Development of an LC-MS/MS-based method for quantification and pharmacokinetics study on SCID mice of a dehydroabietylamine-adamantylamine conjugate, a promising inhibitor of the DNA repair enzyme.

Earlier, it was found that the agent KS-389, a conjugate of dehydroabietylamine and 1-aminoadamantane, possess inhibiting activity with regard to Tdp1. It this study, LC-MS/MS-based methods of quantification of KS-389 in mice blood and several organs (brain, liver and kidney) were developed and validated. Validation of the methods was performed according to the guidelines of U.S. Food and Drug Administration and European Medicines Agency in terms of selectivity, linearity, accuracy, precision, recovery, matrix effect, stability and carry-over. Dried blood spots (DBS) method was used for blood sample preparation. HPLC separation was performed on a reversed-phase column; the total analysis time was 12 min. Mass spectral detection was performed on a 6500 QTRAP mass spectrometer in multiple reaction monitoring mode. Transitions 463.5→135.1/107.2 and 336.2→332.2/176.2 were scanned for KS-389 and 2,5-bis(4-diethylaminophenyl)-1,3,4-oxadiazole used as the internal standard, respectively. Pharmacokinetics of the compound as well as its distribution in the organs were studied on SCID mice after intraperitoneal administration of the substance at a dose of 5 mg/kg, and it was found that its maximum concentration in blood is reached in 1-1.5 h and was 80 ng/mL. The maximum concentration in all organs is reached after the same time and is approximately 1500 ng/g and 1100 ng/g in liver and kidney, respectively. This is the first report on the pharmacokinetics of Tdp1 inhibitor based on dehydroabietylamine and 1-aminoadamantane after a single administration to mice. Also, the substance was found to be able to penetrate the blood-brain barrier which is important for, and its maximum concentration was c.a. 25-30 ng/g. These results are important for glioma treatment and make it promising for this purpose.

Efficacy of the new therapeutic approach in curing malignant neoplasms on the model of human glioblastoma.

OBJECTIVE: Glioma is a highly invasive tumor, frequently disposed in essential areas of the brain, which makes its surgical excision extremely difficult; meanwhile adjuvant therapy remains quite ineffective. METHODS: In the current report, a new therapeutic approach in curing malignant neoplasms has been performed on the U87 human glioblastoma model. This approach, termed "Karanahan", is aimed at the eradication of cancer stem cells (CSCs), which were recently shown to be capable of internalizing fragments of extracellular double-stranded DNA. After being internalized, these fragments interfere in the process of repairing interstrand cross-links caused by exposure to appropriate cytostatics, and such an interference results either in elimination of CSCs or in the loss of their tumorigenic potency. Implementation of the approach requires a scheduled administration of cytostatic and complex composite double-stranded DNA preparation. RESULTS: U87 cells treated in vitro in accordance with the Karanahan approach completely lost their tumorigenicity and produced no grafts upon intracerebral transplantation into immunodeficient mice. In SCID mice with developed subcutaneous grafts, the treatment resulted in reliable slowing down of tumor growth rate (P < 0.05). In the experiment with intracerebral transplantation of U87 cells followed by surgical excision of the developed graft and subsequent therapeutic treatment, the Karanahan approach was shown to reliably slow down the tumor growth rate and increase the median survival of the mice twofold relative to the control. CONCLUSIONS: The effectiveness of the Karanahan approach has been demonstrated both in vitro and in vivo in treating developed subcutaneous grafts as well as orthotopic grafts after surgical excision of the tumor.

Oncolytic Effect of Adenoviruses Serotypes 5 and 6 Against U87 Glioblastoma Cancer Stem Cells.

BACKGROUND/AIM: Oncolytic adenoviruses are promising therapeutic agents against both the bulk of tumor cells and cancer stem cells. The present study intended to test the oncolytic capability of adenovirus serotype 6 (Ad6), which has a lower seroprevalence and hepatotoxicity relatively to adenovirus 5 (Ad5), against the glioblastoma and its cancer stem cells. MATERIALS AND METHODS: Oncolytic efficacy of Ad6 was compared to widespread Ad5 both in vitro and in vivo, using the U87 and U251 human glioblastoma cell lines and subcutaneously transplanted U87 cells in SCID mice, respectively. RESULTS: Ad6 had a dose-dependent cytotoxicity toward glioblastoma cells in vitro and its intratumoral injections lead to a significant (p<0.05) decrease in volume of U87 xenografts, similarly to Ad5. Based on the innate capability of glioblastoma cancer stem cells to internalize a fluorescent-labeled double-stranded DNA probe, the spatial localization of these cells was estimated and it was shown that the number of cancer stem cells tended to decrease under adenovirus therapy as compared to the control group. CONCLUSION: Ad6 was shown to be a promising agent for treating glioblastomas.

Gene expression profiling of tumor-initiating stem cells from mouse Krebs-2 carcinoma using a novel marker of poorly differentiated cells.

Using the ability of poorly differentiated cells to natively internalize fragments of extracellular double-stranded DNA as a marker, we isolated a tumorigenic subpopulation present in Krebs-2 ascites that demonstrated the features of tumor-inducing cancer stem cells. Having combined TAMRA-labeled DNA probe and the power of RNA-seq technology, we identified a set of 168 genes specifically expressed in TAMRA-positive cells (tumor-initiating stem cells), these genes remaining silent in TAMRA-negative cancer cells. TAMRA+ cells displayed gene expression signatures characteristic of both stem cells and cancer cells. The observed expression differences between TAMRA+ and TAMRA- cells were validated by Real Time PCR. The results obtained corroborated the biological data that TAMRA+ murine Krebs-2 tumor cells are tumor-initiating stem cells. The approach developed can be applied to profile any poorly differentiated cell types that are capable of immanent internalization of double-stranded DNA.

Identification of cancer stem cells and a strategy for their elimination.

It has been established previously that up to 40% of mouse CD34(+) hematopoietic stem cells are capable of internalizing exogenous dsDNA fragments both in vivo and ex vivo. Importantly, when mice are treated with a combination of cyclophosphamide and dsDNA, the repair of interstrand crosslinks in hematopoietic progenitors is attenuated, and their pluripotency is altered. Here we show for the first time that among various actively proliferating mammalian cell populations there are subpopulations capable of internalizing dsDNA fragments. In the context of cancer, such dsDNA-internalizing cell subpopulations display cancer stem cell-like phenotype. Furthermore, using Krebs-2 ascites cells as a model, we found that upon combined treatment with cyclophosphamide and dsDNA, engrafted material loses its tumor-initiating properties which we attribute to the elimination of tumor-initiating stem cell subpopulation or loss of its tumorigenic potential.