Pharmacokinetic study of Tdp1 inhibitor resulted in a significant increase in antitumor effect in the treatment of Lewis lung carcinoma in mice by its combination with topotecan.

We have previously shown that the Tdp1 inhibitor, enamine derivative of usnic acid, the agent OL9-116, enhances the antitumor activity of topotecan. In the present study, we developed and validated LC-MS/MS method for the quantification of OL9-116 in mouse whole blood and studied pharmacokinetics of the agent. The substance OL9-116 was shown to be stable in the whole blood in vitro. Sample preparation included two steps: mixing 10 µL of a blood sample with 10 µL of 0.2 M ZnSO4 aqueous solution, followed by protein precipitation with 100 µL of acetonitrile containing internal standard. Quantification of the compound was performed using SCIEX 6500 QTRAP mass spectrometer in MRM mode following chromatographic separation on a C8 reversed-phase column. The method was validated in terms of selectivity, linearity, accuracy, precision, recovery, and stability of the prepared sample. When the agent OL9-116 was administered intragastrically at a dose of 150 mg/kg, the maximum concentration in the blood (about 5000 ng/mL) was reached after 2-4 h followed by the distribution and elimination of the compound. A study of the antitumor activity of a combination of OL9-116 and topotecan against Lewis lung carcinoma revealed that administration of topotecan 3 h after OL9-116 resulted in the most pronounced antitumor effect compared to simultaneous or individual administration of both compounds.

Folate-Equipped Cationic Liposomes Deliver Anti-MDR1-siRNA to the Tumor and Increase the Efficiency of Chemotherapy.

In this study, we examined the in vivo toxicity of the liposomes F consisting of 1,26-bis(cholest-5-en-3-yloxycarbonylamino)-7,11,16,20-tetraazahexacosan tetrahydrochloride, lipid-helper 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine and folate lipoconjugate (O-{2-[rac-2,3-di(tetradecyloxy)prop-1-yloxycarbonyl]aminoethyl}-O'-[2-(pteroyl-L-glutam-5-yl)aminoethyl]octadecaethyleneglycol) and investigated the antitumor effect of combined antitumor therapy consisting of MDR1-targeted siMDR/F complexes and conventional polychemotherapy using tumor xenograft initiated in immunodeficient mice. Detailed analysis of acute and chronic toxicity of this liposomal formulation in healthy C57BL/6J mice demonstrated that formulation F and parent formulation L (without folate lipoconjugate) have no acute and chronic toxicity in mice. The study of the biodistribution of siMDR/F lipoplexes in SCID mice with xenograft tumors formed by tumor cells differing in the expression level of folate receptors showed that the accumulation in various types of tumors strongly depends on the abandons of folate receptors in tumor cells and effective accumulation occurs only in tumors formed by cells with the highest FR levels. Investigating the effects of combined therapy including anti-MDR1 siRNA/F complexes and polychemotherapy on a multidrug-resistant KB-8-5 tumor xenograft in SCID mice demonstrated that siMDR/F increases the efficiency of polychemotherapy: the treatment leads to pronounced inhibition of tumor growth, reduced necrosis and inflammation, and stimulates apoptosis in KB-8-5 tumor tissue. At the same time, it does not induce liver toxicity in tumor-bearing mice. These data confirm that folate-containing liposome F mediated the extremely efficient delivery of siRNA in FR-expressing tumors in vivo and ensured the safety and effectiveness of its action.

A novel pro-apoptotic effector lactaptin inhibits tumor growth in mice models.

Lactaptin, a human milk-derived protein, induces apoptosis in cultured tumor cells. We designed a recombinant analog of lactaptin (RL2) and tested its antitumor activity. The sensitivity of hepatocarcinoma A-1 (HA-1), Lewis lung carcinoma, and Ehrlich carcinoma to RL2 were tested to determine the most reliable in vitro animal model. HA-1 cells, which had the highest sensitivity to RL2, were transplanted into A/Sn mice to investigate RL2 antitumor activity in vivo. Investigation of the molecular effects of RL2 shows that RL2 induces apoptotic transformation of HA-1 cells in vitro: phosphatidylserine translocation from inner side of the lipid bilayer to the outer one and dissipation of the mitochondrial membrane potential. Repetitive injections of RL2 (5-50 mg/kg) for 3-5 days effectively inhibited ascites and solid tumor transplant growth when administered intravenously or intraperitoneally, without obvious side effects. The solid tumor inhibitory effect of RL2 (5 i.v. injections, cumulative dose 125 mg/kg) was comparable with that of cyclophosphamide at a therapeutic dose (5 i.v. injections, cumulative dose 150 mg/kg). In combination therapy with cyclophosphamide, RL2 had an additive antitumor effect for ascites-producing tumors. Histomorphometric analysis indicated a three-fold reduction of spontaneous metastases in the liver of RL2-treated mice with solid tumor transplants in comparison with control animals. Repeated RL2 treatment substantially prolonged the lifespan of mice with intravenously injected tumor cells. Recombinant analog of lactaptin effectively induced apoptosis of tumor cells in vitro and suppressed the growth of sensitive tumors and metastases in vivo.

Short double-stranded RNA with immunostimulatory activity: sequence dependence.

Small interfering RNAs (siRNA) are able to activate the mammalian innate immune system depending on their structure, sequence, and method of delivery. The immunostimulatory activity of double-stranded RNA can be applied to antiviral and antitumor therapy. Here we identified a set of 19-bp RNA duplexes with 3-nucleotid overhangs in the 3' ends that display immunostimulating activity (here and after immunostimulating RNA, or isRNA) and studied their sequence/activity relationships. It was found that the introduction of substitutions in the middle part of the isRNA sequence (10-16 positions counting from the 5' end of strand 1) does not alter the antiproliferative activity, while substitutions in the 3' end region of isRNA substantially reduce it. isRNAs efficiently inhibit the proliferation of human oral epidermoid carcinoma cells [half-maximal inhibitory concentration (IC(50)) values varied from 10 to 100 nM]. Our research demonstrated that antiproliferative effects of isRNAs are related to cell growth arrest, rather than the induction of apoptosis. These isRNAs strongly stimulate the synthesis of interferon-α (IFN-α), and to a lesser extent the synthesis of tumor necrosis factor (TNF-α) and interleukin-6 (IL-6), in adherent peripheral blood mononuclear cells. An intravenous injection of isRNA/Lipofectamine complexes into C57BL mice increases IFN-α and IL-6 levels in the blood serum up to 15-fold and 3-fold, respectively, compared to the control mice. The results obtained clearly demonstrate the pronounced immunostimulatory and antiproliferative properties of the isRNAs under study. Hence, these short double-stranded RNAs can be considered as potential agents for the therapy of oncological and viral diseases.

The siRNA targeted to mdr1b and mdr1a mRNAs in vivo sensitizes murine lymphosarcoma to chemotherapy.

BACKGROUND: One of the main obstacles for successful cancer polychemotherapy is multiple drug resistance phenotype (MDR) acquired by tumor cells. Currently, RNA interference represents a perspective strategy to overcome MDR via silencing the genes involved in development of this deleterious phenotype (genes of ABC transporters, antiapoptotic genes, etc.). METHODS: In this study, we used the siRNAs targeted to mdr1b, mdr1a, and bcl-2 mRNAs to reverse the MDR of tumors and increase tumor sensitivity to chemotherapeutics. The therapy consisting in ex vivo or in vivo application of mdr1b/1a siRNA followed by cyclophosphamide administration was studied in the mice bearing RLS40 lymphosarcoma, displaying high resistance to a wide range of cytostatics. RESULTS: Our data show that a single application of mdr1b/1a siRNA followed by treatment with conventionally used cytostatics results in more than threefold decrease in tumor size as compared with the control animals receiving only cytostatics. CONCLUSIONS: In perspective, mdr1b/1a siRNA may become a well-reasoned adjuvant tool in the therapy of MDR malignancies.

Tempol reduces the therapeutic effect of cyclophosphamide on an experimental tumour model.

The nitroxyl radical 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (Tempol) is reported to elicite some effects on different biological models. This paper studied the influence of Tempol on the therapeutic action of an alkylating agent cyclophosphamide (CP) in the transplantable murine lymphosarcoma LS. When administered exactly before CP, Tempol exerted no influence on efficacy of the tumour therapy but significantly reduced it under a single or multiple preliminary injections 1-3 days before CP. This regimen of Tempol administration is found to elevate the activity of aldehyde dehydrogenase in the liver, the enzyme which is known to reduce the yield of the activated metabolite(s) of CP.