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. 2014 May;16(5):432-40.
doi: 10.1016/j.neo.2014.05.005. Epub 2014 Jun 23.

PARPi-FL--a fluorescent PARP1 inhibitor for glioblastoma imaging

Christopher P Irwin  1 , Yasiri Portorreal  1 , Christian Brand  1 , Yachao Zhang  1 , Pooja Desai  1 , Beatriz Salinas  1 , Wolfgang A Weber  2 , Thomas Reiner  3
Affiliations

PARPi-FL--a fluorescent PARP1 inhibitor for glioblastoma imaging

Christopher P Irwin et al. Neoplasia. 2014 May.

Abstract

New intravital optical imaging technologies have revolutionized our understanding of mammalian biology and continue to evolve rapidly. However, there are only a limited number of imaging probes available to date. In this study, we investigated in mouse models of glioblastoma whether a fluorescent small molecule inhibitor of the DNA repair enzyme PARP1, PARPi-FL, can be used as an imaging agent to detect glioblastomas in vivo. We demonstrated that PARPi-FL has appropriate biophysical properties, low toxicity at concentrations used for imaging, high stability in vivo, and accumulates selectively in glioblastomas due to high PARP1 expression. Importantly, subcutaneous and orthotopic glioblastoma xenografts were imaged with high contrast clearly defining tumor tissue from normal surrounding tissue. This research represents a step toward exploring and developing PARPi-FL as an optical intraoperative imaging agent for PARP1 in the clinic.

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Figures

Figure 1
Figure 1
Structure of Olaparib precursor and PARPi-FL biophysical properties. (A) Molecular structures of Olaparib and PARPi-FL. Green: fluorescent BODIPY-FL component of PARPi-FL; (B) Key pharmacokinetic parameters of PARPi-FL and Olaparib in mice; aMenear et al. 2008 , bReiner et al. 2012 ; cThurber et al. 2013 .
Figure 2
Figure 2
In vitro toxicity of PARPi-FL and Olaparib. (A,B) Percent of surviving U87 and U251 cells incubated with varying amounts of PARPi-FL or the parent drug Olaparib for 7 days based on MTT assays; (C) IC50 values obtained from MTT assays for U87 and U251, treated with PARPi-FL or Olaparib; (D) Representative images of clonogenic assay; (E) Percent surviving U87 cells or (F) U251 cells after incubation in 10 μM PARPi-FL or 10 μM Olaparib for 24 h based on clonogenic assays.
Figure 3
Figure 3
Metabolic stability of PARPi-FL. (A) Fluorescence-HPLC trace of PARPi-FL (green) and metabolites (red) in mouse blood at different time points post injection of PARPi-FL (200 μL of 19.5% 1:1 dimethylacetamide (DMAC):Kolliphor, 3.5% DMSO, 77% PBS); (B) Concentration of PARPi-FL and metabolites at different time points post injection; (C) Percent intact PARPi-FL in mouse blood at different time points after injection.
Figure 4
Figure 4
Pharmacokinetics of PARPi-FL in mice. (A) U87 tumor, (B) brain and (C) muscle fluorescence intensities above baseline measured from 5 to 720 minutes after injection of PARPi-FL (200 μL of 19.5% 1:1 DMAC:Kolliphor, 3.5% DMSO, 77% PBS). Baseline fluorescence was determined by imaging nude mice injected with vehicle only.
Figure 5
Figure 5
PARPi-FL tumor accumulation and PARP1 expression. (A) Representative images of tumor, muscle and brain tissue 2 h after injection of PARPi-FL (200 μL of 19.5% 1:1 DMAC:Kolliphor, 3.5% DMSO, 77% PBS) or vehicle only imaged ex vivo; (B) Fluorescence quantification of U87 xenograft, U251 xenograft, muscle and brain tissue; (C) PARP1 Western Blot of imaged organs.
Figure 6
Figure 6
PARPi-FL imaging of U87 tumors with and without prior injection of olaparib. (A) Representative white light, fluorescence, and overlay images of U87 tumor tissues which were injected with either vehicle alone, PARPi-FL (2.5 mg/kg, 200 μL of 19.5% 1:1 DMAC:Kolliphor, 3.5% DMSO, 77% PBS) or olaparib/PARPi-FL (125 mg/kg olaparib in 100 μL of 7.5% DMSO, 12.5% Cremophor, 80% PBS, followed 30 minutes later by 2.5 mg/kg PARPi-FL in 200 μL of 19.5% 1:1 DMAC:Kolliphor, 3.5% DMSO, 77% PBS); (B) Fluorescence quantification of U87 xenografts from panel 6A; (C–E) Fluorescence microscope imaging of tumor tissues in panel 6A, confirming nuclear uptake of PARPi-FL in non-blocked tumor tissues, but not in the vehicle or Olaparib pre-treatment groups.
Figure 7
Figure 7
Orthotopic brain tumor imaging with PARPi-FL. (A) White light, fluorescence, and overlay images of healthy brain and orthotopic tumor-bearing brain imaged with an IVIS spectrum fluorescence imaging system 1 hour post intravenous injection (200 μL of 19.5% 1:1 DMAC:Kolliphor, 3.5% DMSO, 77% PBS); (B) Profile plot of fluorescence intensity across orthotopic tumor-bearing brain, healthy brain, and background; (C) H&E stained tumor-bearing brain section from A; (D) High resolution image of tumor shown in C; Radiant efficiency = (emission light (photons/sec/cm2/str))/(excitation light (mW/cm2)).
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