS/MS, and tumor b germline BRCA2-mutant breast cancer and CNS Cmax, AUC0-inf, inhibition, rucaparib, rucaparib, T/P day 28, Rucaparib Niraparib Olaparib Talazoparib Veliparib of tumors in the vehicle control mice (12/12 mice, top panel), but not in rucaparib- poly(ADP-ribose) (PAR) levels were measured by ELISA (Trevigen). c mg/kg ng/mL hr×ng/mL % ng/g ng/mL ratio % a involvement who was treated with rucaparib In vitro protein-binding assay: Talazoparib (0.01 and 0.1 µM) and other agents Cmax, plasma ng/mL 1513 6140 3147 869 7603 treated mice (0/11 mice, bottom panel) 2 26 115 37 131 16 8.58 22 a (1 and 10 µM) were mixed with mouse plasma and brain homogenate before loading AUC0-inf, plasma hr×ng/mL 8974 38,600 19,370 4066 24,310 • Additional studies are warranted to investigate the multiple 5 82 221 31 134 27 5.18 30 Exposure and response in the intracranial MDA-MB-436 model onto rapid equilibrium dialysis devices. After 4 hours at 37°C, samples were analyzed C , brain ng/mLa 119 716 62 16 1944 by LC-MS/MS, and the amount of protein binding was determined. max factors that play a role in CNS activity, including potency, 15 173 682 45 340 68 5.81 58 a AUC0-inf, brain hr×ng/mL 1122 7168 348 259 9021 Drug Dose Efficacy Body weight loss Drug concentrations In vivo PK study: CD-1 male mice were given a single dose PO of the indicated 50 2340 6850 86 1982 975 2.63 105 distribution, efflux ratio, and apparent permeability PARP inhibitors, and plasma, brain, and CSF samples were collected at 0.5, 2, 4, 8, Cmax, CSF ng/mL 7 62 30 6 1343 150 3550 20,000 96 3967 1897 2.17 112 T/C Max average Plasma, Brain, B/P and 24 hours (n=3 mice/group). Drug levels were assessed by LC-MS/MS. K ,brain 0.13 0.19 0.02 0.06 0.37 a aPK with nontumor-bearing mice; PK/PD with tumor-bearing mice; samples taken 2 hours after last dose. bPAR levels calculated as a p mg/kg day 57, % BWL, % Day ng/mL ng/mL ratio REFERENCES In vivo intracranial efficacy study: MDA-MB-436 cells were transduced with a percent of the PAR level in the vehicle-treated animals. cMean of T/P ratio of rucaparib levels were calculated on individual T/P ratios. Kpuu,brain 0.03 0.09 0.06 0.27 0.27 Rucaparib 150 BID 0.01 -5.7 27 2206 ± 653 41 ± 21 0.019 lentivector expressing luciferase to enable tumor growth monitoring by AUC , area under the concentration-time curve extrapolated from time zero to infinity; C , maximum concentration; 1. Karginova et al. Mol Cancer Ther. 2015;14:920-30. 4. Murray et al. Br J Cancer. 2014;110:1977-84. 0-inf max 2. Sambade et al. Cancer Res. 2018;78(13 suppl):abst 5. Parrish et al. Mol Cancer Ther. 2015;14: 2735-43. bioluminescence imaging (BLI), and cells (5×105) were intracranially implanted T/P, tumor-to-plasma. Kpuu,CSF 0.01 0.06 0.08 0.18 0.30 a Niraparib 50 QD 0.05 -9.5 48 3760 ± 940 100 ± 57 0.027 nr 2813. through surgical incision over the cranium bregma of NOD/SCID female mice. Total AUC0-inf and total Cmax. • In an in vivo PK/PD study using the MDA-MB-436 orthotopic model, rucaparib K ,brain, AUC (total brain)/AUC (total plasma); K ,brain, AUC (free brain)/AUC (free plasma); aMean of B/P ratio was calculated by dividing mean brain level (ng/mL) by mean plasma level. 3. Kizibash et al. Mol Cancer Ther. 2017;16:2735-46. Treatment started at mean 2.4×107 BLI photons/second, and compounds were given p 0-inf 0-inf puu 0-inf 0-inf demonstrated increased plasma and tumor concentrations with increasing doses Kpuu,CSF, Cmax (free CSF)/Cmax (free plasma). B/P, brain-to-plasma; BWL, body weight loss; T/C, treated tumor volume to control tumor volume ratio. PO for the study duration at doses shown in the graph and table (n=11–12/group). ACKNOWLEDGMENTS • Brain-to-plasma ratios K ,brain, K ,brain, and K ,CSF were determined to evaluate Samples were collected 2 hours after last dose, and drug levels were assessed by • Higher levels of rucaparib were observed in tumors relative to plasma at all doses p puu puu • At the end of the intracranial efficacy study, rucaparib and niraparib were These studies are funded by Clovis Oncology, Inc. Medical writing and editorial LC-MS/MS. evaluated. An inverse, dose-dependent correlation between rucaparib levels and PAR in brain penetration of the PARP inhibitors measured in the brain and plasma at 2 hours post dose. Levels of the inhibitors support were funded by Clovis Oncology and provided by Nathan Yardley and Shannon Davis of Ashfield Healthcare Communications. BID, twice daily; CSF, cerebrospinal fluid; ELISA, enzyme-linked immunosorbent assay; LC-MS/MS, the plasma and tumor was observed in female tumor-bearing NOD/SCID mice, with • Of the 5 PARP inhibitors tested, veliparib consistently had the highest brain and CSF and brain-to-plasma ratios were lower than the Cmax and Kp derived from the PK Copies of this poster obtained through Quick Response (QR) code are for personal liquid chromatography with tandem mass spectrometry, NOD/SCID, nonobese diabetic/severe combined increased PAR inhibition correlating with greater TGI use only and may not be reproduced without written permission from the authors. immunodeficiency; PBS, phosphate buffered saline; PD, pharmacodynamic. exposures, whereas the other 4 showed limited CNS penetration in mice with an intact BBB study with nontumor-bearing mice Corresponding author: Michelle Liao; email: [email protected]. AACR Annual Meeting | March 29–April 3, 2019 | Atlanta, GA