Evaluation of TK1 targeting carboranyl thymidine analogs as potential delivery agents for neutron capture therapy of brain tumors
Introduction
Studies on the design and synthesis of boron-containing nucleoside analogs potential delivery agents for boron neutron capture therapy (BNCT) were initiated in the early 1990s by Soloway and his co-workers (Wyzlic et al., 1994, Soloway et al., 1998). More recently, Tjarks and his research team have designed and synthesized a second generation panel of 3-carboranyl thymidine analogs (3CTAs) as substrates of thymidine kinases (TK), and these are described in a review on the chemistry of the 3CTAs (Khalil et al., 2013). TK1 is widely distributed and expressed in all neoplastic cells, but it is virtually absent in normal cells. TK1 and TK2 catalyze the transfer of γ-phosphate groups from ATP to the 5′ hydroxyl groups of the respective nucleosides. The expression of TK1 is tightly regulated during the cell cycle, and the active enzyme is found only in S-phase cells (Arner and Eriksson, 1995, Persson et al., 1985). Enzyme kinetic and cell culture studies have been carried out on a selected number of 3CTAs designated N4, N5, and N7 and the corresponding dihydroxypropyl derivatives, N4-2OH, N5-2OH and N7-2OH (Al-Madhoun et al., 2004, Barth et al., 2004). Of these compounds, N5-2OH (3-[5-{2-(2,3-dihydroxyprop-1-yl)-o-carboran-1-yl}pentan-1-yl]thymidine) (Fig. 1) had the most favorable in vitro properties, which included high phosphorylation by TK1, low intrinsic toxicity to normal and malignant cells, and high, TK1-dependent, cellular uptake and retention. Based on these properties, we carried out in vivo studies to evaluate N5-2OH as a boron delivery agent for BNCT using the RG2 rat glioma model (Barth et al., 2008) and in that study there was convincing evidence of therapeutic efficacy. A significant increase in mean survival time (MST) was seen in animals that had received intracerebral (i.c.) administration of N5-2OH compared to that obtained with animals that had received intravenous (i.v.) BPA (45.6±7.2 d versus 35.0 ±3.3 d). Furthermore, the combination of i.c. N5-2OH and i.v. BPA resulted in an increased MST of 52.9 ±8.9 d. In the present report we describe our studies using another rat brain tumor model, the F98 rat glioma (Barth and Kaur, 2009), which has been used extensively by us to evaluate a variety of boron delivery agents for BNCT (Barth et al., 1997, Barth et al., 2000), and contrary to our previous report, there was only a modest increase in MST compared to irradiated controls.
Section snippets
Target validation
Target validation was established by means of Western blot analysis of TK1 protein expression in the F98 glioma and the L929 wildtype TK1+ and mutant TK1− cell lines. Total cell lysates, prepared using T-PER tissue protein extraction reagent (ThermoScientific), were resolved by electrophoresis on sodium dodecylsulfate (SDS)-polyacrylamide gel (4–20%) and transferred onto polyvinylidine difluoride membranes (PVDF) (Bio-Rad). Western blotting was performed using TK1 primary antibodies (Novus
Target validation, in vivo biodistribution studies and dosimetry in glioma bearing rats
As shown in Fig. 2, Western blots detected TK1 enzyme expression in the F98 glioma was approximately 2× higher than the TK1(+) L929 cell line (3.6 vs 1.7). In contrast, normal brain cells have low expression of TK1 since they usually are not in the S phase of the cell cycle. Boron concentrations in tumor, brain and blood of F98 glioma bearing rats (Table 1), following i.c. administration of N5-2OH, were carried out in separate groups of animals prior to the initiation of therapy studies. The
Discussion
We previously have reported that N5-2OH was effective as a boron delivery agent for BNCT in RG2 glioma bearing rats (Barth et al., 2008). The MSTs of rats that received i.c. N5-2OH alone or in combination with i.v. BPA were 45.6 ±7.2 d and 52.9 ±8.9 d, respectively, compared to 35.9 d for BPA alone and 28.1 d for irradiated controls. The purpose of the present study was to evaluate N5-2OH using another rat brain tumor model, the F98 glioma, which has been used extensively by us to evaluate varying
Acknowledgments
This paper is dedicated to the memory of our friend and colleague Kent Riley, who passed away in June 2014.
The studies described in this report were supported in part by National Institutes of Health grant 5 R01 CA127935 and funds from the Kevin J. Mullin Memorial Fund for Brain Tumor Research. We thank Michele Swindall for technical assistance, Kevin Tordoff and Melvin L. Moeschberger for statistical evaluation of the animal data, Xiao Koi Mo for evaluation of the unpublished in vitro cell
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- 1
Present address: Changzhou, China.
- 2
Present address: Soil and Environmental Chemistry Laboratory, School of Environmental and Natural Resources, The Ohio State University, USA.
- 3
Present address: College of Pharmacy, Korea University, Sejong, Republic of Korea.
- 4
Present address: Department of Radiology, Mount Auburn Hospital, Cambridge, MA 02138, USA.
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Deceased June 30, 2014.