Vasoactivity of AG014699, a clinically active small molecule inhibitor of poly(ADP-ribose) polymerase: a contributory factor to chemopotentiation in vivo?

Abstract

Purpose: Poly(ADP-ribose) polymerase (PARP) plays an important role in DNA repair, and PARP inhibitors can enhance the activity of DNA-damaging agents in vitro and in vivo. AG014699 is a potent PARP inhibitor in phase II clinical development. However, the range of therapeutics with which AG014699 could interact via a DNA-repair based mechanism is limited. We aimed to investigate a novel, vascular-based activity of AG014699, underlying in vivo chemosensitization, which could widen its clinical application.

Experimental design: Temozolomide response was analyzed in vitro and in vivo. Vessel dynamics were monitored using "mismatch" following the administration of perfusion markers and real-time analysis of fluorescently labeled albumin uptake in to tumors established in dorsal window chambers. Further mechanistic investigations used ex vivo assays of vascular smooth muscle relaxation, gut motility, and myosin light chain kinase (MLCK) inhibition.

Results: AG014699 failed to sensitize SW620 cells to temozolomide in vitro but induced pronounced enhancement in vivo. AG014699 (1 mg/kg) improved tumor perfusion comparably with the control agents nicotinamide (1 g/kg) and AG14361 (forerunner to AG014699; 10 mg/kg). AG014699 and AG14361 relaxed preconstricted vascular smooth muscle more potently than the standard agent, hydralazine, with no impact on gut motility. AG014699 inhibited MLCK at concentrations that relaxed isolated arteries, whereas AG14361 had no effect.

Conclusion: Increased vessel perfusion elicited by AG014699 could increase tumor drug accumulation and therapeutic response. Vasoactive concentrations of AG014699 do not cause detrimental side effects to gut motility and may increase the range of therapeutics with which AG014699 could be combined with for clinical benefit.

Figure 1. The different effects of AG014699 on TMZ-induced growth inhibition of SW620 cells in vitro and xenografts in vivo

(A) TMZ alone (filled circles, solid line) caused a concentration-dependent inhibition of cell growth that was not significantly increased by the combination with 0.4 μM AG014699 (open circles, broken line). Data are mean ± standard deviation of 5 independent experiments. (B) Growth of SW620 xenografts treated with control vehicle (filled circles, solid line), AG014699 (10 mg/kg daily ×5) alone (open circles broken line), TMZ (68 mg/kg daily ×5) alone (filled triangles, solid line) or the combination of TMZ with AG014699 at 1 mg/kg (open triangles, broken line). Data are median RTV from 5 animals/group.

Figure 2. AG014699 and AG14361 are more potent inhibitors of vessel mismatch than the positive control agent nicotinamide

The perfusion markers Hoechst and carbocyanine were administered i.v. 20 minutes apart to SW620 tumour bearing mice that had been pre-treated with AG014699 (1mg/kg), AG14361 (10mg/kg), nicotinamide (1g/kg) or saline (control). Data presented in (A) are average vessel mismatch values (n=4 per group, ± se). *p=0.03 versus saline treated control. (B) shows the impact of the agents on percentage of vessels closing (Hoechst positive only) or opening (carbocyanine positive only; average values ± se).

Figure 3. Real-time analysis of the effects of AG014699 on the accumulation of BSA-labelled with Alexa-647 in SW620 and HT29 tumours established in DWC

(A) Fluorescence (arbitrary units, FU) was monitored every minute in control (saline; open symbols) and AG014699 (1mg/kg, solid symbols) treated tumours. (B) Images correspond to the distribution of BSA-647 immediately after injection (i), immediately before AG014699 or saline administration (ii) and at the end of the experiment (iii) as illustrated on the uptake curves in (A). Bolus injections of saline via i.p. (iv) or i.v. (v) routes do not alter BSA-647 fluorescence.

Figure 4. The PARP inhibitors AG014699 and AG14361 cause relaxation in pre-constricted rat arteries ex vivo

(A) shows a representative trace of the dilatory effect of 100 μM AG014699 in 10 μM PE-constricted rat-tail artery. Evident are the constriction elicited by PE and the relaxant effect of AG014699. Note also the time delay between commencement of perfusion with a particular agent and visualisation of the response, resulting from the transit time of the perfusate through the apparatus. (B) illustrates the dose responses of AG014699, AG14361 and hydralazine in constricted rat-tail artery. (C) shows a representative trace illustrating the lack of an inhibitory effect of 100 μM AG014699 in contracting rat ileum and (D) illustrates the lack of inhibitory effects of AG14361 and AG014699 on the contraction in rat ileum.

Figure 5. AG014699 inhibits myosin light chain kinase activity, but AG14361 does not

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