Heterogeneity of rucaparib activity in arteries isolated from patients having undergone nephrectomy.

<p>Panel A; concentration-response curves that were generated. Tissues were constricted using 10 μM PE, before being treated with PE and the relevant concentration of rucaparib. Panels B1 and B2; responses of two separate arterial sections from a single donor; panel B1 details the relaxant activity when a single tissue segment was constricted using 10 μM PE before being relaxed using 100 μM rucaparib; panel B2 details the slight inhibition of spontaneous oscillation of a single tissue segment that was observed following treatment with 100 μM rucaparib (the tissue contracted spontaneously, so no PE constriction was performed). Points/bars in most cases represent mean of two parallel experiments. Error bars represent SEM. Panels B1 and B2 error bars, as only a single observation was made per tissue section.</p

Effects of rucaparib on tumor vessel perfusion may be dependent on PARP.

<p>Panels A and B; B16 tumours were established in PARP WT or KO female mice. The extent of vessel ‘mismatch’ following the administration of the perfusion markers Hoechst 33342 and carbocyanin was reduced by rucaparib (1 mg/kg) in tumors established in WT but not PARP-1^-/- mice. Panel C; fold change in intratumoral fluorescence above that seen following initial plateau (20 min) in dorsal window chambers implanted with B16 tumors in WT and PARP-1^-/- mice and treated with 1 mg/kg rucaparib. Panel D; representative real time analysis of the accumulation of BSA-647 (administered via iv injection at time 0) in B16 tumors established in dorsal window chambers in PARP WT (closed symbols) and PARP-1^-/- (open symbols) mice. Arrow indicates the administration of rucaparib (10 mg/kg). NS—p >0.05, *p<0.05, **p<0.01 as compared with relevant control. N = 3 mice per condition.</p

Rucaparib may act at multiple P2 receptor subtypes.

<p>Panels A and B; blockade of specific P2 receptor subtypes P2X₄, P2X₇ and P2Y₁ in tail artery (A) and aorta (B) models failed to categorically identify the receptor subtype at which rucaparib elicits its dilatory effect. ^Δ p<0.05 as compared with the degree of dilation achieved by rucaparib in the absence of P2 antagonism. Panel C; summary of the duration of perfusion of tail artery segments necessary for relaxation plateau to be reached. Although the absolute degree of relaxation achieved was similar in the absence and presence of specific P2 receptor antagonism, the time taken for relaxation to complete was prolonged in all cases tested in tail artery. ***p<0.001 compared to time taken for relaxation plateau in the absence of P2 antagonism. Panel D; representative trace of rucaparib-induced relaxation when the P2Y₁ receptor was antagonised using MRS-2179 (top), and rucaparib-induced relaxation (bottom). Bars represent mean of at least three independent experiments. Error bars represent SEM. Arteries from at least three rats were used per test.</p

P2 receptor blockade abrogates rucaparib-evoked vasodilation.

<p>Panel A; broad-spectrum antagonism of P2 receptors using suramin (inverted open triangles) abrogated rucaparib-evoked relaxation in rat tail artery (left) and aorta (right). Panel B; suramin itself was without vasoactivity, demonstrable by its failure to alter the tone of sub-maximally constricted vessels. The shaded region represents the degree of constriction evoked using 10 μM PE alone. Panel C; suramin has little effect on vasodilation elicited by nicotinamide and none on vasodilation elicited by ML-9, but inhibits that elicited by rucaparib in tail artery and aorta. **p<0.01, ***p<0.001 as compared with dilation achieved in the absence of suramin. Arteries from at least three rats were used per test.</p

Rucaparib-mediated vasodilation of rat vascular tissue may be partially dependent on myosin light chain kinase.

<p>Panel A; rucaparib (closed squares), nicotinamide (open squares) and ML-9 (open triangles) inhibit smooth muscle contraction in PE-constricted rat tail artery. Artery sections were constricted using 10 μM PE before perfusion with a solution containing 10 μM PE plus the relevant concentration of drug. Panel B; rucaparib, nicotinamide and ML-9 inhibit smooth muscle contraction in PE-constricted rat aorta. Panel C; inhibition of arterial smooth muscle contraction by rucaparib is dependent on a mechanism in addition to MLCK inhibition. Constricted vessel segments were relaxed to the maximal degree achievable with ML-9, before being challenged with a relaxing cocktail of ML-9 plus rucaparib. The histograms illustrate the additive effects that were observed in the cases of both tail artery (left) and aorta (right). ** p<0.01, *** p<0.001 versus relaxation evoked by rucaparib alone; ^ΔΔ p<0.01 versus ML-9 alone. Bars represent mean of at least three independent experiments. Arteries from at least three rats were used per test. Error bars represent SEM. Panel D; rucaparib (closed squares) inhibits MLCK activity with ten times the potency of ML-9 (open triangles). Kinase activity was analyzed using the Millipore IC₅₀<i>Profiler</i> Express service. Points represent results of duplicate experiments. Error bars represent SEM.</p

Summary of rucaparib activity in PE-constricted human tumor-associated vasculature.

<p>^aData presented represents the magnitude of vessel relaxation in response to the top concentration of rucaparib tested, except A5, where the second highest concentration is summarised.</p><p>^bVessel section B1 was constricted with PE as the ‘A’ vessels; B2 contracted spontaneously, so is not summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118187#pone.0118187.t001" target="_blank">Table 1</a>.</p><p>Summary of rucaparib activity in PE-constricted human tumor-associated vasculature.</p