Rucaparib Phosphate (PF-01367338 phosphate) is an inhibitor of PARP (Ki: 1.4 nM for PARP1) that is used in clinical therapy to sensitize cancer cells to chemotherapy.

PARP:1.4 nM (Ki, cell free)

AG14447 (the phosphate salt of Rucaparib) is the most potent PARP inhibitor in enzyme assays (Ki: 1.4 nmol/L) [1]. In permeabilised D283Med cells, Rucaparib (AG-014699), at concentrations of 0.1, 0.4 and 1?μ inhibited PARP-1 activity by 81.1, 96.8 and 97.1%, respectively [2]. AG014699 (≤10 μM) was cytotoxic to cells with mutated BRCA1/2 or XRCC3 and to UACC3199 cells with epigenetically silenced BRCA1 but not to cells without BRCA1/2 or XRCC3 mutations or that were heterozygous for BRCA2 mutation [3].

When 10 mg/kg Rucaparib was administered in combination with temozolomide, body weight loss was observed at days 4 to 13 posttreatment with the nadir body weight ranging from 83% to 96% of the starting weight. At a dose of 1 mg/kg, Rucaparib also significantly increased temozolomide-induced body weight loss [1]. At 1mg/kg daily five times, AG-014699 alone did not cause any marked toxicity or affect tumour growth compared with vehicle-only controls. Co-administration of AG-014699 with temozolomide also resulted in complete tumour regressions in all mice, of which three out of five were sustained throughout the experiment. The MMR-defective D283Med xenografts grew very rapidly and showed very little response to temozolomide alone (TGD of only 2 days) with no regressions observed in any mice [2].

We measured inhibition of human full-length recombinant PARP-1 by [32P]NAD+ incorporation as described previously. The [32P]ADP-ribose incorporated into acid insoluble material was quantified using a PhosphorImager. Kis were calculated by nonlinear regression analysis [1].

Inhibition of PARP activity in 5000 exponentially growing D283Med cells was measured following treatment with a range of AG-014699 concentrations (0–1?μ), in comparison with DMSO-only controls. Maximally stimulated PARP activity was measured in replicate samples (n?3) of permeabilised cells by immunological detection of the amount of poly(ADP-ribose) (PAR) formed, using 10H anti-PAR antibody, during a 6-min incubation with NAD+ and oligonucleotide (substrate and activator) by reference to a PAR standard curve using a GCLP-validated assay described previously [2].

One or four daily doses of PARP inhibitor AG-014699 (1?mg/kg intraperitoneally (i.p.)) were given to CD-1 nude mice bearing established D283Med xenografts. At 0.5, 2, 6 and 24?h after the initial or fourth daily dose of AG-014699, three animals per time point were bled by cardiac puncture under general anaesthesia, and then killed. Plasma was separated from the blood samples using standard methods and stored at ?80°C. The brains and tumours were removed, snap frozen in liquid nitrogen and stored at ?80°C before analysis. Blood, tumour and brain tissue were removed from three untreated control animals and processed in the same way [2].