Preclinical modeling of a phase 0 clinical trial: qualification of a pharmacodynamic assay of poly (ADP-ribose) polymerase in tumor biopsies of mouse xenografts

Abstract

Purpose: The National Cancer Institute has completed a first-in-human clinical pharmacodynamic trial of the targeted agent ABT-888, a poly (ADP-ribose) polymerase (PARP) inhibitor, under the auspices of the U.S. Food and Drug Administration's Exploratory Investigational New Drug Application. Performance of the study design, needle biopsy procedure, and validated pharmacodynamic assay were evaluated in human tumor xenograft models.

Experimental design: A validated ELISA was used to quantify PAR, a product of the PARP 1/2 enzyme activity. Sampling variability from tumor heterogeneity was determined by comparing PAR content in multiple tumors, and in different areas of the same tumor in a particular animal, collected under anesthesia by needle biopsy or resection before and after administration of nontoxic doses of ABT-888. The degree of PARP inhibition following single-dose treatment was evaluated in the time frame anticipated for biopsy in humans.

Results: Sampling variability around the mean (approximately 50%) for untreated and vehicle-treated animals was random and due to specimen heterogeneity. PAR levels in initial and repeat tumor biopsies, separated by 1 week, were not altered by the stress induced by daily handling of the animals. A single ABT-888 dose (3 or 12.5 mg/kg) reduced intratumor PAR levels by >95%. ABT-888 (1.56-25 mg/kg) significantly decreased PAR levels at 2 h post-dosing.

Conclusion: The detailed methodologies developed for this study facilitated the design of a phase 0, first-in-human clinical trial of ABT-888 and could serve as a model for developing proof-of-principle clinical trials of molecularly targeted anticancer agents.

Fig. 1

Inter- and intra-tumor variability of PAR levels in untreated-Colo829 xenografts. (A) Correlation in PAR levels between the large and small bilateral tumors of individual mice; (B) Correlation in PAR levels between two quadrants cut from each resected large tumor of the bilateral pair; (C) Correlation in PAR levels between two quadrants cut from each resected small tumor of the bilateral pair. Samples were collected 4 hours post dose. Values reported as pg PAR/mL normalized to 100 μg protein. Solid diamond, measured point; line, linear regression fit.

Fig. 2

Measurement of PAR levels from 18-gauge needle biopsies. (A) Direct placement of the 18-gauge biopsy needle into the subcutaneous tumor nodule via a skin flap; animals were under general anesthesia. (B) Typical needle biopsy yield from a subcutaneous tumor nodule. Typical specimen sizes ranged from 5 mm to 20 mm in length and 3 mg to 12 mg in mass (see Table 2). (C) Hematoxylin-stained frozen sections from a needle biopsy.

Fig. 3

Correlation of PAR levels in A375 xenografts. (A) PAR levels in 18-gauge needle biopsies versus surgically excised tumor pieces. Needle biopsies were collected from anesthetized animals, placed in pre-tared, pre-cooled vials, and flash frozen in liquid nitrogen; the remaining xenograft was surgically excised and flash frozen. (B) PAR levels in left versus right side tumors. Biopsies and tumor pieces (left versus right) from the same animal were graphed together. Data points represent either samples from needle biopsies or xenograft tumor pieces. (C) First versus second repeat biopsy samples (collected from 16 xenografts in 8 animals). Values were reported as pg PAR/mL normalized to 100 μg protein. Solid diamond, measured point; line, linear regression fit.

Fig. 4

Inter- and intra-tumor variability of PAR levels in vehicle- and ABT-888-treated Colo829 xenografts. PAR levels were measured 4 hours following drug administration by combining 1 to 2 quadrants cut from each resected tumor. ABT-888 was administered at doses of 3 mg/kg or 12.5 mg/kg as indicated (n=6 animals/group). (A) Correlation of PAR levels between small and large tumors following ABT-888 dosing in mice bearing Colo829 xenografts. Large and small tumors occurred randomly on the right or left flank, thus, xenograft size was randomized. The difference in scale of PAR values in vehicle compared to ABT-888 treatment was due to significant drug suppression of PAR. (B) Correlation of PAR levels between first and second quadrant dissected from resected large tumors (n=6 animals/group). (C) Correlation of PAR levels between first and second quadrant dissected from resected small tumors (n=6 animals/group). Values were reported as pg PAR/mL normalized to 100 μg protein. Solid diamond, measured point; line, linear regression fit.