In vivo PK and efficacy studies

PK and efficacy studies in the orthotopic MDA-MB-436 human cell line-derived breast cancer model, subcutaneous A2780 and OVCAR3 human cell line-derived ovarian cancer models, and subcutaneous OV5308 and OV5311 patient-derived ovarian cancer xenograft models were conducted at Crown Bioscience (Taicang, China or San Diego, USA). PK and efficacy studies in the OVC134 and OVC527 patient-derived ovarian cancer xenograft models and subcutaneous Capan-1 and intracranial Capan-1-luc human cell line derived pancreatic cancer models were performed at Pharmaron, Inc. (Beijing, China). PK studies were conducted with 3 mice at each timepoints. For efficacy studies, typically 6 to 10 mice were randomized to each group. The protocol and any amendment(s) or procedures involving the care and use of animals in these studies were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Pharmaron or CrownBio prior to conduct. During the study, the care and use of animals was conducted in accordance with the regulations of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC).

Seven- to 9-week-old female NOD/SCID mice (Beijing HFK Bio-Technology Co. Ltd., Beijing, China or Shanghai Lingchang Bio-Technology Co. Ltd., Shanghai, China) were inoculated at the right mammary fat pad with the MDA-MB-436 tumor cells (1 × 10⁷) in 0.2 mL of phosphate-buffered saline (PBS) mixed with matrigel (1:1) for tumor development. For the A2780 or OVCAR3 tumor development, 6- to 8-week-old or 8- to 9-week-old female BALB/c Nude mice (Shanghai SIPPR-Bk Lab Animal Co. Ltd., Shanghai, China) were inoculated subcutaneously at the right flank with 5 × 10⁶ tumor cells in 0.1 mL of PBS (1:1 mixed with matrigel) or with 1 × 10⁷ tumor cells in 0.1 mL of PBS (1:1 mixed with matrigel). 6- to 8-week-old female NOD/SCID mice (Envigo Laboratories, USA) were inoculated at the rear flank with the OV5311 tumor cells (3 × 10⁵) in 0.2 mL of PBS mixed with Cultrex ECM (1:1) or with OV5308 tumor cells (9 × 10⁴) in 0.2 mL of PBS mixed with Cultrex ECM (1:1) for tumor development. 6- to 8-week-old female BALB/c nude mice (Beijing AniKeeper Biotech Co. Ltd., Beijing, China) were inoculated subcutaneously on the right flank with a tumor fragment (2 mm × 2 mm × 2 mm) of the OVC134 or OVC527 human primary ovarian cancer for tumor development. For the Capan-1 subcutaneous tumor model development, 6- to 8-week-old female BALB/c Nude mice were inoculated subcutaneously at the right flank with 4 × 10⁶ tumor cells in 0.1 mL of IMDM without serum (1:1 mixed with matrigel). For the Capan-1-luc tumor development, mice were anesthetized by intraperitoneal (IP) injection of ketamine/xylazine (90-120/5-10mg/kg). The skins over the coronal and sagittal sutures were sterilized with iodine followed by alcohol. An incision of 0.5 cm was made along the skin over the midline to expose coronal and sagittal suture junctions. An inoculum of 2 × 10⁵ Capan-1-luc tumor cells (in 2 μL IMDM) were injected into the right forebrain by positioning the needle at 2.0 mm lateral to the sagittal suture, 1.0 mm inferior to coronal suture with the injection depth precisely controlled at 3.0 mm. The injection was slowly proceeding over a one-minute period.

After tumor cell inoculation, the animals were checked daily for morbidity and mortality. At the time of routine monitoring, the animals were checked for any effects of tumor growth and treatments on normal behavior such as mobility, visual estimation of food and water consumption, body weight gain/loss (body weights were measured 2 to 3 times per week throughout the study), eye/hair matting and any other abnormal effect. Any mortality and/or abnormal clinical signs were recorded.

Tumor volumes were measured 3 times weekly in 2 dimensions using a caliper, and the volume was expressed in mm³ using the formula: V = 0.5 a × b² where a and b were the long and short diameters of the tumor, respectively. The entire procedures of dosing as well as tumor and body weight measurement were conducted in a Laminar Flow Cabinet. For the Capan-1-luc tumors, mice were injected intraperitoneally with D-luciferin of 7.5 mg/mL (at 10μl/g BW) and anesthetized with isoflurane inhalation. At 10 minutes after the luciferin injection, mice were imaged with an In Vivo Imaging System (IVIS, Xenogen/Caliper, USA). Bioluminescent signals (photons/s) from the region of interest (ROI) were quantified and used as an indicator of tumor growth. Tumor bioluminescent signals were conducted once a week. For PK studies, the treatment was started when the average tumor size reached approximately 320 to 360 mm³. For the Capan-1-luc intracranial efficacy study, the treatment was started when the mean bioluminescent signal reaches approximately 5.5 × 10⁶ photons/s. For all other efficacy studies, the treatment was started when the average tumor size reached approximately 50 to 200 mm³. The tumor size was used for the calculation of tumor growth inhibition (TGI) = [1 - (T₁-T₀)/(C₁-C₀)] × 100, where C₁= mean tumor volume of control mice at time t, T₁ = mean tumor volume of treated mice at time t, C₀ = mean tumor volume of control mice at time 0, and T₀ = mean tumor volume of treated mice at time 0.

Niraparib, olaparib, or vehicle (0.5% methyl cellulose) was administered orally once daily in the MDA-MB-436 PK, as well as A2780, OV5311, OVCAR3, Capan-1 and Capan-1-luc efficacy studies. Olaparib was administered orally twice daily in both OVC134 PK and efficacy studies, as well as the MDA-MB-436, OV5308, OVC527 efficacy studies as per Institutional Animal Care and Use Committee guidelines.