Synthesis and in vivo evaluation of [11C]PJ34, a potential radiotracer for imaging the role of PARP-1 in necrosis

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Abstract

Poly(ADP-ribose) polymerase-1 (PARP-1) is an abundant nuclear enzyme of eukaryotic cells that has been implicated in response to DNA injury. PARP-1 detects single-strand DNA breaks induced by a variety of genotoxic insults. A hyperactivation of PARP-1 is believed to play a critical role in tissues undergoing cellular death by necrosis. Therefore, a radiotracer that could image PARP-1 levels with PET could provide a useful tool in measuring necrosis in a variety of pathological conditions. The phenanthridinone derivative, 2-(dimethylamino)-N-(5,6-dihydro-6-oxophenanthridin-2-yl)acetamide (PJ34), has a high affinity for PARP-1 (IC50=20 nM) and is a suitable lead compound for PET radiotracer development. The synthesis of [11C]PJ34 was accomplished by base-catalyzed reaction of the corresponding des-methyl precursor, N-(5,6-dihydro-6-oxophenanthridin-2-yl)-2-(methylamino)acetamide with [11C]methyl iodide in DMF. The radiolabeling yield was 60% and the specific activity was ∼2000 mCi/μmol (decay corrected to E.O.B.). The total radiosynthesis time was approximately 50 min. Preliminary in vivo biodistribution studies in a rodent model of diabetes indicate that [11C]PJ34 displays a high uptake in tissues where PARP-1 is hyperactivated. These data indicate that [11C]PJ34 may be a useful radiotracer for imaging tissues undergoing cellular death via necrosis.

Introduction

Cellular death has been shown to occur through two different distinct biochemical pathways, apoptosis and necrosis [1], [2]. Apoptosis is a tightly regulated mode of cellular death that does not result in injury to neighboring cells, nor elicit an inflammatory response. Necrosis, on the other hand, is a catastrophic, unregulated mode of cell death that is followed by the invasion of inflammatory cells [1]. Although apoptosis and necrosis are caused by distinct biochemical pathways, there is a great deal of experimental evidence to suggest that both processes are observed in diseases characterized by abnormal cellular death. The development of noninvasive imaging procedures for measuring apoptosis and necrosis could provide valuable information regarding the relative contribution of each in tissues undergoing abnormal cellular death.

The development of radiotracers for imaging cellular death has largely focused on use of radiolabeled annexin V, which measures phosphatidyl serine (PS) exposed to the extracellular membrane leaflet during the early stages of programmed cell death [2], [3], [4]. However, since PS inversion occurs in both apoptosis and necrosis, this imaging strategy is not able to differentiate between these two functionally different forms of cellular death [2]. The development of imaging strategies that can differentiate apoptosis from necrosis will require targeting enzymatic pathways that are specific for each mechanism of cellular death.

Poly(ADP-ribose) polymerase-1 (PARP-1) is an abundant nuclear enzyme of eukaryotic cells; it consumes nicotinamide adenine dinucleotide (NAD) in response to DNA strand breaks. PARP-1 detects single-strand DNA breaks induced by a variety of genotoxic insults. Upon binding to DNA strand breaks, PARP-1 catalyzes the transfer of ADP ribose units from NAD+ to a variety of nuclear proteins, including DNA polymerase and histones. The physiological function of PARP-1 is not completely understood, but it is currently believed that PARP-1 plays a key role in DNA repair, cell differentiation, DNA replication and control of the cell cycle. On the contrary, the role of increased PARP-1 activation in a variety of pathological conditions has been well established. In conditions of severe DNA injury, hyperactivation of PARP-1 accelerates NAD+ consumption and decreases in cellular ATP content [1], [5]. The hyperactivation of PARP-1 levels and subsequent depletion of NAD+ pools is currently believed to be the mechanism responsible for cellular death via necrosis. Therefore, PARP-1 is thought to play a major role in a variety of pathological conditions in which necrosis has been observed, including ischemia-reperfusion injury (i.e., myocardial infarction and stroke [6]), septic shock [7], [8], inflammation [7], [9], diabetic cardiomyopathy [10], [11], [12], [13], [14], [15], [16], [17] and neurodegeneration [18]. The measurement of PARP-1 activity is currently limited to the use of immunohistochemistry techniques in tissue slices. The development of a radiotracer that can image PARP-1 activity with PET could provide an alternative method for studying the role of this enzyme in a variety of pathological conditions.

Previous studies have shown that the phenanthridinone derivative, PJ34, has a high affinity for PARP-1 [10]. The presence of the N,N-dimethyl moiety in the structure of PJ34 indicates that it could be labeled carbon-11 via N-alkylation of the des-methyl precursor, 2-(methylamino)-N-(5,6-dihydro-6-oxophenanthridin-2-yl)acetamide, 3, with [11C]methyl iodide ([11C]MeI). In this paper, we report the synthesis of precursor 3 and the radiolabeling conditions used to prepare [11C]PJ34 via N-alkylation of 3 with [11C]MeI. Biodistribution studies were also conducted in streptozotocin (SZT)-treated rats, an animal model of type I diabetes that involves a hyperactivation of PARP-1 leading to necrosis in the islet cells of the pancreas [19]. The results of the current study indicate that [11C]PJ34 may be useful in imaging PARP-1 activity in tissues undergoing necrosis.

Section snippets

General

DMF and sodium hydroxide were purchased from Sigma-Aldrich USA (St. Louis, MO). Other chemicals were obtained from standard commercial sources and were of analytical grade. All reactions were carried out under an inert nitrogen atmosphere with dry solvents using anhydrous conditions unless otherwise stated. Reagent grade solvents were used without further purification. Flash column chromatography was conducted using Scientific Adsorbents, silica gel, 60a, “40 Micron Flash” (32–63 μm). Melting

Chemistry

The phenanthridinone derivative, 2-(dimethylamino)-N-(5,6-dihydro-6-oxophenanthridin-2-yl)acetamide, possesses an N-dimethyaminoacetamido group, which gives easy access to 11C-labeling by N-methylation of the corresponding des-methyl compound with [11C]MeI. The synthesis of the N-des-methyl precursor and authentic PJ34 was accomplished from phenanthridin-6(5H)-one, using the sequence of reactions outlined in Scheme 1. Treatment of phenanthridin-6(5H)-one with 90% nitric acid in acetic acid gave

Discussion

Previous studies have shown that the key molecular event in the onset of cellular death by necrosis is the hyperactivation of the enzyme, PARP-1 [1]. Since PARP-1 is cleaved and inactivated by caspase-3 in cells undergoing apoptosis, radiotracers that can measure increased levels of PARP-1 activity relative to surrounding normal tissue should provide a means of detecting cells undergoing cellular death via necrosis [2].

The phenanthridinone derivative, PJ34 [10], inhibits PARP-1 by competing for

Conclusion

The PARP-1 inhibitor PJ34 was radiolabeled with [11C]MeI using sodium hydroxide as a base catalyst. The radiolabeling yield was >60% and the specific radioactivity of the final product was ∼2000 mCi/μmol (decay corrected to E.O.B.). Given the high affinity of this compound for inhibiting PARP-1 activity (IC50=20 nM) and the high uptake of the radiotracer in tissues known to undergo necrosis following treatment with the toxin, STZ, [11C]PJ34 appears to be a promising radiotracer for the

Acknowledgments

This work was supported by grant HL1385 awarded by the National Institutes of Health. The immunochemistry of PAR was supported in part by Digestive Disease Research Cores Center in Washington University School of Medicine with NIH grant P30 DK52574. The authors would like to thank Terry Sharp, Lynne Jones and Pat Margenau for their excellent technical assistance. We would also like to thank Dr. Jinbin Xu for conducting the statistical analysis of the biodistribution data.

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